ISBN: 978-83-60043-10-3 - eurobic9

Faculty of Chemistry 

University of Wrocław 

EUROBIC9 

9th European Biological Inorganic Chemistry Conference 

2-6 September, 2008 

Wrocław, Poland

Eurobic9, 2-6 September, 2008, Wrocław, Poland 

Front cover design: Kinga Kulon 

Front cover picture: S. Klimek (by permission) 

© Copyright by Faculty of Chemistry, University of Wrocław, 

Wrocław 2008 

ISBN: 978-83-60043-10-3 

Editing and DTP: M. Cebrat, K. Kulon, M. Łuczkowski et al. 

Printed by: Wrocławska Drukarnia Naukowa PAN Sp. z o.o. 

ul. Lelewela 4, 53-505 Wrocław; http://www.wdn.pl 

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2

International Steering Committee: 

Miguel Teixeira (Oeiras, Portugal) 

Maria Arménia Carrondo (Oeiras, Portugal) 

Henryk Kozłowski (Wrocław, Poland) 

Leonard Proniewicz (Kraków, Poland) 

Thanos Salifoglou (Thessaloniki, Greece) 

Dimitris Kessissoglou (Thessaloniki, Greece) 

Bernhard Lippert (Dortmund, Germany) 

National Organizing Committee: 

Henryk Kozłowski (Wrocław) - Chairman 

Leonard Proniewicz (Kraków) - Vice Chairman 

Małgorzata Jeżowska-Bojczuk (Wrocław) 

Teresa Kowalik-Jankowska (Wrocław) 

Lechosław Łomozik (Poznań) 

Stanisław Ołdziej (Gdańsk) 

Wanda Radecka - Paryzek (Poznań) 

Grażyna Stochel (Kraków) 

Edward Szłyk (Toruń) 

Jolanta Świątek-Kozłowska (Wrocław) 

Local Organizing Committee: 

Henryk Kozłowski - Chairman 

Justyna Brasuń 

Marek Cebrat 

Anna Janicka-Kłos 

Alicja Kluczyk 

Marek Łuczkowski 

Agnieszka Matera 

Ariel Mucha 

Wojciech Szczepanik 

Secretariat: 

Elżbieta Gumienna-Kontecka 

Kinga Kulon 


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Conference organized under the 

auspicies of the 

Rector of the University of Wrocław 

and 

Mayor of Wrocław 

Exhibitors 

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4

Tuesday 

Wednesday 

Thursday 

Friday 

Saturday 

2 September 

3 September 

4 September 

5 September 

6.09 

9.00-10.00 

PL-3 

PL-5 

PL-6 

PL-8 

10.00-10.30 

Coffee break 

Coffee break 

Coffee break 

Coffee break 

KL1 KL4 KL7 KL9 KL11 KL13 KL15 KL18 KL21 KL24 SL29 KL29 

KL2 KL5 KL8 KL10 KL12 KL14 KL16 KL19 SL21 KL25 SL30 KL30 

S6 S7 S2 

SL1 SL4 SL9 O9 O11 SL13 KL17 SL18 SL22 SL24 SL31 O29 

10.30-12.30 S1 S2 S4 

S8 S10 S11 S12 S1 S6 

O1 SL5 SL10 O10 O12 SL14 SL15 SL19 O20 SL25 SL32 O30 

O2 O4 O5 O13 O17 O21 SL26 O25 O31 

O22 O26 

12.10-13.10 Jezowska-Trzebiatowska Lecture 

12.30-14.00 

Lunch / 13:30 SBIC Council Meeting Lunch Lunch 

KL3 KL6 SL11 SL16 KL20 KL22 KL26 KL27 KL31 

SL2 SL6 SL12 SL17 SL20 KL23 SL27 KL28 KL32 

S3 S5 S9 S10 S11 S13 S14 S11 

SL3 SL7 O6 O14 O18 SL23 SL28 O27 SL33 

O3 SL8 O7 O15 O19 O23 O24 O28 SL34 

O8 O16 

Coffee break 

Coffee break Coffee break 

PL-4 PL-7 PL-9 

Free Afternoon 

PL-10 

Poster Session 1 Poster Session 2 

18.10-18.30 Eurobic9 Medal & Eurobic10 Presentation 

Closing Ceremony 

14.30-15.40 Registration S1 

15.40-16.10 

16.10-17.10 Opening Ceremony 

17.10-18.10 PL-1 

18.10-19.10 PL-2 

20.00-22:30 Banquet 

19.30-21:30 

Welcome Reception 


Metalloenzymes 

Anticancer Agents 

Drugs 

Iron 

His Proteins 

Bioinspired Catalysis 

Copper 

Metallothioneins 

Light & Life 

Metals and Nucleic Acids – Chemical and Biological Aspects 

Biomimetic Systems 

Metal Related Diseases 

Computational Aspects and Metal Containing Molecules 

Metals and Oxidation Processes 

S1 

S2 

S3 

S4 

S5 

S6 

S7 

S8 

S9 

S10 

S11 

S12 

S13 

S14 

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Roland K. O. Sigel (University of Zürich, Switzerland) - 

EUROBIC Medal 2008 

The EUROBIC Award was created when the EUROBIC conferences were established and soon settled a new 

tradition to honor promising young, and other bioinorganic chemists deserving an honor of high calibre. The first 

medallist was Fred Hagen (1994; now professor at Delft, NL), and since then every 2 years a medal was granted, 

after a basic endowment had been constituted. The subsequent medallists are Claudio Luchinat (1996), Fraser 

Armstrong (1998), Simon P. J. Albracht and Juan C. Fontecilla-Camps (2000), Peter M. H. Kroneck (2002), 

Maria Armenia Carrondo (2004), as well as Antonio Xavier (2006). 

When looking at these names of previous EUROBIC medallists in Bioinorganic Chemistry, two things become 

evident: Virtually exclusively they have come from the "Bio" side (or from physical biochemistry), and without 

exception their research was centered around metal-protein chemistry, be it mechanistic, structural, or 

spectroscopic. This year for the first time the EUROBIC medal is awarded to a young scientist who represents 

the second part in the definition of our discipline of "bioinorganic chemistry", namely "Inorganic"; i.e. the 2008 

winner is an inorganic coordination chemist by training. Moreover, and again it is a change compared to 

previous years, the expertise and research interests of this year's medallist are in the area of metal-nucleic acid 

interactions rather than that of the field of metalloproteins. 

Roland K. O. Sigel, working at the University of Zurich, Switzerland, receives the EUROBIC medal 2008 in 

recognition of his contributions to further the basic understanding of metal ion interactions with nucleic acids. 

Born in 1971 in Basel, Switzerland, Roland Sigel received his chemistry education at the University of Basel 

(Diploma) and at the University of Dortmund (Ph.D. degree in 1999). Following a postdoctoral stay at Columbia 

University, New York City, working with Anna Marie Pyle, he became Oberassistent at the Department of 

Chemistry and Biochemistry at the University of Zurich in 2003 and was after a short time promoted to Assistant 

Professor, endowed with a SNF-Förderungsprofessur of the Swiss National Science Foundation. 

As a Ph.D. student in the group of Bernhard Lippert (Dortmund), Roland Sigel has been studying interactions 

between model nucleobases and metal species, mainly of platinum, in an attempt to elucidate the influence of the 

metal on the basic characteristics of the nucleobases, such as acid-base properties and internucleobase hydrogen 

bond formation. As a result, several fundamental aspects of unexpected base pairing schemes between metalcarrying 

nucleobases were unravelled, including experimental evidence on the role of a coordinated metal on the 

strength of a Watson-Crick base pair. During his postdoctoral stay at Columbia University he moved deeply into 

the field of large RNAs and specifically that of the catalytically active ribozymes. Ever since his return to 

Switzerland (in 2003) Roland Sigel has been developing his independent research on the coordination chemistry 

of large nucleic acids, i.e. mainly RNAs, but also DNAs. A major focus of this recent work is thereby the role of 

metal ions on the folding and on the catalysis of group II intron ribozymes. 

As an inorganic chemist he tries to answer very fundamental questions, such as those of selectivity and 

specificity of metal binding and their effects on structure and function. 3-D NMR structures of important RNA 

domains are now routinely performed in the Sigel lab with the goal of finding the exact positioning of the 

nucleotides that are crucial for catalysis and, of course, of identifying the metal ions close by and their 

coordination behaviour. For a number of metal ions intrinsic affinities for particular domains of the group 2 

intron have been derived from 2D-NMR experiments by use of an iterative procedure developed in his group in 

Zürich. It is surprising to see how different metal ions interfere with each other and with the RNA, even at very 

low concentrations of the "wrong" metal ion! To understand in detail the role of metal ions on the consecutive 

steps of folding of large RNA domains is no doubt a major challenge in his future work. Most recent results on 

single group II intron molecules have thereby revealed a new paradigm in RNA folding. 

Finally, one major focus during the last few years has been the study of metal ion binding properties of small 

mono- and dinucleotides in an attempt to compare these findings with our current knowledge on metal binding to 

larger nucleic acids and to extrapolate this information, respectively. Last but not least, other areas of his 

research, often in international collaborations, include projects on a B12-dependent riboswitch of E. coli as well 

as on metal chains in the interior of nucleic acid duplexes. 

_____________________________________________________________________ 

6 

Jan Reedijk, Leiden, the Netherlands


PLENARY LECTURES 

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7


PL1. Physiological Chemistry of Cellular Systems Controlling Intracellular 

Inorganic Chemistry 

T.V. O’Halloran 

The Chemistry of Life Processes Institute, Northwestern University, Evanston, USA 

Living systems concentrate many transition metal ions to precise levels. Extensive machinery maintains an 

intracellular quota for each metal within a narrow range. The ensemble of metal concentrations is then referred 

to as the metallome of the cell. As the nature of the metallome is revealed for different types of cells, we are 

finding that a general pattern of metal ion utilization is highly conserved across evolution; however, specialized 

cells exhibit unique transition metal signatures that suggest specific functions. Intriguingly, these patterns of 

metal utilization are perturbed in many types of stress responses, infectious and neurological diseases and cancer 

cell proliferation. While most of these intracellular metals are bound tightly in a variety of well characterized 

metalloenzyme active sites, recent studies of the emerging class of metal trafficking proteins reveal new types of 

biological coordination chemistry and are opening new questions about how specialized and aberrant cells 

acquire, deploy and store these metal ions. A variety of analytical methods including ICP-MS, X-ray 

fluorescence microscopy and new fluorescent zinc-specific probes, allow for a comparison between the 

subcellular distribution of both total zinc and chelator-accessible zinc pools and thus provide insights into 

intracellular speciation. Several cases of unique metal ion signatures in mammalian physiology will be described 

including infection processes of the malaria causing parasite, Plasmodium falciparum and metal concentration 

within the rat hippocampal formation. The cellular mechanisms and physiological consequences of exceeding 

the canonical metal quotas will be discussed. 

_____________________________________________________________________ 

8

C. Luchinat 


PL2. Biological Inorganic Chemistry at an NMR Infrastructure 

Magnetic Resonance Center, University of Florence, Via Luigi Sacconi, 6, 50019 Sesto Fiorentino, Italy 

NMR is playing an important, but sometimes controversial, role in structural biology. Structures of biological 

macromolecules can be obtained both by NMR and X-ray. X-ray is the preferred technique because i) the size 

limitation is not as severe as in NMR; ii) the accuracy is generally higher; iii) it is less time-consuming. 

However, NMR provides a precious window on dynamics, and proves unique whenever dynamics is an essential 

aspect of biological function [1]. When dealing with metalloproteins, the presence of the metal offers even more 

opportunities to exploit the potential of NMR. If the metal is paramagnetic, or can be substituted with a 

paramagnetic one, the NMR parameters are altered in many ways, and these alterations contain additional 

information on both structure and dynamics [2]. In many cases, we find that the combination of X-ray and NMR 

information provides the best picture, for example to obtain hints on the metal uptake and release of metal 

storage proteins [3], or to address the problem of multidomain proteins that require, or may require, interdomain 

conformational freedom for their function [4]. By this combination of tools we address problems such as the 

mechanisms of intracellular calcium signalling (EF-hand proteins like calmodulin [5,6] and S100 proteins [7,8]), 

of extracellular hydrolytic activities carried out by matrix metalloproteinases [9-11], and of the possible 

extracellular role of EF-hand proteins themselves [8]. 

References: 

[1] M. Fragai, C. Luchinat and G. Parigi, "Four-dimensional" protein structures: examples from metalloproteins, 

Acc.Chem.Res. , 39: 909-917 (2006). 

[2] I. Bertini, C. Luchinat, G. Parigi and R. Pierattelli, NMR of paramagnetic metalloproteins, ChemBioChem, 

6: 1536-1549 (2005). 

[3] V. Calderone, C. Del Bianco, B. Dolderer, H. Echner, H.J. Hartmann, C. Luchinat, S. Mangani and U. 

Weser, The crystal structure of yeast copper thionein: The solution of a long-lasting enigma, 

Proc.Natl.Acad.Sci.USA, 102: 51-56 (2005). 

[4] I. Bertini, C. Del Bianco, I. Gelis, N. Katsaros, C. Luchinat, G. Parigi, M. Peana, A. Provenzani and M.A. 

Zoroddu, Experimentally exploring the conformational space sampled by domain reorientation in calmodulin. 

Proc.Natl.Acad.Sci.USA 101:6841-6846 (2004). 

[5] I. Bertini, Y.K. Gupta, C. Luchinat, G. Parigi, M. Peana, L. Sgheri and J. Yuan, Paramagnetism-Based NMR 

Restraints Provide Maximum Allowed Probabilities for the Different Conformations of Partially Independent 

Protein Domains, J.Am.Chem.Soc., 129, 12786-12794 (2007). 

[6] I. Bertini, P. Kursula, C. Luchinat, G. Parigi, J. Vahokoski, M. Wilmanns, J. Yuan, A combined X-ray and 

NMR structural investigation of calmodulin with DAPk and DRP1 peptides: detection of rearrangement in 

solution, submitted. 

[7] Y. Arendt, A. Bhaumik, R. Del Conte, C. Luchinat, M. Mori and M. Porcu, Fragment docking to S100 

proteins reveals a wide diversity of weak interaction sites, ChemMedChem, 2, 1648-1654 (2007). 

[8] Unpublished results from CERM 

[9] I. Bertini, V. Calderone, M. Cosenza, M. Fragai, Y.-M. Lee, C. Luchinat, S. Mangani, B. Terni and P. 

Turano, Conformational variability of MMPs: beyond a single 3D structure, Proc.Natl.Acad.Sci.USA, 102: 

5334-5339 (2005). 

[10] I. Bertini, V. Calderone, M. Fragai, C. Luchinat, M. Maletta and K.J. Yeo, Snapshots of the Reaction 

Mechanism of Matrix Metalloproteinases, Angew.Chem.Int.Ed , 45: 7952-7955 (2006). 

[11] I. Bertini, V. Calderone, M. Fragai, R. Jaiswal, C. Luchinat, M. Melikian, E. Mylonas and D.I. Svergun, 

Evidence of reciprocal reorientation of the catalytic and hemopexin-like domains of full-length MMP-12, 

J.Am.Chem.Soc., 130, 7011-7021 (2008). 

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9


PL3. Unprecedented DNA Recognition Properties and Anti-cancer Activity 

Using Nano-size Metallo-supramolecular Cylinders 

M. J. Hannon 

School of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK 

e-mail: m.j.hannon@bham.ac.uk 

Within biological systems, sequence specific DNA recognition is achieved by the surface motifs of proteins, 

which generally interact non-covalently with the major groove of DNA. We have been developing metal-based 

agents which also recognise DNA non-covalently. A particular success has come from metallo-supramolecular 

cylinders [1, 2] which are a similar size and shape to the zinc finger motifs found in certain DNA-recognition 

proteins. We have demonstrated that these synthetic tetracationic supramolecular cylinders bind strongly 

(binding constant > 107 M-1) and non-covalently to DNA and induce dramatic and unexpected intra-molecular 

DNA coiling that is unprecedented with synthetic agents and somewhat reminiscent of the effect of histones. 

Still more excitingly they can also bind at the heart of DNA Y-shaped junction structures [3]. This 

unprecedented new mode of DNA recognition not only will transform the way that scientists think about how 

molecules can bind to DNA but is itself an important biomedical target as replication forks are Y-shaped 

junctions. The agents are potent cytostatics yet do not cause unwanted genotoxic DNA damage as cis-platin: 

their effects on cancer cells will be described. 

References: 

[1] M.J. Hannon, Pure and Applied Chemistry, 2007, 79, 2243-2261; M.J. Hannon, Chem. Soc. Rev., 2007, 36, 

280-295 

[2] G. I. Pascu, A. C. G. Hotze, C. Sanchez Cano, B. M. Kariuki, M. J. Hannon, Angew. Chem., Intl. Ed., 2007, 

46, 4374-4378; A.C.G. Hotze, B.M. Kariuki and M.J. Hannon, Angew. Chem., Intl. Ed., 2006, 45, 4839-4842; 

L.J. Childs, J. Malina, B.E. Rolfsnes, M. Pascu, M.J. Prieto, M.J. Broome, P.M. Rodger, E. Sletten, V. Moreno, 

A. Rodger and M.J. Hannon, Chem. – Eur. J., 2006, 12, 4919-4927; I. Meistermann, V. Moreno, M.J. Prieto, E. 

Moldrheim, E. Sletten, S. Khalid, P.M. Rodger, J.C. Peberdy, C.J. Isaac, A. Rodger and M.J. Hannon, Proc. 

Natl. Acad. Sci., USA., 2002, 99, 5069-5074. M.J. Hannon, V. Moreno, M.J. Prieto, E. Molderheim, E. Sletten, 

I. Meistermann, C.J. Isaac, K.J. Sanders and A. Rodger, Angew. Chem., Intl. Ed., 2001, 40, 879-884. 

[3] A. Oleksy, A.G. Blanco, R. Boer, I. Usón, J. Aymami, A. Rodger, M.J. Hannon and M. Coll, Angew. Chem., 

Intl. Ed., 2006, 45, 1227-1231; L. Cerasino, M. J. Hannon, E. Sletten, Inorg. Chem., 2007, 46, 6245-6251; J. 

Malina, M. J. Hannon, V. Brabec, Chem. - Eur. J., 2007, 13, 3871-3877 

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10


PL4. High-Valent Iron(IV)-Oxo Complexes of Heme and Nonheme Ligands 

in Dioxygen Activation Chemistry 

W. Nam 

Department of Chemistry and Nano Science, Center for Biomimetic Systems, Ewha Womans University, Seoul 

120-750, Korea 

e-mail: wwnam@ewha.ac.kr 

Heme iron enzymes catalyze a diverse array of important metabolic transformations that require the binding and 

activation of dioxygen. One primary goal in cytochrome P450 research is to understand the mechanistic details 

of dioxygen activation and oxygen transfer reactions by the enzymes. Extensive mechanistic studies with the 

enzymes and iron porphyrin models resulted in proposing oxoiron(IV) porphyrin cation radical as a sole active 

oxidant that effects metabolically important oxidative transformations. Recent studies from a number of 

laboratories, however, have provided experimental evidence that in addition to the high-valent iron-oxo species, 

other oxidizing species are involved in oxidation reactions. For example, a hydroperoxo-iron(III) porphyrin 

intermediate has been proposed as a second electrophilic oxidant in cytochrome P450-catalyzed oxidations, on 

the basis of site-directed mutagenesis and radical clock experiments. The involvement of multiple oxidants has 

also been proposed in iron porphyrin reactions, mainly on the basis of competitive oxidation experiments. 

Computational studies, however, concluded that the oxidation reactions by the hydroperoxo-iron(III) porphyrin 

intermediate are energetically unfavorable, ruling out the existence of a second electrophilic oxidant. Thus there 

is an intriguing, current controversy on the involvement of multiple oxidizing species in oxygen transfer 

reactions by cytochromes P450 and iron porphyrin models. 

Mononuclear nonheme iron enzymes comprise an important group of dioxygen-activating enzymes that are 

involved in many metabolically important oxidative transformations. The mechanistic details of dioxygen 

activation and oxygen atom transfer reactions by the enzymes and their model compounds have been extensively 

studied over the past two decades, thereby proposing high-valent iron(IV)-oxo intermediates as the active 

oxidizing species. Recently, we have succeeded in obtaining a high-resolution crystal structure of an Fe(IV)=O 

intermediate with a nonheme macrocyclic ligand and reported the synthesis and reactivity studies of a number of 

nonheme iron(IV)-oxo complexes. With nonheme iron(IV)-oxo complexes firmly established by 

crystallography, significant progress has been made in the chemistry of nonheme iron(IV)-oxo intermediates 

over the past five years; ~15 nonheme iron(IV)-oxo complexes appeared in literature at the present time. In this 

presentation, I will present our recent results on the determination of the nature of active oxidant(s) in nonheme 

iron complex-mediated oxygen atom transfer reactions and the generation and reactivity studies of mononuclear 

nonheme oxoiron(IV) complexes having different axial ligands. In addition, the reactivities of mononuclear 

nonheme iron(IV)-oxo complexes in a variety of oxygenation reactions will be discussed (see below). 

Aromatic hydroxylation 

O 

S 

S-oxidation 

OH 

S 

R 

P-O 

P-oxidation 

P 

Aliphatic hydroxylation 

R 

C OH 

C H 

Nonheme Iron(IV)-Oxo Complex 

H3C N 

H 

N + HCHO 

N-dealkylation 

Alkene epoxidation 

O 

C O 

H 

C OH 

Alcohol oxidation 

Alkylaromatic oxidation 

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11


R. Krämer 

PL5. Controlling the Structure and Function 

of Modified Nucleic Acids by Metal Ions 

Anorganisch-Chemisches Institut Universität Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, 

Germany 

e-mail: roland.kraemer@urz.uni-heidelberg.de 

Nucleic acids modified with high-affinity binding sites for metal ions hold considerable potential as tools for 

RNA and DNA processing, as bioanalytical probes and as building blocks in DNA nanotechnology. 

This lecture will focus on oligonucleotides with terminally appended chelating groups, 

in particular terpyridine. These hybrid compounds become highly responsive to 

transition metal ions such as Zn 2+ and Cu 2+ at low micromolar concentration. The metal 

ions trigger the formation of circular structures and off-regulate hybridisation with 

complementary nucleic acids [1]. Application as probes to the PCR-free detection of 

nucleic acid sequences will be discussed [2]. Metal-dependent biological activity is 

exemplified by Zn 2+ -triggered uptake of a modified antisense oligonucleotide by cancer 

cells [3], inspiring the idea of a stimulus-controlled selective accumulation of 

oligonucleotide drugs in zinc-rich tissues. An unprecedented view of the interaction of a 

chelator-modifed oligonucleotide with metal ions will be given at the single molecule level [4]. 

References: 

[1] M. Göritz, R. Krämer, J. Am. Chem. Soc., 127, 18016 (2005). 

[2] N. Graf, M. Göritz, R. Krämer, Angew. Chem., 45, 4013 (2006). 

[3] A. Fuessl, A. Schleifenbaum, M. Goeritz, A. Riddell, C. Schultz, R. Krämer, J. Am. Chem. Soc., 128, 5986 

(2006). A. Fuessl, Dissertation, Universität Heidelberg (2007). 

[4] A. Kiel, J. Kovacs, A. Mokhir, R. Krämer, D.-P. Herten, Angew. Chem. Int. Ed. 46, 3363 (2007) 

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12


PL6. From the Geochemistry of Zn/S Minerals to the Inner Workings of 

Zn/S Proteins 

W. Maret 

PMCH, The University of Texas Medical Branch, 700 Harborside Drive, 77555, Galveston, TX, United States, 

e-mail: womaret@utmb.edu 

In his landmark textbook of chemistry (1827), Jöns Jakob Berzelius drew attention to the fact that the 

chemistry of living matter follows quite different laws than that of dead matter. While arguments over vitalism 

have long since abated and biochemistry is widely acknowledged as the basis for describing life processes, 

bioinorganic chemists continue to discover functional principles that are not evident from pure chemistry. A 

particularly exciting area in this regard is the biology of zinc. In zinc proteins, the interaction of zinc(II) with 

the sulfur donor of the amino acid cysteine generates a rich coordination and redox chemistry that, in the 

context of biological function, gains new significance for catalysis, structure, and regulation. New insights 

from the properties that isolated components acquire in complex biological systems inform future research in 

"bio-inspired" inorganic chemistry. 

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13


PL7. Significance of Error-avoiding Mechanisms for Oxidative DNA 

Damage in Carcinogenesis 

T. Tsuzuki 

Department of Med. Biophys. & Radiat. Biol., Faculty of Medical Sciences, Kyushu University, 1-1, Maidashi 3chome, 

Higashi-ku, 812-8582, Fukuoka, Japan 

e-mail: tsuzuki@med.kyushu-u.ac.jp 

Oxygen radicals are produced through normal cellular metabolism, and formation of such radicals is further 

enhanced by ionizing radiation and by various chemicals. Among various classes of oxidative DNA damage, 8oxo-7, 

8-dihydroguanine (8-oxoG) is the most abundant, and appears to play important roles in mutagenesis and 

carcinogenesis. Enzyme activities which may be responsible for preventing 8-oxoG-evoked mutations were 

identified in mammalian cells [1, 2]. We have focused on following the two enzymes. MTH1 (Mth1) protein is 

the mammalian counterpart of E. coli MutT protein, which hydrolyzes 8-oxo-dGTP to monophosphate in the 

nucleotide pool, thereby preventing occurrence of transversion mutations. On the other hand, MUTYH (Mutyh) 

protein, a counterpart of E. coli MutY protein, having adenine/2-hydroxyadenine DNA glycosylase activity, is 

expected to prevent G:C to T:A transversions, by excising adenine from G:A mismatches induced by 8-oxoG 

and 2-OH-A. To analyze the function of the mammalian Mth1 and Mutyh proteins in vivo, we established geneknockout 

mice for these two enzymes by gene targeting, and investigated spontaneous tumorigenesis as well as 

mutagenesis [3, 4, 5]. I will present data on spontaneous and oxidative stress-induced mutagenesis with these 

mutant mice lines. 

References: 

[1] Sekiguchi, M. & Tsuzuki, T: Oxidative nucleotide damage: consequences and prevention. Oncogene, 21, 

8895-8904 (2002). [2] Tsuzuki, T., Nakatsu, Y. & Nakabeppu, Y.: Significance of error-avoiding mechanisms 

for oxidative DNA damage in carcinogenesis. Cancer Sci., 98, 465-470 (2007). [3] Tsuzuki, T., Egashira, A., 

Igarashi, H., Iwakuma, T., Nakatsuru, Y., Tominaga, Y., Kawate, H., Nakao, K., Nakamura, K., Ide, F., Kura, S., 

Nakabeppu, Y., Katsuki, M., Ishikawa, T. & Sekiguchi, M.: Spontaneous tumorigenesis in mice defective in the 

MTH1 gene encoding 8-oxodGTPase. Proc. Natl. Acad. Sci. USA, 98 (20), 11456-11461 (2001). [4] Egashira, 

A., Yamauchi, K., Yoshiyama, K., Kawate, H., Katsuki, M., Sekiguchi, M., Sugimachi, K., Maki, H. & Tsuzuki, 

T.: Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes. DNA Repair, 1, 881-893 

(2002). [5] Sakamoto, K., Tominaga, Y., Yamauchi, K., Nakatsu, Y., Sakumi, K., Yoshiyama, K., Egashira, A., 

Kura, S., Yao, T., Tsuneyoshi, M., Maki, H., Nakabeppu, Y. & Tsuzuki, T.: MUTYH-null mice are susceptible 

to spontaneous and oxidative stress-induced intestinal tumorigenesis. Cancer Res., 67, 6599-6604 (2007). 

_____________________________________________________________________ 

14


PL8. Cytochromes P450 And Diversity : Adaptation of Living Organisms to 

Their Chemical Environment 

D. Mansuy 

Chemistry and Biochemistry, UNIVERSITE PARIS DESCARTES, 45 rue des Saints-Péres, 75270, PARIS, 

France 

e-mail: daniel.mansuy@univ-paris5.fr 

Living organisms exhibit a spectacular ability to adapt themselves to an always changing chemical environment. 

The pathways that have been selected by life evolution for aerobic organisms to metabolize and eliminate 

xenobiotics are strikingly similar. The first step of xenobiotics metabolism is most often catalyzed, in all aerobic 

organisms, by a multigene family of hemeproteins, the cytochromes P450(P450s). The P450s responsible for 

xenobiotics metabolism are able to catalyze the monooxygenation of a huge amount of compounds exhibiting an 

extreme diversity of structures. Consequently, most often, those P450s exhibit a very poor substrate selectivity ; 

however they act as efficient catalysts and, very often, lead to regioselective hydroxylations of their substrates. 

How is it possible to explain this “paradox”, which is a key element in our adaptation to variable chemical 

environments? Recent X-ray structures of mammalian P450s, published during these last four years allow one to 

start to understand the molecular basis of the adaptation of P450s to xenobiotics for the most possible efficient 

oxidation catalysis. The corresponding recent data will be presented after a brief overview of 50 years of 

research on P450s (P450 has been discovered in 1958), illustrating the species, substrate, reaction, and 

coordination chemistry diversity of these enzymes . Finally, the possible future of P450 research will be 

discussed on the basis of quite recent preliminary results 

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15


PL9. Redox-active Metal Clusters and Free Radicals in Ribonucleotide 

Reductase 

A. Gräslund 

Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories, SE-106 91, Stockholm, Sweden 

e-mail: astrid@dbb.su.se 

Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides, a reaction required for 

DNA replication and repair. Class I RNRs, in e.g. E. coli or eukaryotes, consist of two homodimeric 

components, proteins R1 and R2. R1 contains the active site, whereas R2 has an oxobridged diferric cluster, 

neighboring a tyrosyl free radical. The radical is necessary for enzyme activity, initiating long-range coupled 

electron/proton transfer (PCET) from the R1 active site to the tyrosyl radical in R2, about 35 Å away [1, 2]. 

The tyrosyl radical is generated by cleavage of molecular oxygen after binding ferrous iron at the diiron site. One 

reaction intermediate is an Fe(III)/Fe(IV) state (intermediate X), which oxidizes the neighboring tyrosine to a 

free radical. The tyrosyl radical is stable in the resting enzyme due to its shielded environment. The 

paramagnetic states of the iron cluster and the tyrosyl free radical have been studied by EPR. 

The RNR of the intracellular parasite Chlamydia trachomatis is structurally similar to class I RNRs but lacks 

tyrosyl radical. The Chlamydia enzyme defines a new RNR subclass Ic where the dimetal cluster replaces the 

tyrosyl radical [3-5]. A new mixed manganese/iron cluster in protein R2 confers high catalytic activity to the 

Chlamydia enzyme. The active state is an antiferromagnetically coupled high spin Mn(IV)/Fe(III) cluster. The 1electron 

reduced inactivated form, Mn(III)/Fe(III), gives a characteristic EPR spectrum. 

References: 

[1] Gräslund, A. and Sahlin, M. EPR and NMR studies of class I ribonucleotide reductase. Ann. Rev. Biophys. 

Biomol. Struct. 25 (1996) 259-286. 

[2] Narvaez, A.-J., Voevodskaya, N., Thelander, L. and Gräslund, A. The involvement of Arg 265 of mouse 

ribonucleotide reductase R2 protein in proton transfer and catalysis. J. Biol. Chem. 281 (2006) 26022-26028. 

[3] Högbom, M., Stenmark, P., Voevodskaya, N., McClarty, G., Gräslund, A. and Nordlund, P. The radical site 

in Chlamydial ribonucleotide reductase defines a new R2 subclass. Science 305 (2004) 245-248. 

[4] Voevodskaya, N., Narvaez, A.-J., Domkin, V., Torrents, E., Thelander, L., and Gräslund, A. Chlamydial 

ribonucleotide reductase: tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster. Proc. Natl. 

Acad. Sci. USA 103 (2006) 9850-9854. 

[5] Voevodskaya, N., Lendzian, F., Ehrenberg, A. and Gräslund, A. High catalytic activity achieved with a mixed 

manganese-iron site in protein R2 of Chlamydia ribonucleotide reductase. FEBS Lett. 581 (2007) 3351-3355. 

_____________________________________________________________________ 

16


PL10. Specific Keys to Success for Ribozymes and Riboswitches: Metal Ions 

and Metal Ion Complexes 

R.K.O. Sigel 

Institute of Inorganic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland, 

e-mail: roland.sigel@aci.uzh.ch 

RNAs are involved in many processes within living cells, ribozymes and riboswitches being only two examples 

of functional RNAs. Ribozymes perform catalytic reactions, whereas riboswitches are involved in the regulation 

of gene expression by specifically binding small metabolites. Our research focuses on the self-splicing group II 

intron ribozyme Sc.ai5γ and two riboswitches responsive to either coenzyme B12 or Mg 2+ (Figure). 

Like most group II introns, Sc.ai5γ is highly dependent on Mg 2+ and very sensitive to the presence of other M n+ 

ions [1]: Ca 2+ inhibits splicing already at very low concentrations. By a combination of biochemical [1b], NMR 

[2] and single molecule FRET experiments [3] we are now investigating the origin of Ca 2+ inhibition by 

characterizing the intrinsic metal ion binding properties of these large RNAs and the effect of different M n+ ions 

on catalysis, local structures and folding. 

The btuB riboswitch from E. coli regulates the expression of a B12 transport protein and thus the uptake of B12 

into the cell. We could recently show that the corrin moiety is responsible for the structural change of the 

riboswitch, whereas the axial ligands of B12 regulate the affinity towards the RNA [4]. We are currently 

extending these studies by investigating the role of the corrin side chains on the riboswitch structure. 

Acknowledgement: Financial support by the Swiss Nat. Sci. Found. (SNF-Förderungsprofessur PP002-114759 

and project 200021-117999) is gratefully acknowledged. 

References: 

[1] a) R.K.O. Sigel, Eur. J. Inorg. Chem., 2281 (2005). b) M.C. Erat, R.K.O. Sigel, J. Biol. Inorg. Chem., 13, doi 

10.1007/s00775-008-0390-7 (2008). c) E. Freisinger, R.K.O. Sigel, Coord. Chem. Rev., 251, 1834 (2007). 

[2] a) M.C. Erat, O. Zerbe, T. Fox, R.K.O. Sigel, ChemBioChem, 8, 306 (2007). b) M.C. Erat, R.K.O. Sigel, 

Inorg. Chem., 46, 11224 (2007). 

[3] M. Steiner, D. Rueda, R.K.O. Sigel, submitted for publication. 

[4] S. Gallo, M. Oberhuber, R.K.O. Sigel, B. Kräutler, ChemBioChem, 9, 1408 (2008). 

_____________________________________________________________________ 

17


Prof. B. Jeżowska-Trzebiatowska Lecture: 

Platinum Antitumor Chemistry: from DNA Binding to Drug Design 

J. Reedijk 

Leiden Institute of Chemistry, P.O. Box 9502, 2300 RA Leiden, the Netherlands 

e-mail: reedijk@chem.leidenuniv.nl 

Metal coordination compounds that have metal-ligand exchange rates comparable to cell-division processes, i.e. 

those of Pt and Ru, often appear to be highly active as anticancer agents, as evident from studies of several cell 

lines. The classical compound cis-diamminedichloridoplatinum(II), often abbreviated as cisplatin, and several of 

its derivatives are known to bind to DNA. Despite the fact that such compound on their way to reach DNA may 

meet and bind temporarily to other cellular components, like proteins and peptides. 

Studying the details of the binding (kinetics, structures) of such platinum compounds to nucleic acids, may not 

only lead to a better understanding of the mechanism of action, but may also result in the development of new 

drugs, based on improved knowledge of DNA binding [1]. The major chemistry facts that have led to a 

significant improvement of our insight into the mechanism of action will be discussed. 

Most recently we have been giving special attention to other metals that bind to DNA, like Cu and Ru [2], with 

most recently on the multifunctional binding of such compounds, where even a fluorescent group can be 

attached, to follow the process of the compounds in real time, while in the cells [3]. Combining our work on Cu- 

DNA binding [4] with that of platinum, has resulted in totally new compounds that can be used for both DNA 

cutting [5] and for developing new anticancer chemistry [6]. 

References: 

[1] J. Reedijk; New clues for platinum antitumor chemistry: Kinetically controlled metal binding to DNA; Proc. 

Natl. Acad. Sci. U. S. A., 100, (2003), 3611-3616. 

[2 ] K. van der Schilden, F. Garcia, H. Kooijman, A.L. Spek, J.G. Haasnoot and J. Reedijk; A highly flexible 

dinuclear ruthenium(II)-platinum(II) complex: Crystal structure and binding to 9-ethylguanine; Angew. Chem.- 

Int. Edit., 43, (2004), 5668-5670. 

[3] G.V. Kalayda, B.A.J. Jansen, P. Wielaard, H.J. Tanke and J. Reedijk; Dinuclear platinum anticancer 

complexes with fluorescent N, N'-bis(aminoalkyl)-1, 4-diamino-anthraquinones: cellular processing in two 

cisplatin-resistant cell lines reflects the differences in their resistance profiles; J. Biol. Inorg. Chem., 10, (2005), 

305-315. 

[4] P.U. Maheswari, S. Roy, H. den Dulk, S. Barends, G. van Wezel, B. Kozlevcar, P. Gamez and J. Reedijk; 

The square-planar cytotoxic [Cu II (pyrimol)Cl] complex acts as an efficient DNA cleaver without reductant; J. 

Am. Chem. Soc., 128, (2006), 710-711. 

[5] S. Özalp-Yaman, P. de Hoog, G. Amadei, M. Pitié, P. Gamez, J. Dewelle, T. Mijatovic, B. Meunier, R. Kiss 

and J. Reedijk; Platinated copper(3-clip-phen) complexes as effective DNA-cleaving and cytotoxic agents; 

Chem. Eur. J., 14, (2008), 3418-3426. 

[6] J. Reedijk; Metal-ligand exchange kinetics in platinum and ruthenium complexes; Plat. Metals Rev., 52, 

(2008), 2-11. 

_____________________________________________________________________ 

18


KEYNOTE LECTURES 

_____________________________________________________________________ 

19


KL1. Oxotransferase Enzymes: Molybdenum versus Tungsten 

and the Role of Molybdopterin 

C.D. Garner, T.S. Bhachu, L.J. Stewart, and F. Hine 

School of Chemistry, Nottingham University, University Park, Nottingham NG7 2RD, United Kingdom 

e-mail: dave.garner@nottingham.ac.uk 

W-DMSO reductase was isolated from Rhodobacter capsulatus grown phototropically in a medium 

containing 3µM Na2WO4 and 6nM Na2MoO4 [1]. The Mo and W centres of these two enzymes have the same 

structure (A) with the metal ligated by the dithiolene groups of two molybopterin (B) moieties (with R = guanine 

dinucletide), an oxo-group and the oxygen atom of serine 147. 

O S 

H 

N S 

HN 

OPO 

H2N N N O 

3H(R) 

H 

A B 

A theoretical analysis of the reaction profile for oxygen atom transfer at the Mo and W centres of R. 

capsulatus will be presented [2] and related to the catalytic behaviour observed [3]. 

Possible roles for molybdopterin in the catalyses of oxygen atom transfer by the Mo and W oxotransferase 

enzymes will be considered in the context of the properties of some chemical systems that involve ligands 

related to molybopterin. 

Acknowledgement: We thank the EPSRC and BBSRC for their financial support. 

References: 

[1] L. J. Stewart, S. Bailey, B. Bennett, J. M. Charnock, C. D. Garner and A. S. McAlpine, J. Mol. Biol., 299, 

595 (2000). 

[2] J. P. McNamara, I. H. Hillier, T. S. Bhachu, and C. D. Garner, Dalton Trans., 3572 (2005) 

[3] L. J. Stewart, S. Bailey, D. Collison, G. A. Morris, I. Preece, and C. D. Garner, ChemBioChem, 2, 703 

(2001). 

_____________________________________________________________________ 

20


KL2. Nickel Proteins as Enzymes, Biosensors, and Trafficking Shuttles: 

the Urease Story 

S. Ciurli 

Dept. Agro-Environmental Science and Technology, University of Bologna, Via Giuseppe Fanin 40, 40127, 

Bologna, Italy 

e-mail: stefano.ciurli@unibo.it 

Nickel is an essential component of the active site of several enzymes. The toxicity and scarcity of this metal ion 

in natural environments prompted many organisms to regulate its homeostasis at multiple levels. Urease, a Ni 2+ - 

enzyme of high relevance for human health and agriculture, is the final destination of the greater part of the 

intracellular Ni 2+ . The mechanisms that regulate Ni 2+ trafficking leading to urease activation represent a 

paradigm to understand the general principles of cellular Ni 2+ homeostasis [1]. Regulation of urease activity by 

Ni 2+ occurs both at the transcriptional level and at the protein level. NikR is a metal-sensor that responds to 

different concentrations of intracellular Ni 2+ , controlling the transcription of the urease operon: the latest insights 

into metal-binding and DNA-binding of the Ni 2+ sensor NikR will be illustrated [2]. The mechanism of Ni 2+ 

delivery into the apo-urease precursor will be described on the basis of the available information on the urease 

accessory proteins UreD, UreE, UreF, and UreG, which mediate the insertion of nickel into the enzyme active 

site [3-7]. 

References: 

[1] S. Ciurli, "Urease. Recent insights in the role of nickel" in Nickel and its surprising impact in nature (A. 

Sigel, H. Sigel, R. K. O. Sigel, Eds.), Metal Ions in Life Sciences, Vol. 2, John Wiley & Sons, Ltd., Chichester, 

UK, 2007; pp. 241-278 

[2] B. Zambelli; M. Bellucci; A. Danielli; V. Scarlato; S. Ciurli "The Ni 2+ -binding properties of Helicobacter 

pylori NikR" Chem. Commun. 2007, 35, 3649-3651 

[3] M. Salomone-Stagni; B. Zambelli; F. Musiani; S. Ciurli "A model-based proposal for the role of UreF as a 

GTPase activating protein in the urease active site biosynthesis" 

Proteins: Struct. Funct. Bioinform. 2007, 68, 749-761 

[4] B. Zambelli; F. Musiani; M. Savini; P. Tucker; S. Ciurli "Biochemical studies on Mycobacterium 

tuberculosis UreG and comparative modeling reveal structural and functional conservation among the bacterial 

UreG family" Biochemistry, 2007, 46, 3171-3182 

[5] P. Neyroz; B. Zambelli; S. Ciurli "The intrinsically disordered structure of Bacillus pasteurii UreG as 

revealed by steady-state and time-resolved fluorescence spectroscopy" Biochemistry, 2006, 45, 8918-8930 

[6] M. Stola; F. Musiani; S. Mangani; P. Turano; N. Safarov; B. Zambelli; S. Ciurli "The nickel site of Bacillus 

pasteurii UreE, a urease metallo-chaperone, as revealed by metal-binding studies and X-ray absorption 

spectroscopy" Biochemistry, 2006, 45, 6495-6509 

[7] B. Zambelli; M. Stola; F. Musiani; K. De Vriendt; B. Samyn; B. Devreese; J. Van Beeumen; P. Turano; A. 

Dikiy; D.A. Bryant; S. Ciurli "UreG, a chaperone in the urease assembly process, is an intrinsically unstructured 

GTPase that specifically binds Zn 2+ " J. Biol. Chem. 2005, 280, 4684-4695 

_____________________________________________________________________ 

21


KL3. Binuclear Metallohydrolases: 

Emerging Bioremediators and Drug Targets 

G. Schenk, N. Mitić, L. Gahan, S. Smith, K. Hadler 

School of Molecular and Microbial Sciences, University of Queensland, 68 Cooper Road, 4072, Brisbane, 

Australia 

e-mail: schenk@uq.edu.au 

Binuclear metallohydrolases form a large and diverse group of enzymes that are involved in biological functions 

ranging from bone resorption to signal transduction [1]. Recently, members of this group of enzymes have 

gained attention due to their ability to degrade (i) most commonly known β-lactam-based antibiotics (and thus 

contribute strongly to the emergence of drug-resistant pathogens), and (ii) organophosphate (OP) pesticides and 

nerve agents [2-4]. While the former ability makes these enzymes promising targets for the development of new 

chemotherapeutics, the latter provides the basis for the development of potent bioremediators. Here, recent 

advances in the understanding of the structure and function and biotechnological application of two illustrative 

binuclear metallohydrolases, the purple acid phosphatase (PAP; a target for anti-osteoporotic drugs) and a 

glycerophosphodiesterase (GpdQ; a promiscuous enzyme with potential as a novel bioremediator) are discussed. 

For PAP, the combination of spectroscopic, kinetic and structural data led to the proposal of a comprehensive, 

eight-step catalytic mechanism, whereby the identity of the hydrolysis-initiating nucleophile is dependent on the 

metal ion composition, the pH and the identity of the substrate [5-7]. GpdQ differs markedly from PAP in 

several aspects of its molecular mechanism. In the resting state GpdQ is mononuclear and inactive; the addition 

of a substrate triggers the formation of a catalytically competent binuclear centre. Following hydrolysis and 

product release the enzyme returns to its mononuclear resting state [7]. 

References: 

[1] Mitić, N., Smith, S.J., Neves, A., Guddat, L.W., Gahan, L.R., and Schenk, G. (2006) The catalytic 

mechanism of binuclear metallohydrolases. Chem. Rev., 106: 3338-3363. 

[2] Crowder M.W., Spencer J., and Vila A.J. (2006) Metallo-β-lactamases: novel weaponry for antibiotic 

resistance in bacteria. Acc. Chem. Res., 39: 721-728. 

[3] Raushel, F.M. (2002) Bacterial detoxification of organophosphate nerve agents. Curr. Opinion. Microbiol., 

5: 288-295. 

[4] Ely, F., Foo, J.-L, Jackson, C.J., Gahan, L.R., Ollis, D.L., and Schenk, G. (2007) Enzymatic bioremediation: 

organophosphate degradation by binuclear metallo-hydrolases. Curr. Topics Biochem. Res., 9: 63-78. 

[5] Cox, R. S., Schenk, G., Mitić, N., Gahan, L., and Hengge, A.C. (2007) Diesterase activity and substrate 

binding in purple acid phosphatases. J. Am. Chem. Soc., 129: 9550-9551. 

[6] Smith, S.J., Casellato, A., Hadler, K.S., Mitić, N., Riley, M.J., Bortoluzzi, A.J., Szpoganicz, B., Schenk, G., 

Neves, A., and Gahan, L.R. (2007) The reaction mechanism of the Ga(III)Zn(II) derivative of uteroferrin and 

corresponding biomimetics. J. Biol. Inorg. Chem., 12: 1207-1220. 

[7] Schenk, G., Elliott, T.W., Leung, E.W.W., Mitić, N., Carrington, L.E., Gahan, L.R., and Guddat, L.W. 

(2008) Snapshots of the reaction mechanism of purple acid phosphatase-catalyzed hydrolysis. BMC Struct. Biol., 

8: 6. 

[8] Hadler, K.S., Tanifum, E.A., Yip, S., Mitić, N., Guddat, L.W., Jackson, C.J., Gahan, L.R., Nguyen, K., Carr, 

P.D., Ollis, D.L., Hengge, A.C., Larrabee, J.A., and Schenk, G. Substrate induced formation of a catalytically 

competent binuclear center and regulation of reactivity in glycerophosphodiesterase from Enterobacter 

aerogenes. Submitted for publication. 

_____________________________________________________________________ 

22


KL4. Ruthenium Anticancer Compounds: Challenges and Expectations 

E. Alessio a , I. Bratsos a , T. Gianferrara b 

a 

Chemical Sciences, University of Trieste, Via L. Giorgieri 1, 34127, Trieste, Italy 

e-mail: alessi@units.it 

b 

Pharmaceutical Sciences, University of Trieste, Piazzale Europa 1, 34127, Trieste, Italy 

e-mail: gianfer@units.it 

Since several years the authors have been involved in the development of anticancer Ru-dmso complexes [1, 2]. 

The most advanced representative in this series is the Ru(III) compound NAMI-A which is selectively active 

against metastases of solid tumors and has successfully accomplished a phase I clinical study on humans. 

In general, ruthenium anticancer compounds can be divided in two main classes: one in which ruthenium has a 

structural role, i.e. it is instrumental in determining the shape of the compound, and another in which ruthenium 

has a functional role. Functional ruthenium compounds must have at least one coordination position available for 

binding to the biological target. Most commonly, they are prodrugs and are activated by hydrolysis. NAMI-A is 

believed to belong to this class. Conversely, structural ruthenium compounds must be stable and inert: ruthenium 

itself will not make any coordination bond with the biological target, but the compound as a whole is expected to 

give supramolecular interactions with the target (e.g. Coulombic, hydrogen bonding, p-p stacking, ...). Some 

compounds may belong to both classes, i.e. ruthenium can make coordination bonds (functional role) and it can 

give additional supramolecular interactions with the target (e.g. through its appropriately positioned ancillary 

ligands, structural role). There are also other, less likely, possibilities in which ruthenium acts as a catalyst or as 

a carrier for biologically active ligands that are delivered in vivo. 

After a general introduction, the lecture will give an update of the clinical status of NAMI-A and then will focus 

on new classes of Ru compounds that were developed more recently in the attempt to find new active species 

and establish some general structure-activity relationships [3, 4]. 

References: 

[1] E. Alessio, G. Mestroni, A. Bergamo, G. Sava In Metal Ions in Biological Systems, Volume 42: Metal Ions 

and Their Complexes in Medication and in Cancer Diagnosis and Therapy; Sigel, A., Sigel, H., Eds.; M. Dekker: 

New York, 2004; pp. 323-351. 

[2] E. Alessio, G. Mestroni, A. Bergamo, G. Sava, G. Curr. Topics Med. Chem. 2004, 4, 1525-1535. 

[3] I. Bratsos, A. Bergamo, G. Sava, T. Gianferrara, E. Zangrando, E. Alessio J. Inorg. Biochem. 2008, 102, 606- 

617. 

[4] I. Bratsos, S. Jedner, A. Bergamo, G. Sava, T. Gianferrara, E. Zangrando, E. Alessio J. Inorg. Biochem. 

2008, 102, 1120-1133. 

_____________________________________________________________________ 

23


KL5. Design and Mechanism of Reaction of Metal-Based Antitumor Agents 

and Artificial Metallonucleases 

Z. Guo 

Chemistry, Nanjing University, Hankou Road, No. 22, 210093, Nanjing, China, 

e-mail: zguo@nju.edu.cn 

The side effects of the current clinic drugs have stimulated the research on the novel platinum-based antitumor 

complexes. Improving the tumor-selectivity of these complexes may mitigate the toxic effects and enhance the 

therapeutic index of these drugs. Design of complexes with novel structural features is another major focus of 

the area. In this talk, a strategy that can encapsulate Pt drugs in ferritin cage will be discussed. Conjugation of 

photodynamic therapeutic agent with cisplatin provides the possibility of red light excited cytoxicity of 

platinum-based compounds. Moreover, several new polynuclear Pt(II) complexes with robust structural features, 

designed in our lab, will be also discussed. These complexes have different structural features from cisplatin and 

have demonstrated notable cytotoxic activity against tumour cell lines. Their reactivity towards DNA and GSH 

has been investigated. Selective cleavage of DNA is of paramount interests in medicine and biotechnology. 

Transition metal complexes stand out as candidates for artificial nucleases due to their diverse structural features 

and reactivities. We have designed and structurally characterized a series of polynuclear copper and zinc 

complexes and studied their cleavage activity towards DNA and DNA model compounds. Structural factors that 

determine the activity will be discussed. 

References: 

[1] Yang, Z.; Wang, X.; Diao, H.; Zhang, J. Li, H.; Sun, H.; Guo, Z.J. Chem. Commun. 2007, 3409. [2] Wang, 

X.Y.; Guo Z.J., Dalton Trans., 2008, 1521. 

_____________________________________________________________________ 

24


KL6. Coupling Molecular Recognition and Signaling 

A. Shanzer, R. Kikkeri, G. Melman and Y. Barda 

Department of Organic Chemistry, Weizmann Institute of Science, P.O. Box 26, Rehovot, Israel 

Novel recognition-signaling conjugates, which integrate molecular recognition with signaling provides a 

powerful tool in the study of molecular recognition events in real time. Further improvements are obtained by 

combining spectroscopy and microscopy to generate dynamic probes to follow cellular events as they unfold. 

The generality of these conjugates will be demonstrated in following diverse events including: intracellular 

compartmentalization, molecular trafficking and identification of secondary targets. 

The limitations of using several probes simultaneously will be outlined and means to overcome these limitations 

by developing ‘tunable’ probes capable of emitting at different wavelength will be described. 

Advantages and limitations will be discussed. 

References: 

[1] R. Kikkeri, H. Traboulsi, N. Humbert, E. Gumienna-Kontecka, R. Arad-Yellin, G. Melman, M. Elhabiri, 

A.M. Albrecht-Gary, A. Shanzer. Inorganic Chemistry 46, 2485 (2007). 

_____________________________________________________________________ 

25


KL7. Blood and Iron 

N. de Val a , J.-P. Declercq b , C. Lim c , R.R. Crichton a 

a 

Biochemistry Unit, Department of Chemistry, University of Louvain, Place Croix du Sud, Louvain-la-Neuve, 

Belgium 

e-mail: robert.crichton@uclouvain.be 

b 

Structural Chemistry Unit, Department of Chemistry, University of Louvain, Place Louis Pasteur, Louvain-la- 

Neuve, Belgium. 

c 

MRC Bioanalytical Science Laboratory, School of Biological and Chemical Sciences, Birkbeck College, 

University of London, Malet Street, London WC1 7HX, United Kingdom. 

There are extensive structural similarities between eukaryotic ferritins and prokaryotic ferritins. However, there 

is one essential difference between these two types of ferritins : whereas bacterioferritins bind haem in-vivo (the 

haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two axial Met 52 

residues), eukaryotic ferritins are considered to be non-haem proteins. Studies carried out a number of years ago 

showed that horse spleen apoferritin, isolated by classic procedures, contains a cofactor with many of the 

characteristics of a porphyrin. In a series of in-vitro experiments, it was shown that when horse spleen apoferritin 

[1, 2] or a number of recombinant horse L chain apoferritins are co-crystallised with haemin, the 

metalloporphyrin is demetallated [3-5], and a demetallated porphyrin is found within the same hydrophobic 

pocket as in BFRs. 

In the present study the cofactor has been isolated from horse spleen apoferritin and from crystals of the mutant 

horse L chain E53, 56, 57, 60Q + R59M which had been co-crystallised with haemin. In both cases the 

HMLC/ESI-MS results confirm that the cofactor is the N-alkyl porphyrin, N-ethylprotoporphyrin IX (m/Z 591). 

Crystal structures of wild type L chain horse apoferritin and its three mutants E53, 56, 57, 60Q / R59M and E53, 

56, 57, 60Q + R59M co-crystallised with haemin have been determined to high resolution and in all cases a 

metal-free molecule derived from haemin was found in the hydrophobic pocket, close to the two-fold axis. The 

X-ray structure of the E53, 56, 57, 60Q + R59M recombinant horse L-chain apoferritin has been obtained at a 

higher resolution (1.16 Å) than previously reported for any mammalian apoferritins. Similar evidence for a 

metal-free molecule derived from haemin was found in the electron density map of horse spleen apoferritin (at a 

resolution of 1.5 Å) which had been prepared by classical procedures, which retains the cofactor. However in 

none of the structures, the electron density could be unequivocally fitted to the N-alkyl porphyrin because of 

local disorder. 

We conclude that L-chain ferritins are capable of binding and demetallating haemin, generating in the process Nethylprotoporphyrin 

IX both in vivo and in vitro. While mutation of the cluster of Glu residues previously 

implicated in this process [6], and of the Arg residue in the predominantly hydrophobic porphyrin binding 

pocket, slows down the rate of demetallation (as measured by EPR spectroscopy), it does not prevent it. The 

mechanism by which the demetallation and alkylation of the metalloporphyrin might take place, together with 

the wider implications of this phenomenon in a physiological context will be discussed. 

Acknowledgement: We thank the FNRS for financial support and the EU for supporting access to research 

infrastructure. 

References: 

[1] G. Précigoux, J. Yariv, B. Gallois, A. Dautant, C. Courseille, B. Langlois d’Estaintot, Acta Cryst., D50, 739 

(1994). 

[2] M.A. Michaux, A. Dautant, B. Gallois, T. Granier, B. Langlois d’Estaintot, G. Précigoux, Proteins, 24, 314 

(1996). 

[3] N. Carette, W. Hagen, L. Bertrand, N. De Val, D. Vertommen, F. Roland, L. Hue, R.R. Crichton, J. Inorg. 

Biochem., 100, 1426 (2006). 

[4] N. de Val, H. Herschbach, N. Potier, A. Van Doorsselaer, R.R. Crichton, FEBS Lett., 580, 6275 (2006). 

[5] N. de Val, W. Hagen, R.R. Crichton, Biometals, 20, 21 (2007). 

[6] R.R. Crichton, J.A. Soruco, F. Roland, M.A. Michaux, B. Gallois, G. Précigoux, J.-P. Mahy, D. Mansuy, 

Biochem., 36, 15049 (1997). 

_____________________________________________________________________ 

26


KL8. Mechanistic Studies of Oxidative Halophenol Dehalogenation by 

Heme-Containing Peroxidases 

J.H. Dawson, R.L. Osborne, S. Sumithran, M. Coggins, M. Sono 

Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, 29208, Columbia, SC, United 

States 

e-mail: dawson@sc.edu 

Toxic halophenols are produced through industrial processes and pose both environmental risks and health 

hazards. Curiously, marine worms in coastal sediments produce noxious halophenols, apparently to deter 

predators. To survive in the presence of such poisons, A. ornata uses a catalytically-active globin 

dehaloperoxidase to oxidatively dehalogenate halophenols to the corresponding quinones. Two mechanisms for 

this reaction have been proposed: a direct two-electron oxygen atom transfer or two successive one-electron 

steps via a phenoxy radical. We have also shown that the most versatile heme-containing peroxidase, 

Caldariomyces fumago chloroperoxidase (CCPO) - best known as a halogenation catalyst - and the oxygen 

transport protein myoglobin both catalyze halophenol dehalogenation. With all three enzymes as well as 

horseradish peroxidase (HRP), the mechanism has been probed using para-halophenols, and the product 

distribution is consistent with involvement of a phenoxy radical intermediate. Since CCPO and HRP form 

relatively stable high-valent ferryl intermediates, we have employed rapid-scan stopped-flow techniques to 

differentiate between the two mechanisms. Parallel studies with A. ornata dehaloperoxidase and myoglobin have 

also been pursued. Finally, as phenoxy radicals and quinones are known to bind irreversibly to DNA, the ability 

of myoglobin to oxidatively dehalogenate halophenols may explain their carcinogenicity. 

_____________________________________________________________________ 

27


F. Armstrong 

KL9. Oxygen, the Trojan Horse for Hydrogenases 

Department of Chemistry, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, UK 

e-mail: Fraser.armstrong@chem.ox.ac.uk, Telephone +44 1865 272647/287182, Fax +44 1865 287182 

Hydrogenases are attracting great interest because they offer inspiration and even practical solutions to 

developing a hydrogen economy that would help remove the world’s dependence on fossil fuels. The binuclear 

active sites of [NiFe]- or [FeFe]-hydrogenases have activities that compare with platinum, that is, they behave as 

essentially reversible electrocatalysts requiring only the smallest of overpotentials. Future technologies ranging 

from ‘renewable’ hydrogen production, based either on ‘biohydrogen’ or electrolysis, to efficient hydrogen 

oxidation in low-temperature fuel cells without platinum catalysts –may be based upon microorganisms 

containing these enzymes or upon man-made catalysts that are functional analogues of their active sites. These 

fragile catalytic centres, all of which contain low-spin Fe coordinated mainly by non-protein ligands CO and CN � , 

are deeply buried within the protein. They resemble organometallic compounds and may be among the earliest 

catalysts; hence it is not surprising that they are inactivated by O2. Like H2 and the competitive inhibitor CO, O2 

enters the protein and coordinates to the metal centres by synergic bonding involving a combination of σ-donor 

and π-back donation; however, a major difference is that O2 withdraws rather than provides electrons and in the 

process is likely to attack the active site through reactive oxygen intermediates. 

We have defined ‘O2-tolerance as the capability of a hydrogenase to function in the presence of O2 (not merely 

to resist degradation on the bench) and we are investigating three different ways in which hydrogenases can (and 

may have evolved to survive) the attack of O2 – the ‘Trojan Horse’. Firstly, and most widely acknowledged, is 

that O2 might be prevented from reaching the active site because it is excluded from ‘gas channels’ through the 

protein. Secondly, the rate and extent of damage caused by O2 reduction may be minimized by certain electronic 

properties of the active site. Thirdly, the damage from O2 attack may be repaired extremely rapidly, thus 

allowing the enzyme to resume normal catalysis. All of these options are being investigated by protein film 

voltammetry and some interesting new results are surfacing. 

Recent references 

- Investigating and Exploiting the Electrocatalytic Properties of Hydrogenases K. A. Vincent, A. Parkin and F. 

A. Armstrong. Chemical Reviews. 107, 4366-4413 (2007). 

- Enzymatic Oxidation of H2 in Atmospheric O2: The Electrochemistry of Energy Generation from Trace H2 by - 

Aerobic Microorganisms J. A. Cracknell, K. A. Vincent, M. Ludwig, O. Lenz, B. Friedrich and F. A. Armstrong. 

J. Amer. Chem. Soc. 130, 424-425 (2008). 

- Hydrogen Production under Aerobic Conditions by Membrane-bound Hydrogenases from Ralstonia species. G. 

Goldet, A.Wait, J. A. Cracknell, B. Friedrich, M. Ludwig, O. Lenz and F. A. Armstrong. 

J. Amer. Chem. Soc. 130, in press (2008). 

- The difference a Se makes? Oxygen-tolerant hydrogen production by the [NiFeSe]-hydrogenase from 

Desulfomicrobium baculatum. A. Parkin, G. Goldet, C. Cavazza, J. C. Fontecilla-Camps and F. A. Armstrong. J. 

Amer. Chem. Soc. 130, in press (2008). 

_____________________________________________________________________ 

28


KL10. Preparing Chiral Vanadium Catalysts based on the Vanadium 

Haloperoxidases 

C.J. Schneider a , G. Zampella b , L. De Gioia b , V.L. Pecoraro a 

a 

Department of Chemistry, University of Michigan, Ann Arbor, MI USA 

email: vlpec@umich.edu 

b 

Universita degli Studi di Milano Biocca, Italia 

Vanadium Haloperoxidases catalyze the halogenation of organic substrates using hydrogen peroxide and halides. 

In addition, these vanadium containing enzymes can stereoselectively convert sulfides to sulfoxides. The active 

center contains a single V(V) which is thought to bind peroxide prior to oxidation of halide or sulfur. 

Protonation of this vanadium peroxo species has been proposed as a critical step in the catalytic mechanism. We 

will describe experiments using small molecule models (VO(O2)Heida and VO(O2)noreida) for the reactivity of 

the Vanadium Haloperoxidases that have been useful in developing the presently accepted mechanism for this 

enzyme. Furthermore, we will present recent studies using DFT calculations that provide insight into the site of 

active site protonation and the role of active site residue positioning in the catalytic process. 

_____________________________________________________________________ 

29


KL11. The Role of 2 nd Coordination Sphere in a Blue Copper Protein, 

Pseudoazurin 

T. Kohzuma, R.F. Abdelhamid, and Y. Obara 

Institute of Applied Beam Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Japan 

e-mail: kohzuma@mx.ibaraki.ac.jp 

Noncovalent weak interactions play important roles in biological systems [1]. In particular, such interactions in 

the second coordination shell of metal ions in proteins may modulate the structure and reactivity of the metal ion 

site in functionally significant ways. Recently, π−π interactions between metal ion coordinated histidine 

imidazoles and aromatic amino acids have been recognized as potentially important contributors to the properties 

of metal ion sites [2]. 

Here we would like to demonstrate the π−π interaction between a coordinated histidine imidazole ring and the 

side chains of aromatic amino acids in the second coordination sphere of pseudoazurin significantly influences 

the properties of the blue copper site. Electronic absorption and electron paramagnetic resonance (EPR) spectra 

indicate that the blue copper electronic structure is perturbed, as is the redox potential, by the introduction of a 

second coordination shell π−π interaction. The π−π interaction with the metal ion coordinated histidine 

imidazole ring modulates the electron delocalization at the active site has been suggested, and that such 

interactions may be functionally important in refining the reactivity of blue copper sites [3]. 

On the other hand, several blue copper proteins are known to change the active site structure at alkaline pH 

(alkaline transition). The Alkaline transition experiment of Met16Phe, Met16Tyr, Met16Trp, and Met16Val 

pseudoazurin variants were also performed to investigate more detailed function of the second sphere. The 

visible electronic absorption and resonance Raman (RR) spectra of Met16Phe, Met16Tyr, and Met16Trp variants 

showed the increasing of axial component at pH ~11 like wild-type PAz. The visible electronic absorption and 

far-UV CD spectra of Met16Val demonstrated that the destabilization of the protein structure was triggered at 

pH > 11. RR spectra of PAz showed that the intensity-weighted averaged Cu-S(Cys) stretching frequency was 

shifted to higher frequency region at pH ~11. The higher frequency shift of Cu-S(Cys) bond is implied the 

stronger Cu-S(Cys) bond at alkaline transition pH ~11. The visible electronic absorption and far-UV CD spectra 

of Met16X PAz revealed that the Met16Val variant is denatured at pH >11, but Met16Phe, Met16Tyr, and 

Met16Trp mutant proteins are not denatured even at pH > 11 [4]. These observations suggest that Met16 is 

important to maintain the protein structure through the possible weak interaction between methionine –SCH3 

part and coordinated histidine imidazole moiety. The introduction of π−π interaction in the second coordination 

sphere may be contributed to the enhancement of protein structure stability. 

Very recently, the stabilization of His-Cu(I) bond in the reduced Met16Phe variant has been observed by 244 nm 

excited UV resonance Raman spectroscopy [5]. The UV resonance Raman spectra of Met16Phe variant also 

strongly supports the physilogical meanings of π−π interactions between metal ion coordinated histidine 

imidazole and benzen ring of phenylalanine in the structure of fern plastocyanin, which keeps the electron 

transfer function even at acidi pH condtions unlike to the higher plant plastocyanin [6]. 

Acknowledgement: The authors are grateful to Profs. Osamu Yamauchi (Kansai University) and David M. 

Dooley (Montana State University) for their helpful discussion. The authors also would like to thank to Mr. 

Takayuki Higuchi and Mr. Daisuke Fukushima for their experimental help. A part of this work is supported by 

Research Promotion Bureau, Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan to 

TK, a Grant-in-Aid for Scientific Research from JSPS (No. 18550147), Japan to TK, and the Project of 

Development of Basic Technologies for Advanced Production Methods Using Microorganism Functions by the 

New Energy and Industrial Technology Development Organization (NEDO) to TK. 

References: 

[1] S. K. Burley and G. A. Petsko, Science, 229, 23 (1985). 

[2] O. Yamauchi, A. Odani, M. Takani, J Chem Soc Dalton Trans, 3411 (2002). 

[3] R. F. Abdelhamid, Y. Obara, Y. Uchida, T. Kohzuma, D. E. Brown, D. M. Dooley, and H. Hori, J. Biol. 

Inorg. Chem., 12, 165 (2007). 

[4] R. F. Abdelhamid, Y. Obara, and T. Kohzuma, J. Inorg. Biochem., 102, 1373 (2008). 

[5] T. Kohzuma, unpublished results. 

[6] T. Kohzuma, T. Inoue, F. Yoshizak, Y. Sasakawa, K. Onodera, S. Nagatomo, T. Kitagawa, S. Uzawa, Y. 

Isobe, Y. Sugimura, M. Gotowda, Y. Kai, J Biol Chem, 274, 11817 (1999). 

_____________________________________________________________________ 

30


KL12. Copper Resistance in E. coli. The Multicopper Oxidase PcoA 

Catalyses Oxidation of Copper(I) in Cu I Cu II -PcoC. 

Control of Seven Copper Sites in a Single Catalytic Reaction. 

K.Y. Djoko, M. Zimmermann, L.X. Chong, Z. Xiao, A.G. Wedd 

School of Chemistry and Bio21 Research Institute, University of Melbourne, Parkville, Victoria 3010, Australia 

PcoA and PcoC are two of the soluble proteins expressed to the periplasm as part of the copper resistance 

response of E. coli. PcoC binds both copper(I) and copper(II) to form air-stable Cu I Cu II -PcoC. The blue 

multicopper oxidase PcoA is shown to catalyze oxidation of copper(I) bound in Cu I Cu II -PcoC to less toxic 

copper(II) which is released into solution (Figure) [2] This is consistent with a role for PcoA as a cuprous 

oxidase. These two proteins may interact with the outer membrane protein PcoB to export excess copper from 

the periplasm. 

Figure. The binding modes of copper and their affinities in these systems will be compared with: 

(a) N-terminal domains of copper and zinc transmembrane transporters HMA2, 4 and 7 from the simple plant 

Arabidopsis thaliana: the HMA4 domain binds Cu + with 10 6 higher affinity than it binds Zn 2+ , its putative 

substrate [2]. 

(b) the chaperone protein CopK from the bacterium Cupriavidus metalliduran CH34: remarkably, binding of 

Cu(I) in a [Cu(S-Met)4] + site induces cooperative binding of Cu(II). The affinity for Cu(II) increases by a factor 

of 10 6 upon binding of Cu(I) [3]. 

References: 

[1] K.Y. Djoko, Z. Xiao, A.G. Wedd, ChemBioChem 2008, in press. 

[2] M. Zimmermann, Z. Xiao, A. G. Wedd et al 2008, submitted for publication. 

[3] L. X. Chong, M. J. Maher, M. G. Hinds, Zhiguang Xiao and Anthony G. Wedd 2008, submitted for 

publication. 

_____________________________________________________________________ 

31


KL13. Beyond Platinum: Investigational Metallodrugs for Cancer Therapy 

B.K. Keppler 

University of Vienna, Institute of Inorganic Chemistry, Waehringer Strasse 42, 1090 Wien, Austria 

e-mail: bernhard.keppler@univie.ac.at 

Platinum compounds are a mainstay in chemotherapy of a variety of malignant tumors from testicular carcinoma 

to colorectal cancer. Metaphorically speaking, their pharmaceutical and economical success has caused a lasting 

“platinum rush” in Bioinorganic Chemistry, which brought roughly forty platinum complexes into clinical trials 

and an immense number into preclinical studies. However, there is little reason to assume that platinum is the 

only metal predestined for cancer therapy. Although clinical development of tumor-inhibiting non-platinum 

metallodrugs is still in its infancy, there is a growing body of evidence that concepts making use of 

pharmacologically relevant chemical properties of other metals offer the chance of effectively treating 

malignancies that are not susceptible to platinum drugs. 

Currently, ruthenium and gallium complexes are at the forefront of this development. Obvious from the 

coordination chemist’s point of view, experimental findings confirm that these classes of compounds are indeed 

no more apprehensible by analogies with platinum drugs than cisplatin can be considered an alkylating agent. 

Both metals differ from platinum in their coordination geometry and binding preferences as described by the 

HSAB principle. In the case of ruthenium, redox activity in biological environments is an additional convenient 

feature which can be adjusted by appropriate sets of ligands. The reductive milieu of solid tumors and the 

frequent over-expression of transferrin receptors render tumor cells susceptible to ruthenium complexes with 

appropriate redox potentials and transferrin-binding capacities. Both criteria are met by the complex indazolium 

trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019), which could be corroborated by further 

experimental evidence. The potential role of mitochondria as a target for this compound [1] as well as the 

consequences of serum protein binding for escaping P-glycoprotein-mediated resistance [2] are being discussed. 

Gallium is known to interfere with cellular iron uptake, trafficking and iron-dependent enzymatic processes 

without possessing the redox activity of iron. It was the second metal (after platinum) to prove clinically active 

in malignant diseases, but pharmacological and toxicological impediments could not be overcome for a long 

time. Coordination to a rather lipophilic ligand sphere stabilizing gallium against hydrolysis and improving its 

bioavailability has been recognized as a strategy to develop an oral gallium drug. The complex tris(8quinolinolato)gallium(III) 

(KP46) shows promise not only to provide gallium in an orally available form, but 

also to broaden the spectrum of activity as compared with gallium nitrate, since a clinical phase I trial gave 

preliminary evidence of activity in renal cell carcinoma [3] and preclinical studies suggest suitability for 

treatment of malignant melanoma [4]. 

Recently, the 1, 10-phenanthroline-containing lanthanum complex KP772 was identified as a tumor-inhibiting 

compound in vivo. It shares with gallium salts the capacity of inhibiting the iron-dependent enzyme 

ribonucleotide reductase [5], but otherwise shows unique pharmacological properties different from any other 

metal compound investigated so far. Most remarkably, multidrug resistance mediated by ABC transporters does 

not affect its activity, but is on the contrary associated with increased sensitivity to this compound. Moreover, 

KP772 in subcytotoxic concentrations is capable of sensitizing multidrug-resistant tumor cells to the antitumor 

drugs vincristine and doxorubicine [6]. 

References: 

[1] S. Kapitza, M. Pongratz, M.A. Jakupec, P. Heffeter, W. Berger, L. Lackinger, B.K. Keppler, B. Marian, J. 

Cancer Res. Clin. Oncol., 131, 101 (2005). 

[2] P. Heffeter, M. Pongratz, E. Steiner, P. Chiba, M.A. Jakupec, L. Elbling, B. Marian, W. Körner, F. Sevelda, 

M. Micksche, B.K. Keppler, W. Berger, J. Pharmacol. Exp. Ther., 312, 281 (2005). 

[3] R.-D. Hofheinz, C. Dittrich, M.A. Jakupec, A. Drescher, U. Jaehde, M. Gneist, N. Graf v. Keyserlingk, B. K. 

Keppler, Int. J. Clin. Pharmacol. Ther., 43, 590 (2005). 

[4] S.M. Valiahdi, M.A. Jakupec, R. Marculescu, W. Berger, K. Rappersberger, B.K. Keppler, Proceedings of 

the AACR-NCI-EORTC International Conference “Molecular Targets and Cancer Therapeutics”, 155 (2007). 

[5] P. Heffeter, P. Saiko, M.A. Jakupec, M. Micksche, T. Szekeres, B.K. Keppler, W. Berger, J. Biol. Inorg. 

Chem., 12 (Suppl. 1), S34 (2007). 

[6] P. Heffeter, M.A. Jakupec, W. Körner, P. Chiba, C. Pirker, R. Dornetshuber, L. Elbling, H. Sutterlüty, M. 

Micksche, B.K. Keppler, W. Berger, Biochem. Pharmacol. 73, 1873 (2007). 

_____________________________________________________________________ 

32


KL14. Metal Anticancer Complexes with Novel Mechanisms of Action 

P.J. Sadler 

Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK 

Metal complexes provide versatile platforms for the design of anticancer complexes with novel mechanisms of 

action [1]. 

Photoactivated platinum complexes offer potential advantages over conventional platinum anticancer drugs such 

as cisplatin [2]. Potentially they can be activated locally and selectively in the cancer cells and unwanted toxic 

side-effects can be reduced. Kinetically-inert platinum(IV) prodrugs are suitable candidates. Diazido Pt(IV) 

complexes are stable in the dark and when photoactivated under certain conditions, can be more potent towards 

cancer cells than cisplatin. Moreover they can form novel lesions on DNA [3]. 

Organometallic half-sandwich ruthenium(II) arene anticancer complexes can bind to DNA after activation not 

only by hydrolysis [4, 5], but also by ligand-based redox reactions. Two examples of such redox activation will 

be discussed. The first involving oxidation of coordinated thiolates, formation of unusual sulfenato adducts and 

an unexpected role for glutathione [6, 7], and the second, ligand-based reduction, the catalytic oxidation of 

glutathione and production of reactive oxygen species in cancer cells [8]. Extended arenes in these complexes 

can behave as novel DNA intercalators [9, 10]. 

The subtle differences between ruthenium and osmium involving ligand exchange rates and properties of 

coordinated ligands present challenges in designing osmium analogues of active ruthenium arene anticancer 

complexes. Our progress in achieving this [11, 12] will be discussed. 

Acknowledgements: I thank my research group and our collaborators for their contributions to this work, the 

EC, EPSRC, BBSRC, Wellcome Trust and MRC for funding, and members of COST Action D39 for stimulating 

discussions. 

References: 

[1] P.C.A. Bruijnincx, P.J. Sadler, Curr. Opin. Chem. Biol. 12, 197 (2008). 

[2] P.J. Bednarski, F.S. Mackay, P.J. Sadler, Anticancer Agents in. Med. Chem. 7, 75 (2007). 

[3] F.S. Mackay, J.A. Woods, P. Heringová, J. Kaspárková, A.M. Pizarro, S.A. Moggach, S. Parsons, V. Brabec, 

P.J. Sadler, Proc. Natl. Acad. Sci. USA 104, 20743 (2007). 

[4] Y.-K. Yan, M. Melchart, A. Habtemariam, P.J. Sadler, Chem. Commun. 4764 (2005). 

[5] F. Wang, A. Habtemariam, E.P.L. van der Geer, R. Fernández, M. Melchart, R.J. Deeth, R. Aird, S. 

Guichard, F.P.A. Fabbiani, P. Lozano-Casal, I.D.H. Oswald, D.I. Jodrell, S. Parsons, P.J. Sadler, Proc. Natl. 

Acad. Sci. USA 102, 18269 (2005). 

[6] H. Petzold, J. Xu, P.J Sadler, Angew Chem Int Ed Engl. 47, 3008 (2008). 

[7] H. Petzold, P.J Sadler, Chem. Comm., DOI: 10.1039/b805358h (2008). 

[8] S.J. Dougan, A. Habtemariam, S. McHale, S. Parsons, P.J. Sadler, (2008) in press. 

[9] H.-K. Liu, S.J. Berners-Price, F. Wang, J.A. Parkinson, J. Xu, J. Bella, P.J. Sadler, Angew. Chem. Int. Ed. 

45, 8153 (2006). 

[10] T. Bugarcic, O. Nováková, A. Halámiková, L. Zerzánková, O. Vrána, J. Kašpárková, A. Habtemariam, S. 

Parsons, P. J. Sadler, V. Brabec, submitted. 

[11] A.F.A. Peacock, S. Parsons, P.J. Sadler, J. Am. Chem. Soc. 129, 3348 (2007). 

[12] H. Kostrhunova, J. Florian, O. Novakova, A.F.P. Peacock, P.J. Sadler, V. Brabec, J. Med. Chem. (2008) in 

press. 

_____________________________________________________________________ 

33


M. Vašák 

KL15. Metalloneurochemistry of Metallothionein-3 in the Brain 

Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057, Zürich, Switzerland 

Zinc and copper homeostasis plays a crucial role in brain physiology and pathology. Metallothionein-3 (Zn7MT- 

3), an intra- and extracellularly occurring metalloprotein, is critically involved in the homeostasis of Cu(I) and 

Zn(II) in the brain. Intracellular Zn7MT-3 is present in high amounts in zinc-enriched glutamatergic neurons that 

release zinc from their synaptic terminals. The demonstrated specific binding of Zn7MT-3 to the small GTPase 

Rab3A in complex with GDP indicates that Zn7MT-3 actively participates in synaptic vesicle trafficking 

upstream of vesicle fusion [1]. Aberrant metal-protein interactions and the production of reactive oxygen species 

(ROS) play a key role in Alzheimer's (AD) and prion diseases. In both diseases Zn7MT-3 was found 

downregulated. In AD, the ROS production and neuronal toxicity are linked to the binding of redox-active Cu(II) 

to amyloid-beta (Aβ). Zn7MT-3 protects cultured neurons from the toxicity of Aβ by an unknown mechanism. 

We found that a metal swap between Zn7MT-3 and soluble and aggregated Aβ-Cu(II) is the underlying 

mechanism by which the ROS production and the related cellular toxicity is abolished. In this process, Cu(II) is 

reduced by protein thiolates forming Cu(I)4Zn4MT-3, in which an air stable Cu(I)4-thiolate cluster and two 

disulfide bonds are present [2, 3]. In a similar process Zn7MT-3 removes Cu(II) from prion peptides [4]. These 

findings signify a so far unrecognized protective role of this protein in the brain. 

References: 

[1] M. Knipp, G. Meloni, B. Roschitzki, and M. Vašák, Biochemistry, 44, 3159 (2005). 

[2] G. Meloni, P. Faller, and M. Vašák, J. Biol. Chem., 282, 16068 (2007). 

[3] G. Meloni, V. Sonois, T. Delaine, L. Guilloreau, A. Gillet, J. Teissié, P. Faller, and M. Vašák, Nat. Chem. 

Biol., 4, 366 (2008). 

[4] G. Meloni, G. Fritz, P.M.H. Kroneck, and M. Vašák, (2008), manuscript in preparation. 

_____________________________________________________________________ 

34


KL16. Towards the Structure and Properties of Plant Metallothioneins 

E. Freisinger 

Institute of Inorganic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. 

As their vertebrate relatives, plant metallothioneins (MTs) are small cysteine-rich proteins with a preference for 

metal ions with the electron configuration d 10 . They have been proposed to play a role in the homeostasis of 

metal ions such as Zn II and Cu I . Some plant MTs also seem to function in the detoxification of heavy metals like 

e.g. Cd II , most likely in combination with enzymatically synthesized cysteine-rich peptides called phytochelatins. 

Gene expression studies further show inducibility of mt gene expression in response to stress conditions such as 

draught, leave wounding, and scenescence. 

In the past, research activity on the actual gene products, the plant MT proteins themselves, has been low only 

increasing in the last few years. Accordingly, informations about the structures and functions of these proteins 

are scarce, in particular, no three-dimensional structure is available from the literature so far. This is even more 

surprising considering the amino acid sequence diversity plant MTs display, especially in comparison to the 

vertebrate isoforms. This diversity has let to a further differentiation of the plant MTs into four subfamilies 

mainly based on the respective cysteine-distribution pattern. In part, the low research activity might be due to 

difficulties in isolating enough protein material to perform experiments. However, with cloning and recombinant 

expression techniques becoming more and more common including the use of fusion proteins for the expression 

of small proteins and peptides, the production of highly pure and homogeneous protein material is no longer the 

major bottleneck. 

Current knowledge about plant MTs includes the 

number of divalent metal ions they are able to 

coordinate in form of metal-thiolate clusters, the 

pH stability of the Zn II - and Cd II -thiolate clusters 

based on the apparent pKa values of the cysteine 

thiolate groups in presence of the respective metal 

ions, the presence of secondary structural elements, 

namely �-sheets, in the long cysteine-free linker 

regions connecting cysteine-rich regions, as well as 

the number and arrangement of metal-thiolate 

clusters in selected proteins [1�3]. This contribution 

will summarize the present research status and 

allow new insights into the structures and metal ion 

coordination abilities of plant MTs based on 

spectroscopic methods such as UV-Vis, circular 

dichroism, and NMR spectroscopy, dynamic light 

scattering experiments and limited proteolytic 

digestion reactions to probe the number of metalthiolate 

clusters formed and their arrangement 

along the amino acid chain. In addition, metal ion 

substitution and reconstitution reactions followed 

by mass spectrometry provide exciting informations about the cluster formation processes and reveal metal ion 

coordination sites with reduced as well as increased stabilities. 

Acknowledgement: Financial support from the Swiss National Science Foundation is gratefully acknowledged: 

SNF grant 20-113728/1 and SNF-Förderungsprofessur PP002-119106/1. 

References: 

[1] E. Freisinger, Inorg. Chim. Acta, 360, 369 (2007). 

[2] E. A. Peroza, E. Freisinger, J. Biol. Inorg. Chem., 12, 377 (2007). 

[3] O. Schicht, E. Freisinger, Inorg. Chim. Acta, doi: 10.1016/J.ica.2008.03.097 (2008). 

_____________________________________________________________________ 

35


M. Capdevila a , S. Atrian b 

KL17. Lights and Shades of Metallothioneins 

a 

Departament de Química, Universitat Autònoma de Barcelona, Facultat de Ciències, 08193, Bellaterra 

(Barcelona), Spain 

e-mail: merce.capdevila@uab.cat 

b 

Departament de Genètica, Universitat de Barcelona, Facultat de Biologia, Av Diagonal 645, 08028, 

Barcelona, Spain 

At the beginning of the 90’s, when we started working on the metallothionein field, there was a good body of 

knowledge of particularly two model MTs: the mammalian MT1 isoform and the yeast CUP1 protein. During 

these years we have explored some features of a considerable number of recombinant MTs belonging to the most 

diverse phyla, by always applying the same methodology. This allows us to compare all of them from the same 

perspective. Therefore, we are confident that currently we have a quite realistic picture of the MT family. 

However, it is also our opinion that MT study still generates more questions than answers. Here we will 

highlight the main achievements of our research by outlining the particular features of the studied proteins, the 

information that this provided to understand MTs functionality and the conclusions we have drawn from it. All 

this data, together with the goals we plan to achieve in a near future, are good basis to unveil the physiological 

function of this peculiar family of metalloproteins, how it may have evolved and how its members became 

differentiated, spreading over the tree of life. Finally, some uses of MTs, from a biotechnological point of 

interest will be shown. 

_____________________________________________________________________ 

36


KL18. DNA Repair, Iron Sulphur Clusters and Free Radicals: the Case of 

the Spore Photoproduct Lyase, a Radical-SAM Enzyme 

M. Fontecave 

Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-Université Joseph Fourier 5249, 

CEA Grenoble, 17 Avenue des Martyrs, 38054 Grenoble Cedex 9 – France 

e-mail: mfontecave@cea.fr 

The DNA of all organisms is subject to modifications upon exposure to a wide variety of chemical and physical 

agents. Among them, solar ultraviolet radiation is known to induce dimerization reactions between adjacent 

pyrimidines. In spores of some bacteria such as Bacillus subtilis the only photoproduct generated upon exposure 

to UV light is 5-thyminyl-5,6-dihydrothymine (spore photoproduct, SP). The extreme resistance of spores to UV 

radiation is due to the presence of a specific and very efficient repair enzyme, the spore photoproduct lyase (SP 

Lyase) that directly reverts SP to two unmodified thymines upon germination (scheme). SP Lyase belongs to a 

superfamily of [4Fe-4S] iron-sulfur enzymes, named "Radical-SAM", involved in a great variety of biosynthetic 

pathways and metabolic reactions that proceed via radical mechanisms [1]. Recent biochemical and mechanistic 

studies by W.L. Nicholson's [2], J. Broderick's [3] groups and by our laboratory [4] have provided detailed 

insights into the mechanism of the reaction catalyzed by SP Lyase and how this enzyme controls high potential 

intermediate free radicals. These data will be discussed in the general context of the fascinating chemistry of 

"Radical-SAM" enzymes. 

References: 

[1] Fontecave, M., Ollagnier-de-Choudens,S. & Mulliez, E. (2001) Curr Opin Chem Biol.5, 506-11 ; Wang, S.C. 

& Frey, P.A. (2007) Trends Biochem. Sci. 32,101-10 

[2] Rebeil, R. & Nicholson, W.L. (2001) Proc Natl Acad Sci U S A. 98, 9038-43 

[3] Cheek, J. & Broderick, J.B. (2002) J Am Chem Soc. 124, 2860-1; Buis, J.M., Cheek, J., Kalliri, E. & 

Broderick, J.B. (2006) J Biol Chem 281, 25994-26003 

[4] Friedel, M.G., Berteau, O., Carsten Pieck, J, Atta, M., Ollagnier-de-Choudens, S., Fontecave, M. & Carell, T. 

(2006) Chem. Commun. 445-7; Chandor, A., Berteau, O., Douki, T., Gasparutto, D., Gambarelli, S., Nicolet, Y., 

Sanakis, Y., Ollagnier-de-Choudens, S., Atta, M. & Fontecave, M. (2006) J. Biol. Chem. 281, 26922-26931; 

Chandor, A., Douki, T., Gasparutto, D., Gambarelli, S., Sanakis, Y., Nicolet, Y., Ollagnier-de- Choudens, S., 

Atta, M. & Fontecave, M.(2007) Comptes Rendus de l'académie des Sciences, 10, 756-765 

_____________________________________________________________________ 

37


B. Lippert 

KL19. Ligand-pKa Shifts through Metal Ions: 

Potential Relevance to Ribozyme Chemsitry 

Fakultät Chemie, Technische Universität Dortmund, Otto-Hahn-Str. 6, 44221 Dortmund, Germany 

e-mail: bernhard.lippert@tu-dortmund.de 

There is increasing evidence that reactions of catalytically active RNA molecules in many cases involve acidbase 

chemistry. With metal ions representing important components of RNA structures, the question arises 

whether metal ions, in an indirect way, can contribute to catalysis by influencing acid-base properties of ligands 

bonded to the metals. This applies in particular to metal-bound nucleobases and aqua ligands. Although pKa 

values of isolated nucleobases are usually well outside the physiological pH range, in a suitable environment or 

when chemically modified by metal ions, nucleobases can shift their pKa values easily to values close to 7, hence 

into the physiological pH range. 

In the lecture, the influence of coordinated metal ions on the pKa values of model nucleobases as well as aqua 

ligands will be discussed, and the potential relevance for acid-base catalysis in RNAs will be pointed out [1, 2]. 

Acknowledgement: This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der 

Chemischen Industrie. 

References: 

[1] B. Lippert, Chem. & Biodiv., in press (2008). 

[2] P. M. Lax, M. Garijo Añorbe, B. Müller, E. Y. Bivián-Castro, B. Lippert, Inorg. Chem., 46, 4036 (2007). 

_____________________________________________________________________ 

38


KL20. Base and Sequence Selective Cleavage of Rna Phosphodiester Bonds 

by Zn(II) Azacrown Chelates 

H. Lönnberg a , Q. Wang a , P. Poijärvi-Virta a , K. Ketomäki a , M. Mannila a , T. Niittymäki a , 

P. Virta a , E. Leino a , A. Jancsó, b I. Szilágyi b , T. Gajda b 

a Department of Chemistry, University of Turku, Vatselankatu 2, FIN-20014, Turku, Finland, 

b Department of Inorganic and Analytical Chemistry, University of Szeged, P.O. Box 440, Szeged H-6720, 

Hungary 

e-mail: harlon@utu.fi 

Many of the enzymes catalyzing phosphoryl transfer reactions contain Zn(II) in their catalytic center.[1] For this 

reason, Zn(II)-based cleaving agents have received exceptionally wide interest among researchers attempting to 

create chemical models for the enzyme action [2] and developing artificial restriction enzymes.[3] Among 

various Zn(II) complexes, the azacrowns chekates exhibit two interesting properties: they promote the cleavage 

of RNA phosphodiester bonds [4] and undergo selective binding to nucleic acid bases [5]. The affinity to uracil 

and thymine bases is much higher than to guanine base, and adenine and cytidine bases are not recognized at 

all. The binding of di- and tri-nuclear Zn(II) azacrown chelates to contiguous bases in a nucleic acid strand is a 

co-operative process. The stability of the resultinh ternary complexes may, in fact, be so high that the complex 

formation is possible at intracellular concentrations of Zn(II) ion. Various di- and tri-nucleating azacrown 

ligands have been prepared and studied as base-selective cleaving agents of RNA using dinucleoside 3´, 5´monophosphates 

and short synthetic oligoribonucleotides as model compounds [6-8]. Sequence-selective 

artificial ribonucleases have, in turn, been obtained by tethering azacrown ligands to a 2´-O-methyl 

oligoribonucleotide that recognizes the target sequence [9, 10]. The results of these studies are surveyed. 

References: 

[1] For a recent review, see Weston, J. (2005) Chem. Rev. 105, 2151. 

[2] Mancin, F., Tecilla, P. (2007) New J. Chem. 31, 800. 

[3] Niittymäki, T., Lönnberg, H. (2006) Org. Biomol. Chem. 4, 15. 

[4] Kuusela, S., Lönnberg, H. (1994) J. Chem. Soc. Perkin Trans. 2, 2301. 

[5] Aoki, S., Kimura, E. (2004) Chem. Rev. 104, 769. 

[6] Wang, Q., Mikkola, S., Lönnberg, H. (2004) Chem. Biodiv. 1, 1316. 

[7] Wang, Q., Lönnberg, H. (2006) J. Am. Chem. Soc. 128, 10716. 

[8] Wang, Q., Leino, E, Jancsó, A., Szilágyi, I., Gajda, T., Hietamäki, E., Lönnberg, H. (2008) ChemBioChem, 

in press. 

[9] Niittymäki, T., Lönnberg, H. (2004) Bioconjugate Chem. 15, 1275. 

[10] Niittymäki, T., Virta, P., Ketomäki, K., Lönnberg, H. (2007) Bioconjugate Chem. 18, 1583. 

_____________________________________________________________________ 

39


KL21. New Model Compounds to Help Unravel the Complexities of the 

Oxygen Evolving Centre in PhotosystemII 

A.K. Powell 

Institut für Anorganische Chemie, University of Karlsruhe, Engesserstrasse 15, D76131 Karlsruhe, German, 

e-mail: powell@aoc.uni-karlsruhe.de 

Recent protein X-ray crystallographic studies on Photosystem II present various models for the cluster of metal 

centres within the Oxygen Evolving Centre (OEC), but there are serious discrepancies amongst these with the 

current favoured interpretation assigning the core to a Mn4CaO4 cluster assembly with a cubane motif [1]. We 

recently reported [2] the synthesis and structures of three pentanuclear metal aggregates with the core motifs, 

{CaMn III 3Mn II (µ4-O)L3Cl2(O2CMe) 1.2(H2O)1.5}; 

{NaMn III 3Mn II (µ3-O)L3(N3)2.7(O2CMe)1.3(MeOH)} and 

{NaMn III 4(µ3-O)L’4(N3)3(MeOH)}, 

which provide useful models to help in unravelling the details of the OEC in terms of their structures. It has also 

been acknowledged that the native protein is subject to radiation damage,[3] and thus these models can be used to 

test the susceptibility of such motifs to radiation damage and gauge to what extent this complicates assigning the 

oxidation states of the manganese centres. 

References: 

[1] K.N. Ferreira, T.M. Iverson, K. Maghlaoui, J. Barber and S. Iwata, Science, 303, 1831 (2004) 

[2] I. J. Hewitt, J.-K. Tang, N.T. Madhu, R. Clérac, G. Buth, C. E. Anson and A. K. Powell, Chem. Commun., 

2006, 2650 (2006) 

[3] J. Yano, J. Kern, K.-D. Irrgang, M. J. Latimer, U. Bergmann, P. Glatzel, Y. Pushkar, J. Biesiadka, B. Loll, K. 

Sauer, J. Messinger, A. Zouni and V. K. Yachandra, Proc. Nat. Acad. Sci., 102, 12047 (2005) 

_____________________________________________________________________ 

40


KL22. Probing the Reduced S States of Photosystem II with Mixed -Valence 

Trinuclear and Tetranuclear Manganese Complexes 

A. Dimitrakopoulou, V. Tangoulis, D. P. Kessissoglou 

Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece 

e-mail: kessisog@chem.auth.gr 

The enzyme which produces oxygen is known as photosystem II (PSII). At the heart of this enzyme is a 

tetrameric manganese cluster known as the oxygen evolving 

complex (OEC). The manganese cluster undergoes a series of 

oxidations in a reaction cycle with each different oxidation 

level known as an S state. The starting state, S0, is the most 

reduced and the production of oxygen occurs upon the 

transition of the most oxidized states to the S0 state 

(S3�S4�S0). The exact oxidation states of each manganese 

ion in each S state is still highly debated. To probe the 

oxidation states of the manganese ions, numerous model 

complexes have been synthesized and subjected to a variety of 

techniques including XAS and EPR. A synthetic challenge 

arises to construct complexes of the same overall charge but with a different distribution of oxidation states, and 

then to probe these complexes to see if they can be distinguished under XANES and EPR experimental 

conditions. We have synthesized and studied trimeric and tetrameric manganese clusters with oxidation states 

distributions of Mn(III)Mn(II)Mn(III) and Mn(II)Mn(IV)Mn(II) or Mn(II)Mn(II)Mn(III)Mn(III) and 

Mn(II)Mn(II)Mn(II)Mn(IV) while recently we have isolated (figure) an hexanuclear mixed valence 

[Mn(II)Mn(II)Mn(III)][[Mn(II)Mn(III)Mn(III)] compound consisting of two pseudo-cubane like cores connected 

by an hydroxyl and an oxo groups. 

Acknowledgment. This project is co-funded by the European Union/European Social Fund, “Pythagoras II”. 

References: 

[1]. A. Dimitrakopoulou, C. Dendrinou-Samara, A.A. Pantazaki, M. Alexiou, E. Nordlander, D.P. Kessissoglou, 

J. Inorg. Biochem., 102, 618 (2008) 

[2] C. Zaleski, T-C. Weng, C. Dendrinou-Samara, M. Alexiou, P. Kanakaraki, J. Kampf, J. E. Penner-Hahn, V. 

L. Pecoraro, D. P. Kessissoglou. Inorg. Chem. 47, (2008) 

[3] A. Dimitrakopoulou, V. Psycharis, C. P. Raptopoulou, A. Terzis, V. Tangoulis, D. P. Kessissoglou, Inorg. 

Chem. (2008) 

_____________________________________________________________________ 

41


V. McKee 

KL23. Geometry, Redox and Catalase/SOD Activity in Manganese 

Complexes 

Chemistry, Loughborough University, LE11 3TU, Loughborough, United Kingdom, 

e-mail: v.mckee@lboro.ac.uk 

Geometric constraints can control the aerobic redox state in simple manganese complexes because the electronic 

configurations available render the redox potential particularly susceptible to geometric control. 

Precise control of redox potential is essential for the function of redox-active manganese enzyme systems and 

much of the fine-tuning of redox is achieved by imposition of geometric constraints by the protein. Such effects 

are also likely to be important in the function of manganese-based drugs and oxidation/bleaching catalysts. 

Additionally, cooperative mechanisms can be demonstrated in polymanganese systems, where geometric 

changes following oxidation at one manganese ion are transmitted to the neighbouring ions, causing a change in 

their redox potentials. The same considerations are likely to be exploited in polymanganese metalloproteins. 

Understanding of geometric influence is essential in understanding the natural systems, and should be valuable 

in the development of manganese-based drugs and catalytic systems. The gap between structural data and redox 

chemistry can be bridged by combining solid state electrochemistry with crystallography in order to understand 

the fundamental influence of coordination geometry on redox potential, and hence on catalase and SOD activity. 

_____________________________________________________________________ 

42

B. Meunier 


KL24. Alzheimer Disease: a Bioinorganic Approach 

PALUMED, rue Pierre et Marie Curie, BP 28262, 31682 Labège cedex. 

Among the (unresolved) questions in the pathophysiology of different neurodegenerative diseases, including 

Alzheimer’s disease (AD), one concerns the role of metal ions (zinc, copper and iron) in the formation of 

amyloid plaques. Two of these metal ions are redox-active and are able to generate reactive oxygen species 

(ROS, mainly hydrogen peroxide and hydroxyl radicals) that can create irreversible damage on neurons. 

Since these metal ions are in large excess in the brain of AD patients, the development of a selective chelatotherapy 

is a possible strategy to treate AD. Such approach has been initially developed by A. Bush and coll. by 

using clioquinol, an old antibiotic able to chelate copper ions (1). 

In Toulouse, we have recently developed two different series of new chelating agents (2) that might have a future 

in the treatment of neurodegenerative diseases (3-6). 

References 

[1] Cherny et al., Neuron, 2001, 30, 665. 

[2] Two patents pending (PALUMED-CNRS, 2003 and 2005). 

[3] C. Boldron, I. Van der Auwera, C. Deraeve, H. Gornitzka, S. Wera, M. Pitié, F. van Leuven, B. Meunier, 

ChemBioChem, 2005, 6, 1976. 

[4] C. Deraeve, M. Pitié, B. Meunier, J. Inorg. Chem. 2006, 100, 2117. 

[5] C. Deraeve, M. Pitié, H. Mazarguil, B. Meunier, New J. Chem. 2007, 31, 193. 

[6] C. Deraeve, C. Boldron, A. Maraval, H. Mazarguil, H. Gornitza, L. Vendier, M. Pitié, B. Meunier, Chemistry, 

2008, 14, 682. 

_____________________________________________________________________ 

43


KL25. Specific Cu 2+ Interactions with Fragments of Prion 

and Related Proteins 

D. Valensin a , E. Gaggelli a , H. Kozłowski b , G. Valensin a 

a Department of Chemistry, University of Siena, Via A. Moro 2, 53100, Siena, Italy, 

b Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383, Wrocław, Poland 

e-mail: valensindan@unisi.it 

Prion diseases are fatal neurodegenerations in humans and animals, which manifest as infectious, sporadic or 

inherited [1-3]. All these diseases share the common feature of an aberrant metabolism of the prion protein, PrP, 

resulting in the PrP C →PrP Sc transformation that implies a change in secondary structure elements. Even though 

the role of PrP in the diseases process is generally accepted, its normal function remains elusive. Mammalian 

PrP C , that is mostly expressed in the central nervous system, is a variably glycosylated protein attached via 

glycosylphosphatydylinositol (GPI) anchor located on its C-terminus to the outer surface of the plasma 

membrane. The highly conserved N-terminal domain contains four octapeptide repeats (PHGGGWGQ) that have 

a strong impact on the biology of copper [2, 4-8]. Also the N-terminal region of avian PrP contains mostly 

regular hexapeptide repeats which bind Cu 2+ [9, 10], but its physiological function is much less examined [6]. 

Comparing with more advanced vertebrate PrPs, the family of fish prion proteins contain region(s) with the 

potential of binding Cu 2+ ions [11, 12].The metal binding with prion peptide fragments from different species 

will be discussed. All proteins are His-rich and the obtained results indicates the basic role of imidazole side 

chains and the adjacent amide nitrogen atoms in copper ion binding. Prions represent the family of proteins with 

new mode of Cu 2+ binding which includes the amide nitrogen coordination. The multi-imidazole coordination is 

also likely and it can play a critical role in the antioxidant activity of the copper–prion complexes. 

References: 

[1] Prusiner, S. B. Proc. Natl. Acad. Sci. USA 1998, 95, 363-383. 

[2] Kozłowski, H.; Brown, D.R.; Valensin, G. Metallochemistry of Neurodegeneration 2006, The Royal Society 

of Chemistry, Cambridge. 

[3] Johnson, R.T. Lancet Neurolog 2005, 4, 635-642. 

[4] Brown, D.R.; Wong, B.S.; Hafiz, F.; Clive, C.; Haswell S.J.; Jones, I.M. Biochem. J. 1999, 344, 1–5. 

[5] Taylor, D.R.; Watt, N.T.; Perera, W.S.; Hooper, N.M. J. Cell Sci., 2005, 118, 5141-5153. 

[6] Vassallo, N.; Herms, J. J. Neurochem., 2003, 86, 538-544. 

[7] Miura, T.; Sasaki, S.; Toyama, A.; Takeuchi, H. Biochemistry, 2005, 44, 8712-8720. 

[8] Kozlowski, H.; Janicka-Klos, A.; Stanczak, P.; Valensin, D.; Valensin, G.; Kulon, K.; Coord. Chem. Rev. 

2008, 252, 1069-1078. 

[9] Stańczak, P.; Luczkowski, M.; Juszczyk, P.; Grzonka, Z.; Kozłowski, H. Dalton Trans., 2004, 2102-2007. 

[10] Stańczak, P.; Valensin D.; Juszczyk, P.; Grzonka, Z.; Migliorini, C.; Molteni, E.; Valensin, G.; Gaggelli, E.; 

Kozłowski, H. Biochemistry, 2005, 44, 12940-12954. 

[11] Stańczak, P.; Valensin, D.; Porciatti, E.; Jankowska, E.; Grzonka, Z.; Molteni, E.; Gaggelli, E.; Valensin, 

G.; Kozlowski, H. Biochemistry, 2006, 45, 12227-12239. 

[12] Gaggelli, E.; Jankowska, E.; Kozlowski, H.; Marcinkowska, A.; Migliorini, C.; Stanczak, P.; Valensin, D.; 

Valensin, G. in preparation 

_____________________________________________________________________ 

44


KL26. Accurate Spin State Energies for First-row Transition Metal 

Compounds 

M. Swart a , M. Güell b , J.M. Luis b , M. Solà b 

a Institució Catalana de Recerca i Estudis Avançat, (ICREA), and Universitat de Girona, Institut de Química 

Computacional, Campus Montili, 17071, Girona, Spain, 

b Institut de Química Computacional, Dept. de Química, Universitat de Girona, Campus Montilivi, 17071, 

Girona, Spain 

e-mail: marcel.swart@icrea.es 

An accurate theoretical description of the spinground state of transition-metal complexes is vital for the 

elucidation ofreaction mechanisms of metalloenzymatic reactions.[1] For instance, thecatalytic cycle of 

cytochrome P450 goes from a doublet in the resting state, toa sextet after substrate binding, to a singlet after the 

firstelectron-reduction and binding of molecular oxygen. The subsequent steps, leading to product formation, are 

still being debated, both experimentally andcomputationally. Therefore, to be able to discriminate between e.g. a 

doubletor a quartet pathway, one should be able to trust the methodology to give thecorrect spin ground state. 

In this respect, the OPBE functional has shown promisingresults, [2, 3, 4] unlike other DFT functionals that fail 

for either high or lowspin states. This good performance of OPBE concurs with recent benchmark studiesfor 

reaction barriers [5] and NMR chemical shifts.[6] Moreover, withthe OPBE functional, the spin ground state of 

transition-metal compounds can beeasily understood as a delicate compromise between metal-ligand bonding 

andHund’s rule.[3] Furthermore, not only the functional, also the basis set usedis of the utmost importance.[4] 

Here we will show results for spin-stateenergies of several iron complexes [7], computed with a range of 

computationalmethods, both for vertical and relaxed spin-state splittings. The OPBE results are reliable and 

consistent, in contrast to the other DFT functionals. Alsofor spin-crossover compounds and compounds with 

other first-row transition-metalsdoes OPBE give good results.[8] 

References: 

[1] See e.g.: Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K. J. Am. Chem. Soc. 2007, 129, 6204. 

[2] Swart, M.; Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K. J. Phys. Chem. A 2004, 108, 5479. 

[3] Swart, M. Inorg. Chim. Acta (“The Next Generation” issue) 2007, 360, 179. 

[4] Güell, M.; Luis, J.M.; Solà, M.; Swart, M. J. Phys. Chem. A 2008, accepted. 

[5] Swart, M.; Solà, M.; Bickelhaupt, F.M. J. Comput. Chem. 2007, 28, 1551. 

[6] Wu, A.; Zhang, Y.; Xu, X.; Yan, Y. J. Comput. Chem. 2007, 28, 2431. 

[7] Swart, M. submitted. 

[8] Güell, M.; Luis, J.M.; Solà, M.; Swart, M. in preparation. 

_____________________________________________________________________ 

45


KL27. New Pathways of Nonheme-iron-catalyzed Oxidation Processes 

P. Comba 

Inorganic Chemistry, University Heidelberg, Im Neuenheimer Feld 270, 69120, Heidelberg, Germany 

Bispidine ligands are extremely rigid, easy to synthesize and available in a large variety. They enforce 

coordination geometries derived from cis-octahedral, and the two vacant coordination sites with the tetradentate 

ligand systems are sterically and electronically distinct. Substrates coordinated trans to N3 have strong and short 

bonds, those trans to N7 are labile. Reasons are thoroughly analyzed on the basis of computational work as well 

as experimental structural data, thermodynamics and reactivities. 

Implications with respect to the mechanism of formation and the structure and spin state of high valent iron 

oxidants are analyzed, and possibilities to tune the spin state, structure and reactivity of high valent iron 

complexes are discussed. Novel mechanisms and iron(IV) oxidants will also be presented. 

_____________________________________________________________________ 

46


KL28. Polytopic Metal Complexes With 2, 6-Di-Tert-Butylphenol Pendants 

In Cellular Oxidation Processes 

E. Milaeva a , D. Shpakovsky a , Z. Jingwei a , S. Filimonova a , E. Shevtsova a , S. Bachurin b , 

N. Zefirov a 

a Organic Chemistry, Moscow State Lomonosov University, Lenin Hills, 119992, Moscow, Russia, 

b Neurochemistry of Physiologically Active Compounds, Institute of Physiologically Active Compounds of R, 

Severnyi proezd, 142432, Chernogolovka, Russia 

e-mail: milaeva@org.chem.msu.ru 

The polytopic biometal complexes Rn[L]M (M = Fe, Mn, Co, Cu, Zn) with various chelating ligands L bearing 

2, 6-di-tert-butylphenol groups R as antioxidant fragments were synthesized (Fig.). The physico-chemical 

properties of the compounds and the characteristics of phenoxyl radicals formed in the oxidation of Rn[L]M 

have been studied [1-4]. 

The antioxidative activity of Rn[L]M has been studied in cellular oxidation processes using the lipid 

peroxidation (1) in vitro in intact mitochondria isolated from rat liver, (2) in vitro in rat and fish liver 

homogenates, (3) in the model reactions of (Z)-octadec-9-enic acid peroxidation, and of DPPH reduction. The in 

vivo study has been performed for the most active compounds using fish organism (Asipenser gueldenstaedti 

B.). 

The results suggest that Rn[L]M are membrane active compounds and might be studied in an effort to find novel 

protectors of oxidative stress. 

The financial support of RFBR (08-03-00844, 07-03-00751, 07-03-12110, 06-03-32773), the Program 

"Biomolecular and Medicinal Chemistry" of Russian Academy of Sciences is gratefully acknowledged. 

References: 

[1] Milaeva E., Gerasimova O., Jingwei Zh., Shpakovsky D., Shevtsova E., Bachurin S., Zefirov N. J. Inorg. 

Biochem., 2008, 102, 1348-1358. 

[2] Milaeva E. J. Inorg. Biochem., 2006, 100, 905-915. 

[3] Milaeva E., Tyurin V., Shpakovsky D., Gerasimova O., Jingwei Zh., Gracheva Yu. Heteroatom Chemistry, 

2006, 17, 475. 

[4] Milaeva E., Shpakovsky D., Tyurin V. J. Porphyrins & Phthalocyan., 2003, 8, 701. 

_____________________________________________________________________ 

47


I. Moura 

KL29. The Two Terminal Enzymes of Denitrification: 

Nitric and Nitrous Oxide Reductase 

Departamento de Química, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova De Lisboa, 

Campus de Caparica, 2829-516 Caparica, Portugal 

e-mail: isa@dq.fct.unl.pt 

We present a concise updated review of the bioinorganic aspects of denitrification in particular with emphasis on 

structural and mechanistic aspects of the relevant enzymes involved in the two last steps of this complex 

pathway. The metal diversity detected in this pathway is also acknowledged. Denitrification, or dissimilative 

nitrate reduction, is an anaerobic process used by some bacteria for energy generation. This process is important 

in many aspects, but its environmental implications have been given particular relevance. Nitrate accumulation 

and release of nitrous oxide in the atmosphere due to excess use of fertilizers in agriculture are examples of two 

environmental problems where denitrification plays a central role. The reduction of nitrate to nitrogen gas is 

accomplished by four different types of metalloenzymes in four simple steps: nitrate is reduced to nitrite, then to 

nitric oxide, followed by the reduction to nitrous oxide and by a final reduction to dinitrogen. 

In this process, the nitrate molecule is reduced to molecular dinitrogen in a series of reactions: 

2 NO3 - → 2 NO2 - → 2 NO → N2O → N2 

The structures of these enzymes are known except the one of nitric oxide reductase. 

Biochemical and spectroscopic studies of the two terminal enzymes will be discussed. Nitric oxide reductase 

(NOR), a membrane enzyme contains a catalytic center composed by a non-heme iron coupled to a b type heme. 

The crystal structure of N2OR was solved to a resolution of 2.4 Aº. This enzyme contains one binuclear (CuA) 

and a tetranuclear copper center (CuZ), an unusual structure (catalytic site). CuZ center is a new type of cluster, 

in which four copper ions are ligated by seven histidine residues. A model is proposed for the binding of N2O 

binds to this center. A mechanistic proposal will be presented. 

Keywords: nitric oxide reductases, nitrous oxide reductases, iron, copper, formate electron paramagnetic 

resonance, X-ray crystallography, DFT. 

Acknowledgement: 

BIOIN and BIOPROT Groups at REQUIMTE, and FCT-MCTES. 

References: 

Tavares et al JIB (2006) 1000, 2087-2100. 

_____________________________________________________________________ 

48

J.J.G. Moura 


KL30. Biological Reduction of Nitrate: Mechanistic Aspects 

Departamento de Química, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova De Lisboa 

Campus de Caparica, 2829-516 Caparica, Portugal, 

e-mail: jose.moura@dq.fct.unl.pt 

New coordination sphere of Mo has been recently proposed in Nitrate reductases, mononuclear molybdenum 

containing enzymes, which led to revise the currently accepted reaction mechanisms. One essential aspect is the 

presence of a sulfur in the coordination sphere of Molybdenum (in addition to two MGD and a Cystein) as well 

as the redox interplay of molybdenum and sulfur in the enzyme behavior. Data now available and experiments 

performed on as-prepared, EPR active and inhibited forms of the enzyme, and the high quality of the diffraction 

data, prompted a detailed analysis that has helped to clarify the true nature of the sixth Mo ligand. The 

spectroscopic data, conducted under different experimental conditions, are combined with DFT calculations in 

order to probe the enzyme mechanism. The combination of these spectroscopic studies allowed a deeper 

understanding about the inhibition mechanism of periplasmic nitrate reductases. Also, EPR studies, under 

turnover conditions, have helped to ascertain the nature of the turnover species. The implications of these results 

are discussed also in terms of W containing enzymes (Formate dehydrogenases). 

Keywords: nitrate reductases, formate dehydrogenases, molybdenum, tungsten, electron paramagnetic 

resonance, X-ray crystallography, DFT. 


BIOIN, BIOPROT and X-Tal Groups at REQUIMTE, and FCT-MCTES. 

References: 

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic 

molybdenum. Najmudin S, González PJ, Trincão J, Coelho C, Mukhopadhyay A, Cerqueira NM, Romão CC, 

Moura I, Moura JJ, Brondino CD, Romão MJ (2008) J Biol Inorg Chem. 13, 737-53. 

_____________________________________________________________________ 

49


KL31. Biomimetic Study of Novel Hydroxamate Ligands as Powerful 

Chelating Agents and Efficient Metalloenzyme Inhibitors 

I. Fritsky a , I. Golenia a , E. Gumienna-Kontecka b , H. Kozłowski b , A. Boyko a 

a Chemistry, Kiev National Taras Shevchenko University, Volodymyrska Str., 64, 01601, Kiev, Ukraine, 

b Chemistry, University of Wroclaw, 14, F. Joliot-Curie Str., 50383, Wrocław, Poland 

e-mail: ifritsky@univ.kiev.ua 

Hydroxamic acids are classical chelating ligands which have been traditionally used in analytical chemistry and 

extraction metallurgy. Hydroxamic acids are also important bioligands representing interest not only as 

inhibitors of enzymes (e.g., urease, matrix metalloproteinases, cyclooxygenase) but also as constituting parts of 

the naturally occurring siderophores and compounds used as antibiotics and chelating therapy agents [1]. Most 

recently hydroxamic acids started to be extensively studied as very efficient inhibitors of histone deacetylase, 

which can be used as targeted anticancer agents that have significant anticancer activity at doses well tolerated 

by patients [2]. 

The modern trends in coordination chemistry of hydroxamic acids are mostly directed on elaboration of powerful 

and selective chelators as well as development of novel efficient inhibitors of enzymes and related model studies. 

In this report we present the results of synthetic and biomimetic study of a series of novel hydroxamate ligands 

and their complexes with biometals. The results to be discussed include development of new hydroxamate 

chelators and their speciation solution study; synthesis and structural chemistry of novel polynuclear complexes 

based on hydroxamic acids with unprecedented molecular topology and new coordination modes, in particular, 

the complexes mimicking interaction of hydroxamic inhibitors with enzyme active sites; and study of inhibition 

of tyrosinase by pyridylhydroxamic acids. 

References: 

[1] E. M. F. Muri, M. J. Nieto, R. D. Sindelar, J. S. Williamson, Curr. Med. Chem., 2002, 9, 1631. 

[2] Y. Shao, Z. Gao, P.A. Marks, X. Jiang, Proc. Natl. Acad. Sci., 2004, 101, 18030. 

_____________________________________________________________________ 

50


KL32. New Biomimetic Analagoues of Functional Fe/S-Clusters 

F. Meyer a , J. Ballmann a , M. Fuchs a , S. Dechert a , E. Bill b , E. Bothe b , U. Ryde c 

a 

Institut für Anorganische Chemie, Georg-August-Universität Göttingen, Tammannstrasse 4, D-37077 

Göttingen, Germany 

email: franc.meyer@chemie.uni-goettingen.de 

b 

Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34–36, D-45470 Mülheim an der Ruhr, Germany 

c 

Department of Theoretical Chemistry, Lund University, Chemical Centre, S-22100 Lund, Sweden 

Iron-sulfur cofactors are ubiquitous in biological systems and have been of prime importance in nature since the 

beginning of terrestrial life. Their main role is electron transfer, but other functions (where iron-sulfur clusters 

act as catalytic centers or sensors) are increasingly recognized – iron-sulfur clusters have thus been termed 

"nature’s modular multipurpose structures" [1, 2]. 

The investigation of synthetic model complexes has provided valuable insight into the properties and electronic 

structures of iron-sulfur cofactors [3]. Despite decades of research, however, many challenges and synthetic 

targets have remained even for the smaller clusters, in particular those with a [2Fe-2S] core. Work in the field is 

further stimulated by the recent discovery of various [2Fe-2S] cofactors with unusual ligand environment, 

comprising, e.g., N-donor protein residues such as in biotin synthase [4, 5]. 

Some new developments in synthetic iron-sulfur chemistry will be reported, including the investigation of 

secondary bonding interactions in biomimetic [2Fe-2S] clusters (A) [6], the preparation of new [2Fe-2S] clusters 

with N-donor ligation and of reduced [2Fe-2S] + species [7, 8], as well as the first isolation and full 

characterization of a Rieske-type [2Fe-2S] model complex (B) [9]. 

S 

S 

X 

S 

Fe Fe 

S 

X 

S 

S 

2-/ 3- 

S 

Fe Fe 

S 

A B 

Acknowledgement: This work has been carried out in the framework of the DFG-funded International Research 

Training Group "Metal Sites in Biomolecules: Structures, Regulation and Mechanisms" (IRTG 1422; see 

www.biometals.eu) 

References: 

[1] H. Beinert, J. Biol. Inorg. Chem., 5, 2 (2000). 

[2] H. Beinert, R. H. Holm, E. Münck, Science, 277, 653 (1997). 

[3] P. V. Rao, R. H. Holm, Chem. Rev., 104, 527 (2004); and references therein. 

[4] F. Berkovich, Y. Nicolet, J. T. Wan, J. T. Jarrett, C. L. Drennan, Science, 303, 76 (2004). 

[5] J. Lin, T. Zhou, K. Ye, J. Wang, PNAS, 104, 14640 (2007). 

[6] J. Ballmann, S. Dechert, E. Bill, U. Ryde, F. Meyer, Inorg. Chem., 47, 1586 (2008). 

[7] J. Ballmann, X. Sun, S. Dechert, E. Bill, F. Meyer, J. Inorg. Biochem., 101, 305 (2007). 

[8] J. Ballmann, S. Dechert, E. Bill, E. Bothe, F. Meyer, unpublished. 

[9] J. Ballmann, A. Albers, S. Demeshko, S. Dechert, E. Bill, E. Bothe, U. Ryde, F. Meyer, submitted. 

N 

N 

_____________________________________________________________________ 

51 

S 

S 

2-/ 3-


SESSION LECTURES 

_____________________________________________________________________ 

52


SL1. Temperature Dependent Electrochemistry of Analogous Models for 

Molybdenum and Tungsten Enzymes 

C. Schulzke 

Institut fuer Anorganische Chemie, Georg-August-Universitaet Goettingen, Tammannstr. 4, 37077, Goettingen, 

Germany, 

e-mail: carola.schulzke@chem.uni-goettingen.de 

Molybdopterin containing enzymes are important components of almost any known organism. They catalyse the 

Oxo transfer as a two-electron redox process and are involved in the C, N and S metabolisms. Every enzyme of 

this kind is either a reductase or an oxidase and they show very diverse substrate specifities. In most cases the 

active site metal is molybdenum. Not so in the thermophillic and hyperthermophillic microorganisms which 

utilise tungsten. As reasons for this distribution several issues are discussed in the literature (evolution, metal 

supply, stability, redox potentials [1-5]) but a final conclusion is still to be drawn. If the redox potentials play a 

vital role for the enzymes` choice of metal, this could be due to different temperature dependencies of 

molybdenum and tungsten compounds. To evaluate this hypothesis we investigated known[5-10] and new pairs 

of analogous molybdenum and tungsten complexes (with and without dithiolene ligands) with temperature 

dependent voltammetry methods. The results show that there appears to be indeed a fundamental difference in 

the redox potentials behaviour with changing temperatures. This may be an indication that the evolutionary 

change from tungsten to molybdenum took not place only because of the better availability of molybdenum 

under mesophillic conditions but also because it provides more stable redox potentials[10]. 

References: 

[1] F. A. M. de Bok, P.-L. Hagedoorn, P. J. Silva, W. R. Hagen, E. Schiltz, K. Fritsche, A. J. M. Stams, Eur. J. 

Biochem. 2003, 270, 2476-2485. 

[2] J. Bernholt, E. I. Stiefel, Inorg. Chem., 1985, 24, 1323. 

[3] M. K. Johnson, D. C. Rees, M. W. W. Adams, Chem. Rev., 1996, 96, 2817. 

[4] G. Pappenberger, H. Scheurig, R. Jänicke, J. Mol. Biol., 1997, 274, 676. 

[5] S. K. Das, D. Biswas, R. Maiti, S. Sarkar, J. Am. Chem. Soc., 1996, 118, 1387. 

[6] S. K. Das, P. K. Chaudhury, D. Biswas, S. Sarkar, J. Am. Chem. Soc., 1994, 116, 9061. 

[7] C. A. Goddard, R. H. Holm, Inorg. Chem., 1999, 38, 5389. 

[8] B. S. Lim, J. P. Donahue, R. H. Holm, Inorg. Chem., 2000, 39, 263. 

[9] B. S. Lim, R. H. Holm, Journal of the American Chemical Society, 2000, 123, 1920. 

[10] C. Schulzke, Dalton Trans. 2005, 713. 

_____________________________________________________________________ 

53


SL2. X-ray Radiation Damage on Metallo-enzymes: 

Can Soaked-in Scavengers Protect Metalloprotein Active Sites from 

Reduction During Data Collection? 

S. Macedo a , M. Pechlaner a , W. Schmid b , M. Weik c , K. Sato d , Ch. Dennison d , 

K. Djinović-Carugo a 

a 

Dept. for Biomolecular Structural Chemistry, Max F Perutz Laboratories, University of Vienna, Campus 

Vienna Biocenter 5, 1030 Vienna, Austria 

b 

Institute of Organic Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria 

c 

Laboratoire de Biophysique Moléculaire, Institut de Biologie Structurale J.-P. Ebel, CEA CNRS UJF, UMR 

5075, 41 rue Jules Horowitz, 38027 Grenoble Cedex 01, France 

d 

Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, NE2 

4HH, UK 

One of the first events taking place when a crystal of a metalloprotein is exposed to X-ray radiation is photoreduction 

of the metal centres. The oxidation state of a metal cannot always be determined from routine X-ray 

diffraction experiments alone, but it may have a crucial impact on the metal's environment and on the analysis of 

the structural data when considering the functional mechanism of a metalloenzyme. A combination of UV-vis 

micro-spectrophotometry and X-ray crystallography was used to test the efficacy of 18 selected scavengers in 

reducing the undesirable photo-reduction of the iron and copper centres in myoglobin and azurin, respectively. 

Results of the UV-vis absorption spectra show dramatic metal reduction occurring in the first 60 seconds of 

irradiation with an X-ray beam from a 3 rd generation synchrotron source, and only two scavengers were capable 

of partially mitigating the rate of metal photo-reduction, but not to a sufficient extent that would allow a 

complete data set to be recorded from a fully oxidised crystal. On the other hand, analysis of the X-ray 

crystallographic data confirms ascorbate as an efficient protecting agent against radiation damage, other than 

metal centre reduction. 

_____________________________________________________________________ 

54


SL3. Isolation and Characterization of New Laccases, Verstile Bio-catalysts 

for Water Remediation 

A. Scozzafava 

Department of Chemistry, University of Florence, via della Lastruccia 3, 50019, Sesto Fiorentino, Italy 

Laccases belong to multicopper oxidases, an important and widespread class of enzymes implicated in a 

extensive series of oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms as well 

as in the metabolic turnover of complex organic substances such as lignin, humic matter, and toxic xenobiotics. 

They catalyze the coupling between four one-electron oxidations of a broad range of substrates with the fourelectron 

reduction of dioxygen to water. In our laboratory we have isolated and characterized some new laccases 

from fungi and solved the X ray structure of those from Lentinus Tigrinus and from Trametes Trogiii. In 

particular in the case of Lentinus (Panus) tigrinus, the reduction of the copper ions centers obtained by the longterm 

exposure of the crystals to the high-intensity X-ray synchrotron beam radiation in aerobic conditions and 

pH 8.5 allowed us to trap two intermediates in the molecular oxygen reduction pathway: the “peroxide” and the 

“native” intermediates result of a two- and four-electrons reduction of molecular oxygen, previously 

hypothesized through spectroscopic and kinetic studies. In the case of T. trogii laccase structural model revealed 

the presence of an exogenous p-toluate molecule bound to the T1 active site, mimicking the interaction of 

substrates and/or mediators with this center. The above structures will be presented and discussed. In addition to 

structural characterization, extensive studies on potential application of laccases for water remediation of waste 

waters from textile industry have been also performed, the results being quite promising. 

_____________________________________________________________________ 

55


SL4. The Use of 64-Cu in Radio-diagnostics and Radionuclide Therapy - 

Required Properties of Chelators 

M. J. Bjerrum . a , J. R. Holm-Jørgensen b , K. Jensen , M. Jensen c 

a 

Department of Natural Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, 

Denmark, 

b 

Department of Natural Sciences, University of Copenhagen and Risø DTU, Thorvaldsensvej 40, DK-1871, 

Frederiksberg C, Denmark 

c 

Risø National Laboratory for Sustainable Energy, Risø DTU, Technical University of Denmark, 

Frederiksborgvej 399, DK-4000, Roskilde, Denmark 

e-mail: mobj@life.ku.dk 

Radiodiagnostics and radionuclide therapy are heavily investigated topics. The use of the same compound for 

diagnostic and therapeutic purposes is highly desirable, and among the requirements are: tumor specificity, ease 

and speed of isotope uptake and high inertia with respect to isotope expulsion. Possible candidate isotopes are 

64 Cu (PET imaging and therapy) and 67 Cu (therapy). With copper radioisotopes there is a need for developing 

novel chelators which forms kinetic and thermodynamic stable copper complexes. New candidates could be 

adamanzanes, a class of macrobicyclic tetraamine ligands with unique copper binding properties. [1] 

Cu(II) complexes are known for several of the adamanzane ligands. The uptake of Cu(II) is possible, choosing 

the right conditions, and expulsion has been shown to be extremely slow from the native adamanzanes. 

Furthermore, substituents on the non-bridged amine groups have been designed to function as linkers to a tumortargeting 

protein, and this has been performed in our group with cytochrome c as the model protein. It has been 

shown that it is possible to link the adamanzane ligand in a one-to-one ratio to one single protein site. It remains 

to be shown if the inherent Cu binding stability of the adamanzane complex is retained. Knowing the structures 

is essential to the interpretation of kinetic experiments necessary to prove the inertia with respect to Cu(II) 

expulsion or other unwanted interactions under biological conditions. 

References: 

[1] Springborg J. Adamanzanes - bi- and tricyclic tetraamines and their coordination compounds. Dalton Trans., 

2003, 1653-1665. 

_____________________________________________________________________ 

56


SL5. Unique Properties of DNA Cross-links of Antitumor Oxaliplatin and 

the Effect of Chirality of the Carrier Ligand 

V. Brabec a , J. Malina a , J. Kasparkova b 

a 

Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, 61265, Brno, 

Czech Republic, 

b 

Laboratory of Biophysics, Department of Experiment, Palacky University, tr. Svobody 26, 77146, Olomouc, 

Czech Republic 

e-mail: brabec@ibp.cz 

Downstream processes that discriminate between DNA adducts of a third generation platinum antitumor drug 

oxaliplatin and conventional cisplatin are believed to be responsible for the differences in their biological effects. 

These different biological effects are explained by the ability of oxaliplatin to form DNA adducts more efficient 

as for their biological effects. We examined conformation, recognition by damaged-DNA binding-proteins, 

cellular processing and repair of the major 1, 2-GG intrastrand cross-link formed by oxaliplatin in three sequence 

contexts and of interstrand cross-link with the aid of biophysical and biochemical methods. We found that the 

properties of the cross-links of oxaliplatin and conventional cisplatin were notably different. In addition, the 

chirality at the carrier 1, 2-diaminocyclohexane ligand affected structural properties of cross-links of cisplatin 

analogs. We suggest that the unique properties of DNA cross-links of oxaliplatin are at least partly responsible 

for its unique antitumor effects [1, 2]. 

References: 

[1] Malina, J., Novakova, O., Vojtiskova, M., Natile, G. and Brabec, V. (2007) Conformation of DNA GG 

intrastrand cross-link of antitumor oxaliplatin and its enantiomeric analog. Biophys J, 93, 3950-3962. 

[2] Kasparkova, J., Vojtiskova, M., Natile, G. and Brabec, V. (2008) Unique properties of DNA interstrand 

cross-links of antitumor oxaliplatin and the effect of chirality of the carrier ligand. Chem Eur J, 14, 1330-1341. 

_____________________________________________________________________ 

57


SL6. Bisphosphonates: Coordination Abilities of Potent Chelators for 

Metal Ions 

E. Gumienna-Kontecka a , J. Gałęzowska b and H. Kozłowski a 

a Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wroclaw, Poland. 

b Department of Inorganic Chemistry, Faculty of Pharmacy, Wrocław Medical University, Szewska 38, 

50-139 Wrocław, Poland. 

e-mail: kontecka@wchuwr.pl 

The recent approaches to Alzheimer’s and Parkinson’s diseases reflect the current concepts of copper(II) and 

iron(III) involvement in the etiology of these very common neurological disorders. From the other side 

inexorable increase is observed for cancer diseases causing among others metastases to bones. To find 

therapeutic measures which will slow down the onset of these diseases, the design of novel treatment strategies 

is currently at the focus of attention. 

One of the most promising approaches to search novel ligand systems for development of highly efficient 

chelators lie in rational combination of known donor functions with specific characteristics in one ligand 

molecule. The principal aims of our work is design of novel ligands of different donor and chelating capacity, 

consisting of, among others, bisphosphonate moiety. 

Bisphosphonates are highly hydrophilic and low toxic ligands which may accommodate a large number of ionic 

radii due to their mobility and flexibility. Earlier studies have shown that bisphosphonates are efficient chelating 

agents for Fe(III) [1], as well as Cu(II) [2] but their oral bioavailability is poor. It has inspired us to modify 

phosphonic ligand with hydrophobic, well studied chelating agent – hydroxypyridinone (Ligand 1) [3]. 

The radionuclide complexes of 153 Sm, 166 Ho and 177 Lu with poliaminopolyphosphonates are considered as 

possible therapeutic pharmaceuticals in bone pain palliation [4]. The potential properties of the use of Gd(III) 

complexes with poliaminopolyphosphonic ligands as MRI imaging agents have been also studied [5]. 

Considering both, the binding properties and the results of biological trials we have decided to study the 

influence of the COOH/PO3H2 substitution in CDTP on the binding properties of this ligand towards Ln(III) 

ions (Ligand 2) [6]. 

O 

N 

OH 

C 

O 

NH 

Ligand 1 

CH 2 

CH 2 

CH 

PO 3H 2 

PO 3H 2 

_____________________________________________________________________ 

58 

N 

N 

Ligand 2 

Acknowledgement: E.G.-K. thanks the Polish Ministry of Science and Higher Education for reintegration grant 

(113/6.PR UE/2007/7). 

References 

[1]. E. Gumienna-Kontecka, R. Salvagni, R. Lipinski, M. Lecouvey, F. Cesare Marincola, G. Crisponi, V.M. 

Nurchi, Y. Leroux, H. Kozłowski, Inorg. Chim. Acta, 339, 111-118 (2002). 

[2]. E. Gumienna-Kontecka, J. Jezierska, M. Lecouvey, Y. Leroux, H. Kozłowski, J. Inorg. Biochem., 89, 13-17 

(2002). 

[3]. G. Crisponi, V.M. Nurchi, T. Pivetta, J. Gałęzowska, E. Gumienna-Kontecka, T. Bailly, R. Burgada H. 

Kozłowski, J. Inorg. Biochem., 102, 1486-1494 (2008). 

[4]. J.R. Zeevart, N.V. Jarvis, W.K.A. Louw, G.E. Jackson, J. Inorg. Biochem., 83, 57-65 (2001). 

[5 ] The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, ed. A.E. Merbach and E. Tóth 

Wiley, Chichester (2001). 

[6] J. Gałęzowska, R. Janicki, A. Mondry, R. Burgada, T. Bailly, M. Lecouvey, H. Kozłowski, Dalton Trans, 

4384-4394 (2006). 

PO 3H 2 

PO 3H 2 

PO 3H 2 

PO 3H 2

I. Turel 


SL7. Interactions of Metal Ions with Quinolone Antibacterial Agents. 

Isolation of Metal Complexes, Their Biological Activity and Possible 

Practical Applications 

Faculty of Chemistry, University of Ljubljana, Askerceva 5, 1000, Ljubljana, Slovenia 

e-mail: iztok.turel@fkkt.uni-lj.si 

In the last years quinolones are clinically the most successful synthetic antibacterial agents and one of the 

famous members of this large family-ciprofloxacin (cfH) is a real blockbuster drug [1-3]. 

Unpredictable and never ending battle between bacteria and mankind shows that diseases considered to be 

controlled or even eradicated are appearing again, often in new forms that are multidrug resistant. Therefore it is 

crucial to understand the molecular mode of action of existing drugs which could help us to exploit them even 

more efficiently in the future. 

The selective activity of quinolones is due to the inhibition of the supercoiling of DNA catalyzed by the bacterial 

enzyme DNA gyrase and metal ions (magnesium) are also involved in these processes. It is also known that 

quinolones can easily react with metal ions [4]. In one hand metal-quinolone interactions are disturbing because 

the absorption of these drugs is reduced (due to the formation of sparingly soluble metal complexes) but on the 

other hand it is believed that metal ions are needed for the biological activity of quinolones. From all these facts 

it is obvious that it is extremely important to thoroughly reveal the details of metal-quinolone interactions. In this 

lecture our results from quinolone-metal (magnesium, copper, vanadium, bismuth, europium) systems will be 

shown. 

References: 

[1] L. A. Mitscher, Chem. Rev. 2005, 105, 559-592. 

[2] K. E. Brighty, T. D. Gootz, in: The Quinolones (Ed.: V. T. Andriole), Academic Press, San Diego, 2000, pp. 

33-97. 

[3] G. Sheehan, N. S. Y. Chew, in: Fluoroquinolone Antibiotics, (Eds.: A. R. Ronald, D. E. Low), Birkhauser, 

Basel, Switzerland, 2003, p. 1-10. 

[4] I. Turel, Coord. Chem. Rev. 2002, 232, 27-47, and the references cited therein. 

_____________________________________________________________________ 

59


SL8. Peptide Hydroxamic Acids – Versatile Ligands for Metal Ions 

P. Buglyó a , E. Farkas a , E. Csapó a , E.M. Nagy a , D. Sanna b and G. Pappalardo c 

a 

Department of Inorganic & Analytical Chemistry, University of Debrecen, H-4010 Debrecen, Hungary 

b 

Istituto C.N.R. di Chimica Biomolecolare, Sezione di Sassari, Trav. La Crucca 3 Reg. Baldinca, I-07040 

Sassari, Italy 

c 

Istituto per lo Studio delle Sostanze Naturali di Interesse Alimentare e Chimico Farmaceutico, CNR, Catania, 

Italy 

e-mail: buglyo@delfin.unideb.hu 

Peptide hydroxamic acids consist of a peptide chain and a hydroxamic (-CON(R)OH) function at the C terminus. 

The most remarkable feature of these ligands obtained via hydroxyamidation of the corresponding oligopeptide 

is the selective metal ion binding which is one important reason for their inhibitory effect on numerous 

metalloproteinase enzymes. For design of effective enzyme inhibitors based on peptide hydroxamic acids the 

knowledge of the most important factors determining the strength of interaction with metal ions is crucial. In 

order to design effective inhibitors a systematic study has started in our laboratories with the synthesis of this 

type of ligands and with the study of their metal ion binding capabilities. 

Combining a peptide chain and a hydroxamic function there are numerouos coordination sites available for metal 

binding in these ligands providing high selectivity for the metals. 

Beside the hydroxamate, the terminal amino group, the amide 

nitrogen of the peptide group(s) and the side chain donors present 

in the molecule can also take part in the metal ion binding. 

While primary hydroxamic acids (R3 = H) are capable to form 

hydroximato type chelates with soft metal ions after 

deprotonation and co-ordination of the hydroxamate NH, the 

alkyl substituted secondary hydroxamic acids can only form 

hydroxamato [O, O] type complexes. In order to obtain information about the role of the terminal amino group in 

metal binding the corresponding non-protected and Z-protected ligand pairs were synthesized and studied. 

Beside simple (AlaAla) dipeptide hydroxamic acids, the tripeptide analogues were also prepared and 

investigated to explore the role of the peptide amide(s) of the molecule in the interaction with metal ions. 

Since the imidazole moiety is one of the most important binding site in peptides it may significantly influence 

the strength and selectivity of metal ion binding in peptide hydroxamates too. To explore this field new, 

histidine-containing di- and tripeptide hydroxamates were synthesized and investigated. 

In all cases pH-potentiometry was applied to determine the protonation constants of the ligands as well as the 

stoichiometry and stability constants of the metal complexes formed in aqueous solution. In order to obtain 

reliable information on the most probable solution structure of the associates in the metal ion containing systems 

combined spectroscopic (NMR, EPR, UV-VIS, CD, ESI-MS) techniques were used. 

The lecture will summarise the most important trends which were found in the interaction of these ligands with 

biologically relevant (Fe(III), Cu(II), Ni(II), Zn(II), Al(III), Mo(VI)) metal ions. 

Acknowledgement: This research was supported by the Hungarian Scientific Research Fund (OTKA T 046366, 

T 049612). 

References: 

[1] P. Buglyó, E.M. Nagy, E. Farkas, I. Sóvágó, D. Sanna and G. Micera, Polyhedron, , 26, 1625, (2007). 

_____________________________________________________________________ 

60 

H 2N 

R 1 

O 

H 

N 

R 2 

O 

N 

R 3 

OH 

n n = 1, 2

P. Turano 


SL9. NMR of Ferritin: a 480 kDa Protein 

CERM & Department of Chemistry, University of Florence, Via Luigi Sacconi, 6, 50019, Sesto Fiorentino 

(Florence), Italy 

e-mail: turano@cerm.unifi.it 

We report here about the NMR characterization of the 480 kDa protein bullfrog ferritin M. The protein is a 

homopolymer of 24 identical subunits forming a large hollow sphere. Each subunit has 175 amino acids folded 

into four helix bundles; in living cells the cavity contains concentrated iron (hydrated ferric oxide mineral). The 

protein is assembled as a spherical nanocage with an external diameter of 12 nm, and an inner cavity, 8 nm in 

diameter.A combined solution-solid state approach was developed by us to assign the signals of such a large 

system in its apo form. By exploiting 13 C-resonance-specific chemical shifts and spin diffusion effects, by 13 Cdirect 

detection NMR in solution we identified 75% of the spin patterns of protein amino acids, with intraresidue 

C-C connectivities between nuclei separated by 1-4 bonds [1, 2]. Solid state NMR provided sequence specific 

assignments [3].Paramagnetic protonless NMR in solution was successfully used to identify the channel through 

which iron(III) migrates from the ferroxidase site to the internal nanocompartment where the mineral is formed. 

References: 

[1] Matzapetakis, M., Turano, P., Theil, E. C., and Bertini, I., 13 C- 13 C NOESY spectra of a 480 kDa protein: 

solution NMR of ferritin, J.Biomol. NMR, 38, 237-242, 2007. 

[2] Bermel, W., Felli, I. C., Matzapetakis, M., Pierattelli, R., Theil, E. C., and Turano, P., A method for Cα 

direct-detection in protonless NMR, J.Magn.Reson., 188, 301-310, 2007. 

[3] Bertini, I., Felli, I. C., Klein, R., Lalli, D., Matzapetakis, M., Theil, E. C., and Turano, P. Solid state NMR of 

ferritin, in preparation. 

_____________________________________________________________________ 

61


SL10. FeFe-hydrogenases: Insights into the Structural Basis of Active Site 

Maturation 

Y. Nicolet a , J.K. Rubach b* , M.C. Posewitz c , P. Amara d , C. Mathevon b , M. Atta b , 

M. Fontecave b and J.C. Fontecilla-Camps a 

a Laboratoire de Cristallographie et Cristallogenèse des Protéines; Institut de Biologie Structurale J.P. Ebel; 

CEA; CNRS; Université J. Fourier; 41 rue J. Horowitz, 38027 Grenoble Cedex 1, France. 

b Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques; iRTSV-CB; CEA; CNRS; Université J. 

Fourier; 17 avenue des Martyrs, 38054 Grenoble Cedex 09, France 

c Colorado School of Mines, Environmental Sciences and Engineering Division, Golden, CO 80401, USA. 

d Laboratoire de Dynamique Moléculaire; Institut de Biologie Structurale J.P. Ebel; CEA; CNRS; Université J. 

Fourier; 41 rue J. Horowitz, 38027 Grenoble Cedex 1, France. 

* Life Sciences institute, University of Michigan, 210 Washtenaw Ave, Ann Arbor, MI 48109 

FeFe-hydrogenases mediate both hydrogen oxidation and proton reduction by microorganisms. The maturation 

of the enzyme’s active site depends on at least three genes products called HydE, HydF and HydG. HydF has 

GTPase activity and has been recently shown that is involved in active site insertion [1]. The other two 

components are “AdoMet” radical enzymes and should be involved in the synthesis of the [FeFe] cluster ligands, 

namely, CO, CN - and the small organic, dithiolate-containing, molecule (figure). Very recently, we have 

reported the high-resolution structure of recombinant, reconstituted HydE from Thermotoga maritima [2]. Sitedirected 

mutagenesis studies of the closely related HydE from Clostridium acetobutylicum have allowed the 

mapping of regions in this protein essential for hydrogenase maturation. In addition, from soaking experiments 

we have identified three anion-binding sites inside a large, positive cavity, one of which binds SCN - with very 

high affinity. Recent results will be reported at the meeting. 

FeFe-hydrogenase active site 

(L2: CN - ; L1-L3: CO) 

References 

[1] McGlynn SE, Shepard EM, Winslow MA, Naumov AV, Duschene KS, Posewitz MC, Broderick WE, 

Broderick JB, Peters JW. HydF as a scaffold protein in [FeFe] hydrogenase H-cluster biosynthesis. FEBS Lett. 

2008 582(15):2183-7. 

[2] Nicolet Y, Rubach JK, Posewitz MC, Amara P, Mathevon C, Atta M, Fontecave M, Fontecilla-Camps JC. 

X-ray Structure of the [FeFe]-Hydrogenase Maturase HydE from Thermotoga maritima. J Biol Chem. 2008; 

283(27):18861-18872 

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62


SL11. The Role of Histidine-rich Proteins in Helicobacter pylori 

H. Sun, S. Cun, R. Ge and Y. Zeng 

Department of Chemistry, The University o Hong Kong, Pokfulam Road, Hong Kong 

e-mail: hsun@hku.hk 

Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative spiral-shaped bacterium which causes 

chronic inflammation of the stomach and peptic ulcer formation in humans [1]. Bismuth compounds such as 

colloidal bismuth subcitrate (De-Nol ® ) and ranitidine bismuth citrate (Pylorid ® ) have been widely used for the 

treatment of H. pylori infection together with antibiotics [2,3]. Proteins and enzymes have been thought to be the 

target(s) of bismuth in vivo. Our comparative proteomic data of H. pylori cells before and after treatment with 

colloidal bismuth subcitrate showed that eight proteins are significantly up- or down-regulated. Using 

immobilized-metal affinity chromatography (Bi- and Ni-IMAC), we isolated and subsequently identified seven 

bismuth-binding proteins from H. pylori extracts [4]. One of the binding proteins, the Heat-Shock Protein A 

(HspA), was then overexpressed and purified to confirm its metal-binding properties. Recombinant H. pylori 

HspA was found to bind both Ni 2+ and Bi 3+ at its C-terminal histidine- and cysteine-rich domain of the proteins. 

Binding of bismuth to the protein is much stronger than nickel. Importantly, Bi 3+ induces the structural changes 

of the protein from its native form (heptamer) to a dimer [5]. When cultured in Ni 2+ -supplemented M9 minimal 

media, E. coli BL21(DE3) expressing the wild-type HspA or C-terminal deletion mutant clearly indicated a role 

that the C-terminus might protect cells from higher concentration of external Ni 2+ . In contrast, an opposite 

phenomenon was observed when the same E. coli hosts were grown in Bi 3+ -supplemented media. The histidine- 

and cysteine-rich domain may play a critical role in nickel homeostasis and bismuth susceptibility in vivo. 

Binding of the metallodrug to other histidine-rich proteins was also characterized [6]. Our preliminary 

bioinformatic search has found that there are actually any histidine-rich proteins and motifs in microorganisms 

[7]. Hpn (28 His residues out of 60 aa) is a small cytoplasmic protein in H. pylori and is present as a multimer 

with 20-mer being the predominant species in solution and binds to five Ni 2+ and four Bi 3+ per monomer 

moderately (Kd of 7.1 and 11.1 µM respectively) [6]. Although in vitro, it binds to Cu 2+ stronger than Ni 2+ and 

Bi 3+ , the in vivo protection by the protein is in the order of Ni 2+ > Bi 3+ > Cu 2+ [4]. Hpn may therefore serve to 

buffer intracellular Ni 2+ in much the similar way to that the small and cysteine-rich protein, metallothionein 

interacts with Zn 2+ /Cu + . 

Acknowledgement: This work was supported by Research Grants Council of Hong Kong (HKU7039/04P, 

HKU7043/06P, HKU1/07C), National Science Foundation of China and the University of Hong Kong! 

References: 

[1] B.J. Marshall, J.R. Warren, Lancet 1984, i, 1311. 

[2] S. Suerbaum, P. Michetti, New Engl. J. Med. 2003, 347, 1175. 

[3] (a) H. Sun, L. Zhang, K.Y. Szeto, Met. Ions Biol. Syst. 2004, 41, 333; (b) R.G. Ge, H. Sun, Acc. Chem. Res. 

2007, 40, 267. 

[4] R. Ge, X. Sun, Q. Gu, R.M. Watt, B.C.Y.;Wong, H.H.X. Xia, J. Huang, Q. He, H. Sun J. Biol. Inorg. Chem. 

2007, 12, 831. 

[5] S.J. Cun, H. Li, R. Ge, M.C. Lin, H. Sun, J. Biol. Chem. 2008, 283, 15142. 

[6] (a) R. Ge, Y. Zhang, X. Sun, R.M. Watt, Q.Y. He, J. Huang, D.E. Wilcox, H. Sun, J. Am. Chem. Soc. 2006, 

128, 11330; (b) R. Ge, R.M. Watt, X. Sun, J.A. Tanner, Q.Y. He, J. Huang, H. Sun, Biochem. J. 2006, 393, 

285. 

[7] J.F. Tomb et al (1997) Nature 388, 539-547. 

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63


SL12. N-terminal, Histidine-containing Metal Binding Sites in Proteins: 

Lessons from Model Studies 

T. Gajda a , A. Jancsó a , A. Kolozsi a , A. Battistoni b , Z. Paksi a 

a Dept. of Inorganic and Analytical Chemistry, University of Szeged, Hungary, 

b Dept. of Biology, University of Rome Tor Vergata, Roma, Italy 

e-mail: gajda@chem.u-szeged.hu 

Recent biochemical studies pointed out the presence of relatively short, histidine-containing sequences with 

strong metal binding ability, being independent from the other parts of the biomolecule, in a great number of 

proteins and enzymes. According to the various biological studies, the metal ion coordination to these sites 

determines or substantially contributes to the function of the given protein/enzyme, i.e. they have catalytic, 

structural or some alternative functions, promoting the biological role of these biomolecules. The N-terminal 

fragments of proteins often have no stable preorganized structures, therefore their metal binding and functional 

properties can be assessed by studying oligopeptides with identical sequences. Here we report three examples 

related to the above mentioned three different functions of N-terminal fragments. 

During the last decade several nickel-containing SOD enzymes (Ni-SOD) have been isolated from aerobic soil 

bacteria, with no apparent similarity to other SODs. Their crystal structure [1] indicates, that the metal ions are 

bound to the N-terminal six amino acids in both the oxidized and reduced enzyme, thus the well conserved Nterminal 

sequence 1 HCDXPC– (X=G or L) provides practically all critical interactions for metal ion binding and 

catalysis. This offers a unique possibility for structural and functional modelling of these enzymes, since the 

metal binding sites in metalloenzymes are generally well separated in the amino acid sequences, therefore the 

structure of the active centres is almost impossible to mimic by small peptides. The solution chemical study of 

the nickel(II)–HCDLPCG-NH2 (L 1 ) system indicated the formation of the square planar NiH–1L 1 species above 

pH 6, with {NH2, N – , S – , S – } donor set, being identical with the active site of the reduced enzyme. This species 

possesses high SOD-like activity, in contrast to the majority of nickel(II) complexes. 

Cu, Zn-SOD enzymes of some Gram-negative bacteria are characterized by a His-rich N-terminal extension with 

strong metal binding ability. The N-terminal sequence of the Cu, Zn-SOD from H. ducreyi has been suggested to 

play a chaperoning role during the uptake of active-site copper, promoting the bacterial survival [2]. We have 

undertaken equilibrium and solution structural study in the copper(II)- and zinc(II)- NH2-HGDHMHNHDTK- 

OH (L 2 ) systems, in order to support this assumption and to investigate the possibility of similar function in zinc 

uptake [3]. L 2 possesses extraordinary metal ion sequestering capacity in the neutral pH, provided only by side 

chain donors, which approaches to the metal-trafficking proteins. The picomolar affinity for copper(II) supports 

the proposed chaperoning role of the N-terminal His-rich region, while the nanomolar zinc(II) binding affinity 

may suggest similar role in the zinc(II) uptake, too. Interestingly, the complex CuHL 2 has significant SOD-like 

activity, suggesting multifunctional role of the N-terminal His-rich domain. 

Endostatin, a fragment of collagen XVIII, is a special inhibitor of endothelial cell proliferation and migration, 

and possesses marked anticancer properties without any side effects. Recently it was shown that the anticancer 

activity of the N-terminal 25 amino acids and the protein itself are equivalent [4], which is of crucial importance 

for future therapeutic usage. The presence of zinc(II), which is likely to have structural role, is necessary to exert 

the anticancer effect in both cases, but details on the metal ion interaction of the N-terminal fragment is not 

known. Here we report solution chemical investigation of the zinc(II) and copper(II) complexes of 

HSHRDFQPVLHL–NH2 (L 3 ) peptide, which is identical with the first twelve amino acids of human endostatine, 

and contains all possible metal binding sites of the N-terminal fragment. In presence of zinc(II) the ZnL complex 

is formed in the neutral pH range with {NH2, 3Nim} coordination, creating huge macrochelates. Due to the 

presence of an ATCUN motif, L 3 also binds copper(II) very efficiently. This finding may have biological 

importance, since copper(II) is also deeply involved in angiogenesis. 

The presentation will discuss some properties of the metallopeptides which uncovered some subtle details on the 

functioning of the corresponding metalloproteins. 

Acknowledgement: This work was supported by the Hungarian Scientific Research Found (NI61786, K63606). 

References: 

[1] J. Wuerges, J.-W. Lee, Y.-I. Yim, H.-S. Yim, K.D. Carugo, Proc. Natl. Acad. Sci. USA, 101, 8569 (2004). 

[2] P. D'Angelo, F. Pacello, G. Mancini, O.Proux, J.L. Hazemann, A. Desideri, A. Battistoni, Biochemistry, 44, 

13144 (2005). 

[3] Z. Paksi, A. Jancsó, F. Pacello, N. Nagy, A. Battistoni, T. Gajda, J. Inorg. Biochem., in press (2008). 

[4] R. M. Tjin Tham Sjin, R. Satchi-Fainaro, A. E. Birsner, V.M. S. Ramanujam, J. Folkman, K. Javaherian, 

Cancer Res., 65, 3656-3663 (2005). 

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64


SL13. Antitumour Activity of Transition Metal Complexes with Hydrazone 

Ligands 

D. Sladić 

Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade. Serbia 

e-mail: dsladic@chem.bg.ac.yu 

Research on coordination compounds as antitumor agents has been an important part of bioinorganic and 

medicinal chemistry since the discovery of the antiproliferative activity of cisplatin. In this talk results of 

antitumor activity investigation in vitro of chelate complexes of d-metals (Zn, Cd, Cu, Ni, Pd, Pt, Co, Fe) with 

hydrazone type ligands, namely hydrazones, hydrazide-hydrazones, thiosemicarbazones and 

selenosemicarbazones will be presented. For some of the most active complexes results of investigations of cell 

cycle perturbations, apoptotic assays and gelatin zymography in relation to invasion and metastasis of tumor 

cells will be discussed. 

Acknowledgement: The research presented in this lecture was supported by the Ministry of Science of the 

Republic of Serbia (Grant no. 142026). 

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65


SL14. New Pt(II) and Pt(IV) Complexes with Purine Ligands 

as Precursors for Anticancer Drugs 

I. Łakomska, E. Szłyk 

Faculty of Chemistry, Nicolaus Copernicus University, ul. Gagarina 7, 87 100 Toruń, Poland, 

e-mail: eszlyk@chem.uni.torun.pl 

Presently used antitumor drugs, based on cisplatin mechanism, still need improvement due to the known side 

effects. Hence the synthesis and structural characterization of platinum(II) complexes with new ligands along 

with tests for antitumor activity is the purpose of our research. In our studies we have focused on 1,2,4triazolo[1,5-a]pyrimidine 

derivatives, which have the structure similar to purine. Their fused ring system differs 

in having the pyrimidine nitrogen atom in a bridgehead position with disappearance of the acidic H-proton of the 

five-membered ring. Triazolopyrimidine derivatives ligands display versatility in their interaction with metal 

ion, because they can bind the metal ion via different hetrocyclic nitrogen atoms and due to the impact on the 

other ligands, either by electronic or steric factors. Pt(II) compounds, including cisplatin, are not orally active 

and often lose their effectiveness in prolonged administration. Therefore a six-coordinate octahedral 

platinum(IV) complexes, which reveal better solubility could be one of the possible ways to solve the problem. 

The six-coordinated Pt(IV) predisposes towards ligands substitution by a dissociative mechanism versus the 

associative mechanism for Pt(II). Moreover Pt(IV) compounds are more substitutionally inert, hence this is 

desirable for oral bioavailability and reduction of toxicity, but is unfavorable for DNA intercalation. 

Nonetheless, several platinum(IV) complexes show considerable activity in initial trials. Their functionality 

depend on the in vivo reduction of Pt(IV) to Pt(II), producing reactive intermediates, that can interact with DNA. 

Examination of the structure-activity relationship (SAR) indicates that biological activity of platinum(II) series 

depended on the coordinated ligand. Studied complexes were structurally characterized by: 1 H, 13 C, 15 N, 195 Pt 

NMR, IR, MS spectra and X-ray crystal structure analysis. One can suggests, that complexes with two 

heterocycles in cis position are more active, than their cisplatin analogues. Examination of SAR, suggests, that 

antitumor activity of platinum(IV) compounds containing 1,2,4-triazolo[1,5-a]pyrimidines correlates directly 

with the size of the alkyl groups substituted on the heterocyclic ligands. The highest activity was observed for 

complexes with tertbutyl group in 5,7 positions, in the pyrimidine ring. In the triazolopyrimidine family of 

platinum(II) complexes the best activity against tumors cell lines has been detected for complexes with steric 

hindrances in the triazolopyrimidine ring. In this case environment around platinum(II) ion influence the 

antitumor activity The discussion of the structural parameters of Pt(II) and Pt(IV) complexes in function of their 

antiproliferative activity in vitro against the cells of human cancer cell lines: T47D (breast cancer), A549 (nonsmall 

cell lung carcinoma), HCV29T (bladder cancer) and SW707 (rectal adenocarcinoma) and MCF7, EVSa- 

T, WIDR, IGROV, M19MEL, A498, H226 will be presented. It has been noted, that ID50 values for some of 

the complexes are in range of the international activity criterion for synthetic agents (4 µg/ml), hence these 

compounds may be considered as the agents for potential antitumor application. 

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66


SL15. Mass Spectrometry and NMR for Spectroscopically Silent Metal 

Ions: Lessons from Zinc Metallothioneins from Bacteria and Plants 

C. Blindauer , O. Leszczyszyn 

Chemistry, University of Warwick, Gibbet Hill Road, CV4 7AL, Coventry, United Kingdom, 

e-mail: c.blindauer@warwick.ac.uk 

In contrast to the plethora of spectroscopic techniques that are available to study iron-binding proteins, methods 

for directly observing the metal ion binding properties of zinc-binding proteins, such as coordination mode, 

uptake and release are far more limited. Recent advances in mass spectrometry instrumentation allow 

observation of native metalloproteins including, in favourable cases, following metal binding events in real time, 

as shown in the Figure. Such experiments are aided by the fact that biologically relevant zinc complexes are 

often relatively stable. Similarly, for NMR spectroscopy, the diamagnetic nature of Zn(II) is advantageous, 

allowing the application of a variety of multinuclear NMR techniques. Discussing our recent work concerning 

bacterial metallothioneins from various cyanobacteria [1, 2], as well as the plant metallothionein wheat EC [3], 

we will demonstrate that the combination of these two experimental techniques, together with site-directed 

mutagenesis and molecular modelling, is a powerful approach to understand metal ion binding thermodynamics 

and kinetics in great detail. 

Acknowledgement: We thank the Royal Society (Olga Kennard Fellowship to C.A.B.), the EPSRC, the 

BBSRC, the Wellcome Trust, and the European Commission for support. 

References: 

[1] C. A. Blindauer, M. T. Razi, D. J. Campopiano, P. J. Sadler, J. Biol. Inorg. Chem. 2007, 12, 393-405. 

[2] O. I. Leszczyszyn, C. D. Evans, S. E. Keiper, G. Z. L. Warren , C. A. Blindauer, Inorg. Chim. Acta 2007, 

360, 3-13. 

[3] O. I. Leszczyszyn, R. Schmid, C. A. Blindauer, Proteins 2007, 68, 922-935. 

_____________________________________________________________________ 

67


G. Knör 

SL16. Potential Applications of Multifunctional Coordination 

Compounds in Molecular Photomedicine 

Institute of Inorganic Chemistry, Johannes Kepler University Linz (JKU), Altenbergerstrasse 69, A-4040 Linz, 

Austria 

e-mail: Guenther.Knoer@JKU.at 

Fundamental research on the photochemical and photophysical properties of coordination compounds frequently 

leads to important innovations in the field of modern life sciences. In this context, the application of luminescent 

and light-responsive metal complexes as probes, labels or diagnostic tools, and the interaction of photosensitizers 

with biological systems in general has been widely studied. Photoreactive metal complexes have also been 

applied successfully in the context of biomimetic and bio-inspired systems, including artificial enzyme catalysis, 

which has been demonstrated to compete very well with the performance of native biocatalysts [1]. More 

recently, the potential use of photoreactive coordination compounds for the controlled release of drugs and 

therapeutic agents has also been taken into consideration [2-4]. 

In the present contribution, some novel aspects of light-sensitive multichromophore systems and photoreactive 

metal complexes will be discussed in the context of molecular photomedicine. These examples include artificial 

photonuclease enzymes and systems for the photoinduced release of small bioactive compounds. 

Acknowledgement: The DFG Graduate College 640 “Sensory Photoreceptors in Natural and Artificial 

Systems” is gratefully acknowledged for financial support. 

References: 

[1] G. Knör, Bionic Catalyst Design: A Photochemical Approach to Artificial Enzyme Function, ChemBioChem 

2001, 2, 593-596. 

[2] F. S. Mackay, J. A. Woods, P. Heringová, J. Kašpárková, A. M. Pizarro, S. A. Moggach, S. Parsons, 

V. Brabec, P. J. Sadler, A Potent Cytotoxic Photoactivated Platinum Complex, PNAS 2007, 104, 20743-20748. 

[3] K. Szacilowski, W. Macyk, A. Drzewiecka-Matuuszek, M. Brindell, G. Stochel, Bioinorganic 

Photochemistry: Frontiers and Mechanisms, Chem. Rev.2005, 105, 2647-2694. 

[4] G. Knör, Artificial Enzyme Catalysis Controlled and Driven by Light, Chem. Eur. J. 2008, submitted for 

publication. 

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68


SL17. Light and Inorganic Species in Nanomedicine 

W. Macyk, K. Szaciłowski, M. Brindell, A. Jańczyk, P. Łabuz, G. Stochel 

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Kraków, Poland, 

e-mail: stochel@chemia.uj.edu.pl 

Nanomedicine, the application of nanotechnology and nanomaterials in healthcare, offers numerous very 

promising possibilities to significantly improve medical diagnosis and therapy, leading to higher quality of life. 

Excited states of metal complexes and nanocrystalline wide bandgap semiconductors offer new physicochemical 

properties which can be used for medical purposes. Depending on the type of excited state and its deactivation 

pathway (radiative or nonradiative, energy or electron transfer, direct or sensitised process) inorganic or hybrid 

(inorganic-organic) nanomaterials can be considered for diagnostic, preventive or therapetic applications.[1] 

In this contribution some examples from our latest studies focused on new nanomaterials or strategies for 

photomedicine (photodynamic therapy PDT, photodynamic inactivation PDI, photochemiotherapy, 

photodiagnosis, phototargeting etc.) will be presented.[2-4] 

References: 

[1] K. Szaciłowski, W. Macyk, A. Drzewiecka-Matuszek, M. Brindell and G. Stochel, Chem. Rev., 105, 2647- 

2694 (2005). 

[2] A. Jańczyk, A. Wolnicka-Głubisz, K. Urbanska, H. Kisch, G. Stochel, W. Macyk, Free Rad. Biol. Med., 44 

(2008) 1120-1130. 

[3] D. Mitoraj, A. Jańczyk, M. Strus, H. Kisch, G. Stochel, P.B. Heczko, W. Macyk, Photochem.Photobiol. Sci., 

6 (2007), 642-648. 

[4]M. Brindell, E. Kuliś, S.K.C. Elmroth, K. Urbanska, G. Stochel, J. Med. Chem., 48, (2005) 7298 - 7304 

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69


J. Ciesiołka 

SL18. The Role of Divalent Metal Ions in Functioning of 

the Antigenomic Delta Ribozyme 

Laboratory of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 

Noskowskiego 12/14, 61-704 Poznań, 

e-mail: ciesiolk@ibch.poznan.pl 

In the genomic RNA strand of the hepatitis delta virus (HDV), as well as in its antigenomic counterpart 

generated during virus replication via the double rolling circle mechanism, there are two sequences with 

ribozyme activities, called the delta ribozymes. Despite large progress in elucidation of the structure and 

mechanism of catalysis of delta ribozymes, one of the most important issues, concerning the role of divalent 

metal ions in their functioning, is still a mater of debate [1]. In our earlier studies we have compared the activity 

of closely related variants of the antigenomic ribozyme in the presence of various divalent metal ions [2]. The 

ribozymes differed in regions that were not directly involved in formation of the delta ribozyme catalytic core. 

Thus the role of these peripheral elements in modulating ribozyme activity could be assessed. Interestingly, some 

antibiotics and their complexes with metal ions could inhibit catalytic activity of this ribozyme [3]. 

The existing data on delta ribozymes do not show whether a similar or better ribozyme performance could be 

achieved by catalytic centers that are composed of nucleotides other than the wild-type residues. High sequence 

conservation of ribozyme regions of viral RNAs precludes answering this question. Simultaneous testing of a 

very large number of ribozyme variants with multiple mutations is, however, possible with the use of the in vitro 

selection methodology. 

We used the in vitro selection method to search for catalytically active variants of the antigenomic delta 

ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2 [4]. In the 

initial combinatorial library sixteen nucleotide positions were randomized and the library contained a full 

representation of all possible sequences. Following ten cycles of selection-amplification several catalytically 

active ribozyme variants were identified. It turned out that one-third of the variants contained only single 

mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next onethird 

of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage 

mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position of 

the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial ‘structural plasticity’ 

and the capacity of functional adaptation [4]. In subsequent studies four selected ribozyme variants were 

subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards 

Mg 2+ , Ca 2+ and Mn 2+ ions. In order to localize tight metal ions binding sites within the ribozyme structures we 

used the metal ion-induced cleavage method. Furthermore, in an attempt to analyze the importance of phosphate 

oxygen atoms in both tertiary interactions and coordination of metal ions several NAIM (nucleotide analog 

interference mapping) experiments were performed. The differences in catalytic activity of ribozyme variants 

seem to be a consequence of the different abilities of various metal ions both to perform a chemical reaction as 

well as to aid the formation of ribozyme structural core. 

Acknowledgement: I would like to thank my present and former coworkers for their work with the delta 

ribozymes and members of Prof. M. Jeżowska-Bojczuk group from University of Wrocław for collaboration. 

This work was supported by the Polish Ministry of Science and Higher Education. 

References: 

[1] M.D. Been. CTMI 307, 47 (2006). 

[2] J. Wrzesiński, M. Łęgiewicz, B. Smólska, J. Ciesiołka. Nucleic Acids Res. 29, 4482 (2001). 

[3] J. Wrzesiński, M. Brzezowska, W. Szczepanik, M. Jeżowska-Bojczuk, J. Ciesiołka. Biochem. Biophy. Res. 

Commun. 349, 1394 (2006). 

[4] M. Łęgiewicz, A.Wichłacz, B. Brzezicha, J. Ciesiołka. Nucleic Acids Res. 34, 1270 (2006). 

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70

A. Mokhir 


SL19. Visible Light Activated Nucleic Acid Binders 

Inorganic Chemistry, Heidelberg University, Im Neuenheimer Feld 270, 69120, Heidelberg, Germany, 

e-mail: Andriy.Mokhir@urz.uni-heidelberg.de 

Hairpin structured oligodeoxyribonucleotides containing a singlet oxygen sensitive linker in the loop have been 

prepared. We have demonstrated that these compounds do not bind complementary deoxyribonucleic acids in 

the dark. Upon irradiation with red light in the presence of a photosensitizer the linker within these compounds 

is cleaved and a single stranded oligodeoxyribonucleotide is produced. The latter compound is an efficient 

binder of complementary nucleic acid. This is the first example of red light activated “caged” 

oligodeoxyribonucleotides. 

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71


J. Müller 

SL20. Oligonucleotides with Metal-ion-mediated Base Pairs: 

Examples and Possible Applications 

Faculty of Chemistry, Dortmund University of Technology, Otto-Hahn-Str. 6, 44227, Dortmund, Germany 

e-mail: jens.mueller@tu-dortmund.de 

A recently introduced method for the site-specific functionalization of nucleic acids with metal ions is based on 

the formal substitution of natural nucleobases by artificial ones. The latter are designed in a way that they have 

an increased affinity towards metal ions, resulting in the formation of metal-ion-mediated base pairs (see 

Figure).[1] In such a construct, the oligonucleotide double helix serves as a scaffold for the arrangement of metal 

ions along its helical axis. Due to their expected interesting physical properties, applications of these nucleic 

acids as molecular wires and nanomagnets are envisaged. 

This lecture will report recent examples of such metal-modified oligonucleotides, including the system with the 

to date longest continuous stack of metal-ion-mediated base pairs [2] as well as examples for oligonucleotides 

that undergo a major conformational change in the presence of appropriate metal ions.[3, 4] 

Acknowledgement: Financial support by the DFG (Emmy Noether Programme (JM1750/1-3) 

and ERA-Chemistry (JM1750/2-1)), the Fonds der Chemischen Industrie, COST D39, and the Faculty of 

Chemistry at Dortmund University of Technology is gratefully acknowledged. 

References: 

[1] Review: J. Müller, Eur. J. Inorg. Chem., in press (doi: 10.1002/ejic.200800301). 

[2] F.-A. Polonius, J. Müller, Angew. Chem. Int. Ed., 46, 5602 (2007). 

[3] D. Böhme, N. Düpre, D. A. Megger, J. Müller, Inorg. Chem., 46, 10114 (2007). 

[4] S. Johannsen, S. Paulus, N. Düpre, J. Müller, R. K. O. Sigel, J. Inorg. Biochem., 102, 1141 (2008). 

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72


SL21. Insight into the Electronic Structure of ‘High-valent’ Iron 

Complexes: Addressing Metal- vs Ligand-based Oxidations Using XAS and 

TD-DFT 

S. DeBeer George 

SSRL, SLAC, Stanford University, 2575 Sand Hill Road, 94025, Menlo Park, United States, 

e-mail: debeer@stanford.edu 

Recently, we have used a combination of metal k-edge XAS combined with time-dependent density functional 

theory, in order to probe the electronic structure of Fe(IV), Fe(V) and Fe(VI) complexes. This combination of 

experimental data coupled to theory has also been extended to the controversial Fe(IV)-chloro-corrole 

complexes. These complexes are formally Fe(IV), but may be described as either and S=1 Fe(IV) or an 

intermediate spin Fe(III) antiferromagnetically coupled to a corrole radical. By using a combination of Fe K-, Cl 

K- and N K-edges, the corrole complexes are compared to their porphyrin analogues in order to differentiate the 

two possible corrole electronic structures. These results are coupled to TD-DFT. Implications for reactivity will 

be discussed. 

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73


SL22. A Biomimetic System for Serine Protease Enzymes 

A. AlAgha a , O.R. Nunez a,b , F. Butler a , K.B Nolan a 

a Centre for Synthesis and Chemical Biology, Department of Pharmaceutical & Medicinal Chemistry, Royal 

College of Surgeons in Ireland, St. Stephen’s Green, Dublin 2, Ireland 

e-mail: kbnolan@rcsi.ie 

b Departmento de Procesos y Sistemas, Universidad Simon Bolivar, Caracas, Venezuela. 

In general, peptides undergo very slow hydrolysis, as demonstrated by the fact that the half-time for 

glycylglycine hydrolysis at neutral pH, 25 o C is ~350 years [1]. Similarly glycylserine at pH = ~ 7 (HEPES 

buffer, D2O) undergoes no appreciable hydrolysis after ~40 h. However in the presence of serine protease 

enzymes the hydrolysis of this and related substrates is catalysed by peptide group activation and by 

participation of the nucleophilic serine -OH group and water in hydrolysis. The nucleophilicity of this group is 

further enhanced by hydrogen bonding with the terminal carboxylate. We report here a biomimetic system for 

catalysis by this enzyme. 

In the presence of zinc(II), glycylserine under the same reaction conditions as above undergoes hydrolysis with a 

half-time of ~26h, which is much more rapid than the free peptide. In this system the glycyl residue is bidentate, 

complexing through the amino and peptide O groups. The complex formed produces a 1 H NMR signal upfield 

by ~0.2 ppm for the glycyl –CH2 group relative to the uncomplexed peptide. Even though the 1 H NMR signals of 

the HEPES buffer overlap with some of the peptide signals, the glycylserine –CH group (~4.21 ppm) as well as 

the signals of the serine –CH group (~3.6 ppm) and the glycine –CH2 group (~3.2 ppm) in the hydrolysis product 

do not overlap with buffer signals. This allowed a study of the reaction kinetics by 1 H NMR spectroscopy. 

The catalysis observed in the Zn(II)-glycylserine hydrolysis is due to a combination of electrophilic catalysis by 

the metal ion and an internal general base catalysis of water promoted by the Ser –COO--- H---O - (see Figure). 

This is under further investigation. 

_____________________________________________________________________ 

74 

Zn 

H 2 N 

O 

H 

O 

NH 

Acknowledgement: We thank the Irish Government under its Programme for Research in Third Level 

Institutions for support. 

Reference: 

[1] A. Radizicka, R. Wolfenden, J.Am.Chem.Soc. 1996, 118, 6105. 

H 

O 

O 

H 

O -


SL23. Synthesis and Reactivity Studies of Model Complexes for Dinuclear 

Active Sites in Metalloenzymes 

M. Jarenmark, a H. Carlsson, a M. Haukka b , A.A. Shteinman c and E. Nordlander a 

a Inorganic Chemistry Research Group, Department of Chemical Physics,Center for Chemistry and Chemical 

Engineering, Lund University, Box 124, SE-221 00, Sweden 

b Department of Chemistry, University of Joensuu, Box 111, FI-80101 Joensuu, Finland 

c Institute of Problems of Chemical Physics, Russian Academy of Sciences, Chernogolovka, Moscow 

Region142432, Russia 

e-mail: Ebbe.Nordlander@chemphys.lu.se 

Metalloenzymes with dinuclear active sites are prevalent in Nature. The active sites have common structural 

traits; for example, structural features shared by most such enzymes include the presence of one or two bridging 

carboxylate bridges and one or two exogenous oxygen-containing (oxo-, hydroxo or water) bridges [1]. Despite 

these structural resemblances, the mechanistic diversity of this class of enzymes is striking. Utilizing a number 

of framework ligands that have been designed to permit the structural emulation of most dinucear 

metalloenzymes [2,3], we have prepared a number of active site mimics that are not only structural, but in 

several cases also functional, model complexes for a number of enzymes, in particular hydrolases and 

monooxygenases. This lecture will highlight a number of examples of this research, including model complexes 

for the active sites of soluble methane monooxygenase, urease, zinc phosphotriesterase and purple acid 

phosphatases. 

Acknowledgements: The authors would like to thank the Swedish research council (VR) for financial support. 

This research is carried out within the framework of the international graduate school ‘Metal sites in 

biomolecules: structures, regulation and mechanisms’ (www.biometals.eu). 

References: 

[1] M. Jarenmark, H. Carlsson, E. Nordlander, Comptes Rendus Chimie, 10, 433 (2007). 

[2] H. Carlsson, M. Haukka, A. Bosseksou, J.-M. Latour, E. Nordlander, Inorg. Chem., 43, 8252 (2004). 

[3] M. Jarenmark, S. Kappen, M. Haukka E. Nordlander, Dalton Trans., 993 (2008). 

_____________________________________________________________________ 

75


SL24. Nickel-related Peptide Bond Hydrolysis: from Carcinogenesis to 

Biotechnology 

W. Bal a , E. Kopera a , A. Krężel b , J. Poznański a , A. Wysłouch-Cieszyńska a 

a 

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106, Warsaw, 

Poland, 

b 

Faculty of Biotechnology, University of Wrocław, Tamka 2, 50-137, Wrocław, Poland 

e-mail: wbal@ibb.waw.pl 

Ni(II) is a human carcinogen, with molecular targets in the cell nucleus. A search of Ni(II) binding sites in 

histones – main nuclear proteins – yielded a sequence-specific Ni(II)-dependent reaction of peptide bond 

hydrolysis in model peptides and histone H2A: extTE!SHHKext (! = cleavage site, ext = chain extension). The 

reaction was stoichiometric, with Ni(II) bound as a square-planar (sp) complex with the C-terminal reaction 

product. This links the epigenetic and oxidative concepts in nickel carcinogenesis, as sp Ni(II) peptidic 

complexes are known oxidants [1]. Further studies revealed that the cleavage occurs in nearly all ext!BXHZext 

sequences (B = S or T), upon the formation of a specific “4N” sp complex involving the Ni(II) ion bound to B, 

X, and H residues [2]. The reaction starts with the acyl shift from the B amide to its OH group, followed by 

hydrolysis of the resulting ester. The B residue nitrogen remains bonded to Ni(II). Its amide-to-amine conversion 

promotes the reaction by enhancing Ni(II) binding. The reaction can be made highly sequence-specific at lower 

pH ~8. Bulky and hydrophobic X and Z residues are preferred (e.g. !SRHW), as shown by a combinatorial 

library. The presence of all required residues (!BXHZ) in the C-terminal product suggested an application of the 

reaction for removing affinity tags from recombinant proteins. A successful demonstration of such process 

completed a pathway of our discovery from basic research to practical applications. 

References: 

[1] AA Karaczyn, W Bal, SL North, RM Bare, VM Hoang, RJ Fisher, KS Kasprzak, The octapeptidic end of the 

C-terminal tail of histone H2A is cleaved-off in cells exposed to carcinogenic Ni(II). Chem Res Toxicol 16, 

1555-1559, 2003 and refs. therein. 

[2] A Krężel, M Mylonas, E Kopera, W Bal, Sequence-specific Ni(II)-dependent peptide bond hydrolysis in a 

peptide containing threonine and histidine residues, Acta Biochim Polon 53, 721–727, 2006 

_____________________________________________________________________ 

76


SL25. Copper Binding to the Prion Protein: a Controversial Issue 

M. Remelli a , D. Bacco a , E. Gralka b , R. Guerrini c , H. Kozłowski b , D. Valensin d 

a 

Dipartimento di Chimica, Università di Ferrara, via L. Borsari 46, 44100, Ferrara, Italy 

b 

Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383, Wroclaw, Poland 

c 

Dipartimento di Scienze Farmaceutiche, Università di Ferrara, via Fossato di Mortara 17/19, 44100, Ferrara, 

Italy 

d 

Dipartimento di Chimica, Università di Siena, Via Aldo Moro, 53100, Siena, 

e-mail: rmm@unife.it 

The prion protein (PrPC) is a cell-surface glycoprotein, mainly expressed in liver and brain; its biological 

function is mostly unknown. However, there is class of neurodegenerative disorders, i.e. prion diseases, where 

high amounts of a modified isoform of PrPC, named PrPSc (scrapie form), abnormally accumulate in neuronal 

cells [1]. The PrPC conversion to PrPSc involves only the secondary and tertiary structure of the protein, but it 

deeply modifies its chemical properties; the conformational conversion can be caused by familial mutations, 

sporadic and even infectious factors [2]. It has been widely demonstrated that PrPC is able to bind copper ions 

[3-6]. It can cooperatively bind four copper ions at its unstructured N-terminal, in the so-called “octarepeat 

region” (residues 60–91) [5, 6], while other two binding sites are located in correspondence of His-96 and His- 

111 residues [3, 7-9]. In this regard, some points are still under investigation: the relative strength of these 

binding sites with reference to both the physiologically relevant ligands and the octarepeat domain of the protein 

itself; the possible preference by the CuII ion for His-96 or His-111; the possible simultaneous participation of 

both His to copper binding; the complex geometry and the donor atoms; the role played by the other amino 

acidic residues surrounding the anchoring site. In order to better clarify these points, some fragments of PrPC, 

containing His-96 and/or His-111, and their analogues have been taken as model peptides and investigated by 

means of potentiometric, calorimetric, UV-VIS, CD, EPR and NMR spectroscopic techniques. 

References: 

[1] Prusiner, S.B. Proc. Natl. Acad. Sci. USA 1998, 95, 13363, 

[2] Prusiner S.B., N.Engl. J. Med. 2001, 344, 1516 

[3] Brown, D. R.; Qin, K.; Herms, J.W.; Madlung, A.; Manson, J.; Strome, R.; Fraser, P.E.; Kruck, T.A.; von 

Bohlen, A.; Schulz-Schaeffer, W.; Giese, A.; Westaway D.; Kretzschmar H. Nature, 1997, 390, 684. 

[4] Hornshaw MP, McDermott JR, Candy JM, Lakey JH., Biochem. Biophys. Res. Commun. 1995 214:993–99 

[5] Stockel J, Safar J, Wallace AC, Cohen FE, Prusiner SB.. Biochemistry 1998 37:7185–93 

[6] D. R. Brown and H. Kozlowski, Dalton Trans., 2004, 1907. 

[7] Gaggelli, E.; Bernardi, F.; Molteni, E.; Pogni, R.; Valensin, D.; Valensin, G.; Remelli, M.; Łuczkowski, M.; 

Kozlowski, H. J. Am. Chem. Soc. 2005, 127, 996 

[8] Berti, F.; Gaggelli, E.; Guerrini, R.; Janicka, A.; Kozlowski, H.; Legowska, A.; Miecznikowska, H.; 

Migliorini, C.; Pogni, R.; Remelli, M.; Rolka, K.; Valensin, D.; Valensin, G. Chem. Eur. J. 2007, 13, 1991. 

[9] Klewpatinond, M.; Davies, P.; Bowen, S.; Brown, D.R.; Viles, J.H. J. Biol. Chem. 2008, 283, 1870. 

_____________________________________________________________________ 

77


SL26. Site-specific Interactions of Cu(II) with Alpha-Synuclein: Bridging 

the Molecular Gap Between Metal Binding and Aggregation 

A. Binolfi, a G. R. Lamberto, a R. Duran, b L. Quintanar, c C. W. Bertoncini, d J. M. Souza, e 

C. Cerveñansky, b M. Zweckstetter, f C. Griesinger, f a, f 

and C. O. Fernández 

a Instituto de Biología Molecular y Celular de Rosario, Argentina 

b Institut Pasteur de Montevideo e Instituto Clemente Estable, Uruguay 

c Centro de Investigación y Estudios Avanzados, Mexico 

d Department of Chemistry, University of Cambridge, United Kingdom 

e Facultad de Medicina, Universidad de la República, Uruguay 

f Max Planck Institute for Biophysical Chemistry, Germany 

e-mail: fernandez@ibr.gov.ar; cfernan@gwdg.de 

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson’s disease (PD) and other 

neurodegenerative synucleinopathies. Protein-metal interactions play a critical role in AS aggregation and might 

represent the link between the pathological processes of protein aggregation and oxidative damage. Our previous 

studies established a hierarchy in AS-metal ion interactions, where Cu(II) binds specifically to the protein and 

triggers its aggregation under conditions that might be relevant for the development of PD. 1, 2 In this work we 

have addressed structural unresolved details related to the binding specificity of Cu(II) to AS. The structural 

properties of the Cu(II) complexes were determined by the combined application of Nuclear Magnetic 

Resonance (NMR), Electron Paramagnetic Resonance (EPR), Mass Spectrometry (MALDI-MS), UV-visible 

spectroscopy and Circular Dichrosim (CD). Two independent, non-interacting copper-binding sites could be 

deflected at the N-terminal region of AS, with significant difference in their affinities for the metal ion. MALDI- 

MS provided unique evidences for the direct involvement of Met 1 as the primary anchoring residue for Cu(II). 

A comparative spectroscopic analysis between different variants of the protein allowed us to deconvolute the 

Cu(II) binding modes and to assign unequivocally the high affinity site to the N-terminal amino group of Met1 

and the low affinity site to that involving the imidazol ring of the sole His residue. Using competitive chelators 

the affinity of the first equivalent of bound Cu(II) was accurately determined to be in the submicromolar range. 

Our results prove that Cu(II) binding at the C-terminal region of the protein represents a non-specific, very low 

affinity process. These new insights into the bioinorganic chemistry of PD are central to understand the role of 

Cu(II) in the fibrillization process of AS. 

Acknowledgement 

C.O. Fernández thanks ANPCyT, Fundacion Antorchas, CONICET, Max Planck Society and the Alexander von 

Humboldt Foundation for financial support. C.O. Fernández is the head of a Partner Group of the Max Planck 

Institute for Biophysical Chemistry (Göttingen). 

References 

[1] R.M. Rasia, C.W. Bertoncini, D. Marsh, W. Hoyer, D. Cherny, M. Zweckstetter, C. Griesinger, T.M. Jovin, 

C.O. Fernández, Proc Natl Acad Sci USA, 102, 4294 (2005). 

[2] A. Binolfi, R.M. Rasia, C.W. Bertoncini, M. Ceolin, M. Zweckstetter, C. Griesinger, T.M. Jovin and C.O. 

Fernández, J Am Chem Soc, 128, 9893 (2006). 

_____________________________________________________________________ 

78


SL27. Combined Theoretical and Experimental Studies of the Reaction 

Intermediates in the TauD Enzyme 

F. Neese 

Inst. for Physical and Theoretical Chemistry, University of Bonn, Wegelerstrassee 12, 53115, Bonn, Germany 

e-mail: theochem@thch.uni-bonn.de 

Combined Theoretical and Experimental Studies of the Reaction Intermediates in the TauD Enzyme Shengfa Ye, 

Carsten Krebs, Martin Bollinger, Frank Neese High-valent iron sites play a fundamental role in bioinorganic 

chemistry as reaction intermediate in heme- and nonheme iron enzymes. To elucidate their geometric and 

electronic structure and function is therefore a key in understanding the reaction mechanisms of these enzymes. 

In recent years, we have – in close collaboration with our experimentally working project partners - studied a 

variety of mono- and dinuclear iron sites in proteins and model complexes. The lecture will stress the impact of 

the combination of quantum chemistry and spectroscopy for the elucidation of the structures of short lived 

intermediates that are not amenable to crystallographic studies. 

_____________________________________________________________________ 

79


SL28. The Reaction Mechanism of Heme Peroxidases and Catalases. 

A QM/MM Molecular Dynamics Study 

M. Alfonso-Prieto a , P. Vidossich a , X. Carpena b , I. Fita b , P. Loewen c , E. Derat d , S. Shaik e , 

C. Rovira a,f 

a 

Computer Simulation and Moceling Laboratory, Parc Científic de Barcelona, Baldiri Reixac 10-12, 08028 

Barcelona, Spain. 

b 

Institut de Biologia Molecular (IBMB-CSIC), Institut de Recerca Biomèdica (IRB), Parc Científic de 

Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain. 

c 

Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. 

e 

Laboratoire de Chimie Organique, Institut de Chimie Moléculaire, Université Pierre et Marie Curie-Paris 6, 4 

place Jussieu B. 229, 75005 Paris, France. 

d 

Department of Organic Chemistry and the Lise Meitner-Minerva Center for Computational Quantum 

Chemistry, Hebrew University of Jerusalem, 91904 Jerusalem, Israël. 

f 

Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys, 23, 08018 Barcelona, 

Spain 

e-mail : crovira@pcb.ub.es 

Heme peroxidases and catalases constitute an important group of enzymes that are found in nearly all living 

organisms. Catalases decompose protect the cells from the toxic H2O2 by degrading it into oxygen and water, 

whereas peroxidases use H2O2 to oxidize organic substrates. Both enzymes form a high-valent iron-oxo species, 

called Compound I (Cpd I), which is their primary reaction intermediate. 1,2 

Enz (Por–Fe III ) + H2O2 → Cpd I (Por ●+ -Fe IV =O) + H2O 

An important question concerning the catalytic cycle of these enzymes concerns the properties of the reaction 

intermediates and the mechanism of their formation. Molecular modeling complements experimental 

observations in the attempt to clarify structure-function relationships. We present results of modeling studies of 

the active species in Horseradish Peroxidase (HRP), 3 a classical monofunctional peroxidase, Helicobacter Pylori 

catalase, 4 a monofunctional catalase, as well as KatG, 5 a bifunctional catalase-peroxidase. The calculations are 

performed by means of DFT QM/MM optimizations as well as Car-Parrinello QM/MM molecular dynamics 

simulations. 

References: 

[1] P. Nichols, I. Fita, P. C. Loewen, Enzymology and Structure of Catalases. In Advances in Inorganic 

Chemistry, Sykes, A. G., Mauk, G., Eds.; Academic Press: 2001; pp 51-106. 

[2] P. Jones, H. B. Dunford, J. Inorg. Biochem. 99, 2292 (2006). 

[3] E. Derat, S. Shaik, C. Rovira, P. Vidossich, M. Alfonso-Prieto, J. Am. Chem. Soc. 129, 6346 (2007). 

[4] M. Alfonso-Prieto, A. Borovik, X. Carpena, G. Murshudov, W. Melik-Adamyan, I. Fita, C. Rovira, P. C. 

Loewen, J. Am. Chem. Soc. 129, 4193 (2007). 

[5] P. Vidossich, M. Alfonso-Prieto, X. Carpena, P. C. Loewen, I. Fita, C. Rovira, J. Am. Chem. Soc. 129, 

13436 (2007). 

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80


SL29. Towards Semi-synthetic Metallo-enzymes; Merging Biochemistry 

with Organometallics 

B. Wieczorek, a H. P. Dijkstra, a L. Rutten, b M. R. Egmond, c M. Lutz, b P. Gros, b G. van Koten, a 

and R. J. M. Klein Gebbink. 

a Chemical Biology & Organic Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, Netherlands 

b Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, Netherlands 

c Membrane Enzymology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, Netherlands 

The development of novel anchoring strategies for transition-metal complexes to proteins has high potential for 

future applications in the fields of catalysis, protein structure elucidation (NMR, X-Ray, MS) and medicinal 

chemistry (MRI contrast agents, radiopharmaceuticals). In using transition metal complexes as enzyme 

modifying agents has, the enzyme backbone may act as a chiral scaffold for the transition metal moiety, alter its 

(enantio)selectivity and enhance its water-solubility. Our laboratory has developed a selective anchoring method, 

in which organometallic pincer complexes are covalently attached to the active site of a lipase via a phosphonate 

linker. [1,2] The crystal structure data of different semisynthetic pincer-metalloenzymes have been solved and 

are presented here (Figure 1). 

Coordination studies with different bulky phosphine ligands, show that the metal centre in the semi-synthetic 

enzymes is available for coordination. In addition, the use of these semisynthetic pincer-metalloenzymes as 

abiotic C-C coupling catalysts in aqueous media is explored. 

References: 

[1] Kruithof., C.A., Casado, M.A., Guillena, G.A., Egmond, M.R., van der Kerk-van Hoof, A., Heck, A.J.R., 

Klein Gebbink, R.J.M., van Koten, G. Chem. Eur. J. 11 (2005) 6869. 

[2] Kruithof, C.A., Dijkstra, H.P., Lutz, M., Spek, A.L., Egmond, M.R., Klein Gebbink, R.J.M., van Koten, G. 

Eur. J. Inorg. Chem. accepted for publication. 

_____________________________________________________________________ 

81


A. Ivancich 

SL30. Tryptophanyl Radicals as Reactive Intermediates 

in Mono- and Bi-functional Peroxidases 

Institut de Biologie et des Biotechnologies (iBiTec-S), CEA Saclay and CNRS URA 2096. Centre d’Etudes de 

Saclay. Bat 532. F-91191 Gif-sur-Yvette, France. 

Protein-based radicals are involved in the redox chemistry of metalloproteins. Tyrosyl and tryptophanyl radicals 

have specific roles in electron and PCET process of selected enzymes, a well documented example being 

ribonucleotide reductase [1]. Such radical species can be also directly involved in enzyme catalysis, with a 

specific role in substrate oxidation [2]. In particular, we have shown that the so-called catalase-peroxidases 

(KatGs) form tryptophanyl radicals as alternative oxidizing intermediate(s) to [(Fe(IV)=O) Por •+ ] species 

[3,4,5]. Taken together, the very high number of Trp and Tyr present in these bifunctional peroxidases and the 

apparent fine-tuning of these enzymes for well defined protein-based oxidation sites indicate that some of the 

Trp and Tyr may have a role in accelerating electron transfer between the heme active site and subtrate 

oxidation sites. In contrast to monofunctional peroxidases, the KatGs distal heme side is more crowded [6] thus 

impairing the existence of the substrate binding site typically found in monofunctional peroxidases [7]. 

Defining the number and the chemical nature of radical species associated to the oxidizing intermediates as well 

as those residues related with ET is an important step for understanding the reactivity of these enzymes towards 

substrates. Selected Trp and Tyr mutations related to the different roles will be discussed. A complementary 

approach consisting in engineering a Trp radical site to mimic the naturally occurring site, as in the case of 

lignin peroxidase will also be discussed. Our powerful approach consists on combining multifrequency (9-285 

GHz) EPR spectroscopy and X-ray crystallography with site-directed mutagenesis, deuterium labeling and 

kinetic studies to characterize both radical formation and substrate oxidation. 

References: 

[1] J. Stubbe, D. G. Nocera, C. S. Lee, M. C. Chang. Chem. Rev., 103, 2167 (2003). 

[2] J. Stubbe,W. A. van der Donk. Chem. Rev., 98, 705 (1998). 

[3] A. Ivancich, A., C. Jakopitsch, M. Auer, S. Un, C. Obinger. J. Am. Chem. Soc., 125, 14093 (2003). 

[4] C. Jakopitsch, C. Obinger, S. Un, A. Ivancich. J. Inorg. Biochem., 101, 1091 (2006). 

[5] R. Singh, J. Switala, P. C. Loewen, A. Ivancich. J. Am. Chem. Soc., 129, 15954 (2007). 

[6] T. Deemagarn, B. Wiseman, X. Carpena, A. Ivancich, I. Fita, P. C. Loewen. Proteins, 66, 219 (2007). 

[7] A. Henriksen, D. J. Schuller, K. Meno, K. G. Wellinder, A. T. Smith, M. Gajhede. Biochemistry, 37, 8054 

(1998). 

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82


SL31. Investigating the Post-translational Modification of Cysteine 

Dioxygenase 

T. Kleffmann a , S. M. Wilbanks a , G. N. L. Jameson b 

a Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. 

b Department of Chemistry, University of Otago, PO Box 56, Dunedin, New Zealand 

e-mail: gjameson@chemistry.otago.ac.nz 

It is crucial for healthy cells that the correct concentrations of the amino acid cysteine are maintained. A build up 

of cysteine is observed in Parkinson’s disease [1, 2] and other pathologies when there is a failure to break 

cysteine down. Degradation of cysteine involves a sequence of enzymatic reactions. The first and regulating 

reaction is catalysed by the enzyme cysteine dioxygenase (CDO). 

Present in organisms from bacteria [3] to humans [4], CDO catalyses the oxidation of cysteine to a cysteine 

sulfinate (Scheme). The oxidation occurs by addition of molecular oxygen (O2) to the sulfur atom of the cysteine 

thiol (-SH). The heart of the active site of CDO is a mono-iron site coordinated by three histidine residues. CDO 

is therefore a member of a larger group of proteins known as the non-heme mono-iron proteins, which have 

gained considerable interest in recent years and have been the subject of numerous reviews. This interest stems 

from their ability, even with such a simple active site, to catalyze a wide range of processes that contribute to a 

variety of important biochemical processes. 

CDO’s catalytic site is also unusual in containing a cross-link (Cys93 to Tyr157) observed in only three other 

enzymes. [5-7] Formation of this cross-link converts this enzyme from an immature, less active form to the 

mature, fully active form. The cross-link formation in CDO depends on the substrate cysteine, [8] suggesting a 

novel feedback mechanism for enzyme activation in control of sulfur metabolism. We have isolated immature 

protein and completely determined the cross-link’s structure by mass spectrometry. Although the available X-ray 

crystal structures show “snap shots” of the catalytic site, mechanisms both of cross-link formation and cysteine 

oxidation remain to be determined. 

In this presentation we will bring the present knowledge up-to-date and sketch the way in which our future 

studies will go. 

Acknowledgement: The Chemistry Department and the Division of Sciences of the University of Otago, and the 

University of Otago Research Grant Committee for financial support. 

References: 

[1] M. H. Stipanuk, J. E. Dominy Jr., J.-I. Lee, R. M. Coloso, J. Nutr., 136 (6S), 1652S (2006). 

[2] M. T. Heafield, S. Fearn, G. B. Steventon, R. H. Waring, A. C. Williams, S. G. Sturman, Neurosci. Lett., 110 

(1-2), 216 (1990). 

[3] J. E. Dominy Jr., C. R. Simmons, P. A. Karplus, A. M. Gehring, M. H. Stipanuk, J. Bacteriol., 188 (15), 5561 

(2206). 

[4] S. Ye, X. A. Wu, L. Wei, D. Tang, P. Sun, M. Bartlam, Z. Rao, J. Biol. Chem., 282 (5), 3391 (2007). 

[5] N. Ito, S. E. V. Phillips, C. Stevens, Z. B. Ogel, M. J. McPherson, J. N. Keen, K. D. S. Yadav, P. F. Knowles, 

Nature, 350 (6313), 87 (1991). 

[6] R. Schnell, T. Sandalova, U. Hellman, Y. Lindqvist, G. Schneider, J. Biol. Chem., 280 (29), 27319 (2005). 

[7] M. M. Whittaker, J. W. Whittaker, J. Biol. Chem., 278 (24), 22090 (2003). 

[8] J. E. Dominy Jr., J. Hwang, S. Guo, L. L. Hirschberger, S. Zhang, M. H. Stipanuk, J. Biol. Chem., 283 (18), 

12188 (2008). 

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83


SL32. Spectroscopic and Structural Studies of Iron Center and Tyrosyl 

Radical in Mammalian, Fish and Bacterial Ribonucleotide Reductase 

A.K. Røhr a , A.B. Tomter a ,G.K. Sandvik a , J. Bergan a , A.L. Barra b , G.E. Nilsson . a , K.R. 

Strand a , K.K. Andersson a 

a Department of Molecular Biosciences, Univ of Oslo, PO Box 1041 Blindern, 0316, Oslo, Norway 

b Grenoble High Magnet Field Laboratory GHMFL, CNRS, 25, Rue des Martyres, B.P: 166, FR-38042, 

Grenoble, France 

e-mail: k.k.andersson@imbv.uio.no 

Ribonucleotide reductase (RNR) is the enzyme that converts ribonucleotides to corresponding 

deoxyribonucleotides. The R2 subunit of the enzyme complex reacts with ferrous iron and dioxygen to generate 

a diferric iron-oxygen cluster and a tyrosyl radical that is essential for enzymatic activity [1]. A p53 induced 

isoform of the R2 subunit (p53R2) is proposed to be involved in the production of deoxyribonucleotides for 

DNA repair and mitochondrial DNA. The human (Class Ia) R2, bacterial (Class Ib) R2 and murine (Class Ia) 

p53R2 proteins have been studied by electron paramagnetic resonance (EPR), magnetic circular dichroism 

(MCD) and CD like for mouse R2 [2]. While the studies of the active diferric iron-oxygen cluster and the tyrosyl 

radical (also hydrogen binding to the tyrosyl radical) [1, 3, 4] together with the mixed valent form (Fe(II)-Fe(III) 

cluster) [3] shows little or no variation between the R2 and p53R2 subunits, also in fish, the MCD and X-band 

integer spin EPR studies reveals a difference between the R2 and p53R2 diferrous forms [2] (see also poster by 

Ane B. Tomter of Class Ib RNR-R2) and the p53R2 lacks the cooperatively binding of Fe(II) or Co(II) [1, 2, 3, 

5, 6]. Taken together the interaction with R1 subunit of RNR are similar for both mammalian and fish R2 and 

p53R2 active and possibly mixed valent forms, while the p53R2 do not need to be regulated by cooperatively 

binding of Fe(II) as it is induced upon DNA damage [7]. Novel 3D structures have been determined of other 

RNR related proteins from B. cereus. In Figure is shown in green diferrous cluster with actetate bound and in red 

diferric iron-oxygen cluster demonstrating carboxylate shift in mouse RNR-R2. 

References: 

[1] Andersson K.K.(ed) Ribonucleotide reductase, Nova Science, 2008, ISBN: 978-1-60456-199-9 

[2] Strand, K.R., Yang, Y.-S., Andersson, K.K., Solomon, E.I. Andersson and E.I. Solomon (2003) Circular 

dichroism and magnetic circular dichroism studies of the biferrous form of the R2 subunit of ribonucleotide 

reductase from mouse. Comparison to the R2 from E. coli and other binuclear ferrous enzymes. Biochemistry, 

2003, 42; 12223-12234. 

[3] Kolberg M., Strand K.R., Graff P., Andersson K.K. Radicals in Three Different Classes of Ribonucleotide 

Reductases: Structural and Functional Basis. Biochim. Biophys. Acta- Proteins and Proteomics, 2004, 1699; 1- 

34. 

[4] K.K. Andersson, P. P. Schmidt, B. Katterle, K. R. Stand, A. Palmer, S.-K. Lee, E. I. Solomon, A. Gräslund, 

A.-L. Barra Examples of High Frequency EPR Studies in Bioinorganic Chemistry. J. Biol. Inorg. Chem. 2003, 8; 

235-247. 

[5] K.R. Strand, S. Karlsen, K.K. Andersson. Cobalt substitution of mouse R2 ribonucleotide reductase as a 

model for the reactive diferrous state. Spectroscopic and structural evidence for a ferromagnetically coupled 

dinuclear cobalt cluster. J. Biol. Chem. 2002, 277; 34229-34238. 

[6] K.R. Strand, S. Karlsen, S., M. Kolberg, Å.K. Røhr, C.H. Gørbitz, K. K. Andersson. Crystal Structural 

Studies of Changes in the Native Dinuclear Iron Center of Ribonucleotide Reductase Protein R2 from Mouse J. 

Biol. Chem. 2004, 279; 46794-46801 

[7] Wei P.P, Tomter A.B, Røhr, Å.K. Andersson K.K., Solomon, E.I. Circular dichroism and magnetic circular 

dichroism studies of the active site of p53R2 from human and mouse: iron binding and nature of the biferrous 

site relative to other ribonucleotide reductases. Biochemistry, 2006, 45; 14043-14051 

_____________________________________________________________________ 

84


SL33. Lanthanide Complexes of Schiff Bases Derived from Biogenic 

Diamines as Potential Synthetic Nucleases 

W. Radecka-Paryzek 

Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780, Poznań, Poland, 

e-mail: wrp@amu.edu.pl 

Non-enzymatic hydrolysis of DNA and RNA has attracted much interest, because it is essential for further 

developments in biotechnology, molecular biology, therapy and related fields. There are no natural enzymes 

showing sufficient sequence-specifity in RNA scission. Thus, artificial enzymes, which selectively hydrolyse 

DNA and RNA at the target position with a desired specifity, are crucially important. A few years ago, the 

catalytic activity of complexes containing lanthanide ions for hydrolysis of nucleic acids was discovered. This 

efficiency results from the conjuction of peculiar chemical and structural properties of the lanthanides associated 

with their 4f configuration, and rapid ligand exchange rate. These characteristics make them well-suited to be 

catalytic centers in the development of artificial ribonucleases. The lanthanide complexes obtained by us as the 

first examples of the usefulness of these metal ions as templates in the metal-promoted synthesis of the 

macrocyclic Schiff bases have found to be very effective catalysts for hydrolytic cleavage and transesterification 

of RNA phosphate diester backbone. In this contribution we wish to report the specific properties of lanthanide 

Schiff base complexes derived from biogenic amines (putrescine, cadaverine, spermine and spermine analogues), 

bound to a DNA oligomer (across succinic acid linker) as the sequence-recognizing moieties which are able to 

selectively hydrolyse RNA at the target site. 

_____________________________________________________________________ 

85


SL34. Biochemical and Medicinal Application of Cage Transition 

Metal Complexes 

Yan Z. Voloshin, a Oleg A. Varzatskii, b Yurii N.Bubnov а 

a 

N.A.Nesmeyanov Institute of Organoelement Compounds RAS, 119991 Moscow, Russia 

e-mail: voloshin@ineos.ac.ru 

b 

V.I.Vernadskii Institute of General and Inorganic Chemistry NANU, 03680 Kiev, Ukraine. 

The following main trends and the perspectives of practical application of the transition metal clathrochelates in 

biochemistry and medicine - encapsulation of radioactive metal ion for diagnostics and therapy; detoxifying 

biological systems and prolonged pharmaceuticals; pharmaceuticals for boron-neutron capture therapy; 

antioxidants; membrane transport of the metal ions; interaction of the cage metal complexes with nucleic acids 

and the potential of their self-assembling reactions in immunology and molecular biology (recognition of 

antibodies, antigens and the DNA sites); design of HIV inhibitors for the therapy - will be discussed. 

substratum 

substratum 

linker 

linker 

linker B(OH) 2 

H 

O 

N 

N 

O 

H 

linker 

_____________________________________________________________________ 

86 

H 

O 

N 

N 

O 

H 

HO 

N N O H 

Fe 2+ 

RB(OH) 2 

substratum 

B(OH) 2 

= flourescent substituent 

R = functionalizing substituent 

substratum linker 

N 

Fe 

N 

2+ 

Y 

O O 

N 

O 

O 

N N 

O 

Y O 

R R 

N R 

R R R 

N 

Fe 

N 

2+ 

O 

B 

O O 

N 

N N 

O 

O 

B O 

R 

N 

R 

substratum 

lin 

ker 

N 

Fe 

N 

2+ 

B 

O O 

N 

O 

O 

N N 

O 

B O 

R R 

N R 

R R R 

lin 

ker 

substratum 

Y = H, SbEt 3 

Acknowledgement: This work was supported by RFBR (№06-03-32626, 07-03-12183 and 07-03-12144). 

References: 

[1] Y.Z.Voloshin, N.A.Kostromina, R.Krämer, Clathrochelates: synthesis, structure and properties, Elsevier, 

Amsterdam, 2002. 

[2] A.Mokhir, R.Krämer, Y.Z.Voloshin, O.A.Varzatskii, Bioorg.Med.Chem.Lett., 14, 2927 (2004). 

[3] Y.Z.Voloshin, O.A.Varzatskii et al., Inorg. Chem., 44, 822 (2005); 47, 2155 (2008); Inorg.Chim.Acta, 360, 

1543 (2007); Russ.Chem.Bull., Int.Ed., 55, 22 (2006); Angew.Chem.Int. Ed., 44, 3400 (2005); Polyhedron, 26, 

2733 (2007); 27, 325 (2008). 

[4] Y.Z.Voloshin, O.A.Varzatskii, Y.N.Bubnov, Russ.Chem.Bull., Int.Ed., 56, 579 (2007) (a review).


ORAL PRESENTATIONS 

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87


O1. Molecular Probes for the Active Site of P450cam and iNOS 

D.B. Goodin a , R.F. Wilson a , P. Glazer a , A. Annalora a , C.D. Stout a , and H.B. Gray b 

a Dept. of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA 

b Dept. of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 

Several families of molecular wires have been developed to probe the active site channels of P450cam and 

murine iNOS heme domain. These wires consist of ligand analogs tethered to a reporter, sensitizer or molecular 

surface. This allows photochemically or electrochemically initiated redox reactions to be explored, and offers a 

method for affinity based selection of binding behavior. We have examined the structural response of a series 

P450cam specific probes that vary in the position of hydrogen bonding groups, linker length and composition. 

Crystallographic structures reveal a significant structural plasticity of the distal substrate binding channel that 

arise from combinations of several modes of movements in the F, G, and B' helices. Changes in the hydrogen 

bonding interactions between wire and protein were observed to affect ligand orientation more than binding 

affinity, while the linker length and hydrophobicity have a significant impact on the conformation disorder in 

both the wire and protein. A separate family of wires using analogs of 6(R)-tetrahydro-L-biopterin (H4B) linked 

to Ru based photosensitizers were shown to bind to pterin free iNOS heme domain. Photoinduced reduction of 

ferric NOS was observed by excitation of the Ru(II) center in the presence of reductive quenchers. These wires 

are being examined for their potential to generate unstable intermediates in the NOS reaction cycle. 

_____________________________________________________________________ 

88


O2. Tracking Molecular Conformations of Cu-Zn Superoxide Dismutase 

J.G. Grossmann a , C.W. Yong b , R.W. Strange a , S.V. Antonyuk a , M.A. Hough a , W. Smith b , 

S.S. Hasnain a 

a 

School of Biological Sciences, University of Liverpool, Crown Street, L769 7ZB, Liverpool, United Kingdom 

e-mail: J.G.Grossmann@liverpool.ac.uk 

b 

Computational Science and Engineering Department, STFC Daresbury Laboratory, Keckwick Lane, WA4 

4AD, Warrington, United Kingdom 

More than 100 different mutations in the gene for Cu-Zn superoxide dismutase (SOD1) cause familial forms of 

amyotrophic lateral sclerosis - a fatal neurodegenerative disease in which aggregation of the SOD1 protein is 

considered to be the primary mode of pathogenesis [1]. SOD1 is active as a homodimer, containing one Cu and 

one Zn per monomer. Each monomer folds into an eight-stranded antiparallel β-barrel connected by external 

loops (see figure). Ala4Val, one of the most fatal mutations, causes a decrease in stability of the native 

conformation yet without affecting metal binding or net charge [1]. 

Protein crystallography investigates structures of native and mutant proteins with differing metal content at 

atomistic levels. However, the underlying dynamic mechanisms have to be inferred from these static studies in 

crystalline forms and extrapolated to aqueous, physiological conditions. Hence the integration of X-ray 

scattering [2] and molecular dynamics (MD) simulation [3] techniques offer a crucial complement to high 

resolution crystallographic studies in understanding the molecular basis of protein destabilisation. 

Here we report on MD calculations (to ≈20ns) of the fully solvated wild-type SOD1 and the Ala4Val mutant 

protein in both the metal-free and metal-loaded states. The MD simulations are discussed in the light of X-ray 

scattering data which show significantly larger conformational changes for Ala4Val SOD1 upon metal loss as 

compared to the wild-type protein. 

References: 

[1] Valentine, J.S. et al. (2004) Copper-zinc superoxide dismutase and amyotrophic lateral sclerosis. Annu. Rev. 

Biochem. 74, 563-593 

[2] Hough, M.A. et al. (2004). Destabilisation of the dimer interface in SOD1 may result in disease causing 

properties: Structure of motor neuron disease mutants A4V and I113T. Proc. Natl. Acad. Sci. U.S.A. 101, 5976- 

5981 

[3] Strange, R.W. et al. (2007). Molecular dynamics using atomic-resolution structure reveal structural 

fluctuations that may lead to polymerization of human Cu-Zn superoxide dismutase. Proc. Natl. Acad. Sci. 

U.S.A. 104, 10040-10044 

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89


O3. A Single Mutation in Nitrophorins from Blood-sucking Insects 

Flips the Heme Orientation by 180° around the C meso-α –C meso-α Axis 

M. Knipp a , F. Yang b , R.E. Berry b , H. Zhang b , M. Vašák, c F.A. Walker b 

a 

Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, D-45470, Mülheim an der Ruhr, Germany 

e-mail: mknipp@mpi-muelheim.mpg.de 

b 

Department of Chemistry, University of Arizona, 1306 East University Boulevard, 85721-0041, Tucson, AZ, 

USA 

c 

Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland 

Heme b is the most common of the heme cofactors that are found in heme proteins with various functions. 

Although the core structure (the tetrapyrrole ring) is highly symmetric, the distribution of the eight substituents 

(methyl, propionate, and vinyl groups) around the aromatic macrocycle results in a significantly lower 

symmetry. Insertion into the asymmetric pocket of a protein produces two different isomers A and B (see 

Figure). For most heme b proteins a preference for one orientation exists, but the reasons are not clear [1]. 

Using 1 H NMR spectroscopy, stopped-flow kinetics, and other techniques, we found that two members of the 

class of NO transporting ferriheme proteins termed nitrophorins (NPs) exhibit opposite cofactor orientation, i.e., 

NP2 stabilizes B orientation [2] whereas NP7 prefers A orientation [3]. This is also dramatically shown by CD 

spectroscopy. Examination of the structures of both proteins (61% amino acid sequence identity) identified E27 

in NP7, which is represented by V24 (V25) in NP2/3 (NP1/4), as a candidate to dictate the heme orientation. 

Appropriate mutant proteins were generated, NP2(V24E), NP7(E27Q), and NP7(E27V), and characterized. As a 

result, the heme orientation in NP2 was completely switched to A upon V24→E mutation. In good agreement, 

NP7(E27V) showed an A:B ratio of ~1:3. Overall, we identified a single amino acid residue to be responsible for 

the orientation of the heme b cofactor in the nitrophorins. 

References: 

[1] La Mar, G. N., Satterlee, J. D., De Ropp, J. S., Nuclear Magnetic Resonance of Hemoproteins; In The 

Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Ed.; Academic Press, San Diego (USA), 2000; 

Vol. 5, pp 185-298. 

[2] Berry, R. E., Shokhireva, T. Kh., Filippov, I., Shokhirev, M. N., Zhang, H., Walker, F. A. Biochemistry 2007, 

46, 6830-6843. 

[3] Knipp, M., Yang, F., Berry, R. E., Zhang, H., Shokhirev, M. N., Walker, F. A., Biochemistry 2007, 46, 

13254-13268. 

_____________________________________________________________________ 

90

J. Mattsson, B. Therrien 


O4. Supramolecular Trojan Horse for Cancer Cells 

Department of Chemistry, University of Neuchatel, Case postale 158, 2009, Neuchatel, Switzerland 

e-mail: johan.mattsson@unine.ch 

Combining the "molecular clip" strategy developed by Stang [1] and the "molecular paneling" strategy 

developed by Fujita [2], we recently synthesized the "complex-in-a-complex" cations [(acac) 2 Pd⊂Ru 6 (p- 

Pr i 

C 6 H 4 Me) 6 (tpt) 2 (dhbq) 3 ] 6+ 

and [(acac) 2 Pt⊂Ru 6 (p-Pr i 

-C 6 H 4 Me) 6 (tpt) 2 (dhbq) 3 ] 6+ 

[3]. The cytotoxicity of the two 

host-guest systems, the empty hexaruthenium cage and the free complexes Pd(acac) 2 and Pt(acac) 2 have been 

evaluated as anticancer agent against A2780 human ovarian cancer cells. The difference in cytotoxicity of the 

different systems suggests that like a "Trojan Horse", once inside a cell, passive leaching of the guest from the 

cage accelerates and increases the cytotoxic effect. 

References: 

[1]C. J. Kuehl, Y. K. Kryschenko, U. Radhakrishnan, S. Russell Seidel, S. D. Huang, P. J. Stang, Proc. Natl. 

Acad. Sci. USA, 2002, 99, 4932-4936. 

[2] M. Fujita, M. Tominaga, A. Hori, B. Therrien, Acc. Chem. Res., 2005, 38, 371-380. 

[3] B. Therrien, G. Süss-Fink, P. Govindaswamy, A. K. Renfrew, P. J. Dyson, Angew. Chem. Int. Ed., 2008, 47, 

3773-3776. 

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91


O5. Different Reaction Mechanism of Beta-diketone Cleavage in Heme and 

Non-heme Fe(II) Enzymes 

S. Leitgeb, G. Straganz, B. Nidetzky 

a Institute of Biotechnology and Biochemical Enginee, Graz University of Technology, Petersgasse 12/1, 8010, 

Graz, Austria 

e-mail: sleitgeb@tugraz.at 

Diketone-cleaving enzyme (Dke1) of Acinetobacter johnsonii [1] is a non-heme iron (II) dependent dioxygenase 

that belongs to the superfamily of the cupins [2]. The enzyme coordinates the active site metal by an unusual 3- 

His motif that deviates from the general 2-His-1-carboxylate motif [3] which is widespread and has been 

characterised extensively. We investigated the reaction mechanism of Dke1 in detail and proposed a reaction 

mechanism for the degradation of beta-diketones [4, 5]. 

The heme-dependent enzyme horseradish peroxidase (HRP) has a very broad substrate spectrum and is also 

capable of cleaving beta-diketones. HRP has already been reported to cleave 2, 4-pentanedione in the absence of 

externally added hydrogen peroxide [6]. We made a detailed mechanistic investigation in order to elucidate the 

reaction mechanism. Therefore we used a set of various diketones, characterized the reaction kinetically and 

analyzed the product spectrum. We were able to distinguish between two mechanistic proposals be comparing 

the distribution of two different product pairs. We could show deviations from the reaction route in Dke1 and 

were able to present a different reaction mechanism for the degradation of beta-diketones in Dke1 and HRP. 

References: 

[1] Straganz, G.D. et al., Biochem. J., 369 (2003), 573-81. 

[2] Khuri, S. et al., Mol. Biol. Evol., 18 (2001), 593-605. 

[3] Hegg, E.L., and Que, L., Eur. J. Biochem., 250 (1997), 625-629. 

[4] Straganz, G.D. et al., J. Am. Chem. Soc., 126 (2004), 12202-12203. 

[5] Straganz, G.D. et al., J. Am. Chem. Soc., 127 (2005), 12306-12314. 

[6] Rodrigues, A.P. et al., Biochim. Biophys. Acta, 1760 (2006), 1755-1761 

_____________________________________________________________________ 

92


O6. Electron Paramagnetic Resonance Studies of Copper Binding Sites in 

Peptides Related to Neurodegenerative Diseases 

P. Dorlet a , C. Hureau b , P. Faller c 

a URA 2096- iBiTecS, CNRS, Bat 532 CEA Saclay, 91191, Gif-sur-Yvette, France 

b UPR 824, CNRS, 205 Route de Narbonne, 31077, Toulouse, France 

c CNRS UPR 8241, Universite Paul-Sabatier, 205 route de Narbonne, 31077, Toulouse, France 

The Prion Protein (PrP) and the amyloid-b peptide (Ab) are involved in transmissible spongiform 

encephalopathies and Alzheimer's disease, respectively, which are fatal neurodegenerative disorders. 

We have recently investigated the Cu(II) coordination to the N- and C-protected Ac-GGGTH-NH2 and 

Ac-GGGTHSQW-NH2 peptides [1, 2] as models of the His96 site in PrP, one of the proposed non-octarepeat Cu 

binding sites [3]. We have shown that, at pH 6.7, the Cu(II) coordination mode is 3N1O in the peptides. The 

binding mode becomes quantitatively 4N at pH higher than 8. At physiological pH (7.5), comparison of the EPR 

spectra obtained on the peptides with that recorded on the PrP protein by Burns and coworkers [3] suggests a 

3N1O binding mode in the protein [1] whereas a roughly 1:1 mixture of 3N1O and 4N coordination modes is 

encountered for the peptides [2] in agreement with the previous study of Burns [3]. 

Concerning the Cu(II) coordination to the histidine-rich Ab peptide, there is yet no real coC Hureau, nsensus in 

the literature [4, 5]. We are currently working on the coordination of Cu(II) to the Ab16 peptide 

DAEFRHDSGYEVHHQK, the hydrophilic domain of the Ab peptide that contains all the ligands of the Cu(II) 

ion. We are using advanced EPR techniques in combination to specific amino-acid labeling in order to identify 

unambiguously the coordination sphere of the Cu(II) ion as a function of pH. 

References: 

[1] C. Hureau, L. Charlet, P. Dorlet, F. Gonnet, L. Spadini, E. Anxolabéhère-Mallart, J.-J. Girerd J. Biol. Inorg. 

Chem. 2006, 11, 735-744 

[2] C. Hureau, C. Mathé, P. Faller, T. A. Mattioli, P. Dorlet J. Biol. Inorg. Chem. 2008, in press 

[3] C. S. Burns, E. Aronoff-Spencer, G. Legname, S. B. Prusiner, W. E. Antholine, G. J. Gerfen, J. Peisach, G. 

Millhauser, Biochemistry 2003, 42, 6794-6803 

[4] E. Gaggelli, H. Kozlowski, D. Valensin, G. Valensin, Chem. Rev. 2006, 106, 1995-2044 

[5] C. Hureau, P. Faller, in preparation 

_____________________________________________________________________ 

93


O7. Crystal Structure of PerR: Characterization of the Regulation Site in 

the Active Protein and Unambiguous Identification of 2-oxo-histidine in the 

Oxidized Form 

V. Duarte a , D. Traoré a , A. El Ghazouani a , L. Jacquamet b , F. Borel b , J.-L. Ferrer b , 

D. Lascoux c , J.-L. Ravanat d , G. Blondin a , C. Caux-Thang a , J.-M. Latour a 

a 

iRTSV - LCBM, CEA, 17 av. des Martyrs, 38054, Grenoble, France 

e-mail: victor.duarte@cea.fr 

b 

IBS - LCCP, CEA, 41 rue Jules Horowitz, 38027, Grenoble, France 

c 

IBS - LSMP, CEA, 41 rue Jules Horowitz, 38027, Grenoble, France 

d 

iNAC - SCIB - LAN, CEA, 17 av. des Martyrs, 38054, Grenoble, France 

Oxidative stress is generated by exposure to elevated levels of Reactive Oxygen Species (ROS). To avoid the 

harmful effects of ROS, cells constitutively express proteins to protect themselves. The expression of these 

proteins is under control of specific regulators. In Bacillus subtilis, the PerR protein is a metal-dependent sensor 

of H2O2. PerR is a dimeric zinc protein with a regulatory metal-binding site that coordinates either Fe 2+ (PerR- 

Zn-Fe) or Mn 2+ (PerR-Zn-Mn). While most of the peroxide sensors use redox-active cysteines to detect H2O2, it 

has been shown that reaction of PerR-Zn-Fe with H2O2 leads to the oxidation of one histidine (H) residue that 

binds the Fe 2+ ion. This metal-catalyzed oxidation of PerR leads to the incorporation of one oxygen atom into 

either H37 or H91. However the exact position of the added oxygen is still unknown. The present study reports 

the crystal structure of the active PerR-Zn-Mn protein, which reveals the nature of the regulatory metal binding 

site. We also present the x-ray structure of the oxidized PerR protein (PerR-Zn-ox) that clearly shows a 2-oxohistidine 

residue in position 37. 2-oxo-histidine formation is also demonstrated and quantified by HPLC- 

MS/MS. EPR experiments indicate that PerR-Zn-ox shows a significant affinity for the regulatory metal, albeit 

lower than that of the wild-type protein. However, due to the predominant oxidation of H37, the oxidized PerR 

protein shows a drastically reduced affinity for the DNA. 

_____________________________________________________________________ 

94


O8. Comparison of the Metal Binding Affinities of Prion and Amyloid 

Peptide Fragments 

I. Sóvágó 

Inorganic and Analytical Chemistry, University of Debrecen, Egyetem ter 1., 4010, DEBRECEN, Hungary 

e-mail: sovago@delfin.unideb.hu 

The proteins responsible for the development of various forms of neurodegenerative disorders are generally rich 

in histidyl residues. In the case of human prion protein six histidines are located in the disordered region of the 

protein and they are well separated from each other. The hexadecapeptide Aβ(1-16) is generally considered as 

the metal binding domain of amyloid peptides and it consists of three histidyl moieties; two of them are in 

adjacent while the third one is located in distant positions. The presence of aspartyl and glutamyl residues 

represents another important difference in the amino acid sequences of prion and amyloid fragments. 

In the past few years we performed potentiometric and spectroscopic studies on the copper(II) and zinc(II) 

complexes of a series of peptide fragments of prion and amyloid-β [1-3]. The results revealed that the metal 

binding affinities of the peptides are largely affected by the number and location of histidyl residues. It was 

found that both prion and amyloid fragments can form stable mono- and oligo-nuclear complexes with 

copper(II), while zinc(II) binding affinity of amyloid peptides is much higher than those of the prion fragments. 

Acknowledgements: This work was supported by the Hungarian Scientific Research Fund, OTKA T 048352. 

References: 

[1] G. Di Natale, G. Grasso, G. Impellizzeri, D. La Mendola, G. Micera, N. Mihala, Z. Nagy, K Ősz, G. 

Pappalardo, V. Rigó, E. Razzarelli, D. Sanna, I. Sóvágó, Inorg. Chem., 2005, 44, 7214-7225. 

[2] V. Jószai, Z. Nagy, K. Ősz, D. Sanna, G. Di Natale, D. La Mendola, G. Pappalardo, E. Rizzarelli and I. 

Sóvágó, J. Inorg. Biochem., 2006, 100, 1399-1409. 

[3] K. Ősz, Z. Nagy, G. Pappalardo, G. Di Natale, D. Sanna, G. Micera, E. Rizzarelli, I. Sóvágó: Chem. Eur. J., 

2007, 13, 7129-7143. 

_____________________________________________________________________ 

95


O9. Manganese- Oxo- Complexes as Water Oxidation Catalysts for 

Artificial Photosynthesis 

P. Kurz, H.-M. Berends, F. Tuczek 

Institute for Inorganic Chemistry, Christian Albrechts University Kiel, Otto-Hahn-Platz 6/7, 24098, Kiel, 

Germany 

e-mail: phkurz@ac.uni-kiel.de 

In nature, the oxygen evolving complex (OEC) of Photosystem II, a cluster containing four µ- oxobridged 

manganese atoms, catalyses the four-electron oxidation of water to molecular oxygen. Our aim is to develop 

water oxidation catalysts inspired by the architecture of the OEC for the construction of systems for artificial 

photosynthesis. 

For this purpose, we synthesise µ- oxobridged, dinuclear manganese complexes bearing oxidation stable 

supporting ligands (see Fig. for an example). Despite numerous reports concerning the synthesis and 

characterisation of such compounds, [1] studies about their interaction with water under oxidative conditions - 

and especially their ability to indeed catalyse O2 formation - are rather rare. 

A recent systematic screening of the reactions combinations of different manganese complexes and oxidants 

indicated that a larger number of manganese complexes than known so far catalyse oxygen formation, [2] but 

only if oxygen- transfer oxidants were used. 

We now found that oxygen- transfer is often no prerequisite for oxygen formation any more if the manganese 

complexes are fixed on surfaces (see Fig.), in agreement with a previous report.[3] The important implications of 

these results for both artificial water oxidation catalysis and oxygen formation by the OEC will be presented. 

Figure. left: The µ- oxobridged, dinuclear manganese complexes 1. right: An O2 evolution curve for the 

reaction of adsorbed 1 (16µmol per g kaolin clay) with Ce 4+ (20mM). No O2 is formed for the reaction of the 

clay alone or for 1 in solution. 

References: 

[1] Mukhopadhyay, S. et al. Chem. Rev. 104, 3981 (2004). 

[2] Kurz, P. et al. Dalton Trans. 2007, 4258. 

[3] Yagi, M.; Narita, K. J. Am. Chem. Soc. 126, 8084 (2004). 

_____________________________________________________________________ 

96


O10. Biocatalytic Alkene Cleavage Using Molecular Oxygen 

F.G. Mutti a , M. Lara b , S.M. Glueck b , W. Kroutil b 

a 

Dipartimento di chimica inorganica, metallorganica, University of Milan, via Venezian 21, I-20133, Milano, 

Italy 

e-mail: francesco.mutti@unimi.it 

b 

Department of organic and bioorganic chemistry, University of Graz, Heinrichstrasse 28, A-8010, Graz, 

Austria 

The oxidative cleavage of alkenes is a widely employed method in synthetic chemistry, particularly to 

introduce oxygen functionalities into molecules and remove protecting groups. Ozonolysis[1] is the most 

common way to perform this reaction although it shows some disadvantages as the need of low temperature (- 

78°C) and reducing reagents in molar amount. Alternative protocols envisage the use of heavy metals as Cr, 

Os or Ru. Some peroxidases[2] and dioxygenases[3] display this activity as a minor side reaction. 

An enviromentally friendly, biocatalytic approach is presented here. An enzyme preparation from the fungus 

Trametes hirsuta G FCC 047 was employed for the C=C cleavage of different compounds in aqueous buffer 

and using dioxygen (2 bar) as sole oxidative reagent[4] (Fig. 1). 

A double bond conjugated with a phenyl ring is required for the biocatalytic activity. Quantitative conversion 

was reached with t-anethole on analytical scale, whereas upscaling to 500 mg furnished 81% conversion (57% 

isolated yield). 

Experiments with labelled O2 and H2O using indene as substrate showed that only oxygen atoms from O2 were 

incorporated, although derived from different molecules. Thus, alkene cleavage undergoes neither a 

dioxygenase mechanism nor a monoxygenase one. Reactions in presence of superoxide dismutase did not 

influence the reaction, so free radical superoxide anion is not the active species. Additionally, this study 

indicated that the reaction is catalysed by a single enzyme. 

References: 

[1] Berglund, R.A., in Encyclopedia of Reagents for Organic Synthesis, Vol. 6 (ed.: L. A. Paquette), Wiley, 

New York (1995) 3837-3843. 

[2] a) Ozaki, S. and Ortiz de Montellano, P.R., J. Am. Chem. Soc. 117 (1995) 7056-7064. b) Tuynman, J.L., 

Ingeborg, M.K., Shoemaker, H.E. and Wever, R., J. Biol. Chem. 275 (2000) 3025-3030. 

[3] Bugg, T.D.H., Tetrahedron 59 (2003) 7075-7101. 

[4] a) Mang, H., Gross, J., Lara, M., Goessler, H.E., Shoemaker, G.M., Guebitz, W. and Kroutil W., Angew. 

Chem. Int. Ed. 45 (2006) 5201-5203. b) Mang, H., Gross, J., Lara, M., Goessler, H.E., Shoemaker, G.M., 

Guebitz, W. and Kroutil W., Tetrahedron 63 (2007) 3350-3354. c) Lara, M., Mutti, F.G., Glueck, S.M. and 

Kroutil W., Eur. J. Org. Chem. (2008) in press. 

_____________________________________________________________________ 

97


O11. Dioxygen Activation on a Dicopper Core with a Distorted 

Coordination Environment 

Y. Funahashi, K. Yoshii, T. Nishikawa, Y. Wasada-Tsutsui, Y. Kajita, T. Inomata, T. Ozawa, 

and H. Masuda 

Department of Applied Chemistry, Faculty of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showaku, 

Nagoya 466-8555 

Binding and activating dioxygen, and O-O bond formation and cleavage on dimetal centers are essentially 

important for O2-transportation, catalytic oxidation, and dioxygen-evolution in biological systems. In the 

biomimetic model studies of type III copper proteins, Tolman et. al clearly showed that relevant Cu2-O2 species, 

µ-η 2 :η 2 -peroxo dicopper(II) and bis(µ-oxo) dicopper(III) species have the interconversion equilibrium in the 

solution. In these valence isomers, the degree of O2-reduction and bond order between these oxygen atoms can 

be changed, inversely. 

In this study, we used (-)-sparteine (Sp) and α-isosparteine (αSp) as supporting ligands of central copper ions 

with distorted coordination. The corresponding copper(I) complexes of Sp isomers reacted with O2 at �80°C to 

form bis(µ oxo) dicopper(III) species, which can be transformed to a bridged and butterfly-shaped µ-η 2 :η 2 -peroxo 

dicopper(II) species by addition of benzoate (OBz). After extensive studies of this system, we succeeded in 

constructing a non-equilibrium Cu2-O2 system using dioxygen and hydrogen peroxide as an oxidant. The 

carboxylate-bridged and butterfly-shaped µ-η 2 :η 2 -peroxo dicopper(II) species potentially has much relevance to 

the reaction intermediates on stepwise O2-reduction in non-heme diiron proteins, and O2-evolving in 

photosystems. 

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98


O12. Comparative Structural, Spectral, and Acid Inertness Studies of 

Copper(II) Adamanzane Coordination Compounds: 

Effect of pH and Chloride Ions 

K. Jensen a, b , P.W. Thulstrup a , I. Søtofte c , M. Jensen b and M.J. Bjerrum a 

a Bioinorganic Chemistry, Department of Natural Sciences, Faculty of Life Sciences, University of Copenhagen. 

Thorvaldsensvej 40, 1871 Fredriksberg C. Denmark. (krje@life.ku.dk) 

b Hevesy Laboratory, Radiation Research Department, Risø DTU, National Laboratory for Sustainable Energy. 

Technical University of Denmark. Frederiksborgvej 399, 4000 Roskilde. Denmark. 

e-mail: Kristian.jensen@risoe.dtu.dk 

c Department of Chemistry, Technical University of Denmark. Kemitorvet 207, 2800 Kgs. Lyngby. Denmark 

An increased interest for the use of positron emission tomography (PET and PET/CT) scanners for “molecular” 

imaging has become more common at the hospitals along with the medical cyclotrons. Various copper isotopes 

have favorable decay properties for the use in both imaging and targeted radiotherapy. 

With copper radioisotopes there is a need for novel chelators which can form kinetically and thermodynamically 

stable coordination compounds, in order to avoid untimely dissociation of the radiolabelled compound. Thus, the 

stability of the copper compounds is of considerable importance in ligand design aimed at in vivo delivery of 

copper-radioisotopes. 

Adamanzanes [1] are a class of bicyclic tetraaza ligands, which are well-suited for this type of application. 

Adamanzane ligands were designed to form highly stable coordination compounds with Cu(II), among others 

metals. 

We have compared adamanzanes (A and B) and adamanzanes functionalized with carboxymethyl groups (C and 

D). The latter should be able to engage in six-coordination of the metal-ion, and furthermore neutralize the 

dicationic charge of Cu(II). We have applied cyclic voltammetry, UV-VIS and FT-IR spectroscopy in the study 

of the stability, particularly with regards to reduction, chloride ion concentration and inertness towards acidic 

decomposition in order to mimic in vivo conditions. Analyses are compared to solid state structures obtained via 

x-ray crystallography. 

A: [3 5 ]adz, B: [Cu([3 5 ]adz)Br] + C: (N’-CH2COOH)2[3 5 ]adz and D: [Cu((N’-CH2COO)2[3 5 ]adz)] 

References: 

[1]: Springborg J. Adamanzanes - Bi- and Tricyclic Tetraamines and Their Coordination Compounds. Dalton 

Transactions 2003;(9):1653-1665. 

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99


O13. Interaction of Zn7metallothionein-2 with Platinum-modified 

5’-guanosine Monophosphate (GMP) and DNA 

A.V. Karotki, M. Vašák 

Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland 

e-mail: karotki@bioc.unizh.ch 

The Cys- and Zn-rich proteins, metallothioneins (Zn7MT), represent a resistance factor in anticancer treatment, 

due to the strong reactivity of Pt drugs with S-donor ligands. Previously, we demonstrated that transdichlorodiammineplatinum 

(trans-DDP), but not cis-DDP in the reaction with Zn7MT retains the N-donor 

ligands [1]. In this study, we show by immunochemical analyses of human MT that platinum-modified DNA 

forms DNA−cis-/ trans-Pt−MT cross-links and that in the case of trans-DDP cross-links platinated MT is 

released with time. Kinetic studies using cis- and trans-DDP−GMP as a model showed that the initial rate of 

the reaction between Zn7MT and cis-DDP−GMP was 4-times higher than the trans-isomer. Quantification of 

Pt−S bonds, GMP, and Pt bound to MT revealed one specific binding site for cis-DDP−GMP. In the binding 

process the fast initial formation of 2 Pt−S bonds was followed by the slow formation of an additional Pt−S 

bond yielding an unusual S3NPt(II) coordination with N7-GMP as the only N-donor. The protein structure, 

closely spaced thiolate ligands and noncovalent interactions are likely responsible for the formation of such 

complex. In the reaction with trans-DDP−GMP the initial formation of 1 Pt−S bond was followed by a GMP 

release, due to the strong trans effect of sulfur, and the formation of a second Pt−S bond. Thus, besides Pt(II) 

sequestration, Zn7MT modulates the potency of anticancer drugs through the formation of DNA−Pt−MT crosslinks. 

References: 

[1] Knipp, M., Karotki, A. V., Chesnov, S., Natile, G., Sadler, P. J., Brabec, V., and Vasak, M. (2007), J. Med. 

Chem. 50, 4075−4086. 

_____________________________________________________________________ 

100

E. Feese, R.A. Ghiladi 


O14. Exploring New Treatment Options for Tuberculosis: 

Photodynamic Inactivation of Mycobacterium Smegmatis 

Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, 27695-8204, Raleigh, NC, 

United States, 

e-mail: efeese@ncsu.edu 

Tuberculosis (TB) is one of the leading causes of death due to a single disease with 9.2 million new infections 

and 1.7 million deaths reported for 2006 alone. Efforts to control TB infection have been hampered by the rise of 

multiple-drug resistant strains, thereby necessitating research into new treatment options. Herein, we explore the 

feasibility of photodynamic inactivation (PDI) as an alternative approach to the current antibiotic-based 

tuberculosis treatments. Non-pathogenic Mycobacteria smegmatis was employed as a surrogate for 

M. tuberculosis and the reduction in colony forming units (CFU) was examined as a function of the 

photosensitizer (PS) concentration and light dose. Several commercially available photosensitizers were 

examined at micromolar to nanomolar concentrations. The most promising results were achieved using the 

cationic tetrakis-(1-methyl-4-pyridinio)porphyrin (146 nM), showing a 5-6 log unit reduction of CFU after 

irradiation (400-700 nm) for 5 minutes at 60 mW/cm 2 s. Longer irradiation times resulted in no CFUs being 

detected. Generally, positively charged photosensitizers showed PDI against M. smegmatis, whereas negatively 

charged PS were ineffective. Further data obtained using other photosensitizers, along with a comparison to 

analogous experiments with E. coli, will be presented. The data show that mycobacteria can be 

photodynamically inactivated, suggesting that PDI may be an attractive treatment option for drug-resistant 

tuberculosis. 

_____________________________________________________________________ 



O15. Lanthanide Metallacrowns as Anion Receptors and Potential MRI 

Contrast Agents 

M. Tegoni a , M. Remelli b , F. Dallavalle a , L. Marchiò a 

a 

Department of General and Inorganic Chemistry, University of Parma, Viale G.P. Usberti 17A, 43100, Parma, 

Italy 

e-mail: matteo.tegoni@unipr.it 

b 

Department of Chemistry, University of Ferrara, Via L. Borsari 46, 44100, Ferrara, Italy 

Metallacrowns (MCs) are a class of metallamacrocycles structurally related to crown ethers with extraordinary 

capabilities of encapsulating various metal ions into the central cavity of a self-assembled supramolecule. [1] 

Peripheral divalent cations and bridging ligands form the metallacycle, while examples of core ions are Cu(II) in 

12-MC-4 and Ln(III) in 15-MC-5. Known since a decade, the 15-MC-5 complexes are capable to encapsulate 

selectively Ln(III) in presence of Ca(II) or uranyl, and to coordinate anions to the peripheral and core ions on 

both faces of the metallamacrocycle. [2] 

The capability of 15-MC-5 to bind the hydroxide ion arise from the high acidity of coordinated water molecules, 

one of which deprotonates at pH 4. These results obtained by thermodynamic studies in aqueous solution 

allowed also to establish that the 15-MC-5 formation is a real self-assembly and that the 15-MC-5 species are the 

only stable Ln(III) complexes up to pH 7. 

The remarkable ability of 15-MC-5 to coordinate carboxylates was studied by means of fluorescence and PGSE 

– NMR in water. The results demonstrate on a thermodynamic basis the observation that 15-MC-5 complexes of 

phenylalaninehydroxamate coordinate preferentially aromatic than aliphatic carboxylates in their hydrophobic 

pocket. 

The stability of Gd(III) 15-MC-5 with potential applications as MRI contrast agents in presence of competing 

ligands and endogenous metals has also been interpreted on the basis of thermodynamic data. 

References: 

[1] G. Mezei, C.M. Zaleski, V.L. Pecoraro, Chem. Rev., 107(11), 2007, 4933-5003. 

[2] C.S. Lim, A. Cutland Van Noord, J.W. Kampf, V.L. Pecoraro, Eur. J. Inorg. Chem., 10, 2007, 1347-1350. 

_____________________________________________________________________ 



O16. Genetically Encoded Fluorescent Sensors for Intracellular Imaging of 

Transition Metal Homeostasis 

M. Merkx 

Biomedical Engineering,Laboratory of chemicla biology,PO box 513,5600 MB,Eindhoven,Netherlands 

e-mail: m.merkx@tue.nl 

The ability to image the concentration of transition metals in living cells in real time is important for 

understanding transition metal homeostasis and its involvement in diseases. Genetically encoded sensors that use 

fluorescence resonance energy transfer (FRET) between two fluorescent proteins are attractive because they 

allow ratiometric detection, do not require cell-invasive procedures, and can be targeted to different locations in 

the cell. Here we present the development of several Zn(II) sensors with affinities ranging from 30 fM to 50 

microM Sensor proteins with a very high and tunable affinity (Kd = 30 fM -1.4 pM) were created by connecting 

two fluorescently labeled metal binding domains, CFP-Atox1 and WD4-YFP, using a series of flexible peptide 

linkers [1,2]. A conformational switch approach was employed to improve the ratiometric change of these 

sensors from ~0.15 to 2 [3], making them ideally suited to probe the very low free Zn(II) concentrations present 

in the cytosol. A second type of sensor was developed in which de novo Zn(II) binding sites were introduced 

directly on the surface of both fluorescent proteins (see figure). This sensor displayed an impressive, 9-fold 

increase in emission ratio in the presence of Zn(II) and allowed detection of Zn(II) from 10 nM to 1 mM. [4]. 

The insights obtained from this work are generally applicable and can easily be extended to design FRET-based 

sensor proteins for other transition metal ions. 

References: 

[1] van Dongen, Dekkers, Spijker, Meijer, Klomp and Merkx (2006) J. Am. Chem. Soc. 128, 10753-10762 

[2] van Dongen, Evers, Dekkers, Meijer, Klomp and Merkx (2007) J. Am. Chem. Soc. 129, 3494-3495 

[3] Vinkenborg, Evers, Reulen, Meijer and Merkx (2007) ChemBioChem 8, 1119-1121 

[4] Evers, Appelhof, de Graaf-Heuvelmans, Meijer, and Merkx (2007) J. Mol. Biol. 374, 411-425 

_____________________________________________________________________ 



O17. Synthesis And Dna Binding Studies Of End-Functionalised Metallo- 

Supramolecular Cylinders 

L. Cardo and M.J. Hannon 

School of Chemistry, University of Birmingham, B15 2LH, Birmingham, United Kingdom 

e-mail: lxc577@bham.ac.uc 

The design and development of new metallo-based drugs, capable of interacting and selectively recognising the 

DNA, is a major challenge in the field of bioinorganic chemistry. We recently reported that di- and tetra-cationic 

supramolecular “cylinders” not only are able to bind in the major groove of natural polymeric DNA [1] and at 

the heart of DNA 3-way junctions [2], but they also exhibit a remarkable cytotoxic activity against cancer cell 

lines [3]. 

These results motivated us to examine useful modifications of these complexes, particularly to explore whether 

DNA recognition motifs [4], such as amino acids or short peptides, might be attached to the cylinders to provide 

a route to targeting the cylinder activity to particular genes on the DNA. Furthermore, mono- and di-capping of 

the cylinders are both being investigated (Figure 1). 

Herein, we report a versatile procedure to end-functionalise the cylinders by amido bonds. This allow us to 

obtain our first hybrids [5]: one di-Fe(II) triple-stranded, two di-Cu(I) and one di-Ag(I) double-stranded 

cylinders have been conjugated to Gly-Gly-Ser peptide sequences; a di-Fe(II) triple stranded complex has also 

been fuctionalised with arginine residues and a very interesting effect on the chirality of the resulting helicate has 

been observed. DNA-binding and cleavage studies confirm that the end-functionalisation does not prevent the 

inherent cylinder DNA recognition properties from being expressed. 

Figure 1: Representation of designed end-functionalised triple stranded cylinder. 

Acknowledgement: University of Birmingham for funding. 

References: 

[1] M.J. Hannon, V. Moreno, M.J. Prieto, E. Molderheim, E. Sletten, I. Meistermann, C.J. Isaac, K.J. Sanders 

and A. Rodger, Angew. Chem., Intl. Ed., 40, 879 (2001); I. Meistermann, V. Moreno, M.J. Prieto, E. 

Moldrheim, E. Sletten, S. Khalid, P. M. Rodger, J.C. Peberdy, C.J. Isaac, A. Rodger and M.J. Hannon, Proc. 

Natl. Acad. Sci., 99, 5069 (2002); C. Uerpmann, J. Malina, M. Pascu, G.J. Clarkson, V. Moreno, A. Rodger, A. 

Grandas and M.J. Hannon, Chem. Eur. J. 11, 1750 (2005). 


Intl. Ed., 45 1227 (2005). 

[3] A.C.G. Hotze, B.M. Kariuki, M.J. Hannon, Angew. Chem., Int. Ed. 45, 4839 (2006); G.I. Pascu, A.C.G. 

Hotze, C. Sanchez Cano, B.M. Kariuki, M.J. Hannon, Angew. Chem., Int. Ed. 46, 4374 (2007). 

[4] S. Neidle, Nucleic acid structure and recognition, Oxford University Press. Oxford (2002). 

[5] L. Cardo, M.J. Hannon, Inorg. Chim. Acta (2008), special issue, doi:10.1016/j.ica.2008.02.050, in press. 

_____________________________________________________________________ 



O18. X-ray Crystallographic Studies on DNA Binding Modes of Polyaminebridged 

Polynuclear Pt(II) Complexes 

S. Komeda a , A. Odani b , M. Chikuma c , N. Farrell d , L. Williams e 

a Department of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minamitamagaki-cho, 

513-0816, Suzuka, Japan 

e-mail: komedas@suzuka-u.ac.jp 

b School of Pharmaceutical Sciences, Kanazawa University, Kakuma-cho, 920-1192, Kanazawa, Japan 

c Graduate School of Pharmaceuticak Sciences, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, 

569-1094, Takatsuki, Japan 

d Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, 23284-2006, Richmond, 

VA, United States 

e School of Chemistry and Biochemistry, Georgia Institute of Technology, 30332-0400, Atlanta, GA, United 

States 

The extensive pre-associations of polyamine-bridged polynuclear Pt(II) complexes with DNA presumably arise 

from the high positive charge (+2 ~ +8) and would be involved in the mechanism of their anticancer actions [1]. 

The results obtained from pre-association studies will allow us to estimate critical parameters in biological 

mechanism [2]. 

Here we present three of X-ray crystal structures of a double-stranded B-DNA dodecamer, 

[d(CGCGAATTCGCG)]2 (Deckerson-Drew dodecamer: DDD), each bound non-covalently to different 

polynuclear platinum(II) complexes, [{cis-Pt(NH3)2(NH2(CH2)6NH3 + )}2-µ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}] 8+ 

(AH78), [{Pt(NH3)3}2-µ-{trans-Pt 

(NH3)2(NH2(CH2)6NH2)2}] 6+ (AH44) and [{Pt(NH3)3}2-µ-{NH2(CH2)3NH2 + 

(CH2)4NH2 + (CH2)3NH2)}] 6+ (AH59). In the Pt-DDD crystal structures the phosphate backbone attracts the Pt 

complexes by a DNA binding unit, we call "Phosphate Clamp". A Phosphate Clamp is a cyclic structure with 

single OP, which accepts two hydrogen bonds, one from each of two am(m)ine ligands of a single Pt(II) center. 

By two sets of Phosphate Clamps polynuclear Pt(II) complexes track the DNA backbone along the single strand 

of the DDD or bridges two strands across the minor groove Implications of these findings are discussed. 

References: 

[1] Qu, Y.; Harris, A.; Hegmans, A.; Petz, A.; Kabolizadeh, P.; Penazova, H.; Farrell, N. J. Inorg. Biochem. 

2004, 98, 1591-1598 

[2] Komeda, S.; Moulaei, T.; Woods, K. K.; Chikuma, M.; Farrell N. P.; Williams, L. D. J. Am. Chem. Soc. 

2006, 128, 16092-16103. 

_____________________________________________________________________ 



O19. DNA-based Asymmetric Catalysis - a Covalent Approach 

N. Sancho Oltra, G. Roelfes 

Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The 

Netherlands 

e-mail: N.Sancho.Oltra@rug.nl; http://Roelfes.fmns.rug.nl/ 

The helical structure of DNA represents an attractive chiral scaffold for bio-inspired asymmetric catalysis.[1, 

2, 3] It has been demonstrated that when a copper complex is bound non-covalently to DNA, the chiral 

information is transferred directly from the DNA to the product of the catalyzed reaction resulting in high 

enantiomeric excesses for several reactions. 

Here, we present the first example of asymmetric DNA-based catalysis in which a metal complex is attached 

covalently to a well-defined position in the DNA. Covalent attachment allows precise control over the 

structure and geometry of the catalyst and, hence, activity and selectivity. To achieve this we introduce a novel 

modular approach towards the assembly of this new generation of DNA-based catalysts (see figure). Indeed, 

the enantiomeric excesses obtained for the copper(II) catalyzed Diels-Alder reaction in water proved to be very 

dependent on the design of the catalyst. Important factors included the distance between the complex and the 

DNA helix, the template length and the DNA sequence. Using an optimized design, ee's of up to 94% have 

been obtained. 

References: 

[1] G. Roelfes, B. L. Feringa, Angew. Chem. Int. Ed., 2005, 44, 3230 

[2] G. Roelfes, A. J. Boersma, B. L. Feringa, Chem. Commun., 2006, 635 

[3] D. Coquière, B. L. Feringa, G. Roelfes, Angew. Chem. Int. Ed., 2007, 46, 9308 

_____________________________________________________________________ 



O20. Biosynthetic Exchange of Bromide for Chloride and Strontium for 

Calcium in the Photosystem-II Oxygen Evolving Complex 

N. Ishida a , M. Sugiura b , F. Rappaport c , T.-L. Lai a , A.W. Rutherford a , A. Boussac a 

a 

DSV, iBiTec-S, URA CNRS 2096, CEA Saclay, 91191, GIf-sur-Yvette, France 

e-mail: alain.boussac@cea.fr 

b 

Department of Plant Biosciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, 599-8531, Sakai, 

Osaka, Japan 

c 

Université Pierre et Marie Curie, Institut de Biologie Physico-Chimique, CNRS UMR 71, 13 rue Pierre et 

Marie Curie, 75005, Paris, France 

Light-driven water oxidation by Photosystem II (PSII) is responsible for the O2 on Earth and most of the 

biomass. Refined 3D X-ray structures at 3.5 Å and at 3.0 Å resolution have been obtained by using PSII isolated 

from the thermophilic cyanobacterium Thermosynechococcus elongatus [1, 2]. The active site for water 

oxidation in PSII goes through five sequential oxidation states before O2 is evolved. It consists of a Mn4Cacluster 

close to a redox-active tyrosine residue and possibly Cl - as cofactor. To study the role of Ca 2+ and Cl - , 

T. elongatus was grown in the presence of Sr 2+ instead of Ca 2+ and Br - instead of Cl - , in order to biosynthetically 

substitute the Ca 2+ and Cl - for Sr 2+ and Br - , respectively. Irrespective of the combination of the non-native ions 

used (Ca/Br, Sr/Cl, Sr/Br), the PSII could be isolated in a state that was fully intact but kinetically limited. The 

step(s) of the enzyme mechanism affected by the exchanges were identified then investigated by using timeresolved 

UV-visible absorption spectroscopy, time-resolved O2 polarography, thermoluminescence and EPR 

spectroscopy. The effect of the Ca/Sr and Cl/Br exchanges were additive and the magnitude of the effects varied 

with the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. All the observations indicate that Cl - is involved in the 

water oxidation mechanism. If so, the lack of a Cl - binding site in the current 3D models of the enzyme from 

X-ray crystallography may be ascribable to insufficient resolution. 

References: 

[1] Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J., and Iwata, S. (2004) Science 303, 1831-1838. 

[2] Loll, B., Kern, J., Saenger, W., Zouni, A., and Biesiadka, J. (2005) Nature 438, 1040-1044. 

_____________________________________________________________________ 



O21. Scorpiand-Like Macrocycles As Funtional Models For Molecular 

Machines. Kinetic And Mechanistic Studies On Molecular Movements 

Induced By pH Gradients 

C.E. Castillo Gonzalez, a B. Verdejo, b A. Ferrer, a S. Blasco, b J. González, b J. Latorre, c 

M.A. Máñez, a M.G. Basallote, a C. Soriano b , E. García-España b 

a 

Dpto. de Ciencia de los Materiales e Ingeniería Metalúrgica y Química Inorgánica, Univ. de Cádiz, Apdo 40, 

Puerto Real, 11510 Cádiz. 

b 

Instituto de Ciencia Molecular, Univ. de Valencia, Apdo 22085, 46071 Valencia. 

c 

Instituto de Materiales de la Universidad de Valencia, C/Dr. Moliner 50, 46100 Burjassot, Valencia 

e-mail: esther.castillo@uca.es 

Biological motors use chemical energy to effect stepwise linear or rotatory motion, and they are essential in 

controlling and performing a wide variety of biological funtions. Thus, a genuine molecular machine is involved 

in the synthesis and hydrolysis of ATP, and other fascinating example is the flagelar motor that enables bacterial 

movements. Interestingly, the movements in both of these biological machines are asociated with gradients in the 

concentration of protons. 

Despite their interest, the number of examples in which the kinetics of controlled molecular motions of this kind 

has been identified in small model molecules is still not large. 

The present study is focused on the kinetics and mechanism of formation, decomposition and reorganization 

processes associated with pH changes for a series of scorpiand-like complexes. Some DFT studies have been 

also made to obtain information about these molecular movements. The systems considered contain a moving 

part, whose motion can be reversibly and repeatedly carried out: i.e. they convert the chemical energy into 

mechanical work and can be therefore considered as machines operating at the molecular level. 

Our results show that molecular movements associated to the changes of pH can be induced in both the ligands 

and their metal complexes. Moverover, the kinetics of the formation processes is strongly conditioned by the 

charge of reactants and by the steric characteristics of the ligand, which is controlled by hydrogen bond 

formation. The results of the theoretical study also help to understand the kinetic results. 

_____________________________________________________________________ 



O22. Zinc-thiolate Group: Reactivity and Alkylation Mechanism 

D. Picot, G. Ohenessian, G. Frison 

Department of Chemistry - Laboratoire des Mécanis, CNRS - Ecole Polytechnique, Route de Saclay, 91128, 

Palaiseau Cedex, France 

e-mail: frison@dcmr.polytechnique.fr 

Alkylation of zinc-bound thiolates occurs in both catalytic and structural zinc sites of enzymes. Recent 

biomimetic studies have led to a controversy as to which mechanism is operative in thiolate alkylation. 

Furthermore, this alkylation reaction has raised question about the nucleophilicity of thiolates located in the zinccoordination 

sphere.[1, 2] Building on one of these biomimetic complexes, we have devised a series of models 

that allow for an appraisal of the roles of charge, ligand nature and hydrogen bonding to sulfur on reactivity. The 

reactions of these complexes with methyl iodide, leading to thioethers and zinc iodide complexes, have been 

examined by density functional theory (DFT) calculations, in the gas phase as well as in aqueous solution. In all 

cases, a SN2 reaction is favoured over sigma-bond metathesis. Both the net electronic charge and the H bond 

play a significant role on the nucleophilicity of the thiolate. We find that the mechanistic diversity observed 

experimentally can be explained by the difference in the net charge of the complexes. Finally, we were able to 

determine correlation between the reactivity of these systems and their thermodynamic and structural properties. 

This allows us to widen these works to real biological systems. 

References: 

[1] G. Parkin, Chem. Rev., 2004, 104, 699. 

[2] J. Penner-Hahn, Curr. Opin. Chem. Biol., 2007, 11, 166. 

_____________________________________________________________________ 



O23. Iron Complexes Mimicking the Active Site of the Iron-Sulfur Cluster- 

Free Hydrogenase 

X. Hu and B. Obrist 

Laboratory of Inorganic Synthesis and Catalysis, Institute of Chemical Science and Engineering, Ecole 

Polytechnique Fédérale de Lausanne, EPFL-ISIC-LSCI, BCH 3305, Lausanne, CH 1015, Switzerland. 

e-mail: xile.hu@epfl.ch 

Three types of phylogenetically unrelated hydrogenases are known, including the [NiFe]- and [FeFe]hydrogenases 

and the iron-sulfur cluster-free hydrogenase (Hmd). Hmd is a unique hydrogenase in that it 

requires one single iron for function and it contains an interesting pyridone cofactor. Our lab is developing the 

coordination chemistry of iron and pyridones to probe their roles in the enzymatic H2 activation by Hmd. 

Current data (Shima and Thauer et al.) suggest that in the active form of Hmd, the iron center is coordinated by 

two cis-CO ligands, one cysteine S atom, one nitrogen/oxygen atom from the pyridone portion of the cofactor, 

and one unknown fifth ligand (Figure inset, top). We choose to use simple pyridone ligands to mimic the 

pyridone cofactor and thiolate ligands to mimic the cysteine S ligand (Figure inset, bottom). Starting from 

Fe(CO)5, we were able to synthesize iron complexes containing a pyridone ligand together with two ciscarbonyls. 

These complexes were characterized by a variety of spectroscopic methods and provided important 

reference data points for the geometric and electronic structure of Fe in Hmd itself. Furthermore, the solid-state 

structure of some of these model complexes was determined and the binding of pyridone to iron was revealed. 

We will present our synthetic, spectroscopic, structural, and reactivity studies on these iron model complexes 

together with the implications for Hmd. 

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110


O24. The Influence of Metal Cation Binding to Aromatic Side Chain on 

Hydrogen Bond System in Alpha Helical Peptides 

R. Wieczorek 

Department of Chemistry, University of Wroclaw, Poland. 

The secondary structure of proteins depends on fragile equilibrium between non-covalent intra and 

intermolecular interactions between protein and solvent/soluted compounds. The common motifs of peptides – 

alpha helical peptides have been explored both experimentally and theoretically [1-4]. The modern 

computational chemistry methods allow to investigate selected effect e.g. interaction between side chain and ions 

present in solvent. The alpha helical peptides contain three chain-organized hydrogen bond groups. The 

interaction between aromatic side chain and small cation influence on helix by strong modification of hydrogen 

bonds. The ion – peptide interaction significantly changes stability of the peptide. 

Acknowledgement: Polish Ministry of Science and Higher Education, grant number: N N204 216834. 

References: 

[1]. Z. Shi, C. A. Olson, G. D. Rose, R. L. Baldwin, and N. R. Kallenbach "Polyproline II structure in a sequence 

of seven alanine residues", Proc. Nat. Acad. Sci. 2002; 99: 9190-9195 

[2]. Wallimann P., Kennedy R.J., Miller J.S., Shalongo W., Kemp D.S. "Dual wavelength parametric test of twostate 

models for circular dichroism spectra of helical polypeptides: anomalous dichroic properties of alanine-rich 

peptides", J. Am. Chem. Soc. 2003;125:1203–1220 

[3]. Wieczorek R. and Dannenberg J.J. "The Energetic and Structural Effects of Single Amino Acid 

Substitutions upon Capped r-Helical Peptides Containing 17 Amino Acid Residues. An ONIOM DFT/AM1 

Study", J. Am. Chem. Soc. 2005; 127: 17216-17223 

[4]. Salvador P., Wieczorek R. and Dannenberg J.J. "Direct Calculation of trans-Hydrogen-Bond 13C-15N 3- 

Bond J-Couplings in Entire Polyalanine alpha-Helices. A Density Functional Theory Study", J. Phys. Chem. B 

2007; 111: 2398-2403 

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111


O25. Modulation of the Ligand–Field Anisotropy in a Series of Ferric Low 

Spin Cytochromes c Mutants from Pseudomonas aeruginosa c–551 

and Nitrosomonas europaea c–552 

E. Harbitz a , G. Zoppellaro a , R. Kaur b , A.A. Ensign b , K.L. Bren b , K.K. Andersson a 

a Department of molecular biosciences, University of Oslo, Box 1041, Blindern, 0316, Oslo, Norway, 

c Department of Chemistry, University of Rochester, Rochester, 14627–0216, New York, United States 

C-type cytochromes with histidine-methionine axial heme ligation play important roles in electron-transfer 

reactions and in enzymes. In this work we study cyt c from Pseudomonas aeruginosa (Pa c-551) [1], 

Nitrosomonas europaea (Ne c-552) [2] and two methane oxidizing bacteria. Point mutations were induced in a 

key residue (Asn64) near the Met axial ligand that have a considerable impact on both heme ligand-field strength 

and in the Met orientation and dynamics (fluxionality). Ne c-552 has a ferric low spin (S=1/2) EPR signal 

characterized by large g anisotropy with gmax at 3.34. In Ne c-552, deletion of Asn64 (NeN64∆) changes the 

heme ligand-field from more axial to rhombic and also hindered the Met fluxionality present in the wild-type 

enzyme. In Pa c-551 (gmax at 3.20) replacement of Asn64 with valine induces a decrease in the axial-strain and 

changes the Met configuration. Other mutants, resulting in modifications in the length of the axial Met-donating 

loop, did not result in appreciable alterations of the original ligand field, but had an impact on Met orientation, 

fluxionality and relaxation dynamics. Comparison of the electronic fingerprints of these proteins reveals a linear 

relation between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or 

dynamics. Thus, for these His-Met axially coordinated Fe(III) the large gmax value EPR signal does not 

represent a special case as is observed for bis-Histidine coordinated iron [3, 4, 5]. 

References: 

[1] Wen, X., Bren, K. L. (2005) Heme axial methionine fluxion in Pseudomonas aeruginosa Asn64Gln 

cytochrome c-551. Inorg. Chem. 44, 8587-8593 

[2] Zoppellaro G., T. Teschner, E. Harbitz, S. Karlsen, V. Schünemann, A. X. Trautwein, D.M. Arciero, A.B. 

Hooper, S. Ciurli, and K. K. Andersson (2006) EPR and Mössbauer Spectroscopical Studies of two c-type 

Cytochromes, exhibiting HALS EPR signals. ChemPhysChem 7, 1258 - 1267 

[3] Hederstedt L. and K.K. Andersson (1986) Electron Paramagnetic Resonance Spectroscopy of Bacillus 

subtilis cytochrome b-558 in Escherichia coli Membranes and in Succinate Dehydrogenase Complex from B. 

subtilis Membranes. J. Bacteriol. 167, 735-739 

[4] Friden H., M.R. Cheesman, L. Hederstedt, K.K. Andersson, and A.J. Thomson (1990) Low temperature EPR 

and MCD studies on cytochrome b-558 of the Bacillus subtilis succinate:quinone oxidoreductase indicate bishistidine 

coordination of the heme iron. Biochem. Biophys. Acta 1041, 207-215 

[5] Walker, F. A. (2004) Models of the bis-histidine-ligated electron-transferring cytochromes. Comparative 

geometric and electronic structure of low-spin ferro and ferrihemes. Chem. Rev. 104, 589-615 

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112


O26. Kinetics of Gas Diffusion in Hydrogenase: Experimental Approaches 

F. Leroux a , B. Burlat a , S. Dementin a , L. Cournac b , A. Volbeda c , B. Guigliarelli e , 

P. Bertrand a , J. Fontecilla-Camps c , M. Rousset a , C. Léger a 

a BIP, CNRS, 31 ch. J. Aiguier, 13009, Marseille, France 

e-mail: leger@ibsm.cnrs-mrs.fr 

b LBBBM, CEA,, 13108, St Paul-lez-Durance, France 

c LCCP, CEA, 41 rue Jules Horowitz, 38027, Grenoble, France 

Hydrogenases, which catalyze H2 to H + conversion as part of the bioenergetic metabolism of many 

microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described using 

crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion 

kinetics is unexplored. 

Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses 

protein film voltammetry [1-3] to resolve the kinetics of binding and release of the competitive inhibitor CO; 

the other is based on interpreting the yield in the isotope exchange assay. 

We study structurally-characterized mutants of a NiFe hydrogenase, and we show that two mutations, which 

significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO 

and H2 toward and from the active site by up to two orders of magnitude. This proves the existence of a 

functional channel which matches the hydrophobic cavity found in the crystal. However, the changes in 

diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the X-ray 

structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of sidechain 

dynamics in determining these [4]. 

References: 

[1] C. Léger at al. J. Am. Chem. Soc. 126, 12162 (2004) 

[2] C. Baffert et al. Angewandte Chemie Int. Ed. 47, 2052 (2008) 

[3] C. Léger et al. Chemical Reviews. In press http://dx.doi.org/10.1021/cr0680742 (2008) 

[4] F. Leroux et al. Submitted (2008) 

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113


O27. The Major EPR Signature of Periplasmic Nitrate Reductases Arises 

from a Species that Activates Upon 

V. Fourmond a , B. Burlat a , S. Dementin a , P. Arnoux b , M. Sabaty b , S. Boiry b , 

B. Guigliarelli a , P. Bertrand a , D. Pignol b , C. Léger a 

a BIP, CNRS, 31, ch Joseph Aiguier, 13009, Marseille, France 

e-mail: vincent.fourmond@ibsm.cnrs-mrs.fr 

b Laboratoire de Bioénergétique Cellulaire, Instit, CEA,, 13108, St Paul-lèz-Durance, France 

Enzymes of the DMSO reductase family use a mononuclear Mo-bis(molybdopterin) cofactor (MoCo) to catalyze 

a variety of oxo-transfer reactions[1]. Regarding nitrate reductases, which are among the most studied members 

of this family, much functional information has been gained from EPR spectroscopy[2, 3, 4], but this technique 

is not always conclusive because the signature of the MoCo is heterogeneous, and which signals correspond to 

active species is still unsure. We use site-directed mutagenesis, EPR and protein film voltammetry[5] to 

demonstrate that the MoCo in Rh. sphaeroides periplasmic nitrate reductase (NapAB) is subject to an irreversible 

reductive activation process that correlates with the disappearance of the so-called "high-g" MoV EPR signal. 

Therefore, this most intense and commonly observed signature of the MoCo arises from an inactive state that 

gives a catalytically competent species only after reduction. This proceeds, even without substrate, according to 

a reduction followed by an irreversible non-redox step, both of which are pH independent. An apparently similar 

process occurs in other nitrate reductases (both assimilatory and membrane-bound[6]) and this also recalls the 

redox cycling procedure which activates DMSO reductases and simplifies their spectroscopy[7]. 

References: 

[1] Hille, R. Trends in Biochemical Sciences 2002, 27, 360-367. 

[2] Butler, C. S.; Charnock, J. M.; Garner, C. D.; Thomson, A. J.; Ferguson, S. J.; Berks, B. C.; Richardson, D. J. 

Biochem. J. 2000, 352, 859-864. 

[3] Arnoux, P.; Sabaty, M.; Alric, J.; Frangioni, B.; Guigliarelli, B.; Adriano, J.-M.; Pignol, D. Nat. Struct. Mol. 

Biol. 2003, 10, 928-934. 

[4] Gonzàlez, P.; Rivas, M.; Brondino, C.; Bursakov, S.; Moura, I.; Moura, J. J. Biol. Inorg. Chem. 2006, 11, 

609-616. 

[5] Léger, C.; Bertrand, P. Chem. Rev., in press. 

[6] Field, S. J.; Thornton, N. P.; Anderson, L. J.; Gates, A. J.; Reilly, A.; Jepson, B. J. N.; Richardson, D. J.; 

George, S. J.; Cheesman, M. R.; Butt, J. N. Dalton Trans. 2005, 3580-3586. 

[7] Bray, R.; Adams, B.; Smith, A.; Bennett, B.; Bailey, S. Biochemistry 2000, 39, 11258-11269. 

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114


O28. Enzyme-like Oxygenation and Oxidation of Catechols with Molecular 

Oxygen via Mn(II)-Semiquinonate Complexes 

T. Funabiki, E. Tanigawa, M. Shimomaki, A. Mochizuki, K. Teramaoto, Y. Hitomi, 

M. Kodera 

Department of Molecular Chemistry and Biochemistry, Doshisha University, Tatara, 610-0321, Kyotanabe, 

Japan 

e-mail: funabiki@m3.dion.ne.jp 

We have developed a new Mn(II)-semiquinone complex, [Mn(L)(DTBSQ)] + (1, DTBSQ: 3, 5-di-tert-butyl-1, 2benzosemiquinonate, 

L: TPA), which is analogous to the intermediate species, Fe(II)-semiquinonate, proposed in 

the oxygenations by Fe 3+ -intradiol catechol dioxygenases. UV-VS and ESI/MS spectroscopies of the solution of 

1 after the alternate O2 and Ar babblings suggested the intermediaate formation of [Mn(L)(DTBSQ)(O2)] + (2). In 

case of alcoholic solvents, intradiol oxygenation products were obtained, indicating that oxygen attached to 

Mn(II) in 2 reacts with the semiquinonate ligand to give intradiol oxygenation products. In acetonitrile, quinone, 

DTBQ, was selectively formed, and a m-oxo-dimer, [Mn(L)(m-O)]2 2+ (3), was isolated as an intermediate [1]. In 

the presence of the excess catehol, DTBCH2, DTBQ was catalytically formed. Noteworthily, O2 is reduced to 

H2O similarly to the enzyme system, while in the most model studies for catechol oxidases O2 is reduced to 

H2O2. The oxidation system was applied to catechols other than DTBCH2, but 3 was used as the starting 

complex in place of semiqunonate complexes which could not be prepared by the same way applied to 1. When 

4-t-Butylcatechol (TBCH2), 4-methylcatechol (MeCH2), and pyrocatechol (HCH2) were added to 3, peaks 

characteristic to the Mn(II)-semiquinonate complexes were first observed, followed by the peaks for quinones. 

The reactivity was in the order DTBCH2 > TBCH2 > MeCtH2 > HCH2 

References: 

[1] Y. Hitomi, A. Ando, H. Matsui, T. Ito, T. Tanaka, S. Ogo, T. Funabiki, Inorg. Chem. 2005, 44, 3473-8. 

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115


O29. Spectroscopic Insights into the Oxygen-tolerant Hydrogenase from 

Ralstonia eutropha in its Native Membrane Environment 

and Immobilized on a Gold Surface 

I. Zebger a , N. Wisitruangsakul a , D. Millo a , M. Saggu a , M. Ludwig b , O. Lenz b , 

B. Friedrich b , P. Hildebrandt a , F. Lendzian a 

a 

Institute of Chemistry, PC 14, Technical University of Berlin, Strasse des 17 Juni 135, 10623, Berlin, Germany, 

e-mail: ingo.zebger@tu-berlin.de 

b 

Institute of Biology / Microbiology, Humboldt University of Berlin, Chausseestr. 117, 10115, Berlin, Germany 

[NiFe] hydrogenases catalyze the reversible cleavage of molecular hydrogen. The enzymes contain a [NiFe] 

active center and various iron-sulfur clusters, which serve as electron transfer cofactors [1]. While most of the 

well-studied [NiFe] hydrogenases are strictly anaerobic, some organisms like Ralstonia eutropha (R.e.) exhibit 

[NiFe] hydrogenases, which are remarkably oxygen-tolerant, a feature, which makes them extremley interesting 

for biotechnological applications. We have investigated the oxygen-tolerant membrane-bound [NiFe] 

hydrogenase (MBH) from R.e. (H16) [2] at different steps of the catalytic cycle using FTIR and EPR 

spectroscopy [3]. Isolated MBH was immobilized via His-tag to an Au surface, while its catalytic behaviour in 

different gas atmosphere was monitored with surface-enhanced infrared absorption spectrocopy (SEIRAS) [4]. 

Complementary FTIR and EPR-studies were performed of MBH in solution. MBH of R.e. shows close similarity 

with anaerobic [NiFe] hydrogenases. One Co and two CN - ligand are bound to the iron of the MBH active[2]. 

Most catalytic redox states (Nir-B, Nir-S, Nia-C, Nia-R) were reversibly switched in the immobilized enzyme and 

in the MBH attached to the cytoplasmic membrane. However, two remarkable differences were observed as 

compared with anaerobic [NiFe] hydrogenases, which might be related to the oxygen tolerance: The absence of 

the oxygen inhibited Niu-A state and a "split" [3Fe4S] EPR signal at higher redox potentials (+290 mV), which 

was converted into the normal narrow [3Fe4S] EPR signal at + 40 mV. This finding indicates coupling to an 

additional high potential paramagnetic center, which may be related to the proximal [4Fe4S] cluster, involving 

two addtional cysteines. 

The SEIRA spectroscopic results demonstrate that binding of the enzyme via a his-tag to AU surfaces is possible 

without affecting the native protein structure and reactivity towards hydrogen. Using the metal support as an 

electrode, further studies will be directed to optimize the electronic coupling of the surface with the catalytic 

center of the immobilzed enzyme. This is a prerequisite for optimizing the functioning of hydrogenase-based 

bioelectronic devices. 

References: 

[1] S. Kurkin, S.J. George, R.N.F. Thorneley, S.P.J. Labracht Biochemistry 43 (2004) 6820-6831 

[2] K.A. Vincent, J.A. Cracknell, O. Lenz, I. Zebger, B. Friedrich, F.A. Armstrong Proc. Natl. Acad. Sci. USA 

102 (2005) 16951-16954 

[3] M. Saggu et al., to be published 

[4] N.Wisitruangsakul et al., submitted 

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116


O30. The Enzyme Mechanism of Nitrite Reductase Studied 

at Single Molecule Level 

G. Zauner a , S. Kuznetsova a , T. Aartsma b , H. Engelkamp c , N. Hatzakis c , A.E. Rowan c , 

R.J.M. Nolte c , P. Christianen c and G.W. Canters a 

a 

Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502 

2300 RA Leiden, The Netherlands 

e-mail: g.zauner@chem.leidenuniv.nl 

b 

Leiden Institute of Physics, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands 

c 

Institute for Molecules and Materials, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, 

The Netherlands 

A generic method is described for the fluorescence "read out" of the activity of single redox enzyme molecules 

based on Förster Resonance Energy Transfer from a fluorescent label to the enzyme cofactor. The method is 

applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass 

surface. The parameters extracted from the single molecule fluorescent time traces can be connected to and agree 

with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type-1 to 

the type-2 centre and back during turnover exhibit a distribution, which is related to the disorder in the catalytic 

site. The described approach opens the door to single-molecule mechanistic studies of a wide range of redox 

enzymes and the precise investigation of their internal workings. 

Figure: The enzyme immobilization scheme. 

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117


O31. Electron Transfer and Electrocatalytic Properties of Covalently 

Immobilized Laccases 

M. Siwek, M. Borsari, G. Battistuzzi, S. Monari, A. Ranieri, M. Solà 

a Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41100, Modena, Italy 

e-mail: michal.siwek@unimore.it 

Electrochemical studies of covalently immobilized laccases have been performed. The electron transfer (ET) of a 

small laccase (SLAC) from Streptomyces coelicolor and a fungal laccase A from Trametes versicolor on a 

SAM-coated Au electrode was investigated [1]. The best protein immobilization was obtained for 1mM 11mercapto-1-undecanoic 

acid (MUA). It is shown that the T1 copper site is the electroactive redox center and 

play crucial role in ET [2]. Scan rate and temperature dependent measurements were exploited to calculate the 

kinetic and thermodynamic parameters of heterogenus ET [3]. Ionic strength and oxygen did not affect the signal 

properties. However, the redox behavior was pH-dependent. SLAC and fungal laccase were both able to yield 

reductive electrocatalysis of nitrite and hydrogen peroxide. 

References: 

[1] Machczynski M.C., Vijgenboom E., Samym B., Canters G.W., ; Protein Science (2004), 13:2388-2397 

[2] Klis M., Maicka E., Michota A., Bukowska J., Sek S., Rogalski J., Bilewicz R., ; Electrochimica Acta 

(2007), 52:5591-5598 

[3] Battistuzzi G., Borsari M., Canters G.W., de Waal E., Loschi L., Warmerdam G., Sola M., ; Biochemistry 

(2001), Vol. 40, No. 23:6707-6712 

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118

POSTERS 


Posters with odd numbers (P1, P3, P5...) will be presented at Poster Session 1, 

on Wednesday, 3 September 2008. 

Posters with even numbers (P2, P4, P6...) will be presented at Poster Session 2, 

on Friday, 5 September 2008. 

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119


P1. Molybdenum and Tungsten (VI) Complexes with Sulfur and Selenium 

Containing Ligands: New Models for the Active Site in Molybdopterin 

Cofactors 

C.E Abad Andrade , C. Schulzke 

Institute for Inorganic Chemistry, Georg-August-University of Goettingen, Tammannstr. 4, D-37077, 

Goettingen, Germany 

e-mail: carlos.abad@chemie.uni-goettingen.de 

Molybdenum and tungsten are present at the active sites of a wide range of enzymes and participate in a variety 

of biological reactions [1]. In the molybdenum containing oxidoreductases the coordination of the peptide chain 

to the metal occurs through specific amino acids as serine (O) [2], aspartate (O) [3], cysteine (S) [4] or 

selenocysteine (Se) [5] as it is shown in the figure. In order to compare the effect of the coordinated atom (S or 

Se) and their influence on the enzyme's properties such as redox potential and catalytic performance several 

complexes with thio- and seleno functional ligand systems were investigated [6, 7]. However, these kinds of 

models were dimers, in contrast to the monomeric systems normally found in the enzymes. Therefore a novel 

way of monomerization by a silylation reaction of dimeric MoO2-S complex was developed using R3SiCl in 

presence of MeOH [8]. In this work we present experiments to confirm the proposed monomerization 

mechanism of this Mo complex. Additionally, the silytation reaction was tested on the monomerization of Se 

analogues as well as other Mo and W complexes showing that this reaction is reproducible in general for dioxo 

metal complexes. 

References: 

[1] “Molybdenum and Tungsten: Their role in Biological Process”; A. Sigel, H. Sigel, Eds.; “Metal Ions in 

Biological Systems” 39; Marcel Dekker: New York, 2002. 

[2] George, J. Hilton, C. Temple, R. C. Prince and K. V. Rajagopalan. J. Am. Chem. Soc., 1999, 121, 1256– 

1266. 

[3] M. G. Bertero, R. A. Rothery, M. Palak, C. Hou, D. Lim, F. Blasco, J. H. Weiner and N. C. J. Strynadka. Nat. 

Struct. Biol., 2003, 10(9), 681–687. 

[4] C. S.Butler, J. M. Charnock, C. D. Garner, A. J. Thomson, S. J. Ferguson, B. C. Berks and D. J. Richardson. 

Biochem. J., 2000, 352, 859–864. 

[5] J. C. Boyington, V. N. Gladyshev, S. V. Khangulov, T. C. Stadtman and P. D. Sun, Science, 1997, 275, 

1305–1308. 

[6] X. Ma, C. Schulzke, Z. Yang, A. Ringe, J. Magull. Polyhedron, 2007, 26, 5497-5505. 

[7] X. Ma, C. Schulzke, H.-G. Schmidt and M. Noltemeyer. Dalton Trans., 2007, 1773–1780. 

[8] X, Ma, Z. Yang, C. Schulzke, A. Ringe and J. Magull. Z. Anorg. Allg. Chem. 2007, 633, 1320-1322. 

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120


P2. Synthesis, Structure and Spectroscopic Studies on New Derivatives of 2acetyl-1, 

3-indandione, Coupled with Crown Ether, 

as Potential Sensors for Metal Ions 

A. Ahmedova a , N. Burdjiev a , S. Ciattini b , M. Mitewa a 

a Faculty of Chemistry , University of Sofia, J. Bourcher av. 1, Sofia 1164, Bulgaria 

e-mail: Ahmedova@chem.uni-sofia.bg 

b Dipartimento di Chimica, Universita` degli Studi di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino 

(FI), Italy 

The parent compound, 2-acetyl-1, 3-indandione (2AID), is known for its physiological activity and interesting 

photophysical properties. Recently the potential application of cinnamoyl derivatives of 2AID as anti HIV agents 

has been suggested. Moreover, 2-acyl-1, 3-indandiones are very good chelating agents for heavy and transition 

metal ions due to the β–dicarbonyl fragment they posses and have found practical application as extracting 

agents for metal ions. 

Present report deals with a 2-cinnamoyl derivative of 2AID (depicted in the Figure and R = N(Me)2; compound 

1) and a 1, 3-indandione derivative directly conjugated with N-penylaza-15-crown-5 (compound 2). As might be 

expected, the conjugation of a strong electron acceptor, such as 2AID, with a strong electron donor groups, as 

dialkyl amino groups, results in very strong absorption in the visible region of the spectrum. 

In combination with their very good ability for chelation with metal ions the studied organic compounds can 

be exploited for development of real-time methods for metal ion sensing and their quantitative determination. 

In this respect, the synthesis of the ligands and their metal complexes is described as well as structural 

elucidation of the obtained compounds. Further on the optical (absorption and emission) properties of the 

organic compounds are studied in detail accounting for various effects, such as aggregation, pH, solvent effect 

and mainly the presence of metal ions. 

As a result of all the data obtained a final assessment of the new compounds as potential optical sensors for 

metal ions is given. All potential fields of applications and their limitations are discussed. 

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121


P3. Oligomolecular Ternary Cu(II) Complexes 

with Bridging µ-N6, N7- or µ-N7, N9-(2, 6-diaminopurine) 

C. Alarcón-Payer a , M. Brandi-Blanco b , D. Choquesillo-Lazarte c , A. Castiñeiras d , 

J. M. González-Pérez a , J. Niclós-Gutiérrez a 

a 

Department of Inorganic Chemistry, University of Granada, Fac. Pharmacy, Campus Cartuja, E-18071 

Granada, Spain 

e-mail: jniclos@ugr.es 

b 

Fakultät Chemie, Lehrstuhl für Bioanorganische Chemie, Technische Universität Dortmund, Otto-Hahn- 

Strasse 6, D-44227 Dortmund, Germany 

c 

Laboratorio de Estudios Cristalográficos, IACT-CSIC, Edif. Inst Lopez-Neyra, PTCS. Avda. del Conocimiento 

s/n, E-18100 Armilla, Granada, Spain 

d 

Department of Inorganic Chemistry, University of Santiago, Fac. Pharmacy, Campus Sur, E-15782 Santiago 

de Compostela, Spain 

2,6-diaminopurine (Hdap) takes part of a nucleoside analogue, the anti-HIV pro-drug amdoxovir. The 

coordination behaviour of its free purine base is poorly documented. Working with free Hdap we have obtained 

two oligo-molecular compounds with different bridging µ2-Hdap modes: {[Cu(µ2-EGTA)Cu(H2O)(µ-N7, N9- 

H(N3)dap)]·nH2O}2 (1) and {[Cu(pdc)(µ-N6, N7-H(N9)dap)]·H(N9)dap ·0.5H2O}2 (2) where EGTA and pdc are 

ethylene-bis(oxyethylenenitrilo)-tetraacetate(4-) or 2, 6-pyridine-dicarboxylate(2-) ligands, respectively. In 1 the 

Cu-N9 and Cu-N7 bonds are reinforced by N3-H···O(coord.) and N6-H···O(coord.) interligand interactions. In 2 

the –N6H2 group is involved in both the binucleating Cu-N6 bond and the N6-H···O(coord.) interligand 

interaction, which reinforces the Cu-N7 bond. 

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122


P4. Fluorescence Correlation Spectroscopy in the Study of Fast Biological 

Electron Transfer Reactions 

A. Andreoni a , A. W. J. W. Tepper a , S. Kuznetsova a , L. C. Tabares a , T. J. Aartsma b , 

G. W. Canters a 

a 

Leiden Institute of Chemistry, Leiden University, Einsteinweg, 55, 2333CC, Leiden, Netherlands 

e-mail: aandreoni@chem.leidenuniv.nl 

b 

Leiden Institute of Physics, Leiden University, Niels Bohrweg, 2, 2333CA, Leiden, Netherlands 

Azurin is a 14 kDa blue-copper protein that serves as electron carrier in cells. Oxidised Azurin can donate an 

electron to different partners including reduced Azurin. By using chemically cross-linked Cu(I)/Cu(II)-Azurin 

dimers it is possible to measure the electron self-exchange between the monomers[1]. In the present work a 

novel method to measure this electron transfer process is presented. Oxidized Azurin has a strong absorption 

band at 628nm that disappears upon reduction. By using a covalently attached fluorescent dye it was possible to 

translate this change in absorption to a change in fluorescence by means of Forster Resonance Energy Transfer 

(FRET): while Azurin is reduced no FRET occurs but after oxidation part of the energy absorbed by the dye was 

transfer to the Cu(II) resulting in a decrease of the emitted fluorescence. This effect allowed the detection of the 

Cu redox state in Azurin dimers at single molecule level by Fluorescence Correlation Spectroscopy. Experiments 

were performed on Cy5 labelled Cu- or Zn-Azurin dimers. While for the redox inactive Zn-dimers the 

autocorrelation curve fit well to a simple diffusion model an extra parameter was necessary to fit the Cu-dimers 

data. A model to explain this different behaviour that includes the electron self-exchange between the Cu centres 

was developed and the kinetic data obtained from it are presented. The results show that this method is suitable 

for the investigation of electron transfer processes in proteins. 

Figure: Illustration of the FRET effect behaviour while electron transfer within the dimer occurs 

References: 

[1] van Amsterdam IM, Ubbink M, Einsle O, Messerschmidt A, Merli A, Cavazzini D, Rossi GL, Canters GW, 

Nat Struct Biol.; 9(1):48-52 (Jan 2002) 

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123


P5. New Species Displaying Antibacterial and Antifungal Activities Based 

on Acrylate Complexes 

M. Badea a , R. Olar a , D. Marinescu a , G. Vasile b , V. Lazar c , C. Chifiriuc Balotescu c 

a 

Faculty of Chemistry, University of Bucharest, Panduri, 050663, Bucharest, Romania 

e-mail: e_m_badea@yahoo.com 

b 

Agrochemistry, University of Agronomic Sciences and Veterinary Me, Marasti, , Bucharest, Romania 

c 

Faculty of Biology, University of Bucharest, Aleea portocalelor, 060101, Bucharest, Romania 

The interest in complexes having as mixed ligands an aromatic amine and an organic derivative, which possesses 

a vinyl group, potentially polymerizable, was generated by the possibility of their inclusion in polymeric matrix. 

The purpose of this study was the synthesis of four new complex compounds of Cu(II), Ni(II) and Zn(II) with 

mixed ligands having the general formulae M(C10H8N2)(C3H3O2)2·xH2O. The acrylate presence into their 

composition gives us the possibility to use these compounds as monomers in the co-polymerization reaction with 

traditional organic monomers. These compounds were characterized on the basis of chemical analysis, IR, 

1 H NMR, electronic spectra, X-ray single crystal diffraction as well as thermal behavior. 

The in vitro antimicrobial testing were performed in order to establish the minimal inhibitory concentration 

(MIC), against Gram-positive (Bacillus subtilis, Listeria monocytogenes, S. aureus), Gram-negative 

(P. aeruginosa, Escherichia coli, Klebsiella pneumoniae, Salmonella enteritidis), as well as Candida sp., using 

multidrug resistant strains. 

Our studies demonstrated that the acrylate complexes exhibit selective and effective antimicrobial properties that 

could lead to the selection and use of these as efficient antimicrobial agents, especially for the treatment of 

multidrug resistant infections. 

Acknowledgements: This work was partially supported by the PNII grants 61-42 and 61-48/2007 of the 

Romanian Ministry of Education and Research. 

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124


P6. Fe-Fe Hydrogenases: Activity Does not Correlate with Oxygen 

Sensitivity 

C. Baffert a , M. Demuez b , L. Girbal b , I. Meynial-Salles b , P. Soucaille b , F. Leroux a , 

B. Burlat a , P. Bertrand a , B. Guigliarelli a , C. Léger a , 

a 

Laboratoire de Bioénergétique et Ingénierie des, Université de Provence/ CNRS, 31 Chemin J. Aiguier, 13402, 

Marseille, France 

e-mail: carole.baffert@ibsm.cnrs-mrs.fr 

b 

Laboratoire d'Ingénierie des Systèmes Biologique, INSA-CNRS-INRA, 135, avenue de Rangueil, 31077, 

Toulouse, France 

Hydrogen metabolism is a field in expansion because of the potential utilisation of micro-organisms in 

dihydrogen production. Hydrogenases are the metalloenzymes that catalyse the production and oxidation of H2. 

They are classified as Fe-Fe and Ni-Fe according to the structure of their active site [1]. They usually react 

quickly with inhibitors such as O2 and CO [2], whereas applications of hydrogenases require that the enzyme 

can work in the presence of O2. For this study, we selected the Fe-Fe hydrogenases from the bacterium 

Clostridium acetobutylicum (Ca) because it is one of the most active hydrogenases, and because biochemical and 

molecular biology procedures are available in our group to engineer and purify this enzyme [3]. To measure its 

activity, we use Protein Film Voltammetry, whereby the hydrogenase is immobilized onto an electrode and 

electron transfer is direct. The redox state of the enzyme depends on the electrode potential and the measured 

current is proportional to the turnover frequency [4.5]. We studied the O2 sensitivity of this enzyme and the 

inhibition mechanism. We showed that different inhibition processes coexist and we quantified their kinetics [6]. 

These results could be compared to there obtained with the Fe-Fe hydrogenase from Desulfovibrio desulfuricans 

[2]: despite the fact that the two enzymes have very similar structure, they react with O2 in different manners. 

The inhibition of Ca hydrogenase by O2 is surprisingly slow but partly irreversible. 

References: 

[1] De Lacey A. L., et al., Chem. Rev. 107, (2007), 4304. 

[2] Vincent K. A., et al., J. Am. Chem. Soc. 127 (2005) 8179. 

[3] Demuez M., et al., FEMS Microbiology Letters 275 (2007)113. 

[4] Léger, C., et al., J. Am. Chem. Soc., 126 (2004) 38 

[5] Léger, C., Bertrand, P., Chem Rev, in press (july 2008). 

[6] Baffert C., et al., Angew. Chem. Int. Ed. 47 (2008) 2052. 

_____________________________________________________________________ 

125


P7. Structural features and oxidative stress towards plasmid DNA of 

apramycin copper complex 

D. Balenci a , G. Bonechi a , N. D’Amelio a , E. Gaggelli a , N. Gaggelli a , E. Molteni a , 

G. Valensin a , W. Szczepanik b , M. Dziuba b , J. Skała c and M. Jeżowska-Bojczuk b 

a Department of Chemistry, University of Siena, Via Aldo Moro, 53100 Siena, Italy 

b Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, Wrocław, 50-383, Poland 

c Microbiological Institute, University of Wrocław, Przybyszewskiego 63, 51-148, Wrocław, 

Poland 

Apramycin is an aminocyclitol antibiotic belonging to the aminoglycoside family. It contains a bicyclic 

aminooctodiosyl sugar, which is the only substituent of the 2-deoxystreptamine (2-DOS) moiety, at the 4 

position. Apramycin is unique among aminoglycosides both for its molecular structure and for its mode of 

action, inhibiting the elongation by blocking the ribosome translocation. The mechanism underlying its 

pharmacological effects has been extensively studied, showing that this antibiotic strongly interacts with the A 

site in 16S rRNA. First isolated from Streptomyces tenebrarius, apramycin is used to treat infections caused by 

Gram-negative bacteria. 

Toxicological pattern of apramycin as well as other aminoglycosides has not been fully understood. Some 

evidence has been collected suggesting that toxicological effects of aminoglycosidic antibiotics could be 

connected to the catalytic action exerted by copper ions [1, 2]. Copper complexes of several aminoglycosides 

induce oxidative stress towards nucleic acids, through formation of reactive oxygen species by the redox active 

metal center [3]. In vivo cleavage of DNA and RNA by copper aminoglycosides has been observed [4]. 

Copper(II) complexes of aminoglycosides have been extensively studied by spectroscopic and potentiometric 

techniques, and they were found to be the strongest with respect to complexes with different metal ions [5, 6]. 

The frequent occurrence of vicinal amine and hydroxyl groups in such antibiotics constitutes a potential metalchelating 

motif; the resulting chelates are more stable than the monodentate one. All mentioned aminoglycosides 

bind copper by deprotonated amino groups and/or deprotonated hydroxyl groups, depending on the pH value. 

This study is aimed at reporting the interaction of apramycin with copper(II) ions, which has not been 

characterized up to now, defining also a structural model of the obtained complex at nearly physiological pH. A 

second goal is devoted to show the effects of apramycin-Cu(II) complex on plasmid DNA. 

Acknowledgements: We acknowledge the MIUR (FIRB RBNE03PX83_003) and the C.I.R.M.M.P. (Consorzio 

Interuniversitario Risonanze Magnetiche di Metalloproteine Paramagnetiche) for financial support. 

References: 

[1] M. Jeżowska-Bojczuk, W. Szczepanik, W. Leśniak, J. Ciesiołka, J. Wrzesiński, W. Bal, Eur. J. Biochem. 

269, 5547 (2002) 

[2] W. Szczepanik, J. Ciesiołka, J. Wrzesiński, J. Skała and M. Jeżowska-Bojczuk, Dalton Trans., 1488 (2003) 

[3] A. Patwardhan and J.A.Cowan, Chem.Commun., 1490 (2001) 

[4] C.A. Chen, J.A. Cowan, Chem. Commun., 196 (2002) 

[5] N. D’Amelio, E. Gaggelli, N. Gaggelli, E. Molteni, M.C. Baratto, G. Valensin, M. Jeżowska-Bojczuk, W. 

Szczepanik, Dalton Trans., 363 (2004) 

[6] W. Szczepanik, A. Czarny, E. Zaczyńska and M. Jeżowska-Bojczuk, J. Inorg. Biochem. 98, 245 (2004) 

_____________________________________________________________________ 

126


P8. Spectroscopic Study of the Interaction of Ni II -5-triethyl Ammonium 

Methyl Salicylidene orto-phenylendiiminato with Native DNA 

G. Barone a , N. Gambino a , A. Ruggirello b , A. Silvestri a , A Terenzi a , V. Turco Liveri b 

a Dipartimento di Chimica Inorganica e Analitica “S. Cannizzaro”, Università di Palermo, Viale delle Scienze, 

Parco d’Orleans II, Edificio 17, 90128 Palermo, Italy 

e-mail: gbarone@unipa.it. 

b Dipartimento di Chimica Fisica “F. Accascina”, Università di Palermo, Viale delle Scienze, Parco d’Orleans 

II, Edificio 17, 90128 Palermo, Italy. 

The interaction of native calf thymus DNA with cationic complexes of 5-triethyl ammonium methyl salicylidene 

orto-phenylendiimine (ML 2+ ), in 1 mM Tris-HCl aqueous solutions at neutral pH (M=Cu II and Zn II ) [1], and in 

inverse micelles to simulate the intracellular solution environment (M=Cu II ) [2], has been recently reported. 

The interaction has been monitored as a function of the metal complex-DNA molar ratio by UV absorption 

spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. Here we report on preliminary 

results of analogous studies performed on the interaction of DNA with the cationic Ni(II) complex of the same 

ligand (NiL 2+ ). 

The dramatic modification of the DNA CD spectrum, the appearance of a broad induced CD band in the range 

350-450 nm, the strong increase of the DNA melting temperature (Tm) and the fluorescence quenching of 

ethidium bromide-DNA solutions, in the presence of increasing amounts of the NiL 2+ metal complex, support the 

existence of a tight intercalative interaction of NiL 2+ with DNA, analogous to that recently found for both ZnL 2+ 

and CuL 2+ [1]. The intrinsic binding constant (Kb) and the interaction stoichiometry (s), determined by UV 

spectrophotometric titration, are equal to 4.3x10 6 M -1 and 1.0 base pair per metal complex, respectively. 

Interestingly, the value of Kb is slightly higher than and more than 10 times higher than that relative to the 

CuL 2+ -DNA and the ZnL 2+ -DNA systems, respectively. 

Speculations can be performed of the observed trend, on the basis of the electronic and geometrical structures of 

the three complexes of the same ligand, characterized by a square planar coordination geometry of the metal 

centre. 

Analogously to what observed for CuL 2+ , the shape of the CD of the NiL 2+ -DNA system, at NiL 2+ -DNA molar 

ratios higher than 0.5 is indicative of the formation of supramolecular aggregates in solutions, as a possible 

consequence of the electrostatic interaction between the cationic complex and the negatively charged phosphate 

groups of DNA. 

References: 

[1] A. Silvestri, G. Barone, G. Ruisi, D. Anselmo, S. Riela and V. Turco Liveri, J. Inorg. Biochem. 101, 841 

(2007). 

[2] G. Barone, A. Longo, A. Ruggirello, A. Silvestri, A. Terenzi, V. Turco Liveri, Dalton Trans., in press. 

_____________________________________________________________________ 

127


P9. Synthesis, Structural Characterization, DNA Reactivity and 

Antiproliferative Behaviour of cis/trans Ruthenium(II) Compounds 

F. Barragán a , M. Montaña a , M. Prieto b , V. Moreno a , V. Noe c , C. Ciudad c , H. Garcia d 

a 

Inorganic Chemistry, University of Barcelona, Martí i Franquès 1-11, 08028, Barcelona, Spain 

e-mail: flavia.barragan@qi.ub.es 

b 

Microbiology, University of Barcelona, Diagonal 645, 08028, Barcelona, Spain 

c 

Biochemistry and Molecular Biology (Pharmacy), University of Barcelona, Diagonal 643, 08028, Barcelona, 

Spain 

d 

Chemistry, University of Lisbon, Campo Grande, 1749-016, Lisbon, Portugal 

The platinum anti-tumour compounds era began with the cisplatin fortuitous finding by Rosenberg[1] and has 

slowly opened the door to the new ruthenium compounds age. Some of them have successfully reached the final 

stages of the clinic phases and others have shown promising anti-tumour activity. The insignificant side effects 

of these compounds in comparison with those of platinum compounds and their anti-metastatic behaviour have 

been the motor behind the rapid and extensive growth of this research field[2]. 

The synthesis of two new isomers are presented here; cis and trans complexes of ruthenium(II) with 

thieno[3, 2-e][1]benzothiophene-2-carbonitrile (tbc) and 1, 2-Bis(diphenylphosphino)ethane, (dppe). The 

compounds were characterized by spectroscopic analysis ( 1 H and 31 P NMR) and x-ray diffraction. The 

interaction with DNA was studied by electrophoretic mobility and atomic force microscopy (AFM). 

"In vitro" antiproliferative assays were carried out with three different tumour cell lines: HeLa (cervix), MiaPaca 

(pancreas) and LoVo (colon). Although both isomers, cis and trans exhibit a remarkable anti-proliferative 

activity against the three cell lines assayed (HeLa cell line: isomer cis IC50 (µM)=1.23, trans 0.77; MiaPaca cell 

line: isomer cis IC50(µM) = 1.6, trans IC50(µM) = 0.4; LoVo cell line: isomer cis IC50(µM) = 1.5, trans IC50(µM) 

= 0.9) the isomer with trans geometry has shown to be more active than the cis isomer. 

References: 

[1] B.Rosenberg, L. Van Camp, T. Trigas, Inhibition of cell division in E. Coli by electrolysis products from a 

platinum electrode, Nature 205, 698-699 (1965) 

[2] W.H. Ang, P.J.Dyson, Classical and Non-classical Ruthenium-based Anticancer Drugs: Towards Targeted 

Chemotherapy (review), Eur. J. Inorg. Chem. 20, 4003-4018 (2006) 

_____________________________________________________________________ 

128


P10. Silanethiolate Complexes of Bivalent Manganese, Cobalt, Iron and 

Zinc with Water or Methanol as the Only Co-ligands 

B. Becker, A. Kropidłowska 

Chemical Faculty, Gdańsk University of Technology, 11/12 Narutowicza Str., 80-952 Gdańsk, Poland 

e-mail bbecker@chem.pg.gda.pl 

Zinc, with its d 10 electronic configuration and the peculiar properties of its coordination compounds plays a 

specific role in bioinorganic processes. Zn(II) lacks the preference for a special coordination number and in 

many metalloenzymes acts as Lewis acid, without changing its oxidation state. It means, e.g., that water bonded 

to the Zn(II) center becomes more acidic and prone to dissociation. This is true for zinc and several other metals. 

Cysteine is frequently found within coordination sphere of metaloproteins. Zinc fingers, liver alcohol 

dehydrogenase or even metalothioneins may serve as examples. Being interested in metal thiolate chemistry we 

asked ourselves how stable are complexes containing simultaneously these two ligands – thiolate and water. Can 

they be prepared and stored? The search of Cambridge Crystallographic Database [1] revealed that despite the 

simplicity of thiolate – water system, complexes bearing both these ligands are extremaly rare and in fact almost 

unknown. The same was true for the complexes with such co-ligand as the simplest alcohol – methanol. 

We limited our attention to four bivalent, essential trace elements: Mn(II) with high-spin d 5 configuration, d 6 

Fe(II), d 7 Co(II) and closed shell d 10 Zn(II). As source of the thiolate ligand we used tri-tert-butoxysilanethiol 

[2], stable, sterically encumbered and, because of oxygen atoms, able to serve as hydrogen bond acceptor. First 

syntheses were performed for Zn(II) almost 15 years ago [3], and although we did success in isolation of 

crystalline compounds they were not pure. Later we isolated and characterized structurally a Co(II) ionic 

complex [4] [Co{SSi(OBu t )3}3(H2O)] – and only recently four Zn(II) complexes. One of them, shown in Fig. 1, 

was isomorphous with the above mentioned Co(II) complex. Three others were neutral bimetallic molecules with 

a formula [Zn{SSi(OBu t )3}2(H2O)]2. One of them is depicted in Fig. 2. In all complexes water was held in a 

cavity formed by the proximal, spatially encumbered SSi(OBu t )3 silanethiolate ligands, and stabilized by 

probably strong OSi…H–O–H…OSSi hydrogen bonds [5]. 

We did not prepare Co(II) and Zn(II) thiolates with methanol as the sole co-ligand, but it was possible in the case 

of Mn(II) [6] and Fe(II) [7]. Again isomorphous complexes of formula [M{SSi(OBu t )3}2(MeOH)4] are stabilized 

by a formation of four O–H … OSi hydrogen bonds – see Fig. 3. 

Acknowledgement: The research was supported by the grant of the Polish Ministry of Education and Science 

(No. 1 T09A 117 30). A. Kropidłowska thanks The Foundation for Polish Science for the fellowship. 

References: 

[1] Cambridge Structural Database, ver. 5.29, Cambridge 2008 

[2] W. Wojnowski, B. Becker, L. Walz, K. Peters, E.-M. Peters, H.G. von Schnering 

Polyhedron 11 (1992) 607-612. 

[3] B. Becker, K. Radacki, W. Wojnowski, J.Organomet.Chem. 521 (1996) 39-49. 

[4] B. Becker, A. Pladzyk, A. Konitz, W. Wojnowski, Appl. Organometal. Chem., 16 (2002) 517-524. 

[5] A. Kropidłowska, J. Chojnacki, B. Becker, XVth Winter School on Coordination Chemistry, 4-8 December 

2006, Karpacz - Poland, Proceedings, 34-35. 

[6] A. Kropidłowska, J. Chojnacki and B. Becker, Inorg. Chem. Commun. 9 (2006) 383-387. 

[7] L.Aparici-Plaza, K. Baranowska, B. Becker, 50 Crystallographic Meeting, Wrocław, 26-28.06.2008. 

Abstracts, in print. 

_____________________________________________________________________ 

129


P11. New Model Complexes of Relevance to Artificial Photosynthesis 

G. Berggren a , A. Thapper, P. Huang a , L. Eriksson b , M.F. Anderlund a , S. Styring a 

a 

Department of Photochemistry and Molecular Science, Uppsala University, Box 579, S-751 21 Uppsala, 

Sweden 

e-mail: Gustav.berggren@fotomol.uu.se. 

b 

Division of Structural Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91, Stockholm, Sweden 

Inspired by nature’s photosynthesis the Swedish Consortium for Artificial Photosynthesis works towards 

molecular assemblies capable of producing H2 from H2O, using sunlight to drive the reaction [1]. A key part of 

the natural system is a tetranuclear manganese-based catalyst, which provides the system with electrons via 

stepwise oxidation of H2O [2, 3]. 

A model complex capable of fulfilling this role has been a long-standing goal of the scientific community. 

Recently McKenzie and co-workers showed that a Mn-complex of a mononucleating, pentadentate, N4O ligand 

(HL1) evolved oxygen when treated with Ce 4+ , a one electron oxidant [4]. To further investigate this system a 

family of new ligands, mono- as well as dinucleating, based on this framework have been synthesized. The 

corresponding Mn-complexes have been characterized by X-ray crystallography, MS, electrochemical methods, 

magnetic susceptibility and EPR. Their capacity as catalysts for water oxidation using various chemical oxidants 

has also been studied. 

N 

N 

N 

N 

O 

OH 

HO 

HL1 H 2L2 

O 

N 

_____________________________________________________________________ 

130 

N 

N 

N 

N 

N 

N 

N 

O 

OH 

References: 

[1] Sun, L.; Hammarström, L.; Åkermark, B.; Styring, S., Chem. Soc. Rev., 2001, 30, 36-49 

[2] McEvoy, J. P.; Gascon, J. A.; Batista, V. S.; and Brudvig, G. W., Photochem. Photobiol. Sci., 2005, 4, 940- 

949 

[3] Ferreira, K. N.; Iverson, T. M.; Maghlaoui, K., Barber, J., Iwata, S., Science, 2004, 6, 1831-1838 

[4] Poulsen, A. K.; Rompel, A.; McKenzie, C. J., Angew. Chem. Int. Ed., 2005, 44, 6916-6920 

N 

N 

N 

N 

HL3 

N 

N 

HL4 

N 

N 

O 

OH 

O 

OH 

HO 

N 

O 

N 

N 

N 

H 2L5 

N 

N 

N 

O 

OH 

N


P12. Copper(II) Complexes of Neurokinin A and Its Derivative 

Ł. Biega, a T. Kowalik-Jankowska, a E. Jankowska, b Z. Grzonka b 

a 

Faculty of Chemistry, University of Wrocław, Joliot-Curie14 , 50-383 Wrocław, Poland 

e-mail: lukaszbiega@gmail.com 

b 

Faculty of Chemistry, University of Gdańsk, Sobieskiego 18, 80-952 Gdańsk, Poland 

In addition to the classical neurotransmitters, acetylcholine and noradrenaline, a wide number of peptides with 

neurotransmitter activity have been identified in the past few years. Among them, the tachykinins substance P 

(SP), neurokinin A (NKA) and neurokinin B (NKB) appear to act as mediators of nonadrenergic, noncholinergic 

(NANC) excitatory neurotransmission. The mammalian tachykinins share the same conserved hydrophobic Cterminal 

region, FXGLM-NH2 where X is always a hydrophobic residue that is either an aromatic or a betabranched 

aliphatic. The C-terminal region is central to the activation of each of the three known mammalian 

tachykinin receptors, NK1, NK2 and NK3. 

Copper is a redox-active nutrient that is needed at unusually high bodily levels for normal brain function. Owing 

to the large oxygen capacity and oxidative metabolism of brain tissue, neurons and glia alike require copper for 

the basic respiratory and antioxidant enzymes cytochrome c oxidase and Cu/Zn superoxide dismutase, 

respectively. In addition, copper is a necessary cofactor for many brain-specific enzymes that control the 

homeostasis of neurotransmitters, neuropeptides, and dietary amines. Included are dopamine β monooxygenase, 

peptidylglycine α-hydroxylating monooxygenase, tyrosinase, and various copper amine oxidases. 

Results from potentiometric and spectroscopic (UV-Vis, CD and EPR) studies of the protonation constants and 

Cu(II) complex stability constants of neurokinin A (HKTDSFVGLM-NH2) and its derivative (Ac- 

HKTDSFVGLM-NH2) are reported. With neurokinin A, the formation of a dimeric complex Cu2H2L2 was found 

in the pH range 5.5 – 8.5, in which the coordination of copper(II) is glycylglycine-like, while the fourth 

coordination site is occupied by the imidazole N(3) nitrogen atom, forming a bridge between two copper(II) 

ions. The formation of dimeric species does not prevent the deprotonation and coordination of the next amide 

nitrogens and in pH above 7 the 3N {NH2, 2N - } and 4N {NH2, 3N - } complexes are formed. For the Ac- 

Neurokinin A the His imidazole is an anchoring binding site, then the adjacent amide nitrogen coordinates as a 

second donor. At a pH of about 7.4 the major binding sites involve the imidazole nitrogen and one and two 

amide nitrogens of Lys or Thr residues. 

Acknowledgements 

This work is supported by KBN grant N N204 249534. 

_____________________________________________________________________ 

131


P13. Copper(II) Complexes of Alloferons 1 and 2; a Combined 

Potentiometric and Spectroscopic Studies 

Ł. Biega, T. Kowalik-Jankowska, M. Kuczer, D. Konopińska 

Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland, 

e-mail: TerKow@wchuwr.chem.uni.wroc.pl 

Among the bioactive peptides/polypeptides that have already been characterized from insects, antimicrobial 

peptides are fascinating scientists for their potential use as therapeutic agents. However, relatively little data are 

available on molecules from insects with antiparasitic, antiviral, and/or antitumoral activities. Most available 

antiviral and antitumor agents have been derived from plant, microbes, and, to a lesser extent, animal secondary 

metabolites.. Two peptides were isolated from the blood of an experimentally infected insect, the blow fly 

Calliphora vicina (Diptera), with the following amino acid sequences: HGVSGHGQHGVHG (alloferon 1) and 

GVSGHGQHGVHG (alloferon 2). 

Many essential metal ions act as the important factor influencing the structure of natural and synthetic 

oligopeptides and as a consequence they may have critical impact on their biological activity. 

In this presentation we report the results of combined spectroscopic and potentiometric studies on the copper(II) 

complexes of the alloferons 1 and 2 and their analogues with N-terminal amine protected group by acetylation. 

The peptides involved in the study are: alloferon 1, HGVSGHGQHGVHG; alloferon 2, GVSGHGQHGVHG; 

Ac-alloferon 1, Ac-HGVSGHGQHGVHG and Ac-alloferon 2, Ac-GVSGHGQHGVHG. This study was 

performed in order to examine the binding ability, especially the effect of the N-terminal amine group on the 

formation of complexes with copper(II) ions by the peptides containing three and four histidine residues in the 

peptide chain. The presence of four (Ac-alloferon 1) or three (Ac-alloferon 2) histidyl residues provides a high 

possibility for the formation of macrochelates via the exclusive binding of imidazole-N donor atoms. The 

macrochelation suppresses, but cannot preclude the deprotonation and metal ion coordination of amide functions. 

The N-terminal amino group of the alloferons 1 and 2 takes part in the coordination of the metal ion. 

_____________________________________________________________________ 

132


P14. New Insights into the Classification of Metallothioneins: a Gradation 

Between Zn- and Cu-thionein Features 

R. Bofill a , S. Atrian b , M. Capdevila a 

a 

Departament de Química, Universitat Autònoma de Barcelona, Facultat de Ciències, 08193, Bellaterra 

(Catalonia), Spain 

e-mail: roger.bofill@uab.cat 

b 

Departament de Genètica, Universitat de Barcelona, Avda. Diagonal 645, 08028, Barcelona, Spain 

Over the last years we studied many recombinant MTs, from several phyla. A comprehensive consideration of 

all our new data allows us a fine-tuning of our previous MT classification [1] by considering a gradation between 

Zn- and Cu-thioneins (Zn-th & Cu-th). We formerly proposed as Zn-th those that required Zn(II) for in vivofolding 

in the presence of high copper, while Cu-th yielded homometallic Cu-MT species. Now, we propose a 

gradient in the Cu-th character, since homometallic Cu-species are obtained only under low aeration of cultures 

for some of them, but both under low and high O2 conditions for others, thus the latter MTs exhibiting a stronger 

Cu-th character. Noteworthy, all the in vivo-obtained Cu-MT species can be reproduced by Zn/Cu in vitroreplacement, 

the weaker the Cu-th character of an MT, the lesser the Cu(I) eq required to in vitro reproduce the 

in vivo-obtained complexes. 

The gradation in the Zn-th character of MTs is envisaged from their recombinant synthesis in Zn- and Cd-rich 

media. We attribute a major Zn-th character to those MTs that either give rise to Zn, Cd-MT complexes when 

synthesized in Cd-supplemented cultures, or show a clear in vitro reluctance to total Zn/Cd exchange. 

Interestingly, the gradation from both ends (strict Zn-ths and Cu-ths) converge in a group of MTs with 

intermediate properties, which have in common the formation of recombinant Cd-S 2- -MT species, with amounts 

of sulfide increasing in direct relation to their Cu-th character. 

References: 

[1] A New Insight into Metallothionein (MT) Classification and Evolution. The in vivo and in vitro metal binding 

features of Homarus americanus recombinant MT, M. Valls, R. Bofill, R. González-Duarte, P. González-Duarte, 

M. Capdevila, S. Atrian, J. Biol. Chem., 276 (35), 32835-32843 (2001) 

_____________________________________________________________________ 

133


P15. Ca 2+ and Zn 2+ Modulate the Conformation and Stability of the S100A2 

Tumor Suppressor 

H. M. Botelho a , M. Koch b , G. Fritz b , C. M. Gomes a 

a Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal 

b Department of Biology, University of Konstanz, Germany 

The S100 proteins are small Ca 2+ binding proteins which regulate processes such as cell cycle, growth, 

differentiation and mobility in vertebrates [1]. Human S100A2 binds and activates tumor suppressor p53 in a 

Ca 2+ dependent manner [2]. It is an homodimer containing two Ca 2+ and two Zn 2+ binding sites per subunit: Ca 2+ 

binds at the EF-hands exposing a docking site for downstream signaling proteins; Zn 2+ binds at two surface sites 

and regulates the oligomeric state and Ca 2+ affinity in an unique manner in the S100 family [3]. The molecular 

determinants for such regulation are currently unknown. The conformation and stability changes associated with 

metal binding to S100A2, discriminating the contribution of each Zn 2+ site, were investigated by circular 

dichroism spectroscopy using variants with none, one or both Zn 2+ sites. Both metals affected the secondary 

structure content without changing the overall α-helical fold. The apo wild type S100A2 exhibited an unfolding 

free energy (∆GU) of 89.9 kJ/mol and a midpoint transition temperature (Tm) of 58.4ºC. The two metal ions had 

opposite effects towards stability, being Ca 2+ a stabilizer and Zn 2+ a destabilizer [4]. This antagonistic effect, 

which suggests a synergy between Ca 2+ activation/stabilization and Zn 2+ inactivation/destabilization, supports 

the hypothesis in which increased Zn 2+ levels occurring in some cancer cells [5] may promote the progression of 

the disease by impairing S100A2 function. 

References: 

[1] Fritz, G., and Heizmann, C.W. 2004. 3D structures of the calcium and zinc binding S100 proteins. In 

Handbook of metalloproteins. (eds. A. Messerschmidt, R. Huber, T. Poulos, and K. Wieghardt). John Wiley & 

Sons. 

[2] Mueller, A., Schäfer, B.W., Ferrari, S., Weibel, M., Makek, M., Höchli, M., and Heizmann, C.W. 2005. The 

calcium-binding protein S100A2 interacts with p53 and modulates its transcriptional activity. J Biol Chem 280: 

29186-29193. 

[3] Koch, M., Bhattacharya, S., Kehl, T., Gimona, M., Vasak, M., Chazin, W., Heizmann, C.W., Kroneck, P.M., 

and Fritz, G. 2007. Implications on zinc binding to S100A2. Biochim Biophys Acta 1773: 457-470. 

[4] Botelho, H.M., Koch, M., Fritz, G., and Gomes, C.M. 2008. Metal ions modulate the folding and stability of 

the tumor suppressor S100A2: insights into functional implications. Submitted. 

[5] Ionescu, J.G., Novotny, J., Stejskal, V., Latsch, A., Blaurock-Busch, E., and Eisenmann-Klein, M. 2006. 

Increased levels of transition metals in breast cancer tissue. Neuro Endocrinol Lett 27 Suppl 1: 36-39. 

_____________________________________________________________________ 

134


P16. Novel Cyclic Peptides Design Modelling Structure and Reactivity of 

Zn(Cys)4 Reactive Site of Protein Hsp33 

E. Bourles, O. Sénèque, J. M. Latour 

iRSTV/LCBM/PMB UMR 5349, CEA-Grenoble, 17 rue des martyrs, 38054 cedex 9, Grenoble, France 

e-mail: emilie.bourles@cea.fr 

Tetracoordinated zinc sites in metalloproteins, in which zinc is coordinated to cysteines and/or histidines, can 

have multiple roles such as catalytic, structural or redox. In particular, Zn(Cys)4 sites, which are present in 3% of 

proteins, were considered as structural sites since it was found that Hsp33, a molecular chaperone, as well as 

Trx2, the mitochondrial thioredoxin, were regulated by the oxidation of there Zn(Cys)4 sites into disulfides 

concomitant with the release of the zinc ion [1, 2, 3]. We have developed the synthesis of cyclic peptides to 

reproduce the structure of Zn(Cys)4 sites in proteins, such as the structural site of PerR or the reactive site of 

Hsp33. Those de novo twenty amino-acids cyclic peptides contain two CXnC motifs, one in the cycle and 

another one in a linear tail grafted on the cycle, and fit quasi-perfectly the structure of the biological sites [4, 5]. 

Then, this new design represents an interesting approach for modelling metallic sites in protein. 

Here, we present the coordination properties, the structural properties and the reactivity toward oxidation of 

several peptides designed to model the Zn(Cys)4 sites of PerR and Hsp33. The behaviour of Hsp33’s closest 

peptidic structural model toward complexation with metallic cations (Co 2+ , Zn 2+ ) and toward H2O2-mediated 

oxidation is very closed to what is observed in the protein [6, 7]. 

References: 

[1] Jakob U.; Muse W.; Eser, M.; Bardwell J.C.A.; Cell, 1999, 96, p.341. 

[2] Won, H.S.; Low, L.Y.; Guzman, R.D.; Jakob, U.; Dyson, H.J.; J. Mol. Biol., 2004, 341(4), p.893. 

[3] Collet J-F.; D’Souza J.C.; Jakob U.; Bardwell J.C.A.; J. Biol. Chem., 2003, 278(46), p.45325. 

[4] Janda, I. ; Devedjiev, Y. ; Derewenda, U. ; Dauter, Z.; Bielnicki, J.; Cooper, D.R.; Graf, P.C.F.; Joachimiack 

A.; Jakob, U.; Derewenda, Z.S.; Structure, 2004, 12, p.1901. 

[5] Traore D.A.; El Ghazouani A.; Ilango S.; Dupuy J.; Jacquamet L.; Ferrer J.L.; Caux-Thang C.; Duarte V.; 

Latour J-M.; Mol. Microbiol. 2005, 61, p.1211. 

[6] Jakob U.; Eser, M.; Bardwell J.C.A.; J. Biol. Chem., 2000, 275, p.38302. 

[7] Ilbert, M.; Horst, J.; Ahrens, S.; Winter, J.; Graf, P.C.F.; Lilie, H.; Jakob, U.; Nat. Struct. Mol. Biol., 2007, 

14, p.556. 

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135


P17. Non-isotype Crystals in Salts of 2, 6-diaminopurinium(1+) 

and bis(pyridine-2, 6-dicarboxylate)metal(II) Anions (M = Co or Cu) 

M. Brandi-Blanco a , D. Choquesillo-Lazarte b , J. M. González-Pérez c , A. Castiñeiras d , 

J. Niclós-Gutiérrez c 

a 

Fakultät Chemie, Lehrstuhl für Bioanorganische Chemie, Technische Universität Dortmund, Otto-Hahn- 

Strasse 6, D-44227 Dortmund, Germany 

e-mail: pbrandi@correo.ugr.es 

b 



c 



d 



Adeninium(1+) cations generate an iso-structural series of compounds (H2ade)2[M II (pdc)2]·3H2O with M = Mn, 

Co, Ni Cu or Zn. Hade + exists as nearly coplanar pairs of tautomers A:B (with protons on N1 and N9 or N3 and 

N7) H-bonded in (A:B)n ladders. In the crystal of (H2dap)2[Cu II (pdc)2]·4H2O the 2, 6-diaminopurinium(1+) 

cations only have dissociable protons in N3 and N7 forming non-equivalent homo-pairs, {H2dap(1) + }2 and 

{H2dap(2) + }2 using N1 as acceptor and an N6-H or N2-H as donors. These homo-pairs alternate in laddered 

chains (see A). The crystal of (H2dap)2[Co II (pdc)2]·6H2O has also two non-equivalent H2dap(1) + and H2dap(2) + 

which form homo-pairs with or without the mediation of water (B and C). 

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136


P18. The Electrochemical Studies of the Complexes of Histidine Analogues 

of Vasopressin and Oxytocin 

J. Brasuń a , M. Cebrat b , B. Fuglewicz a , S. Plińska a , J. Świątek-Kozłowska a 

a 

Department of Inorganic Chemistry, Wroclaw Medical University, Szewska 38, 50-139 Wroclaw, Poland 

e-mail: stasia.plinska@wp.pl, 

b 

Faculty of Chemistry, Wroclaw University, F. Joliot-Curie 14, 50-363 Wroclaw, Poland 

The coordination abilities of different oligopeptides with many metal ions like Cu(II), Ni(II) and Zn(II) have 

been studied since many years [1, 2]. Peptide hormones like oxytocin (OXT) and vasopressin (AVP) play an 

important role in a human organism. They contain disulphide bridge and are able to form stable complexes e.g. 

with Cu(II) and Ni(II) [3]. 

The biologicaly active peptides are often used as the pharmaceuticals. Some new analogues of OXT and AVP 

with higher selectivity, limited side effects and higher activity, are investigated [4]. 

In this work the results for some the Cu(II) and Zn(II) complexes with histidine-AVP analogue (His-Tyr-Phe- 

Gln-Asn-His-Pro-Leu-Gly-NH2) and OXT analogue (Ac-His-Tyr-Ile-Gln-Asn-His-Pro-Leu-Gly-NH2) are 

presented. The stability constants of investigated complexes have been determined by the analysis of the voltamperometric 

results. 

References: 

[1]. H. Kozłowski, W. Bal, M. Dyba, T. Kowalik-Jankowska, Specific structure–stability relations in 

metallopeptides, Coordination Chemistry Reviews, 184, 319–346 (1999). 

[2]. I. Sovago, K. Osz, Metal ion selectivity of oligopeptides, J. Chem. Soc., Dalton Trans., 3841–3854 (2006). 

[3]. H. Kozłowski, B. Radomska, G. Kupryszewski, B. Lammek, C. Livera, L. D. Petit, S. Pyburn, J. Chem. 

Soc., Dalton Trans., 173 (1989). 

[4]. E. Trzepałka, M. Oleszczuk, M. Maciejczyk, B. Lammek, Solution structure of conformationally restricted 

vasopressin analogues, A. Biochem. Pol., 51, 33-49 (2004). 

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137


P19. Potentiometric and Spectroscopic Studies of Coordination Abilities of 

Dehydrotripeptides Gly-∆Phe-His and His-Gly-∆Phe 

J. Brasuń a , M. Makowski b , O. Gładysz a J. Świątek-Kozłowska a 

a 


e-mail: olimpia@chnorg.am.wroc.pl 

b 

Department of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland 

∆-aminoacids are unsaturated analogues of α-aminoacids and their biological activities and structural properties 

were investigated [1-2] because they are constituents of many microbial proteins (fungal and bacterial 

metabolities) as well antibiotics [3]. Also the coordination abilities compared to parent peptides were widely 

studied [4-8]. These studies showed that ∆-peptides display unusual binding ability towards metal ions such as 

Cu 2+ , Ni 2+ , Co 2+ , Zn 2+ [6-7]. 

The purpose of the present studies was to examine the stability of copper (II) complexes with dehydrotripeptides 

Gly-∆Phe-His and His-Gly-∆Phe . 

Using pH-metric titrations the protonation constants and stability constants of these ligands were found out and 

also UV-Vis spectra were recorded. 

Both investigated dehydro peptides form stable complexes with Cu (II) ions and the the stability constants 

derived from potentiometric titrations were obtained with high accuracy. The potentiometric and spectrosciopic 

results show that Gly-∆Phe-His forms six types of complexes with Cu (II) ions and since pH 4 complex CuL2 is 

created. Although His-Gly-∆Phe forms also six complexes all of them involve only one ligand. The species 

distribution of Gly-∆Phe-His/Cu(II) and His-Gly-∆Phe/Cu(II) solution (Fig.1 and Fig.2) will be discussed as well 

as some likely complexes structure. 

%Cu 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

CuL 

free Cu 

CuL 

CuH -1L 

CuH -1L 2 

CuH -2L 2 

CuH -3L 2 

3 4 5 6 7 8 9 10 11 

pH 

_____________________________________________________________________ 

138 

%Cu 


90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

free Cu (II) 

CuHL 

CuL 

CuH -1L 

CuH -2L 

CuH -3L 

3 4 5 6 7 8 9 10 11 

pH 

Fig. 1 Species distribution curves for Gly-∆Phe-His/Cu(II) Fig.2 Species distribution curves for His-Gly-∆Phe/Cu(II) 

Refrences: 

[1] B. Rzeszotarska, Z. Kubica, J. Tarnawski, Post. Biochem., 33, 533, (1987) 

[2] T. P. Singh, P. Narula, H.C. Patel, Acta Cryst.Sect. B, 46, 539, (1990) 

[3] A.F. Spatola, in: B. Weinstein, editor. Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, 

vol.VII, New York, , M. Dekker, 267, (1983). 

[4] J. Świątek-Kozłowska., J. Brasuń, M. Łuczkowski., M. Makowski, J. Inorg. Biochem., 90, 106 (2002) 

[5] M. Z. Siddiqui, Inter. Journal of Biol. Macromolecules, 26, 17, (1999); 

[6] M. Jeżowska-Bojczuk, H. Kozłowski, Polyhedron, 10, Vol.19, 2331, (1991) 

[7] M. Jeżowska-Bojczuk, H. Kozłowski, Polyhedron, 13, Vol.18, 2683, (1994) 

[8] M. Jeżowska-Bojczuk, K. Varnagy, I. Sovago, G. Pitrzyński, M. Dyba, Y. Kubica, B. Rzeszotarska, L. 

Smełka, H. Kozłowski, J.Chem. Soc., Dalton Trans., 3265 (1996) 

[9] R. Hay, M. M. Hassan, C. You-Quan, Journal of Inorganic Biochemistry, 52, 17 (1993). 

CuH - 

L


P20. The Coordination Abilities of the 14-membered Cyclic Tetrapeptide 

with the c(β 3 homoLysDHisβ-AlaHis) Sequence 

J. Brasuń a* , A. Matera-Witkiewicz a , S. Ołdziej b , A. Pratesi c , M. Ginanneschi c , L. Messori d 

a* 

Department of Inorganic Chemistry, Wrocław Medical University, Szewska 38, 50-139 Wrocław, Poland 

e-mail: jbrasun@chnorg.am.wroc.pl 

b 

Laboratory of Biopolymer Structure, Intercollegiate Faculty of Biotechnology, University of Gdańsk and 

Medical University of Gdańsk, Kładki 24, 80-822 Gdańsk, POLAND 

c 

Laboratory of Peptides and Proteins Chemistry and Biology, Department of Organic Chemistry “Ugo Schiff”, 

University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy 

d 

Department of Chemistry, University of Florence, Via della Lastruccia 3, Sesto Fiorentino, Firenze, Italy 

A new, 14-membered, tetraza cyclic tetrapeptide containing histidine and lysine side-chains, 

c(β 3 homoLysDHisβ-AlaHis), was designed, synthesized and characterized; its copper(II) binding properties were 

investigated in dependence of pH by potentiometric and spectroscopic methods. In line with previous studies of 

similar systems, the progressive involvement of amide nitrogens in copper(II) coordination was evidenced upon 

raising pH. At physiological pH the dominant species consists of a copper(II) center coordinated by two amide 

nitrogens, an imidazole nitrogen and a water molecule. In contrast, at pH above 8.7, a copper(II) coordination 

environment consisting of four amide nitrogens in the equatorial plane and the axial imidazole ligands is formed 

as clearly indicated by spectroscopic data and theoretical calculations. The behavior of this 14-membered cyclic 

tetrapeptide is compared to that of its 12-membered cyclic analog, particular attention being paid to the effects of 

ring size on the respective copper(II) binding abilities. 

%Cu 2+ 


80 

60 

40 

20 

0 

CuH 2 L 

Cu 2+ 

CuHL 

CuH -1 L 

CuH -2 L 

CuH -3 L 

4 6 8 10 

pH 

CuH -4 L 

Figure 1. Species distribution curves for Cu 2+ - 

DK14 (solid line) and Cu 2+ -DK12 data from [1] (dashed 

line) complexes at 25°C and I=0.1 mol dm -3 KNO3. The 

ligand concentration 1×10 -3 mol dm -3 . Ligand to metal 

ratio 1.5:1. 

Scheme 1. The structure of CuH-3L complex 

obtained from theoretical calculations. All hydrogen 

atoms are removed for clarity. The copper(II) ion is 

shown in cyano, oxygen, carbon and nitrogen atoms 

are shown in red, black and blue respectively. 

References: 

[1] J.Brasuń, A.Matera, S.Ołdziej, J.Świątek-Kozłowska, L.Messori, Ch.Gabbiani, M.Orfei, M.Ginnaneschi, J. 

Inorg. Biochem., 101 452 (2007) 

_____________________________________________________________________ 

139


P21. The Reduction of (ImH)[trans-Ru III Cl4(dmso)(Im)] and Preferential 

Reaction of the Reduced Complex with Human Serum Albumin. 

M. Brindell, a, b I. Sawoska, a G. Stochel a , R. van Eldik b 

a 

Department of Inorganic Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 

Krakow, Poland 

e-mail: brindell@chemia.uj.edu.pl 

b 

Inorganic Chemistry, Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, 

Egerlandstrasse 1, 91058 Erlangen, Germany 

NAMI-A a novel anti-metastatic Ru(III) complex, viz. (ImH)[trans-RuCl4(dmso)(Im)] has successfully 

completed phase I clinical trials and undergoes further stages of clinical tests.[1] It can be administered 

intravenously and the pharmacokinetic analysis of the Ru content of blood plasma has revealed that most of the 

Ru in blood plasma is accumulated in the protein-bound form (> 97%). Extensive binding of this drug to the 

plasma proteins may significantly influence its biodistribution and bioavailability, and therefore the 

understanding of this process is of great importance. Considering the physiological conditions in blood plasma 

(pH 7.4, 0.1-0.15 M NaCl, 37 o C), it is expected that NAMI-A undergoes relatively fast hydrolysis. It was 

proposed that under such conditions the stepwise dissociation of two Cl – and one dmso ligands occurs.[2, 3] 

Moreover, one should take into account the redox environment present in the blood. The presence of ascorbic 

acid in blood serum can lead to reduction of NAMI-A. [3-6] 

Based on this information, we can assume that at least two major transformations of NAMI-A, namely 

hydrolysis and reduction, can occur immediately after administration, and they can precede the reaction with 

serum proteins. Therefore, the form of the complex that actually reacts with serum albumin can differ 

significantly from the complex introduced into the organism. In this context, the question arises if NAMI-A 

(Ru(III) complex) really reacts with albumin or rather its reduced form, or maybe even the reduced form of the 

hydrolytic derivatives of NAMI-A. This presentation aims to answer this question in the most precise way. 

References: 

[1] Rademaker-Lakhai, J. M.; van_den_Bongard, D.; Pluim, D.; Beijnen, J. H.; Schellens, J. H. M. Clin. Cancer 

Res. 2004, 10, 3717-3727. 

[2] Bacac, M.; Hotze, A. C. G.; van_der_Schilden, K.; Haasnoot, J. G.; Pacor, S.; Alessio, E.; Sava, G.; Reedijk, 

J. J. Inorg. Biochem. 2004, 98, 402-412. 

[3] Brindell, M.; Stawoska, I.; Supel, J.; Skoczowski, A.; Stochel, G.; van_Eldik, R. J. Biol. Inorg. Chem. 2008, 

DOI 10.1007/s00775-008-0378-3. 

[4] Sava, G.; Bergamo, A.; Zorzet, S.; Gava, B.; Casarsa, C.; Cocchietto, M.; Furlani, A.; Scarcia, V.; Serli, B.; 

Iengo, E.; Alessio, E.; Mestroni, G. Eur. J. Cancer 2002, 38, 427-435. 

[5] Ravera, M.; Baracco, S.; Cassino, C.; Zanello, P.; Osella, D. Dalton Trans. 2004, 2347-2351. 

[6] Brindell, M.; Piotrowska, D.; Shoukry, A. A.; Stochel, G.; van_Eldik, R. J. Biol. Inorg. Chem. 2007, 12, 809- 

818. 

_____________________________________________________________________ 

140


P22. Accumulation and Biotransformation of Arsenic by Embryos of 

Zebrafish (Danio rerio) 

M.A. Bryszewska a , S. E. Hannam b , R. Munoz Olivas c , C. Camara c 

a Technical University of Lodz, Faculty of Biotechnology and Food Sciences, Institute of General Food 

Chemistry, ul. Stefanowskieg 4/10, 90-924 Lodz, Poland 

e-mail: malbrysz@snack.p.lodz.pl 

b University of Waterloo, Chemistry Department, 200 University Avenue West, Waterloo, Ontario, Canada 

c Universidad Complutense Madrid, Facultad de Ciencias Quimicas, Dpto. Quimica Analitica, Madrid, Avda. 

Complutense s/n, 28040 Madrid, Spain 

Anthropogenic activities and natural sources cause that arsenic is ubiquitous element detected in low 

concentrations in virtually all environmental media. Investigations performed over the last 25 years revealed a 

large number of naturally occurring arsenic compounds. It is clear that arsenic metabolism is complex, moreover 

it was demonstrated that pathways of biotransformation varies in the different organisms. Embryos of zebrafish 

have consistently demonstrated their usefulness as a model organism for studies vertebrate development and 

their responses to external stimulus, therefore are an ideal system to study the effects of arsenic exposure on its 

accumulation levels and organisms ability of biotransformation the element. The aim of the present work was to 

estimate arsenic accumulation by single embryos, to observe individual differences in the element accumulation 

and trace element biotransformation. The ability to measure arsenic content in single embryos is important as it 

allows for the determination of differences in uptake between each embryo within a group and between embryos 

in different replicas. For the purposes of this work a method to directly introduce whole single embryos into the 

graphite furnace (ETAAS) was elaborated. The significant matrix effects due to complexity of the sample were 

overcome by the use of a palladium modifier (0.8 g L -1 ) and hydrogen peroxide (12 %) as an oxidizing agent to 

aid in the decomposition of the sample. The results obtained from this direct method of total arsenic 

measurement were in agreement with those from more common sample preparation methods of acid and 

ultrasonic digestion when measured using ETAAS and ICP-MS. Arsenic content was measured for embryos that 

were exposed to the solution containing AsO3 3- or AsO4 3- in the concentrations of 1 mg L -1 and 0, 05 mg L -1 . 

Measurement of total arsenic content in the single embryos showed that there is a large variability in arsenic 

content between single individuals 

- embryos of age 24hpf (hour post fertilisation): control group: 0.027÷0.046 ngAs/embryo (RSD 17, 94), 

enriched group 0.045÷0.110 ngAs/embryo (RSD 26, 51); 

- embryos of age 48hpf: control group: 0.042÷0.079 ngAs/embryo (RSD 21, 69), enriched group 

0.068÷0.202 ngAs/embryo (RSD 32, 94). 

This is likely caused by biological factors that differ between embryos which may have an impact on As uptake 

and its accumulation. Arsenic speciation analysis performed using HPLC ICP MS, revealed that the zebrafish 

embryos exposed to 1 mg AsO4 3- L -1 were able to reduce arsenate to arsenite. Relation of pentavalent form to the 

trivalent form was decreasing during the time reaching 75% of AsO3 3- and 25% of AsO4 3- of detected in the 

extracts arsenic, at the age of 120 hpf. Lack of the other forms like methylated form is suprising. It is well 

documented and observed for the different kinds of the organisms that reduction As V is a initial stage on the 

metabolitical path leading to the methylated form like monomethylarsenic acid or dimethylarsenic acid. 

_____________________________________________________________________ 

141


P23. Synthesis, X-ray Structure of New Pt(II), Pd(II) and Cu(II) Complexes 

with 5-amino-8-methyl-chromone 

E. Budzisz a , I. P. Lorenz b , P. Mayer b , A. Jozwiak c 

a 

Department of Cosmetic Raw Materials Chemistry, Medical University of Lodz, Muszynskiego 1, 90-151, Lodz, 

Poland 

e-mail: elora@ich.pharm.am.lodz.pl 

b 

Department of Chemistry and Biochemistry, Ludwig Maximilian University, Butenandtstr. 5-12 (D), D-81377, 

Munich, Germany 

c 

Department of Organic Chemistry, University of Lodz, Narutowicza 68, 90-136, Lodz, Poland 

The metal complexes which contain chromone derivatives as a ligand show anticoagulant properties [1, 2] and 

antitumor activity. [3, 4] In particular, complexes with palladium(II), copper(II), and platinium(II), exhibit 

pronounced in vitro cytotoxicity. [5, 6] 

In this study, we present the synthesis and characterization of metal complexes of 5-amino-8-methyl-chromone. 

Elemental analysis, FT-IR, UV-Vis spectroscopy and X-ray crystallography have been used to characterize the 

complexes. 

Acknowledgements. Financial support from Medical University of Lodz (grant No 503-3066-2). 

References: 

[1] Jiang, D.; Deng, R.; Wu, J., Wuji Huaxue, 1989, 5, 21-28. 

[2] Deng, R.; Wu, J.; Long, L., Bull. Soc. Chim. Belg., 1992, 101, 439-443 

[3] Kostova, I.; Manolov, I.; Konstantinov, S.; Karaivanova, M., Eur. J. Med. Chem., 1999, 34, 63-68. 

[4] Manolov, I.; Kostova, I.; Netzeva, T.; Konstantinov, S.; Karaivanova, M., Arch. Pharm. Pharm. Med. Chem., 

2000, 333, 93-98. 

[5] E. Budzisz, M. Malecka, I-P. Lorenz, P. Mayer, R. Kwiecień, P. Paneth, U. Krajewska, M. Rozalski, Inorg. 

Chem., 2006, 45(24), 9688-9695. 

[6] E. Budzisz, M. Malecka B. Keppler V.B. Arion, G.Andrijewski, U. Krajewska, M. Różalski, Eur. J. 

Inorg.Chem., 2007, 3728-3735. 

_____________________________________________________________________ 

142


P24. Comparison of the Spectroscopic Characteristics of Two Different 

Fungal Laccases 

C. Bukh and M. J. Bjerrum 

University of Copenhagen, Faculty of Life Sciences, Department of Natural Sciences, Thorvaldsensvej 40, DK- 

1871 Frederiksberg C, Denmark 

e-mail: bukh@life.ku.dk 

Laccase (E.C. 1.10.3.2), a blue multi-copper oxidase found in many plants and fungi, catalyzes single electron 

oxidation of a broad range of substrates, coupled to the four-electron reduction of dioxygen to water. Laccase 

contains four copper ions in three fully conserved binding sites (T1, T3 and T2) and belongs to a sub-group of 

the blue multi-copper oxidases (MCOs), which includes ascorbate oxidase, ceruloplasmin CotA and Fet3p. 

Despite having fully conserved active copper binding domains, the absorption spectrum arising from the bluecopper 

site differs among the different laccase species. We have studied the spectroscopic properties of several 

fungal laccases as a function of pH. Furthermore a change in pH has been shown to cause a time dependent 

change in the behavior of the evaluated laccases. 

The responses to pH changes exhibited by different laccases will be presented using a combination of 

spectroscopic techniques like UV-Vis, CD and EPR spectroscopy. Furthermore in silico models will be included 

in the evaluation of the results. 

Acknowledgement: The enzymes were kindly donated by Novozymes A/S, Bagsværd, Denmark. Jesper Bendix 

is thanked for technical assistance with the EPR measurements. Danish Chemical Society for financial support to 

this conference. 

_____________________________________________________________________ 

143


P25. New Model Systems for Binuclear Nonheme Iron-Oxo Proteins 

B. Burger a , S. Wöckel a , M. Jarenmark b , S. Dechert a , E. Nordlander b , F. Meyer a 

a Institute for Inorganic Chemistry, University of Göttingen, Tammannstr. 4, D-37077, Göttingen, Germany 

e-mail: boris.burger@chemie.uni-goettingen.de 

b Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00, Lund, Sweden 

Binuclear nonheme Iron-oxo proteins are widespread in nature and possess a variety of biochemical functions 

[1]. Most of the carboxylate-bridged diiron active centers of those proteins can actually react with, and activate 

dioxygen. Certain organisms use that in, e. g., dioxygen carrier proteins like Hr, others make use of it to perform 

impressive oxygenation reactions of biological substrates [2]. The interest to develop model systems for those 

diiron-proteins is of course enormous [3], but there is still need to design suitable ligands to reach functionality. 

Therefore nature is the best archetype. 

The active center of, e. g., the enzyme Methane Monooxygenase in its reduced state is a diferrous core ligated by 

histidine and carboxylic residues from surrounding amino acids. Moreover, most of the diiron active centers 

have a ligation that is comprised by only histidine- and carboxylic residues of glutamate or aspartate, so they 

exhibit Nitrogen and Oxygen donor atoms in the ligand sphere [1]. 

The actual work presents some newly developed Pyrazole-based ligands, which have the potential to mimic the 

natural ligation of the carboxylate-bridged diiron active centers. Therefore we have designed different sidearms 

for the Pyrazole, which comprise either Imidazole- or aliphatic Nitrogen donor atoms as well as a carboxylic 

residue. Here we present some of the initial results on the way to functional biomimetic diiron-oxo complexes. 

References: 

[1] D. M. Kurtz Jr., J. Biol. Inorg. Chem. 1997, 2, 159. 

[2] A. L. Feig, S. J. Lippard, Chem. Rev. 1994, 94, 759. 

[3] E. Y. Tshuva, S. J. Lippard, Chem. Rev. 2004, 104, 987. 

_____________________________________________________________________ 

144


P26. Cobalt-porphyrin Containing γ Subunit of Desulfoviridin of D.gigas 

S.A. Bursakov a , A. V. Kladova a , O. Yu. Gavel a , J. Calvete b , V. Cabral a , I. Moura a , J.J.G. 

Moura a 

a REQUIMTE, Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e 

Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 

e-mail: sergey@dq.fct.unl.pt 

b Instituto de Investigaciones Biomédicas, C.S.I.C., Valencia, Spain 

A cobalt- porphyrin containing protein (CoPf) was isolated from the sulphate-reducing bacteria Desulfovibrio 

gigas [1, 2]. The monomeric violet coloured protein contains one molecule of cobalt per molecule of protein in a 

non covalently bound Co (III) porphyrin-like cofactor and exhibits UV-visible spectrum with peaks at 279 nm, 

420 nm and 590 nm with shoulders at 300 nm, 395 nm and 550 nm. 

CoPf was cloned and overexpressed in E. coli cells. The apo-form consist of 105 amino acids residues with 

molecular weight around 11.9 kDa. CoPf contains only few aromatic amino acid residues that are responsible for 

low extinction coefficient at 280 nm. Thus, the pure protein has the ratio A590/280 and the ratio A420/A280 is 

around 1.48 and 3.22, respectively. Alignment of the CoPf with proteins from the database BLAST show very 

high homology from 72 to 82% of this protein with gamma subunit of dissimilatory sulfite reductase from 

Desulfovibrio vulgaris (Hildenborough), Desulfovibrio desulfuricans G20, Thermodesulforhabdus norvegica and 

Desulfotalea psychrophila. According to the results of mass spectrometry and modification by iodoacetamide 

(IA) and vinylpyridine (VP), CoPf has two cysteines linked by one bridge and one of them only accessible to IA 

and VP, in presence denaturing agent (5 M ClGu). As isolated the protein is EPR silent, suggesting the presence 

of diamagnetic Co 3+ not easly reduced by sodium dithionite. 

Acknowledgement: POCI/QUI/59119/2004 (FCT), No E-62/06 (CRUP), SFRH/BPD/28380/2006 and 

SFRH/BD/24744/2005. 

References: 

[1] J.J.G. Moura, I. Moura, M. Bruschi, J. Le Gall and A. V. Xavier, Biochem Biophys Res Commun. 92, 962 

(1980). 

[2] E.C. Hatchikian, Biochem Biophys Res Commun. 103, 521 (1981). 

_____________________________________________________________________ 

145


P27. Orange Protein Mo-Cu Cluster Reconstitution 

M.S. Carepo a , S.R. Pauleta a , A. Duarte a , D.S. Figueiredo a , J.G. Graff a , A.G. Wedd b , 

A.S. Pereira a , J.J.G. Moura a , I. Moura a 

a 

Requimte – Departamento de Quimica, CQFB, Faculdade de Ciências e Tecnologia – UNL, 2825 Monte da 

Caparica, Portugal 

e-mail: marta.carepo@dq.fct.unl.pt 

b 

School of Chemistry, University of Melbourne, ParkVille, Victoria 3010, Australia 

The orange protein (ORP) isolated from Desulfovibrio gigas is a monomeric protein of approximately 12 kDa. 

X-ray absorption fine structure (EXAFS) studies revealed the presence of a mixed-metal sulfide linear cluster 

[S2MoS2CuS2MoS2] 3- which is a quite unusual protein bound heterometallic cluster [1,2]. Tetrathiomolybdate 

and copper are known to form sulphide metal complexes and their antagonism has been exploited for the 

treatment of Wilson’s disease and breast cancer by dietary supplement on tetrathiomolybdate [3]. 

ORP was heterologously expressed in E. coli as an apo-protein, the holo protein can be reconstitute in vitro by 

the addition of copper and tetrathiomolybdate (TM4) or tetrathiotungstate (TW4). The apo-ORP reconstitution 

using TM4 and Cu 2+ (4Mo:2Cu/ protein) is dependent on the incubation order of the metals with ORP. When 

TM4 is added to ORP followed by the addition of Cu 2+ the metal ratio obtained is 1Mo:1Cu the protein whereas 

when Cu 2+ is first incubated with ORP followed by TM4 addition the metal ratio is 2Mo:1Cu, as observed for the 

native protein. The percentage of reconstitution was determined by 1H-15N HSQC. 

In this work we present UV-visible titrations of (ORP+TM4) with Cu 2+ and (ORP+ Cu 2+ ) with TM4. Different 

stages of the interaction between ORP and the metals to form de metal cluster were followed by EPR. 

The two metals were also allowed to react first and then the reaction mixture was incubated with the protein. 

Titrations of the two metals in the absence of ORP were also performed in order to investigate the role of ORP in 

the cluster formation. The results obtained can give some insights concerning the ORP cluster assembling in the 

native protein. 

Acknowledgement: We thank Fundação para a Ciência e a Tecnologia for financial support 

References: 

[1] G.N. George, I. J. Pickering, E. Y. Yu, R.C. Prince, S.A. Bursakov, O.Y. Gavel, J.J.G. Moura and 

I. Moura, JACS, 122, 8321, (2000). 

[2] S.A. Bursakov, O.Y. Gavel, G. Di Rocco, J. Lampreia, J. Calvete, A.S. Pereira, J.J.G. Moura and 

I. Moura, J Inorg Biochem, 98, 833 (2004). 

[3] H. Laurie, Eur. J. Inorg. Chem., 2000, 2443 (2000). 

_____________________________________________________________________ 

146


P28. Complexes of Histidine Analogues of Oxytocin and Vasopressin with 

Zn(II) and Cu(II) ions Studied by ESI-MS Mass Spectrometry 

M. Cebrat, A. Sochacka, P. Stefanowicz 

Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland 

e-mail: lukasz@wchuwr.pl 

Oxytocin (OT) and arginine-vasopressin (AVP) belong to a group of neurohypophysial peptide hormones 

present in virtually all vertebrates and many other species. They are nonapeptides with a disulfide bridge 

between Cys residues 1 and 6. OT is associated with reproductive functions, stimulation of uterine contractions 

during labor and milk ejection during lactation, whereas AVP facilitates water reabsorption by the kidney and 

the contraction of smooth muscle cells in arteries. Divalent metal ions appear to be important element of the 

OT/AVP system [1]. 

Some extracellular proteins employ disulfide bridges to stabilize topologies that are similar to the intracellular 

zinc-stabilized motifs [2, 3]. Therefore, we decided to check whether it is possible to retain the conformation and 

the biological activity of OT and AVP peptides by replacing the disulfide bond in the Cys-Cys pair by the His- 

M-His complex (M = metal ion). The binding abilities of the His-analogue of AVP towards Cu(II) were also 

studied by potentiometric techniques [4]. Here we report the formation and fragmentation pattern of the 

complexes of the OT and AVP analogues with both Cys residues substituted by His with Zn(II) and Cu(II) ions 

as observed by ESI high resolution mass spectrometry: 

1. Ac-His-Tyr-Phe-Gln-Asn-His-Pro-Arg-Gly-NH2 [His 1,6 ]AcAVP OS-1 

2. H-His-Phe-Phe-Gln-Asn-His-Pro-Arg-Gly-NH2 [His 1,6 ,Phe 2 ]AVP OS-2 

3. Ac-His-Phe-Phe-Gln-Asn-His-Pro-Arg-Gly-NH2 [His 1,6 ,Phe 2 ]Ac-AVP OS-3 

4. H-His-Tyr-Ile-Gln-Asn-His-Pro-Leu-Gly-NH2 [His 1,6 ]OT OS-4 

5. Ac-His-Tyr-Ile-Gln-Asn-His-Pro-Leu-Gly-NH2 [His 1,6 ]Ac-OT OS-5 

6. His-Phe-Ile-Gln-Asn-His-Pro-Leu-Gly-NH2 [His 1,6 ,Phe 2 ]OT OS-6 

7. Ac-His-Phe-Ile-Gln-Asn-His-Pro-Leu-Gly-NH2 [His 1,6 ,Phe 2 ]Ac-OT OS-7 

8. H-His-Tyr-Phe-Gln-Asn-His-Pro-Arg-Gly-NH2 [His 1,6 ]AVP OS-8 

At pH 6-7 (NH4Ac-buffered peptide solution) a complexes of a type [peptide +M] 2+ are visible in all recorded 

mass spectra. The intensity of the peak is rather low as compared to [peptide +2H] 2+ and significantly lower in 

case of Zn(II) than for Cu(II) complexes. During MS/MS fragmentation of this complex a respective C-terminal 

tripeptide Pro-Aaa-Gly-NH2 (Aaa = Arg or Leu) is easily cleaved while the metal ion is retained by the Nterminal 

fragment. Further fragmentation results in subsequent cleaving of the amino acid residues mostly from 

the C-terminus of the peptide. Typicaly yn ions are formed during this process, but xn fragments are often 

observed as well. 

Of the two His residues, His 1 seems to chelate metals more efficiently. Even in the case of the acetylated 

peptides we can see N-terminal tripeptide fragments containing Cu (Zn) ions. 

Acknowledgement: ESI MS and MS/MS spectra were recorded on Bruker apex ultra 7T FTMS mass 

spectrometer in the Mass Spectrometry Laboratory at the Faculty of Chemistry, University of Wroclaw. 

References: 

[1] T. Wyttenbach, D. Liu, and M.T. Bowers, J. Am. Chem. Soc., 130, 5993 (2008). 

[2] C.A. Orengo, J.M. Thornton, Structure, 1, 105 (1993). 

[3] I.Z. Siemion, Z. Szewczuk, Wiadomości Chemiczne, 45, 755 (1995). 

[4] J. Brasuń, M. Cebrat, A. Sochacka, O. Gładysz, J. Świątek-Kozłowska, accepted by Dalton Trans. 

_____________________________________________________________________ 

147


P29. 1.5 Å Crystal Structure of the Heterodimeric Nitrate Reductase from 

Cupriavidus necator 

C. Coelho, P. J.González, J. Trincão, A. L.Carvalho, S. Najmudin, I. Moura, 

J. J. G. Moura and M. João Romão 

REQUIMTE, Departamento de Química, CQFB, FCT-UNL, 2829-516 Caparica, Portugal, 

e-mail: catarinacoelho@dq.fct.unl.pt 

Nitrate Reductases belong to the DMSO reductase family of mononuclear Mo-containing enzymes. Periplasmic 

nitrate reductase (Nap) catalyses the reduction of nitrate to nitrite. This enzyme is responsible for initiating 

anaerobic ammonification and also participates in the cellular redox balancing, by which nitrate is used to 

dissipate excess reducing power [1]. Until recently only three crystal structures of Nap were available: NapA 

from Desulfovibrio desulfuricans (Dd NapA, to 1.9 Å), NapA from Escherichia coli (E.coli NapA, to 2.5 Ǻ) and 

NapAB from Rhodobacter sphaeroides (Rs NapAB, to 3.2 Å) [2, 3, 4]. We report here the crystal structure of a 

heterodimeric Nap from Cupriavidus necator (formerly Ralstonia eutropha). CnNapAB comprises a 91 kDa 

catalytic subunit (NapA) and a 17 kDa subunit (NapB) involved in electron transfer. The larger subunit contains 

a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe–4S] 

cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme 

were obtained using PEG 3350 as precipitant. A single crystal grown at the High Throughput Crystallization 

Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 Å at the ESRF (ID14-1), the highest resolution 

reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 Å and β= 

100.7º, space group C2, and one heterodimer is present per asymmetric unit [5]. One clear solution was obtained 

by Molecular Replacement using DdNap as a search model and the structure was refined to a final R factor/Rfree 

of 0.17/0.20. 

References: 

[1] J.J.G. Moura, C.D. Brondino, J. Trincão and M J. Romão, J Biol Inorg Chem, 9, 791 (2004). 

[2] P. Arnoux, M. Sabaty, J. Alric, B. Frangioni, B. Guigliarelli, J. Adriano and D. Pignol, Nature Structural 

Biology, 10, 928 (2003). 

[3] J.M. Dias, M.E. Than, A. Humm, R. Huber, G.P. Bourenkov, H.D. Bartunik, S. Bursakov, J. Calvete, J. 

Caldeira, C. Carneiro, J.J.G. Moura, I. Moura and M.J. Romão, Structure, 7, 65 (1999). 

[4] B J.N. Jepson, S. Mohan, T.A. Clarke., A.J. Gates, J.A. Cole, C.S. Butler, J.N. Butt, A.M. Hemmings, D.J. 

Richardson, J. Biol. Chem., 282, 6425 (2007). 

[5] C. Coelho, P.J. González, J. Trincão, A.L.Carvalho, S. Najmudin, I. Moura, J.J.G. Moura, T. Hettman, S. 

Dieckman and M.J. Romão, Acta Cryst., F63, 516 (2007). 

_____________________________________________________________________ 

148


P30. Coordination Chemistry of a New Cyclam-based Dinucleating Ligand: 

Relevance to Purple Acid Phosphatase Model Systems 

P. Comba, M. Zajaczkowski 

Institute of Inorganic Chemistry, University of Heidelberg, INF 270, 69120 Heidelberg, Germany 

Purple acid phosphatases (PAP) are heterodinuclear enzymes that catalyze the hydrolysis of phosphomonoesters. 

Their biological functions are divers and still are not fully understood.[1] Nevertheless, in mammalians there was 

found a correlation between high PAP levels and osteoporosis.[2] Therefore, the thorough understanding of the 

PAP mechanism is crucial. 

The active site of the enzyme contains, independently of the source, seven invariant amino acid residues (see 

Scheme 1). However, the metal composition is variable. Mammalian PAP has a Fe(III)/Fe(II) core, whereas 

plants and funghi have rather Zn(II) or Mn(II) as divalent metal ion (M2 in Scheme 1).[3] 

Scheme 1: Active site of purple acid phosphatases 

Our aim is to find a suitable model system to mimick the active site of PAP. We have developed a new ligand 

based on cyclam (see Scheme 2), with two distinct coordination sites that may form a (hydr)oxo-bridged 

dinuclear complex. 

Scheme 2: Cyclam-based ligand for PAP mimicks 

The advantages to known model systems are that the cyclam unit can mimick the second coordination sphere of 

the enzyme, which stabilizes the substrate coordination, and that the bridging oxygen is not part of the ligand and 

therefore may act as nucleophile in the hydrolysis mechanism. 

The experimental results on the coordination chemistry of the new ligand are supported by computational 

studies. 

References: 

[1] N. Mitic, S.J. Smith, A. Neves, L.W. Guddat, L.R. Gahan, G.Schenk, Chem. Rev, 106, 3338 (2006). 

[2] D.W. Moss, F.D. Raymond, D.B. Wile, Crit. Rev. Clin. Lab. Sci., 32, 431 (1995). 

[3] T. Klabunde, N. Strater, B. Krebs, J. Mol. Biol., 259, 737 (1996); L.W. Guddat, A. McAlpine, D. Hume, S. 

Hamilton, J. De Jersey, J.L. Martin, Structure Fold Des., 7, 757 (1999). 

_____________________________________________________________________ 

149


P31. Effects of Mannitol or Erythritol on Human Bronchial Epithelial Cells 

Treated with Chromium(VI) 

A. Costa a , V. Moreno a , M. Prieto b , C. Alpoim c 

a 

Inorganic Chemistry, University of Barcelona, Marti i Franquès 1-11, 08028, Barcelona, Spain 

e-mail: andrenunocosta@gmail.com 

b 

Microbiology, University of Barcelona, Diagonal 645, 08028, Barcelona, Spain 

c 

Biochemistry, University of Coimbra, Box 3126, 3001-401, Coimbra, Portugal 

Toxicity of Cr(VI) and its carcinogenic effects have been extensively studied[1]. Attempts of minimizing these 

biological consequences have led to many researches to study the mechanisms of action inside the cell[2]. 

Here, we present results on the behaviour of Cr(VI) in presence or absence of mannitol and erythritol on human 

bronchial epithelial cells. The evolution of solutions of Cr(VI), Cr(VI) with ascorbate anion, Cr(VI) with 

mannitol or erythritol and Cr(VI) with ascorbate anion and mannitol or erythritol, at room T, pH= 7.4, was 

followed by UV-visible spectroscopy. The presence of Cr(V) intermediate product of reduction of Cr(VI) by 

mannitol, was detected by EPR spectroscopy. A typical sharp signal centered at g= 1.98, showing a 

superhyperfine splitting, 1 H aiso = 1,022 x 10 -4 cm -1 , corresponding to the coupling of four protons from two 

mannitol molecules coordinated to the metal ion was observed. In the EPR spectrum, four broad signals due to 

hyperfine coupling with 53 Cr nucleus (I = 3/2), Aiso = 17.75 x 10 -4 cm -1 , were also detected. AFM images of the 

human bronchial epithelial cells treated with Cr(VI) and Cr(VI)/alditol solutions were obtained. The cells were 

cultured on polymer cover slips coated with gelatine type B and treated with the corresponding solution. 

Samples were fixed with glutaraldehyde prior to visualization in air. The images obtained allowed us to observe 

the morphology of the cells surface and the integrity of the membrane. 

References: 

[1] IARC (1990) Monographs on the evaluation of carcinogenic risks to humans, Chromium, Nickel and 

Welding, Vol. 49, World Health Organization, Lyon, pp. 49-256. 

[2] B.D. Martin, J.A. Schoenhard, J.M. Hwang, K.D. Sugden, Mutation Research, 610, 74 (2006) 

_____________________________________________________________________ 

150


P32. Manganese Carbonyls as CO Releasing Molecules 

S. Crook a , B. E. Mann a , D. Scapens a , P. Sawle b , R. Motterlini b 

a 

Department of Chemistry, The University of Sheffield, Brook Hill, Sheffield, S3 7HF, UK 

e-mail: chp06shc@sheffield.ac.uk. 

b 

Vascular Biology Unit, Department of Surgical Research, Northwick Park Hospital, Harrow, HA1 3UJ, 

Middlesex, UK 

The role of CO in mammals and the use of CO and CO-releasing molecules (CO-RMs) as therapeutic agents 

have recently been described. 1 There are very few applications on CO-RMs containing manganese reported in 

the literature. The released CO from [Mn2(CO)10] was shown to cause vasodilatation of an isolated section of rat 

aorta. 2 Subsequently, [Mn2(CO)10] has been used as a light-induced CO-RM in a number of other biological 

applications. 3-7 Recently, a new manganese CO-RM, [(HBpz3)Mn(CO)3][PF6] has been shown to be cytotoxic 

for HT29 human colon cancer cells. 8 

We have developed a range of manganese carbonyls that release CO rapidly when added to myoglobin. 9 Some of 

these compounds, e.g., [NMe4][Mn{SC(O)Me}2(CO)4] and K[Mn2(µ-OAc)3(CO)6] show low cytotoxicity and 

inhibit nitrite formation when tested on endotoxin-stimulated RAW264.7 macrophages. K[Mn2(µ-OAc)3(CO)6] 

loses CO to myoglobin with a t1/2 of 9 min while [NMe4][Mn{SC(O)Me}2(CO)4] loses CO slower with t1/2 of 32 

min. A range of similar compounds of the type [Mn(S2CE)(CO)4], E = OR or NR2, also show rapid CO loss and 

their toxicity on cells is controlled by varying R. For example, [Mn(S2CNMeCH2CO2H)(CO)4] readily dissolves 

in aqueous buffer at pH 7.4, loses at least 3 moles of CO to myoglobin with a t1/2 of 6 min, no loss of cell 

viability or significant cytotoxicity and significant inhibition of nitrite formation at 100 µM. 

The properties of these molecules in water will also be reported 

References: 

[1] B. E. Mann and R. Motterlini, Chem. Commun., 4197 (2007). 

[2] R. Motterlini, J. E. Clark, R. Foresti, P. Sarathchandra, B. E. Mann and C. J. Green, Circ.Res., 90, E17 

(2002). 

[3] E. Fiumana, H. Parfenova, J. H. Jaggar and C. W. Leffler, Am. J. Physiol.-Heart Circul. Physiol., 284, 

H1073 (2003). 

[4] E. Barkoudah, J. H. Jaggar and C. W. Leffler, Am. J. Physiol.-Heart Circul. Physiol., 287, H1459 (2004). 

[5] B. Arregui, B. Lopez, M. G. Salom, F. Valero, C. Navarro and F. J. Fenoy, Kidney International, 65, 564 

(2004). 

[6] P. Koneru and C. W. Leffler, Am. J. Physiol.-Heart Circul. Physiol., 286, H304 (2004). 

[7] Q. Xi, D. Tcheranova, H. Parfenova, B. Horowitz, C. W. Leffler and J. H. Jaggar, Am. J. Physiol.-Heart 

Circul. Physiol., 286, H610 (2004). 

[8] J. Niesel, A. Pinto, H. W. P. N’Dongo, K. Merz, I. Ott, R. Gustb and U. Schatzschneider, Chem. Commun., 

1798 (2008). 

[9] R. Motterlini, B. E. Mann and D. A. Scapens, Application: WO Pat., 2007-GB2483, WO28003953 A2, 

2008. 

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151


P33. Effect of Sodium Cations on the Conformational Preferences 

of Peptides Bridged by PEG Linkers 

M. Cydzik, P. Pasikowski, A. Kluczyk, M. Biernat, M. Lisowski Z. Szewczuk 

Faculty of Chemistry, University of Wrolaw, F. Joliot-Ciurie 14, Wroclaw, Poland 

Ubiquitin is a small protein (8 kDa) that occurs in all eukaryotic cells. Its main known function is to mark other 

proteins for proteolytic degradation. According to our previous results, decapeptide fragment of the ubiquitn 

with LEDGRTLSDY sequence exhibits a very strong immunosuppressive activity, comparable to that of 

cyclosporin [1]. Recently, we revealed that proper dimerization of some other immunosuppressory peptides 

enhanced their biological activity [2,3]. It has been proposed that the dimerized peptides enhance simultaneous 

interaction with two hypothetic receptors located in close proximity on T-cells. 

We synthesized a series of new analogs of the immunosuppressive decapeptide fragment of ubiquitin. We used 

set of polyethylene glycol (PEG) linkers to connect monomeric analogs in various way. The PEG bridge serves 

not only as a dimerizer but also as a group which improves the solubility in water. 

Three different methods of dimerization of the ubiquitin immunosuppressory fragment. Bold line represents PEG linkers. 

The interaction of sodium ions with PEG in the designed dimers results in the creation of noncovalent adducts, 

that may affect overall conformation of the dimeric peptides in solution. We performed conformational analysis 

by CD spectroscopy to evaluate effect of sodium ions on the conformation of the analogs pegylated in different 

ways. 

References: 

[1] Z. Szewczuk, P. Stefanowicz, A. Wilczyński, A. Staszewska, I.Z. Siemion, M. Zimecki, Z. Wieczorek, 

Biopolymers, 74, 352 (2004). 

[2] Z. Szewczuk, M. Biernat, M. Dyba, M. Zimecki, Peptides, 25, 207 (2004). 

[3] M. Biernat, P. Stefanowicz, M. Zimecki, Z. Szewczuk, Bioconjugate Chem., 17, 1116 (2006). 

_____________________________________________________________________ 

152


P34. Synthesis and Characterization of Biodegradable Polylactide- 

Functionalized Graft Copolymers 

I. Czeluśniak 

Faculty of Chemistry,University of Wroclaw, 14 F. Joilot Curie Str., 50-383 Wrocław, Poland 

e-mail: czel@wchuwr.chem.uni.wroc.pl 

Biodegradable polymers and copolymers prepared from cyclic esters, such as lactide (LA), glycolide (GL) or ε– 

caprolactone (CL) have been widely used as sutures, drug delivery carriers and implants to ensure a temporary 

mechanical or therapeutic function as well as cell scaffolds in tissue engineering.[1-3] All the practical uses of 

those materials involve their biodegradable character and thus their decomposition profile has to be practically 

matched to the requirements of the application. The best way for production of the multicomponent materials 

with unusual technologies is to tailor their molecular architectures. 

The synthesis and characterization of the biodegradable graft copolymers with the polar polylactide side chains 

and acetylene or oxanorbornene [4] backbones is presented (Scheme). The method, in which polylactide 

macromonomers with acetylene/oxanorbornene end groups were subjected to polymerization, was chosen so that 

the properties of the macromonomers could be evaluated prior the synthesis of copolymers. Because the physical 

properties of the polymeric materials are tied directly to its molecular weight and control of polymer molecular 

weight is of utmost importance in the synthetic procedure, the well-defined ruthenium initiators were used as 

catalysts in polymerization reactions. The influence of the length and composition of the main chain as well as 

degradable side chain on the polymerisation reactions and degradation behaviour of graft copolymers will be 

discussed. 

: 

O 

O 

O 

O 

O 

CH2OH R 

n 

O H 

or 

HC 

[Ru] 

C R 

R: CH 3 or CH 2 OH R: (CH 2 ) n OH n= 0, 1, 2 

CH(CH 3 )OH; C(CH 3 ) 2 OH 

Scheme 

O 

n n n 

O O 

Acnowledgment: The work concerning research of properties of polylactide-functionalized polyoxanorbornenes 

was supported by Marie-Curie Intra-European Fellowship (MEIF-CT-2003-500919). The research of synthesis 

and properties of polylactide-functionalized acetylenes was supported by Marie-Curie European Reintegration 

Grants (MERG-CT-2005-030757). Author thanks the European Commission for the Marie-Curie Grants. 

References: 

[1] K.A. Athanasiou, G.G. Niederauer, C.M. Agrawal, Biomaterials, 17, 93 (1996). 

[2] J.C. Middleton, A.J. Tripton, Biomaterials, 21, 2335 (2000). 

[3] M. Vert, S.M. Li, G. Spenlehauer, J. Mater. Sci.: Mater. Med.,3, 432 (1992). 

[4] I.Czelusniak, E. Khosravi, A. M. Kenwright, C. W. G. Ansell, Macromolecules, 40, 1444 (2007). 

_____________________________________________________________________ 

153 

O 

O 

H 

O 

O 

O 

O 

H 

O 

O 

O 

O 

O 

H 

O


P35. Pseudomonas nautica Cytochrome c552 is the Electron Donor to Nitrous 

Oxide Reductase (N2OR), a Kinetic and Docking Study 

S. Dell’Acqua a , S.R. Pauleta a , A.S. Pereira a , E. Monzani b , L. Casella b , I. Moura a , 

J.J. G. Moura a 

a 

REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de 

Lisboa, 2829-516 Caparica, Portugal 

e-mail: simone.dellacqua@dq.fct.unl.pt 

b 

Dipartimento di Chimica Generale, Università di Pavia, Via Taramelli 12, 27100 Pavia, Italy 

The multicopper enzyme nitrous oxide reductase (N2OR) catalyses the final step of denitrification, the twoelectron 

reduction of N2O to N2. This enzyme is a functional homodimer containing two different multicopper 

sites: CuA and CuZ, where CuA is a binuclear copper site that transfers electrons to the tetranuclear coppersulfide 

CuZ, the catalytic site. 

The new activity assay presented here separates the activation of N2OR from the catalytic reduction of N2O and 

allowed to identify Pseudomonas nautica cytochrome c552 as the physiological electron donor to N2OR. The 

kinetic data presents differences when comparing physiological with artificial electron donors (cytochrome 

versus methylviologen). In the presence of cytochrome c552, the reaction rate is dependent on the ET reaction and 

independent of the N2O concentration. With MV, the electron donation is faster than the substrate reduction. The 

pH effect on the kinetic parameters is different when MV or cytochrome c552 are used as electron donors 

(pKa=6.6 and 8.3, respectively). The kinetic study also suggested the hydrophobic nature of the interaction. The 

formation of the electron-transfer complex was observed by 1 H-NMR protein-protein titrations and was 

modelled with a molecular docking program (BiGGER) [1]. The proposed docked complexes corroborated with 

the ET studies, giving a cluster of solutions that places cytochrome c552 nearby a hydrophobic patch located 

around the CuA center [2]. 

Acknowledgement: S.D.is supported by a FCT-PhD grant (SFRH/BD/30414/2006) 

References : 

[1] P.N. Palma et al., Proteins, 39, 372 (2000). 

[2] S. Dell’Acqua et al., Biochemistry, submitted. 

_____________________________________________________________________ 

154


P36. Diruthenium(II,III)-ibuprofen Metallodrug: Interaction with Human 

Serum Albumin and Effects on Leukemic Tumor Cells 

D. de Oliveira Silva, R.R.P. Santos 

a Departamento de Quimica Fundamental, Instituto de Quimica da Universidade de São Paulo, Av. Prof. Lineu 

Prestes, 748, B2T, 05508-000, São Paulo, SP, Brazil 

e-mail: deosilva@iq.usp.br 

Ruthenium compounds are of current interest for anticancer chemotherapy. Chemical and biological properties 

of Ru-NSAIDs (nonsteroidal anti-inflammatory drugs) have been studied in our laboratory. Recently, 

a diRu(II,III)-ibuprofen (Ruibp) complex was found to inhibit proliferation of C6 rat glioma cells with induction 

of cell death [1,2]. Interestingly, it also acts as anti-inflammatory with reduced gastrointestinal ulceration [3]. 

Here, the interaction with human serum albumin (HSA) and a novel biological property for Ruibp are reported. 

HSA conformational change was detected by UV circular dichroism and percentages of secondary structure were 

calculated for various HSA:Ruibp molar ratios. Fluorescence quenching of HSA induced by Ruibp in 

physiological condition was observed by monitoring (excitation, 295 nm) the emission of tryptophan, indicating 

that the conformation of the hydrophobic pocket in sub domain IIA is affected. The electronic spectrum of Ruibp 

in solution of histidine suggests that Ru can bind to HSA by imidazole N of this amino acid. SDS-PAGE 

electrophoresis shows no cleavage of HSA in the presence of complex. Preliminary MTT assays show that 

Ruibp exhibits cytotoxic effect on K562 human myeloid leukemic cells at relatively low concentrations 

(60 µmol/L). It is an interesting result since NSAIDs can induce apoptosis and inhibit proliferation of leukemic 

cells at higher concentrations (indomethacin: IC50= 310 µmol/L [4]). 

Acknowledgment: FAPESP, CNPq. 

References: 

[1] G. Ribeiro, M. Benadiba, A. Colquhoun, D. de Oliveira Silva, Polyhedron, 27, 1131 (2008). 

[2] M. Benadiba, G. Ribeiro, D. de Oliveira Silva, A. Colquhoun. Neuron Glia Biology S1- Glia Cells in Health 

and Disease 3, S127, B148 (2007) 

[3] A. Andrade, S.F. Namora, R.G. Woisky, G. Wiezel, R. Najjar, J.A.A. Sertie, D. de Oliveira Silva. J. Inorg. 

Biochem., 81, 23 (2000). 

[4] G.S. Zhang, C.Q. Tu, G.Y. Zhang, G.B. Zhou, W.L. Zheng, Leukemia Research, 24, 385 (2000). 

_____________________________________________________________________ 

155


P37. Protein Engineering of Repressor of Primer (Rop): Construction of 

Molecular Scaffolds for the Introduction of New Functions 

G. Di Nardo a , A. Di Venere b , A. Ortolani a , G. Mei b , S. Sadeghi a , G. Gilardi a 

a Department Of Human And Animal Biology, University Of Turin, via Accademia Albertina,, Turin, Italy 

b Department Of Experimental Medicine And Biochemical Sciences, University Of Rome 'Tor Vergata, via di 

Tor Vergata 135, Rome, Italy 

Rop (repressor of primer) is a dimeric 14 kDa protein of E. coli with a stable four helix bundle structure. 

Protein engineering of Rop has been used to: 1- introduce a heme binding site into the four helix bundle scaffold; 

2- create a new three helix bundle molecular scaffold. 

1- Heme ligands were introduced into an engineered monomeric Rop in two different positions. The mutants 

rop-56H/113H and rop-L63M/F121H were obtained, expressed and purified. They showed the ability to bind 

heme with KD = 1.1±0.2 µM and KD = 0.47±0.07 �M for rop-L63M/F121H and rop-56H/113H respectively. 

The unfolding of heme bound and unbound mutants in presence of guanidine hydrochloride was monitored and 

the total free energy change for heme bound constructs resulted 7.0±1.0 kcal/mol and 7.5±1.1 kcal/mol, similar 

to that of the initial construct. 

Spectroelectrochemical titrations demonstrated that the redox potential resulted positively increased from -154±2 

mV in rop-56H/113H to –87.5±1.2 mV in rop-L63M/F121H. 

2- The last helix of the monomeric ROP protein was removed by PCR and the resulting protein was purified. 

The far-UV circular dichroism spectrum showed a high helical content. Analysis in gel filtration and native 

electrophoresis showed a dimeric behaviour of the protein. Molecular modeling was used to predict the structure 

of the protein. 

The results suggest that it is possible to turn Rop into a redox protein and to create new molecular scaffolds with 

the use of protein engineering. 

_____________________________________________________________________ 

156


P38. Structural Models of LADH Active Site. Pentacoordination of 

Catalytic Zinc Derived from Model Studies 

A. Dołęga a , K. Baranowska a , A. Herman a , D. Gudat b 

a 

Gdańsk University of Technology,Department of Inorganic Chemistry,Narutowicza St. 11/12, 80-952, Gdańsk, 

Poland 

e-mail: ania@chem.pg.gda.pl. 

b 

Institut für Anorganische Chemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany 

Zinc in horse liver alcohol dehydrogenase (EC 1.1.1.1, LADH) supports a reversible oxidation of alcohols to 

aldehydes [1]. The detailed mechanism of the action of LADH is still under discussion. The coordination 

environment of the zinc ion located in the active site is an example of an unsolved problem. The geometry of the 

ligands is usually described as pseudotetrahedral [1], but there are also data, which point to the presence of five 

ligands in the first coordination sphere [2]. 

Close structural models of the LADH active site have been obtained and characterized by FT-IR, NMR and Xray 

diffraction. It has been shown that, similar to a manganese complex [3], zinc and cadmium tri-tertbutoxysilanethiolates 

with 2-methylimidazole as a co-ligand are able to bind alcohol. Comparison of the 

geometry of model complexes with the crystal data for ADH proteins indicates five-coordinated catalytic zinc 

ion in LADH. Our studies support the idea of Ryde [4], that glu68 participates in the reaction catalyzed by 

LADH as a fifth ligand to zinc. 

Both geometrical and electronic features of the active site ligands are reproduced in the presented models. The 

113 Cd NMR shift of one of the cadmium silanethiolates is identical with the shift of a Cd-substituted LADH- 

NAD + complex [5,6] on a 1000 ppm scale of 113 Cd NMR shifts. 

Quantum mechanical calculations with the zinc complex 1 as a starting model show a 20% decrease in the 

enthalpy of ethanol deprotonation due to complexation with Zn 2+ . 

Acknowledgement: The financial support of Polish Ministry of Science and Higher Education - Grant No. N 

N204 274835 - is acknowledged. 

References: 

[1] G. Parkin, Chem. Rev., 104, 699 (2004). 

[2] R. Meijers, R. J. Morris, H. W. Adolph, A. Merli, V. S. Lamzin, E. S. Cedergren-Zeppezauer, J.Biol.Chem., 

276, 9316 (2001). 

[3] A. Kropidłowska, J. Chojnacki, B. Becker, J. Inorg. Biochem., 101, 578 (2007). 

[4] U.Ryde, Protein Sci., 4, 1124 (1995). 

[5] B.R. Bobsein, R.J. Myers, J. Biol. Chem., 256, 5313 (1981). 

[6] A. Dołęga, K. Baranowska, J. Gajda, S. Kaźmierski, M. Potrzebowski, Inorg. Chim. Acta, 360, 2973 (2007). 

_____________________________________________________________________ 

157


P39. Looking 7-azaindole as 1,6,7-trideazaadenine for Metal-complex 

Formation: Structure of [Cu(IDA)(H7azain)]n 

A. Domínguez-Martín a , D. Choquesillo-Lazarte b , C. Sánchez de Medina-Revilla a , 

J.M. González-Pérez a , A. Castiñeiras c , J. Niclós-Gutiérrez a 

a 

Department of Inorganic Chemistry, University of Granada, Fac. Pharmacy, Campus Cartuja; Granada 

(18071), Spain 

e-mail: alidmm@correo.ugr.es 

b 

Edif. Inst Lopez Neyra, University of Granada, Laboratorio de Estudios Cristalográgicos, IACT, Avda. del 

Conocimiento; Armilla, Granada (18100), Spain. 

c 

Department of Inorganic Chemistry, University of Santiago, Fac. Pharmacy, Campus sur; Santiago de 

Compostela (15782), Spain. 

7-azaindole (H7azain = HL) can be looked as 1,6,7-trideazaadenine. The crystal structures reveal that one, two 

or four HL can be coordinated to the same Cu(II) centre occupying equatorial [1] or apical [2,3] sites, in all cases 

forming Cu-N3 bonds. We report the structure of [Cu(IDA)(HL)]n (293 K, final R = 0.026), where the Cu- 

N3(HL) bond (1.992(2) Å) is reinforced by an intramolecular H-bonding interaction (2.88 Å, 120.7º). A similar 

recognition mode Cu-N3 + H-bonding interaction exist in ternary Cu(II)-acetato-HL complexes [2,3]. The new 

compound grows as a polymer due to a syn-anti carboxylate bridge also reinforced by an N-H····O interaction 

(2.92 Å, 155.5º) between adjacent IDA ligands in the complex chain, along the b axis. 

Acknowledgement: Financial support from ERDF-EC, MEC-Spain (Project CTQ2006-15329-C02/BQU) is 

acknowledged. The project “Factoría de Cristalización, CONSOLIDER INGENIO-2010” provided X-ray 

structural facilities for this work. ADM thanks to the MEC for a Collaboration research grant. CSMR thanks to 

the CACOF for a research grant. DChL thanks CSIC-EU for an I3P postdoctoral research contract. 

References: 

[1] J. Poitras, A.L. Beauchamp, Can. J. Chem., 70, 2846 (1992). 

[2] Shie-Ming Peng, Chien-Hsien Lai, J.Chin. Chem. Soc. (Taipei), 35, 325 (1988). 

[3] Y. Kani, M. Tsuchimoto, S. Ohba, Acta Crystallogr., Sect. C:Cryst. Struct. Commun., 56, e193 (2000). 

_____________________________________________________________________ 

158


P40. Interaction Between Re(CO)3 Fragments and a PNA Monomer. 

D. Donghi a , M. Panigati a , G. D’Alfonso a , G. Prencipe b , E. Licandro b , S. Maiorana b , 

L. D’Alfonso c 

a 

Dipartimento di Chimica Inorganica, Metallorganica, Università degli Studi di Milano, Via Venezian 21, 

20133, Milano, Italy 

e-mail: daniela.donghi@unimi.it 

b 

Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, 20133, 

Milano; 

c 

Dipartimento di Fisica, Università di Milano-Bicocca, Piazza delle Scienze 6, 20126 Milano, Italy 

Peptide nucleic acids (PNA) are mimics of DNA, with pseudo-peptide backbones based commonly on 

N-(2-aminoethyl)-glycine, which show high binding affinity and specificity for DNA and RNA.[1] The 

conjugation of organometallic complexes to biomolecules finds applications both in diagnostic and therapeutic 

fields. The incorporation of Re(I) fragments into PNA fragments can offer a double advantage, due both to its 

radiochemical [2] and photo-emitting properties. [3] 

In this work we have investigated two different approaches for binding the thymine-PNA monomer with 

Re(I)complexes. 

We synthesized the novel compound [Re2(CO)6(µ-Cl)2(µ-4-COOH-pdz)], belonging to a recently developed 

family of dimeric luminescent Re(I) complexes,[4] and conjugated it to the NH2 end of a thymine-PNA 

monomer, obtaining the adduct shown in Figure. It exhibits photoluminescence with both one and two photon 

excitation, but low quantum yield (~0.003) and short lifetime. Studies on the binding with the homo-thymine 

PNA decamer are in progress, as well as attempts to synthesize ligands with a spacer between the pyridazine ring 

and the COOH group, to increase lifetime and quantum yields. 

A different conjugation approach involved coupling of the easily obtainable anionic dimer [Re2(CO)6(µ-OH)3] - 

with a thymine-PNA monomer bearing a carboxylic acid function. The interaction gives an adduct, characterized 

by NMR spectroscopy, in which the binding occurs through a bridging carboxylate group. 

References: 

[1] P.E. Nielsen, Editor Peptide Nucleic Acids: Protocols and Applications, Second Editions, Horizon 

Bioscience, 2004, Wymondham, UK. 

[2] R. Alberto, Coord. Chem. Rev., 901, 190 (1999). 

[3] J. Zubieta, J.F. Valliant et al, J.Am. Chem. Soc., 126, 8598 (2004). 

[4] D. Donghi, G. D'Alfonso, M. Mauro, M. Panigati, P. Mercandelli, A. Sironi, P. Mussini, L. D'Alfonso., 

Inorg. Chem., DOI: 10.1021/ic7023692 (2008). 

_____________________________________________________________________ 

159


P41. Preclinical Evaluation of New Radiolabeled CCK Ligands as 

Molecular Imaging Agents for CCK2 Receptor Targeting 

S. Dorbes a , S. Brillouet b , L.P. Delord c , F. Courbon b , M. Poirot a , S. Silvente-Poirot a 

a 

Métabolisme, oncogénèse et différenciation cel, Institut Claudius Regaud, 20-24 rue du pont Saint-Pierre 

31059, Toulouse, France 

e-mail: dorbes@chimie.ups-tlse.fr 

b 

Département de Service de Médecine Nucléaire, Institut Claudius Regaud, 20-24 rue du pont Saint-Pierre, 

31059, Toulouse, France 

c 

EA3035 Laboratoire de Pharmacologie Clinique et Ex, Institut Claudius Regaud, 20-24 rue du pont Saint- 

Pierre, 31059, Toulouse, France 

Somatostatin receptor scintigraphy has been proven as a valuable tool for staging gastrointestinal endocrine 

tumors. But, its sensitivity and accuracy in other cancers, such as metastatic medullary thyroid cancer (MTC), is 

limited. Actually, either the somatostatin receptors are not expressed in these tumors and metastasis or their 

expression change during the evolution of the pathology. 

The cholecystokinin/gastrin receptor (CCK2R) is overexpressed in MTC up to 90% but not in the corresponding 

healthy tissues [1]. Thus, the CCK2R represents a potential target for the diagnosis and internal radiotherapy of 

these tumors. Previously studies had demonstrated the feasibility of radiolabeled CCK/gastrin ligands to target 

MTC in animals and patients. Different adverse effects using these radioligands were reported indicating that 

tumor uptake, biodistribution and stability of the radioligand must be improved for clinical application[2]. 

The aim of this study was to synthesise new radioligands of the CCK2R with optimized properties to target the 

CCK2R in different tumor models : E151A-CCK2R-NIH-3T3 [3] and TT (MTC) cells. 

Chemical synthesis, EC50 studies, SPECT (Single Photon Emission Computed Tomography) imaging and 

biodistribution of optimized CCK2R radioligands will be presented in this communication. 

References: 

[1] J. C. Reubi, J. C. Schaer, Cancer Res., 57, 1377 (1997). 

[2] M. Béhé, T. Behr, Biopolymers (Pept. Sci.), 66, 399 (2002). 

[3] C. Gales et al, Oncogene, 22, 6081 (2003). 

_____________________________________________________________________ 

160


P42. The Investigation of Chelation of Isoflavones with Transition Metals 

S. Dowling a , H. Hughes a , F. Regan b 

a 

Department of Chemical & Life Sciences, School of Science, Waterford Institute of Technology, Brownes Road, 

Waterford, Ireland. 

b 

National Centre for Sensor Research, School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 

9, Ireland 

Flavonoid metal chelates come from the chelating ability of flavonoids with metals such as Cu(II) and Fe(III). 

The investigation of chelation with flavonoids, such as quercetin, has been performed in great detail but little has 

been done with investigation of chelation of isoflavones with metals. The investigation of isoflavone metal 

chelates would be useful to explain how isoflavones can mediate metal overload disorders such as Wilson’s 

Disease [1]. Flavonoid metal chelates have often shown to have enhanced antioxidant potential in comparsion to 

their free flavonoid forms so knowledge of their stoichiometries may optimise their antioxidant potentials. [2] 

The aim of this work was the investigation of metals that can chelate with isoflavones and the stoichiometry of 

successful isoflavone metal chelates. The chelation of isoflavones Genistein, Daidzein and Biochanin A were 

investigated with respect to the metals Cd(II), Cu(II), Fe(III), Zn(II), Ni(II), Pb(II), Co(II) and Ge(IV). 

Isoflavone metal chelate stoichiometry was determined with Cu(II) and Fe(III) using the mole ratio method and 

UV/VIS spectroscopy. 

References 

[1]. Mira L., Fernandez M.T., Santos M., Rocha R., ncio M.H., Jennings K.R. 2002. 

Interactions of Flavonoids with Iron and Copper Ions: A Mechanism for their Antioxidant Activity. Free Radical 

Research 36:1199-1208. 

[2]. Malesev D., Kuntic V. 2007. Investigation of metal-flavonoid chelates and the determination of flavonoids 

via metal-flavonoid complexing reactions. Journal of the Serbian Chemical Society 72:921-939. 

_____________________________________________________________________ 

161


P43. Using Density Functional Theory to Correlate g and A( 95 Mo) 

Non-coincidence with Mo V - dithiolate Folding Angle – Relevance to 

Molybdenum Enzyme Structure and Function 

S.C. Drew a, b, c , G.R. Hanson d 

a 

Department of Pathology, The University of Melbourne, Parkville, Victoria, 3010, Australia. 

b 

Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, 3010, 

Australia. 

c 

School of Physics, Monash University, Clayton, Victoria, 3800, Australia. 

d 

Centre for Magnetic Resonance, The University of Queensland, St. Lucia, Queensland, 4072, 

Australia. 

Molybdenum enzymes typically contain mononuclear Mo active site coordinated by one or two bidentate pterindithiolene 

ligands, known as molybdoterin (MPT), together with a complement of oxo, sulfido, water-based, 

and/or amino-acid ligands. The enzymes cycle through the formal molybdenum oxidation states +6, +5, and +4 

and catalyze a variety of net oxygen atom transfer reactions. Electron Paramagnetic Resonance (EPR) is 

routinely used to interrogate the Mo V intermediate to provide structural and mechanistic information. 

The ‘non-innocence” of MPT has been suggested to play a role in tuning the redox potential of enzyme active 

sites, controlling the reactivity of co-ligands, stabilizing the multiple Mo oxidation states, and facilitating 

electron transfer between the Mo active site and other redox partners. This ability to fine tune the electron 

density at the active site has been linked to a “dithiolate-folding-effect” involving overlap of the metal in-plane 

and sulfur-π orbitals, which depends on the orientation of the MPT ligand. 

The relationship between experimental EPR spectra and the electronic and geometric structure of the active site 

can be difficult to establish, not least because of the low molecular symmetry. Using density functional theory, 

we have carried out a systematic study of the relationship between the metal-dithiolate fold angle and the spin 

Hamiltonian (SH) parameters of a prototypical monoclinic Mo V model complex. The results are compared with 

experimentally determined SH parameters and used to predict the fold angle adopted in solution. This may 

provide a useful method for probing the local structure of the active site of mononuclear molybdenum enzymes 

in the Mo V state. 

_____________________________________________________________________ 

162


P44. Oligonucleotides with a Bipyridine-based Nucleoside: Aggregation in 

the Presence of Metal Ions 

N. Düpre a , L. Welte b , J. Gómez-Herrero c , F. Zamora b , J. Müller * a , 

a 

Inorganic Chemistry,Dortmund University of Technology,Otto-Hahn-Strasse 6,44227,Dortmund,Germany 

e-mail: nicole.duepre@uni-dortmund.de 

b 

Departemento de Química Inorgánica,Universidad Autónoma de Madrid,,28049,Madrid,Spain 

c 

Departemento de Física de la Materia Condensada,Universidad Autónoma de Madrid,,28049,Madrid,Spain 

Research on nucleic acids that contain novel base pairing schemes involving artificial nucleobases has been 

vigorously pursued in recent years. These artificial nucleobases can serve as ligands for metal ions and therefore 

replace hydrogen bonding between complementary bases by coordinative bonds to a central metal ion. 

One of the motivations for synthesizing and characterizing nucleic acids with metal-ion-mediated base pairs is 

their proposed application as a molecular wire. Therefore molecular buildings blocks that are able to connect two 

or three of these wires by coordinating one metal ion are highly desirable. 

Having in mind the formation of nanoscale DNA-based networks that are able to assemble only in the presence 

of appropriate transition metal ions (Scheme 1), we incorporated the 5-methyl-2,2´-bipyridine nucleoside 

(denoted X) at the 5´-end of the self complementary Dickerson-Drew dodecamer d(CGCGAATTCGCG), giving 

d(XCGCGAATTCGCG). This leads to the formation of double helices with single-nucleotide 5´-overhangs that 

are only able to become sticky ends in the presence of appropriate metal ions. The formation of DNA-based 

networks by addition of octahedrally coordinating metal ions such as Fe(II) should now be feasible. Atomic 

force microscopy (AFM) was chosen for studying the formation of DNA-based networks in the presence of 

different metal ions. 

References: 

N. Düpre, L. Welte, J. Gómez-Herrero, F. Zamora, J. Müller; Inorg. Chim. Acta 2008, 361, in press (doi: 

10.1016/j.ica.2007.12.005). 

_____________________________________________________________________ 

163


P45. Complexes of Triazole Derivatives with Copper(II). 

Study of DNA Damage and HDV Ribozyme Activity Inhibition 

M. Dziuba a , J. Wrzesiński b , J. Ciesiołka b , W. Szczepanik a , L. Z. Ciunik a , M. Barys a 

and M. Jeżowska-Bojczuk a 

a 

Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, 

e-mail: magdadz@eto.wchuwr.pl 

b 

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland 

1, 2, 4-triazoles and their derivatives are very interesting compounds due to their role in medicinal, agricultural 

and industrial fields. They were proved to reveal anti-cancer, anti-fungal and anti-inflammatory activity [1-3]. 

We have studied a number of novel chiral 1, 2, 4-triazole derivatives (i.e. 4-amino-1, 2, 4-triazol-2, 4dinitrobenzaldehyde 

(2, 4dnbald), 4-amino-1, 2, 4-triazol-2-nitrobenzaldehyde (2nbald), 4-amino-1, 2, 4-triazol- 

3-nitrobenzaldehyde (3nbald) and 4-amino-1, 2, 4-triazol-4-nitrobenzaldehyde (4nbald)). The newly 

synthesized compounds were active against bacteria and fungi [4]. Ligands alone, as well as their copper 

complexes were also examined for their ability to interact with both RNA and DNA. 

The studied derivatives showed relatively weak inhibitory properties towards cleavage of antigenomic delta 

ribozyme. However, their Cu 2+ complexes reduced ribozyme cleavage kinetics by a factor of 2 - 3.5 in 

comparison to the uncomplexed compounds. The strongest effect with 2nbald and 2, 4-dnbald-Cu 2+ complex for 

Mg 2+ or Mn 2+ -promoted cleavage was observed, respectively. Chemical probing showed preferential binding of 

the complexes to the J4/2 region of HDV ribozyme. Our results suggest that RNA molecules are potential targets 

for binding of triazole-Cu 2+ complexes [5]. 

Metal complexes of various bioavailable ligands and xenobiotics are known to induce cleavage of DNA. We 

studied pBluescriptSK+ plasmid damage caused by the Cu 2+ -nbald species. We observed rather weak influence 

of Cu 2+ -nbald complexes on nucleic acid reflected by a moderate amount of single strand nicks. Only Cu 2+ -2, 

4dnbald in presence of H2O2 was able to induce the double strand scission of plasmid DNA. 

References: 

[1] M.C. Hosur, M.B. Talawar, R.S. Bennur, S.C. Bennur and P.A. Patil, Indian J. Pharm. Sci 55, 86 (1993) 

[2] Y.S. Wu, H.K. Lee, S.F.Y., Li, J. Chromatogr. A 912, 171 (2001) 

[3] D.R. Williams, Chem. Rev. 72, 203 (1972) 

[4] M. Barys, Z. Ciunik data not published 

[5] J. Wrzesiński, M. Dziuba, J. Ciesiołka, W. Szczepanik, M. Jeżowska-Bojczuk submitted 

_____________________________________________________________________ 

164


P46. Synthesis, Spectroscopic Characterization and Catalytic Activity of 

Neuromelanin Models Containing Albumin and Metal Ions 

M. Engelen, E. Ferrari, E. Monzani, L. Casella 

Department of Chemistry, University of Pavia, Via Taramelli 12, 27100, Pavia, Italy 

Neuromelanin is a partially characterized pigment found primarily in catecholaminergic neurons[1,2]. Although 

its composition and function are not completely understood, the pigment seems to be involved in the 

development of neurodegenerative diseases such as Alzheimer′s and Parkinson′s disease [3,4]. One of the 

difficulties concerning the complete characterization of neuromelanin is the scarcity of biological material, as 

well as the poor solubility in non-destructive solvents [5]. 

To gain a better understanding of neuromelanin structure, several synthetic melanins, containing different 

amounts of albumin and iron or copper ions to simulate the natural product, have been synthesized and 

characterized by NMR, UV-VIS, EPR and CD spectroscopy. At higher concentrations of albumin the products 

remain soluble in H2O, which semplifies the characterization process. Surprisingly, the synthetic melanins 

display catalytic activity in the oxidation of dopamine to dopamine quinone. The presence of Fe II or Cu II ions in 

the pigment increases the catalytic activity, but metal ions sequestered by the macromolecule have a much lower 

activity compared to free metal ions in solution. This seems to confirm the hypothesis that neuromelanin might 

play a protective role by sequestering redox active metals from its surroundings[6]. However, since the activity 

of the metal ions is not completely blocked, the pigment can not be considered inert and over long periods of 

time might cause oxidative stress. 

References: 

[1] L. Zecca, P. Costi, C. Mecacci, S. Ito, M. Terreni, S. Sonnino, J.Neurochem., 74, 1758 (2000). 

[2] L. Zecca, T. Shima, A. Stroppolo, C. Goj, G.A. Battistoni, R. Gerbasi, T. Sarna, H.M. Swartz, Neuroscience, 

73, 407 (1996). 

[3] M. Fasano, B. Bergamasco, L. Lopiano, J. Neural Transm., 113, 769 (2006). 

[4] L. Zecca, F.A. Zucca, H. Wilms, D. Sulzer, Trends in neurosciences, 26, 578 (2003). 

[5] K.L. Double, et al., J. Neurochem., 75, 2583 (2000). 

[6] L. Zecca, D. Tampellini, M. Gerlach, P. Riederer, R.G. Fariello,D. Sulzer, J. Clin. Pathol.: Mol. Pathol., 54, 

414 (2001). 

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165


P47. Diiron-containing Metalloprotein: Structural and Functional 

Characterization of DF3, a Catalytic Model 

M. Faiella a , C. Andreozzi a , O. Maglio a , V. Pavone a , W. F. DeGrado b , F. Nastri a , 

A. Lombardi a 

a 

Department of Chemistry, University of Napoli Federico II, Via Cinthia 80126 Napoli, Italy, 

e-mail: angelina.lombardi@unina.it 

b 

Department of Biochemistry & Biophysics, University of Pennsylvania , 19104-6059 Philadelphia, PA, – USA 

Diiron-oxo proteins are a class of macromolecules that catalyze different reactions, from ferroxidation to 

hydroxylation, despite the same structural motif, the four-helix bundle [1]. 

To elucidate how the protein matrix tunes the properties of a single metal cofactor to obtain such a wide diversity 

of functions, we designed minimal models called DFs [2]. The first model DF1 was de novo designed from a 

retro-structural analysis of 6 different carboxylate-bridged diiron proteins. DF1 is made up of two 48-residue 

helix-loop-helix (α2) motifs, able to specifically self-assemble into an antiparallel four-helix bundle, with a 

Glu4His2 liganding environment for the diiron center, housed within the center of the structure. Although DF1 

adopted the intended helical conformation and was able to coordinate different metal ions, it showed low water 

solubility and did not support any catalytic activity. DF1 structural characterization [3] suggested that the interhelical 

loop adopted a strained conformation, which may account for the molecule insolubility, while a pair of 

symmetrically related hydrophobic Leu residues at position 13 of the sequence blocked the access to the active 

site, preventing any activity. 

Here we present DF3, a new DF model, designed to improve thermodynamic and functional properties of DF1. 

The mutations in DF3 sequence are: 

1. two glycine residues in position 9 and 13 to allow easy access of exogenous ligands to the metal site (Figure); 

2. a new inter-helical loop, introduced to evaluate its influence on the overall folding and solubility. 

DF3 is highly water soluble and the NMR characterization of the Zn(II)-complex [4] reveals that it is able to fold 

into a stable native-like four-helix bundle structure. The loop region is very-well defined and adopts a unique 

conformation. DF3 binds Co, Mn and Zn in the expected stoichiometry and coordination geometry. Chemical 

denaturation, by Gdn·HCl, shows that Leu 9 and 13 substitutions destabilize the protein, as expected; 

nevertheless, the newly designed loop positively affects the thermodynamic properties of DF3. 

UV-Vis characterization reveals that DF3 reversibly oxidizes Fe(II) to Fe(III) and its diferric complex is stable in 

water at pH=7. Kinetic experiments were performed to explore the catalytic activity of di-Fe(III)-DF3 using 

different substrates, demonstrating a high selectivity in diphenolase reactions. 

These data confirm that DF3 is a promising candidate in the development of catalytic artificial proteins. 

Active site cavity in DF3 

References: 

[1] S.J. Lange and L. Que, Jr., Curr. Opin. Chem. Biol., 2, 159 (1998). 

[2] O.Maglio, F. Nastri, R.T.M. de Rosales, M. Faiella, V. Pavone, W.F. DeGrado, A. Lombardi, Comptes 

Rendus – Chimie, 10, 703 (2007). 

[3] A. Lombardi, C.M. Summa, S. Geremia, L. Randaccio, V. Pavone, W.F. DeGrado, Proc. Natl. Acad. Sci. 

USA, 97, 6298 (2000). 

[4] Faiella et al. manuscript in preparation 

_____________________________________________________________________ 

166


P48. NeuroInorganic Chemistry of Alzheimer’s Disease: Structure, 

Reactivity and Metal Transfer of Amyloid-β 

P. Faller a , L. Guilloreau a , C. Talmard a , V. Sonois a , M. Vignes a , G. Meloni b , M. Vašák b , 

A. Mockel c , M.L. Maddelein c , J. Teissie c 

a 

Laboratoire de Chimie de Coordination, CNRS, 205, route de Narbonne, 31077, Toulouse, France 

e-mail: peter.faller@lcc-toulouse.frb 

b 

Biochemistry, University of Zürich, Winterthurerstr. 190, Zürich, Switzerland 

c 

IPBS, CNRS, 205, route de Narbonne, 31077, Toulouse, France 

A large body of evidence indicates that the essential metal ions zinc, copper and iron are involved in Alzheimer's 

disease (AD). Amyloid-beta (Aβ) is the major constituent of one of the hallmarks of AD, the so called amyloid 

plaques. According to the widely held amyloid cascade hypothesis, increased Aβ production and accumulation 

leads to its aggregation. First oligomers/polymers are generated and at the end amyloid plaques. The 

oligomers/polymers of Aβ are supposed to provoke neuronal dysfunction and later to the onset of dementia via 

the production of reactive oxygen species (ROS). 

Zinc, copper and iron are found in the amyloid plaques at high concentrations (~mM) and metal-binding to Aβ 

has been proposed for Zn and Cu. In vitro and in vivo studies showed that these metal ions influence the amyloid 

cascade and the neurotoxicity. They are considered as important cofactors in AD. 

Our research group is interested in the bioinorganic aspects of the metal-binding to Aβ complex with other 

metalloproteins, including metallothionein-3 (MT3) [5] and human serum albumin. MT3 is linked to AD and a 

protective role against Aβ. In particular in the coordination chemistry, the role of the metal ions, the reactivity of 

theses complexes in terms of production of ROS and the interaction with metalloproteins [1-4]. The present 

contribution focuses interaction of the metal-Aβ neurotoxicity has been demonstrated. We were able to show, 

that metallothionein-3 is capable to withdraw copper from Aβ and inhibit the production to the radical HO°. This 

mechanism could explain the protective role of metallothionein-3 against Aβ. This could be a potential lead for 

the design of drugs for AD. 

References: 

[1] C. Talmard, A. Bouzan, P. Faller, Biochemistry, 46, 13658 (2007). 

[2] L. Guilloreau, S. Combalbert, A. Sournia-Saquet, H. Mazarguil, & P. Faller, ChemBioChem, 8, 1317 (2007). 

[3] C. Talmard, L. Guilloreau, Y. Coppel, H. Mazarguil & P. Faller, ChemBioChem, 8, 163 (2007). 

[4] L. Guilloreau, L. Damian, Y. Coppel, H. Mazarguil, M. Winterhalter & P. Faller, J. Biol. Inorg. Chem., 11, 

1024 (2006) 

[5] G. Meloni, V. Sonois, T. Delaine, L. Guilloreau, A. Gillet, J. Teissié, P. Faller & M. Vašák, Nat. Chem. 

Biol., 2008, in press 

_____________________________________________________________________ 

167


P49. Photoactivatable Platinum Anticancer Complexes 

N.J. Farrer a , J.A. Woods b , H-C. Tai a , R.J. Deeth a , P.J. Sadler a 

a Chemistry, University of Warwick, CV4 7AL, Coventry, United Kingdom 

e-mail: n.farrer@warwick.ac.uk 

b Photobiology Unit, University Department of Dermat, Ninewells Hospital & Medical School, DD1 9SY, 

Dundee, United Kingdom 

Platinum complexes such as cisplatin are well-established anti-cancer drugs. Adverse side-effects and 

development of resistance are serious limitations of these treatments [1]. Targeting of drugs, can minimise sideeffects 

and resistance can be overcome by novel mechanisms of action. 

The non-toxic photoactivable platinum(IV) complex trans-trans-trans-[Pt(N3)2(OH)2(NH3)(pyridine)] is 13-80 

times more cytotoxic to cancer cells, and ca. 15 times more cytotoxic towards cisplatin-resistant cancer cells than 

cisplatin. It also demonstrates little or no dark toxicity [2]. We will discuss the possible mechanism of action of 

this complex and some of its derivatives. Insight into photochemical reaction pathways have been obtained 

through the use of a variety of analytical techniques including 14 N, 15 N, 195 Pt and 2D NMR spectroscopy. 

Modification of these complexes to enhance tumour targeting and to achieve activation at longer wavelengths of 

light will also be described. 

Acknowledgement: We thank the Medical Research Council (MRC) for support and members of COST Action 

D39 for stimulating discussions. 

References: 

[1] L. Kelland, Nat. Rev., Cancer, 7, 573 (2007). 

[2] F. S. Mackay, J. A. Woods, P. Heringová, J. Kašparková, A. M. Pizzaro, S. A. Moggach, S. Parsons, V. 

Brabec, P. J. Sadler, Proc. Natl. Acad. Sci. USA, 104, 20748, (2007). 

_____________________________________________________________________ 

168


P50. Effect of the Rhombicity on the Peroxidase Activity of Manganese(III) 

Complexes with Schiff Bases and Ambidentade Ligands 

I.M. Fernandez-Garcia a , A. Vazquez-Fernandez a , M.J. Rodríguez-Doutón a , M. Maneiro a , 

A.M. González-Noya b , M.R. Bermejo b 

a 

Quimica Inorganica, Universidad de Santiago de Compostela, Facultad de, Alfonso x, 27002, Lugo, Spain 

e-mail: isaferga@lugo.usc.es 

b 

Quimica Inorganica, Universidad de Santiago de Compostela, Facultad de, Avda Ciencias, 15006, Santiago 

Compostela, Spain 

In the past few decades manganese chemistry has attracted much attention because of its role as an important 

cofactor of several electron-transfer metalloproteins. For example, the manganese peroxidases (MnP) catalyse 

the oxidation of a wide variety of substrates by H2O2. 

Recently, we have tried to establish a correlation between the peroxidase activity and the structural motifs of the 

complexes [1]. As an extension of our investigation on this catalytic behaviour, we have prepared and studied 

new Mn(III) complexes, derived from five Schiff bases and one ambidentade ligand (NCS - ). In these complexes 

the metal centre exhibits an octahedral environment, with the N2O2 donor set of the ligand bound to the Mn(III) 

ion in the equatorial plane and one molecule of solvent and other of NCS - along the symmetry axis. This solvent 

axial molecule constitutes a quite labile ligand, which could generate a vacant position in the coordination sphere 

to accommodate a substrate. In order to quantify the tetragonal elongation we have employed the "rhombicity" of 

the structures as the ratio between the manganese-axial-oxygen distances and the manganese-equatorial-oxygen 

distances. Peroxidase activity was expressed as ratio between ABTS •+ absorbance and manganese complexes 

absorbance at 415 nm. The peroxidase activity of these Mn(III) complexes versus the rhombicity is shown in 

figure 1. 

References: 

[1] M. R. Bermejo, M. I. Fernández, A. M. González-Noya, M. Maneiro, R. Pedrido, M. J. Rodriguez, J. C. 

Monteagudo, B. Donnadieu; J. Inorg. Biochem., 100, 1470 (2006). 

_____________________________________________________________________ 

169


P51. Protein and Electrode Engineering for the Covalent 

Immobilization of P450 BMP on Gold 

V. E. V. Ferrero a , L. Andolfi b , G. Di Nardo a , S. J. Sadeghi a , A. Fantuzzi c , S. Cannistraro b and 

G. Gilardi a 

a 

Department of Human and Animal Biology, University of Turin , Via Accademia Albertina 13, 10123, Torino, 

Italy 

e-mail: valentina.ferrero@unito.it 

b 

Biophysics and Nanoscience Centre, CNISM, Science Faculty, University of Tuscia, 01100, Viterbo, Italy 

c 

Division of Molecular Biosciences, Biochemistry Building, Imperial College London, SW7 2AZ, 

London, United Kingdom. 

Site-directed mutagenesis and functionalization of gold surfaces have been combined to obtain a stable 

immobilization of the haem domain of cytochrome P450 BM3 from Bacillus megaterium (BMP) [1]. 

Immobilization experiments were carried out using the wild type BMP bearing the surface C62 and C156, and 

the site-directed mutants C62S, the C156S and the double mutant C62S/C156S (no exposed cysteines). The gold 

surface was functionalized using two different spacers: cystamine-N-succinimidyl 3-maleimidopropionate 

(CST-MALM) and dithio-bismaleimidoethane (DTME), both leading to the formation of maleimide-terminated 

monolayers capable to covalent linkage to cysteine. Tapping mode atomic force microscopy (TMAFM) 

experiments carried out on CST-MALM derivatized gold led to good images with expected molecular heights 

(5.5-6.0 nm) for the wild type and the C156S mutant. 

These samples also gave measurable electrochemical signals with midpoint potentials of -48 and -58mV for wild 

type and C156S respectively. On the other hand, the DTME spacer led to variability on the molecular heights 

measured by TMAFM and the electrochemical response. This is interpreted in terms of lack of homogeneous 

DTME monolayer on gold. Furthermore, results from TMAFM show that the double mutant and the C62S did 

not lead to stably immobilized BMP, confirming the necessity of the solvent exposed C62. 

Acknowledgement: G. Gilardi acknoledges EU Marie Curie project EdRox-MRTN-035649, the Regione 

Piemonte (IT) and PRIN-MIUR 2006. L. Andolfi acknowledges the MIUR project “Rientro dei cervelli”. 

References: 

[1] S.Govindraj, T. L. Poulos, Journal of Biological Chemistry 272, 7915 (1997). 

_____________________________________________________________________ 

170


P52. Interference of Zinc in the Activation of Human Neutrophils and 

Subsequent Oxidative Burst 

M. Freitas a , G. Porto b , J.L. Fontes Costa Lima a , E. Fernandes a 

a 

Química-Física, Faculdade de Farmácia da Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030, Porto, 

Portugal 

e-mail: marisafreitas@ff.up.pt 

b 

Serviço de Hematologia Clínica, Hospital Geral de Santo António, Largo Professor Abel Salazar, 4099-001, 

Porto, Portugal 

Zinc is considered a non-toxic metal. Nevertheless, in spite of its safety, some reports suggest that zinc may 

disturb the innate host defense response, by interfering in the activation of neutrophils and subsequent oxidative 

burst. However, the exact role of zinc still needs clarification since some authors reported an activation effect 

while in turn, others revealed that zinc inhibits superoxide radical (O2 -. ) formation. Thus, the main objective of 

the present study was to provide clarification on the role of zinc on the activation human neutrophils and the 

consequent oxidative burst. For this purpose, different detection methods [luminol amplified 

chemiluminescence, cytochrome c reduction (UV/Vis spectrometry), Amplex Red and 2-[6-(4'-amino)phenoxy- 

3H-xanthen-3-on-9-yl]benzoic acid (APF) (fluorescence)] were used to evaluate the interference of zinc in the 

neutrophils's oxidative burst in order to understand the apparent contradictory results reported in literature. It was 

clearly demonstrated that zinc, at physiologic concentrations (5-12.5 µM) activate NADPH oxidase leading to 

the formation of O2 -. . On the other hand, higher concentrations of zinc may originate misleading results due to its 

catalytic role on the interconversion among reactive oxygen species. 

Acknowledgement: Marisa Freitas acknowledges Fundação para a Ciência e Tecnologia (FCT) and Fundo 

Social Europeu (FSE) her PhD grant (SFRH/BD/28502/2006). 

_____________________________________________________________________ 

171


P53. Biomimetic Models for Fe/S Clusters Involved in Radical Reactions 

M.G.G. Fuchs a , S. Dechert a , U. Ryde b , F. Meyer a 

a 

Institut für Anorganische Chemie, Georg-August-Universität Göttingen, Tammannstraße 4, D-37077 

Göttingen, German 

e-mail: michael.fuchs@chemie.uni-goettingen.de 

b 

Department of Theoretical Chemistry, Lund University, Chemical Center, S-22100 Lund, Sweden 

Fe/S clusters are discussed as the sulphur sources in biochemical radical reactions in which S atoms are inserted 

into substrates. A [4Fe–4S] cluster is the probable sulphur source in the reactions catalysed by lipoyl synthase 

(LipA) [1] and tRNA-methylthiotransferase (MiaB)[2] whereas in biotin synthase (BioB), a [2Fe–2S] cluster 

delivers the S atom for insertion into the substrate dethiobiotin. [3] This [2Fe–2S] cluster carries an unusual 

arginine ligand which might enhance reactivity by providing additional interactions around one of the Fe atoms. 

[4] 

Model reactions using biomimetic Fe/S clusters and organic radicals are investigated in order to emulate the 

proposed enzyme mechanisms. Furthermore, Fe/S clusters with unusual coordination are synthesised and 

characterised [5] to clarify the role of the unprecedented arginine ligand of the biotin synthase Fe/S cluster. 

Acknowledgement: Financial support from Fonds der Chemischen Industrie (Kekulé fellowship for M. G. G. 

F.) and Deutsche Forschungsgemeinschaft (IRTG 1422) is gratefully acknowledged. 

References: 

[1] J. R. Miller, R. W. Busby, S. W. Jordan, J. Cheek, T. F. Henshaw, G. W. Ashley, J. B. Broderick, J. E. 

Cronan Jr., M. E. Marletta, Biochemistry, 39, 15166 (2000). 

[2] H. L. Hernández, F. Pierrel, E. Elleingand, R. García-Serres, B. H. Huynh, M. K. Johnson, M. Fontecave, M. 

Atta, Biochemistry, 46, 5140 (2007). 

[3] N. B. Ugulava, C. J. Sacanell, J. T. Jarrett, Biochemistry, 40, 8352 (2001). 

[4] F. Berkovitch, Y. Nicolet, J. T. Wan, J. T. Jarrett, C. L. Drennan, Science, 303, 76 (2004). 

[5] J. Ballmann, S. Dechert, E. Bill, U. Ryde, F. Meyer, Inorg. Chem., 47, 1586 (2008). 

M. G. G. Fuchs, S. Dechert, U. Ryde, F. Meyer, unpublished results. 

_____________________________________________________________________ 

172


P54. Zinc(II) Complex for Selective and Rapid Scission of Protein Back 

Bone 

Y. Fujii a , M. Yashiro b , Y.Kawakami a , T. Jyunichi a 

a 

Department of Material and Biological Science, Ibaraki University, 2-8-9 Bunkyo, 310-8512 , Mito, Japan 

e-mail: yuki@mx.ibaraki.ac.jp 

b 

Department of Nano Chemistry, Tokyo Polytechnic University, 1583 Iiyama, 243-0297, Atzugi, Japan 

Zinc(II) is an essential element in living organisms. Enzymes, involving carboxypeptitase A, thermolysin, 

leucine aminopeptidase, require Zn(II) in the catalytic center [1]. Studies on nonenzymatic hydrolysis of 

a protein backbone using a metal ion or its complex, involving Cu(II), Fe(II), Ni(II), Pd(II), Mo(IV) have been 

carried out. However, little success has been reported with a Zn(II) complex [2, 3]. 

Recently, we found that a Zn(II) complex of L1 (Zn(II)-L1) promoted the decomposition of bovine serum 

albumin (BSA) and elastase from porcine pancreatic more effectively than free Zn(II). The decomposition rate of 

BSA was 4.4 x 10-2 h-1 at pH 11, 50 °C, which was 3.3 times and 8 times higher than those with and without 

free Zn(II), respectively. The decomposition rate of elastase was 6.3 x 10-1 h-1 at pH 8, 50 °C, which was 3.5 

times and 7 times higher than those with and without free Zn(II), respectively. 

The SDS-PAGE analysis of the reaction solution of BSA (66 kDa, 583 mer, 59 Lysine residues) showed trace 

amounts of 9 fragments in the range of 66-14 kDa, indicating that the decomposition dominantly yielded low 

molecular weight fragments. In contrast, no fragment was observed in the case of free Zn(II). The 9 fragments 

corresponded to the scission fragments at near K93, K499, K136, K439, K159, K187, K396, K556, K239, K350, 

K312, K273. The MALDI-TOF/MS of the reaction solution of BSA showed no clear fragment in 20000-66000 

(m/z), but several broad peaks in 5000-20000 (m/z). A broad peak of ca.75000 (m/z) suggested the Schiff base 

formation between BSA and Zn(II)L1. 

The SDS-PAGE of the reaction product of elastase (25.9 kDa, 240 mer, 3 Lysine residues) showed bands of ca. 8 

kDa and 6 kDa. MALDI-TOF/MS of the product showed dominant peaks at 8868, 9094, 5878. Both analyses 

were very consistent with each other, and well corresponded to the scission fragments at near K76, K169, K219. 

While, no decomposition occurred in the reaction of human elastase (218 mer) which involves no Lysine 

residue. 

The above facts indicates that Zn(II)-L1 reacts with a protein having a Lysine residue to form Schiff base 

between the CHO group of Zn(II)-L1 and the NH2 group of Lysine residues, selectively promotes the scission of 

the protein back bone at near Lysine site. 

References: 

[1] L.R.Croft, Handbook of Protein Sequence Analysis, 2nd edn., Wiley, Chichester (1968). 

[2] R.Beynon, J.S.Bond, Eds., Proteolytic Enzumes, 2nd edn., Oxford Unversity Press, New York (2001). 

[3] X.Chen, L.Zhu, H.Yan, X.You, N.M.Kostic, J. Chem. Soc., Dalton Trans., 2653-2658 (1996) 

_____________________________________________________________________ 

173


P55. Fac-Tc(Co)3 + And Fac-Re(Co)3 + Complexed by Histidine Derivatives - 

Potential Precursors of Radiopharmaceuticals 

L. Fuks a , E. Gniazdowska a , D. Papagiannopoulou b , P. Kozminski a , M. Papadopoulos c , 

M. Pelecanou c , I. Pirmettis c , C. Raptopoulou c , N. Sadlej-Sosnowska d , C. Tsoukalas c 

a Institute of Nuclear Chemistry and Technology, Warsaw, Poland 

e-mail: egniazdo@ichtj.waw.pl 

b School of Pharmacy, Aristotle University, Thessaloniki, Greece 

c National Centre of Scientific Research "DEMOKRITOS, Athens, Greece 

d National Medicines Institute, Warsaw, Poland 

Studies on the Tc(CO)3 + and Re(CO)3 + complexes in the last years have received special momentum since they 

give chance to prepare a series of novel radiopharmaceuticals [1]. Kinetic inertness of the tricarbonylrhenium(I) 

core containing cation of the d6 electronic configuration gives ground for the interest in its complexes, especially 

those obtained by substitution of two or three labile water molecules by bi- or tridentate chelating ligands. 

The aim of our studies is to find novel ligands, which form stable complexes with Tc(CO)3 + and Re(CO)3 + , that 

after conjugation to a biomolecule can be used for the development of 2 nd generation targeted 

radiopharmaceuticals. In the present paper we describe the synthesis and characterization of novel 

[Tc/Re(SNO)(CO)3] complexes where SNO is the 4-methoxybenzyl derivative of the 

3-(1H-imidazol-4-yl)-2-mercaptopropanoic acid - a tridentate SNO ligand, synthesized from L-histidine. 

The IR spectrum of the [Re(SNO)(CO)3] complex indicates the characteristic bands of three facially coordinated 

CO groups. 1 H NMR studies show typical pattern of diasterotopic protons of the SNO backbone after 

complexation. X ray analysis of the complex shows octahedral structure with SNO ligand coordinated in a 

tripodal fashion. The presence of complex diasteromers was also explored by quantum chemical calculations that 

resulted in two potential configurations. Quantum chemical calculations have shown that they can be related to 

different complex structures with comparable energy (∆ ≈ 0.3 and 0.4 kcal/mol for Tc I and Re I respectively). 

Successful labeling of the SNO ligand with suitable fac-[ 99m Tc/ 188 Re(CO)3] + cores led to the formation of stable 

complexes at tracer level, indicating the suitability of this system for the development of novel 

radiopharmaceuticals. 

Fig.: cis- and trans-configurations (left and right, respectively) of ligand complexing the Re I (CO)3 + moiety. 

Conclusion: 3-(1H-imidazol-4-yl)-2-[(4-methoxybenzyl)thio]propanoic acid is a potent chelator for the fac- 

[ 99m Tc/ 188 Re(CO)3] + core and it could be applied for the design of novel bioactive fac-[ 99m Tc/ 188 Re(SNO)(CO)3] 

complexes - potential diagnostic or therapeutic radiopharmaceuticals, respectively. 

Acknowledgement 

Part of the work was performed within the Maria Curie Transfer of Knowledge training contract MTKD-CT- 

2004-509224 (POL-RAD-PHARM: Chemical studies for design and production of new radiopharmaceuticals). 

References: 

[1] U. Abram, R Alberto, Technetium and Rhenium - Coordination Chemistry and Nuclear Medical 

Applications. J. Brazil. Chem. Soc., 2006, 17(8), 1486-1500. 

_____________________________________________________________________ 

174


P56. Complexes Containing the [99mTc(CO)3] + Core for the Targeted 

Radiotherapy 

L. Fuks a , K. Kothari a , M. Neves b 

a 

Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195, Warsaw, Poland 

e-mail: lfuks@ichtj.waw.pl 

b 

Technological and Nuclear Institute (ITN), Sacavem, Portugal 

99m Tc is the radionuclide of choice for nuclear medicine imaging by SPECT. Because of the nuclear properties, 

complexes containing the monovalent 99m Tc(CO)3 + core and chelating organic ligands received special attention 

as radiopharmaceutical precursors [1]. The aim of this study is to find novel ligands, which form stable 

complexes with the Tc(CO)3 + cation and after further functionalization could serve as precursors for the 

synthesis of radiopharmaceuticals of the 2 nd generation. 

In the presentation we show our preliminary results on application of the tricarbonyltechnetium(I) complexes as 

therapeutic agents. Recently it was found, that technetium-99m nuclide is an Auger electron emitter and if 

located in the close proximity of the cancer cell nucleus can induce the DNA strand breaks [2]. Such 

requirements fulfill group of compounds being so called ‘2+1' or ‘3+0' complexes (see, Scheme 1).Please 

prepare your abstract in English language. Leave 2.5 cm (top), 2.5 cm (bottom), 2.5 cm (left) and 2.5 cm (right) 

margins on the A4 page. 

Scheme 1. 99mTc(CO)3+ ‘2+1' complex containing the biguanide ligand 

Presented work describes our recent studies on rhenium (non radioactive congener of technetium) and 

technetium-99m complexes with the biguanide and thiourea derivatives as the ligands in order to prepare new 

agents for targeted radiotherapy. 

Acknowledgement: The work was undertaken within the Polish-Portuguese bilateral scientific agreement between 

the Institute of Nuclear Chemistry and Technology and Technological and Nuclear Institute (ITN) 

financed by Polish and Portuguese Ministries of Science and Higher Education. Polish Ministery of Science 

and Higher Education is also acknowledged for the grant 122/N-Portugal/2008/0. 

References: 

[1]. U. Abram, R Alberto, Technetium and rhenium - coordination chemistry and nuclear medical applications, 

J. Brazil. Chem. Soc., 2006, 17(8), 1486-1500. 

[2]. (a) P. Haefliger, N. Agorastos, A. Renard, G. Giambonini-Brugnoli, C. Marty, R. Alberto, Cell uptake and 

radiotoxicity studies of an nuclear localization signal peptide-intercalator conjugate labeled with [ 99m Tc(CO)3] + , 

Bioconjugate chemistry, 2005, 16(3), 582-587; (b) F. Marques, A. Paulo, M.P. Campello, S. Lacerda, R.F. Vitor, 

L. Gano, R. Delgado, I. Santos, Radiopharmaceuticals for targeted radiotherapy, Rad. Prot. Dosim., 2005, 

116(1-4 Pt. 2), 601-604. 

_____________________________________________________________________ 

175


P57. Metal Ions and Oxidative Stress in Parkinson’s Disease 

E. Gaggelli, D. Valensin and G. Valensin 

Departmentof Chemistry, University of Siena, Via A.Moro 2, Siena 53100, Italy. 

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, arising from the progressive 

loss of dopaminergic neurons in the substantia nigra pars compacta. 

In the surviving neurons, abnormal proteinaceous aggregates called Lewy bodies and Lewy neurites serve as 

neuropathological hallmarks of the disease. 

Point mutations in the a-synuclein (aS) gene cause rare forms of autosomaldominant familial PD, and wild-type 

aS is the major component of the pathologic lesions characteristic of spontaneous PD. 

aS is a 140 amino acid protein, which in solution adopts an ensemble of conformations that are stabilized by 

long-range interactions and act to autoinhibit oligomerization and aggregation. 

Reactive oxygen species are important in the pathogenesis of sporadic PD, since derangements in mitochondrial 

complex I clearly lead to aggregation and accumulation of aS, and other forms of oxidative stress promote aS 

aggregation. 

A possible link between abnormal copper homoeostasis and the onset of PD was suggested on the basis that 

copper(II) is the most effective metal ion in affecting selfoligomerization of aS. 

With the aim of delineating the role of Cu(II) in chemical and biochemical properties of aS, preliminary data will 

be herein presented that provide detailed structural delineation of the Cu(II) binding sites of aS. 

_____________________________________________________________________ 

176


P58. Synthesis and Characterization of Dibutyltin(IV) Complexes with 

O-donor Ligands Derivatives 

M. Gajewska, a M. F.C. Guedes da Silva, a, b A. J. L. Pombeiro a 

a 

Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, TU Lisbon, Av. Rovisco Pais, 

1049–001 Lisbon, Portugal 

e-mail: magigajewska@yahoo.co.uk 

b 

Universidade Lusófona de Humanidades e Tecnologias, ULHT Lisbon, Av. do Campo Grande, 376, 

1749-024, Lisbon, Portugal 

Among main group metal compounds organotin(IV) derivatives occupy a relevant position in view of their 

potential antitumour effects, since some of them show biological activity with relatively low toxicity [1, 2]. A 

large number of organotin(IV) derivatives with bidentate O-donor ligands, [1-7] including N-substituted 

hydroxamic acids, has been prepared and their in vitro antitumor activities (against a series of human tumor cell 

lines) which, in some cases, are identical to, or even higher than, that of cisplatin, are well recognized. Although 

mononuclear dibutyltin complexes, i.e. those with the organo-ligands having a long carbon chain, are the lead 

compounds in terms of activity [4, 5] the search for other tin compounds with improved solubility mainly in 

alcohols and/or even in water, is essential in view of their possible biological application. 

In pursuit of our interest [3, 4] on diorganotin(IV) acceptors and O-donor ligands and derivatives, we extended 

our studies to other hydroxamic-type molecules as well as N-, P- containing ligands. In our work we are using 

bifunctional hydroxamic acid, which combines both oxime and hydroxamic HON-groups in one molecule. The 

obtained compounds were characterized by FT-IR, 1 H, 13 C, and 119 Sn NMR. Evidence for the formation of 

polynuclear organotin derivatives will be presented. 

Acknowledgement: This work has been partially supported by the Foundation for Science and Technology 

(FCT), grant BPD/32522/2006 and its POCI 2010 programme (FEDER funded). 

References: 

[1] A.J. Crowe, Antitumour activity of tin compounds, in: S.P. Fricker (Ed.), Metal Compounds in Cancer 

Therapy, Chapman & Hall, London, 1994, pp. 147–179. 

[2] M. Gielen, Coord. Chem. Rev. 151 (1996) 41. 

[3] Q. Li, M.F.C. Guedes da Silva, A.J.L. Pombeiro, Chem. Eur. J. 10 (2004) 1456. 

[4] Q. Li, M.F.C. Guedes da Silva, Z. Jinghua, A.J.L. Pombeiro, J. Organometal. Chem. 689 (2004) 4584. 

[5] M. Gielen, App. Organomet. Chem. 16 (2002) 481, and references therein. 

[6] X. Shang, J.Cui, J.Wu, A. J.L. Pombeiro, Q. Li, J. Inorg. Biochem. 102 (2008) 901 

[7] X. Shang, J.Wu, A.J.L. Pombeiro, Q. Li, Appl. Organometal. Chem .21 (2007) 919 

_____________________________________________________________________ 

177


P59. Tetranuclear Pt II Complexes Prepared for DNA Binding Studies 

A. Galstyan a , W-Z. Shen a , E. Freisinger b , H. Alham a , B. Lippert a 

a Technische Universität Dortmund, Fakultät Chemie, Otto-Hahn-Str. 6, 44227 Dortmund, Germany 

b University of Zuerich, Institute of Inorganic Chemistry, Winterthurerstrasse 190, Zuerich CH-8057, 

Switzerland 

Non-covalent interactions between positively charged metal complexes (mono- or multinuclear) with negatively 

charged nucleic acids are a highly topical issue [1]. For example, Pt containing metallacalix[4]arenes, initially 

developed in our group, give rise to unprecedented conformational changes of DNA [2], and cyclic Pd trimers 

with bridging 1-methylcytosinato ligands [3] induce DNA coiling [4]. 

In line with these observations, we are interested in the effects of molecular architectures (triangles, vases, 

squares, boxes, containers etc) derived from metal ions (Pt II , Pd II ) and N-heterocyclic ligands, including 

nucleobases, on DNA structure and, eventually, function as well. Applying 2, 2’-bipyrazine (bpz) as a ligand and 

enPt II or enPd II as metal entities, we have previously charecterized a series of multinuclear complexes of 

different shapes[5]. More recently, we have obtained within the same system also tetranuclear open boxes of 

charge +8, e.g. {cis-[Pt(NH3)2(bpz)]4} 8+ with different counter ions. 

The synthesis, X-ray crystal structures and host-guest chemistry of these tetranuclear compounds with anions are 

reported. Work on their interactions with DNA by means of AFM is planned. 

Acknowledgment: This work was supported by the Deutsche Forschungsgemeinschaft and International Max- 

Planck Research School in Chemical Biology 

References: 

[1] See, e.g. (a) B. M. Zeglis, V. C. Pierre, J. K. Barton, Chem.Comm. 2007, 4565. (b) A. Olesky, A. G.Blanco, 

R. Boer, I. Uson, J. Aymami, A. Rodger, M. J. Hannon, M. Coll, Angew. Chem. Int. Ed. 2006, 45, 1227 

[2] M. A. Galindo, D. Olea, M. A. Romero, J. Gomez, P. del Castillo, M. J. Hannon, A. Rodger, F. Zamora, 

J. A. R. Navarro, Chem. Eur. J. 2007, 13, 5075. 

[3] (a) W.-Z.Shen, D. Gupta, B. Lippert, Inorg. Chem. 2005, 44, 8249. (b) W.-Z.Shen, B. Lippert, J. Inorg. 

Biochem. 2008, 102, 1134. 

[4] W.-Z.Shen, B. Lippert, unpublished results 

[5] R.-D. Schnebeck, E. Freisinger, F. Glahe, B. Lippert, J. Am. Chem. Soc. 2000, 122, 1381 and refs. cited. 

. 

_____________________________________________________________________ 

178


P60. Mixed-ligand M(II) Complexes with Malonate and Adenine 

I. García-Santos a , A. Castiñeiras a , J.M. González-Pérez b , J. Niclós-Gutiérrez b 

a 

Inorganic Chemistry, University of Santiago de Compostela, Faculty of Pharmacy, E-15782, Santiago de 

Compostela, Spain 

e-mail: qiisags@usc.es 

c 

Inorganic Chemistry, University of Granada, Faculty of Pharmacy, E-18071, Granada, Spain 

Adenine (Hade) can bind to metals in cationic (H2ade+), neutral (Hade) or anionic (ade-) forms. The malonate 

ligand (mal) can display both terminal (monodentate and chelating bidentate) and bridging coordination. Thus 

adenine and malonate have versatile coordinating behaviours leading from mono- to poly-nuclear species, with 

diverse nuclearity and dimensionality. By reaction of mal and Hade with different M(II) salts we have obtained 

compounds with a wide coordinating variety, depending on the metal: [Ni(mal)(Hade)(H2O)]2•2H2O (1), 

[Co(Hade)2(H2O)4][Co(mal)2(H2O)2].4H2O (2), [Cu(mal)(Hade)(H2O)]n, (3) (H2ade)2[Cu(Memal)2(H2O)](4) 

and [Zn(mal)(Hade) (H2O)]2 2H2O (5). In 1 (Figure) each metal atom is six-coordinated and 

both mal and Hade act as bridging ligands, thus contributing to the binuclear structure. Noteworthy Hade acts as 

a µ-N3, N9(H(N7))- bridge. This complex can be used like a core to its controlled-expansion, for example by 

addition of two ML chelates, after the dissociation of both Hade to remain as µ -N3, N7, N9-ade- bridges. The 

degree of protonation and possibilities of different tautomeric forms in Hade and additional water molecules 

present in the complexes favour the formation of many intra- and inter-molecular H-bonding interactions and a 

strong three-dimensional network is formed. In 2 and 3 there are π, π-stacking interactions that further reinforce 

their crystals. 

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179


P61. Peroxidase from Royal Palm Tree Roystonea Regia: Structure and 

Stability 

O. Gavel a , L. Zamorano b , L. Sanz c , J. Calvete c , S. Bursakov a , A. Zhadan d , J. Arellano e , 

M. Roig b , I. Polikarpov f , V. Shnyrov g 

a 

Química, REQUIMTE, CQFB/FCT, Universidade Nova de Lisboa, Quinta da Torre, 2829-516, Caparica, 

Portugal 

e-mail: olga.gavel@dq.fct.unl.pt 

b 

Química Física, Facultad de Química, Universidad de Salamanca, , 37008, Salamanca, Spain 

c 

Instituto de Biomedicina, C.S.I.C., Jaime Roig 11, E-46010, Valencia, Spain 

d 

Institute for Biological Instrumentation of the Ru, 142290, Pushchino, Moscow region, Russia 

e 

Instituto de Recursos Naturales y Agrobiologia, C., 37008, Salamanca , Spain 

f 

Instituto de Física de São Carlos, Universidade, SP CEP 13560-970, Brazil, 

g 

Bioquímica y Biología Molecular, Universidad de Salamanca, Plaza Doctores de la Reina, 37007, Salamanca, 

Spain 

Heme-binding peroxidases (EC 1.11.1.) carry out a variety of biosynthetic and degradative functions using 

hydrogen peroxide as the electron acceptor. In this work we present data about primary structure (amino acid 

sequence, carbohydrate composition and places of its binding) together with results of structural stability study 

of dimeric peroxidase from leafs of royal palm tree Roystonea regia (RPTP). The sequence of peroxidase is quite 

conserved and attains 55-61 % identity with Oryza sativa (Rice), Zea mays (Maize), Vitis vinifera (Grape) and 

Spinacia oleracea (Spinach). The structural stability of the peroxidase was studied by differential scanning 

calorimetry circular dichroism and steady state tryptophan fluorescence. The thermal and chemical 

folding/unfolding of royal palm peroxidase (RPTP) at pH 7 is reversible processes that involve a highly 

cooperative transition between folded dimer and unfolded monomers with very high value of free energy 

stabilization -near of 23 kcal per mol of monomer- at 25 oC. At pH 3 where ion pairs have disappeared due to 

protonation, thermally induced denaturation of RPTP is irreversible and strongly dependent upon scan rate, 

suggesting that this process is under kinetic control. Thermodynamic information was extracted in this case by 

extrapolation kinetic transition parameters to infinite heating rate. Obtained in this manner value of RPTP 

stability at 25 oC is ca. 8 kcal per mole of monomer lower than at pH 7. With a big reliability this quantity 

reflects contribution of ion pair interactions in the structure stability of RPTP. From comparison of RPTP 

stability with other plant peroxidases it was proposed that responsible for unusual high stability of RPTP that 

enhance its potential use for biotechnological purposes is its dimerization. 


This work was partially supported by the projects SA-06-00-0 ITACYL-Universidad de Salamanca and SA 

129A07 (Junta de Castilla y León) and BFU2004-01432 (Ministerio de Educación y Ciencia) Spain. L.S.Z and 

O. G. are fellowship holders from Junta de Castilla y León, Spain (Ref. EDU/1490/2003) and from Fundação 

para a Ciência e a Tecnologia, Portugal (Ref. SFRH/BPD/28380/2006), respectively. 

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180


P62. Preparation of Ethyl β-arylamino Crotonates Using Preyssler 

Heteropolyacids Preyssler”s Acid H14NaP5W30O110 , H3PMo12O40 and 

H14NaP5W29 MoO110 and its supported as catalysts 

A. Gharib a, b* , M. Roshani a , M. Jahangir a 

a b 

Department of Chemistry , School of Sciences, Islamic Azad University, Mashhad, Iran Agricultural 

Researches&Services center, Mashhad, Iran 

e-mail: aligharib5@yahoo.com 

The catalysts based on heteropolyacids types and related compounds are less corrosive and produce lower 

amount of wastes than conventional acid catalysts, so they can be used as replacement in ecofriendly 

processes.The HPA were supported on several carriers in order to use these catalysts in heterogeneous liquid 

reactions[1], with the advantage of easy product recovery. 

On the other hand, 4-quinolones are important compounds and valuable synthetic 

Intermediates for derivatives that have biological activities belonging to various types, e.g.tuberculostatic [2]. 

Some , 4-quinolones are used as antibacterials 3 , e.g. ciprofloxacine and other 6-fluoroquinolones.The Conrad- 

Limpach reaction between anilines and a β-ketoester is a general method to synthesize , 4-quinolones [3]. 

The aromatic amines react with methyl acetoacetate yielding alkyl β-arylaminocrotonates, acetoacetanilides, 

diphenylureas or , 4-quinolones , depending on the temperature, solvent and molar ratio of the reactants [4]. 

These compounds can be cyclized , 4-quinolones by heating in diphenyl ether. 

R 1 

R 2 

R 3 

+ 

NH 2 

O 

O 

H 3C OC 2H 5 

preyssler catal. 

R 1 

R 2 

R 3 

N 

H 

CH 3 O 

OC 2H 5 

Refrences: 

[1] L. Pizzio, C. Caceres, M. Blanco, Appl. Catal. A: General 167, 283 (1998) . 

[2] U. Holzgrabe, M. Steinert, Pharmazie. 56, 11 (2001). 

[3] W. Werner, Tetrahedron 25, 255 (1969). 

[4] B. Reddy, synth.commun. 29, 2789 (1999). 

_____________________________________________________________________ 

181 

R 1 

R 2 

O 

R 3 N H 

CH 3


P63. Synthesis and Structure af a Thiosemicarbazonecopper(II) Complex 

with Cytosine 

R. Gil-Garcia, a B. Donnadieu, b J. Garcia-Tojal a 

a 

Department of Chemistry, University of Burgos, Pza. Misael BaĂąuelos s/n, 9001 Burgos, Spain 

e-mail: rgil@beca.ubu.es 

b 

Department of Chemistry, University of California, Riverside, CA 92521, USA 

Thiosemicarbazones and their metal complexes raise a great interest due to their ability of inhibit the DNA 

synthesis. Although these compounds have been widely investigated, as far as we are aware, there is no evidence 

of structures with thiosemicarbazonemetal entities and nucleobases. 

Here we present a new thiosemicarbazonecopper(II) complex with cytosine [CuL(Hcyt)](ClO4) that contains 

[CuL(Hcyt)] + monomeric units and perchlorate counterions. The two crystallographically independent cationic 

entities present in the asymmetric unit are related through a pseudo center of inversion, as it is shown in 

Figure 1. The coordination around the metal center shapes a distorted square–planar topology. The 

thiosemicarbazone ligand exhibits the usual NNS tridentate behavior, while the cytosine links to the copper(II) 

ion by the N13 atom. Notwithstanding, there is a pseudocoordination (4+1) between the metal centre and the 

oxygen of the cytosine. 

The title structure proves the affinity of the [CuL] + cations for cytosine nucleobases. On the other hand, the 

existence of multiple non-covalent interactions spreads the possibilities for the interaction of 

pyridine-2-carbaldehyde thiosemicarbazonatocopper(II) entities with nucleotides and DNA, including mono- or 

polynuclear π-stacking and anion-π interactions with phosphate groups. In addition, other modes of action 

cannot be discarded, e. g. coordination to other nucleobases or even phosphates. 

_____________________________________________________________________ 

182


P64. Hydrogen Production by Hydrogenases in the Presence of Oxygen 

G. Goldet, F. Armstrong 

Inorganic Chemistry Laboratory, Univerity of Oxford, South Parks Road, OX1 3JP, Oxford, United Kingdom 

e-mail: gabrielle.goldet@chem.ox.ac.uk 

Investigating biological, photosynthetic H2 production is a complex process and the subject of intense, exciting 

research.[1] The question is: could biological systems be harnessed in a future technology for solar energy 

capture and fuel production? The hydrogenases are a family of metalloenzymes which reversibly catalyse H2 

from water and electricity.[2] As one of the terminal electron acceptors in the photosynthetic apparatus, they 

produce H2 by reducing protons with electrons harvested from light-activated water splitting. Elechtrochemistry 

has been used to probe and refine our understanding of H2 production by hydrogenases and to investigate the 

capacity for H2 production in the presence of O2, H2 and CO[3, 4] by quantifying any resulting inhibition, all 

physiologically relevant inhibitors. Hydrogenases vary in their tolerance to O2 depending on the metals present 

in their bimetallic active site; the so-called [NiFe]-hydrogenases are generally thought to be more tolerant to O2 

whilst the [FeFe]-hydrogenases are permanently damaged by O2. A novel electrochemical experimental design 

has been used to show that both types of hydrogenase are capable of H2 production in the presence of O2, [3, 4] 

activity which was previously considered to be impossible in the case of the [FeFe]-hydrogenase. This is thus the 

first proof that hydrogenases have this capacity. These findings open up a whole new area of electrochemical 

study of H2 evolution and give hope for technological applications of hydrogenase molecules in their native 

environments or inorganic hydrogenase mimics as biological H2 producers. 

References: 

[1] [1] M.L. Ghirardi, M.C. Posewitz, P.-C. Maness, A. Dubini, J. Yu, and M. Seibert, Annu. Rev. Plant Biol., 

2007, 58, 71 

[2] K.A.V Vincent, A. Parkin, and F.A. Armstrong, Chem. Rev., 2007, 107, 4366 

[3] G. Goldet, A.-M. Wait, J.A. Cracknell, K.A. Vincent, M. Ludwig, O. Lenz, B. Friedrich, and F.A. 

Armstrong, J. Am. Chem. Soc., Accepted, 15/05/08 

[4] A. Parkin, G.Goldet, C. Cavazza, J. Fontecilla-Camps and F. A. Armstrong, J. Am. Chem. Soc. Submitted 

16/05/08 

_____________________________________________________________________ 



P65. Nitrate reduction by periplasmic nitrate reductase from Desulfovibrio 

desulfuricans 

P. J. González a , S. Najmudin a , J. Trincão a , C. Coelho a , A. Mukhopadhyay a , C. C. Romão b , 

M. J. Romão a , C. D. Brondino c , I. Moura a and J. J. G. Moura a 

a 

Departamento de Quimica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica 

2829-516, Portugal 

e-mail: pablo.gonzalez@dq.fct.unl.pt 

b 

Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, Oeiras, Portugal 

c 

Departmento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 

Santa Fe 3000, Argentina 

The periplasmic nitrate reductase (Nap) from Desulfovibrio desulfuricans ATCC 27774 is a molybdenumcontaining 

enzyme from the DMSO reductase family. Recently, it was reported that the Mo ion at the active site 

is coordinated by six sulfurs without any OH/H2O molecule directly bound to the Mo ion [1, 2]. EPR 

spectroscopy was used to corroborate this key result that determines that Naps would catalyze the nitrate 

reduction different to Nar and Euk-NR. Several EPR active Mo(V) species were identified and its role in 

catalysis was analyzed [3]. The finding of a new paramagnetic Mo(V) species of the enzyme obtained in 

catalytic conditions (turnover species) was used to study the oxidation state and coordination environment of the 

Mo-site before it interacts with the substrate. 

Refrences: 

[1] Najmudin et al. J Biol Inorg Chem 2008, 13(5):737-753. 

[2] Dias et al. Struct Fol Des 1999, 7, 65-79. 

[3] González et al. J Biol Inorg Chem 2006, 11(5):609-616. 

_____________________________________________________________________ 

184


P66. A Family of Ternary Copper(II) Complexes 

with Cu-N9(H(N7)hypoxanthine) Coordination Bond 

J. M. González-Pérez a , D. Kumar Patel a , D. Choquesillo-Lazarte b , A. Castiñeiras c , I. García- 

Santos c , J. Niclós-Gutiérrez a 

a 



e-mail: jmgp@ugr.es 

b 



c 



N9 is believed to be the most basic donor atom of hypoxanthine (H(N9)hyp). In crystals, various coordination 

modes are known for Hhyp: M-N7(H(N9)hyp), Cu II -µ2-N3, N7(H(N9)hyp)-Cu II and M II -µ2-N3, N9(H(N7)hyp)- 

M II . However, any structural evidence exists for complexes with only a metal-N9(Hhyp) bond. We report 

structures of five novel Cu II -(IDA-like)-Hhyp compounds with N-methyl-, N-benzyl-, N-(p-Fbenzyl)- and, Nphenethyl-IDA 

or p-xylylene-di(IDA) ligands: [Cu(MIDA)(Hhyp)(H2O)]·H2O 1, [Cu(NBzIDA)(Hhyp)]n 2, 

[Cu(FBIDA)(Hhyp)(H2O)]·2.5H2O 3, [Cu(pheida)(Hhyp)(H2O)] ·2H2O 4 and [Cu2(p- 

XDTA)(Hhyp)2(H2O)2]·2H2O 5 (see Figure), respectively. These compounds feature are 4+1 Cu(II) coordination 

and a Cu-N9(H(N7)hyp) coordination bond, but not an intra-molecular interligand N-H···O(IDA-like) interaction 

[1], because the tautomer H(N7)hyp has not the required N3-H moiety. 

Reference 

[1] D. Choquesillo-Lazarte, M.P. Brandi-Blanco, I. García-Santos, J. M. González Pérez, A. Castiñeiras, J. 

Niclós-Gutiérrez, Coord. Chem. Rev. 252 1241-1256 (2008). 

_____________________________________________________________________ 

185


P67. Interaction of Cu(II) Ions and Neurotoxic Fragment of Chicken Prion 

Peptide 

E. Gralka, a D. Valensin, b G. Valensin, b W. Kamysz, c H. Kozłowski a 

a 

Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, 

Poland 

e-mail: ewagral@wcheto.chem.uni.wroc.pl 

b 

Dipartimento di Chimica, Università di Siena, Siena, Italia 

c Faculty of Pharmacy, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416 Gdańsk, Poland 

Prion proteins are a group of pathogenic glycoproteins which have several characteristic features: 

a highly helical and ordered C-terminal domain, a disordered 

N-terminal domain composed of multiple repeat units and hydrophobic neurotoxin fragment. Residue 106-126 

has been shown to be highly fibrillogenic, resistant to proteinase K and toxic to neurons [1]. 

Recent studies brings evidence that prion proteins (PrP) are involved in the Cu(II) metabolism. In contrast to 

mammalian PrP avian prion proteins have a considerably different N-terminal copper binding region and most 

interestingly they are not able to undergo the conversion proces into an infectious isoform. 

The unstructured region between the N-terminal domain and the structured C-terminal domain may play an 

important role in amyloid formation and infectivity in prion desises. A presence of Cu(II) ions could have 

profound implication in the structure of this PrP region. 

There are secondary and tetriary structural similarity in the C-terminal domain between mammalian (hPrP) and 

avian (chPrP) prion proteins dispite the low overall sequence identity (30%).The affinity, selectivity and Cu(II) 

coordination of the hPrP and chPrP repeat domain are now well determine[2]. 

Definitely less information is available about coordination ability of the region called fifth Cu(II) binding site, 

i.e. a fragment comprising His96, His111 in hPrP and His110, His 124 in chPrP. The affinity of His96 and 

His111 for Cu(II) is similar to each other, although His111 seems to display higher affinity to metal ions [3]. 

Coordination of Cu(II) ions to chPrP peptides is not yet well established [4]. 

The aim of this study is to obtain speciation, affinity, bonding details and conformational features of chPrP 

Cu(II) complexes. 

References: 

[1] B. Belosi, E. Gaggelli, R. Guerrini, H. Kozłowski, M. Łuczkowski, F.M. Mancini, M. Remelli, 

D. Valensin, G. Valensin. ChemBioChem 2004, 349-359 

[2] P.Stańczak, M. Łuczkowski, P. Juszczyk, Z. Grzonka, H. Kozłowski. Dalton. Trans. 2004, 2102-2107 

[3] P. Davis, D. R. Brown, Biochem. J. 2008, 237-244 

[4] G. Di Natale, G. Grasso, G. Impellizzeri, D. La Mendola, G.Micera, N. Mihala, Z. Nagy, K. Osz, 

G. Pappalardo, V. Rigo, E. Rizzarelli, D. Sanna, I. Sovago. Inorg. Chem, 44, 2005, 7214-7225. 

_____________________________________________________________________ 

186

P68. [3Fe-4S] Cluster Reduced States 

Electrochemical and EPR Studies 

R. Grazina, P. P. Sousa, M. Carepo, I. Moura and J. J. G. Moura 


REQUIMTE, Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e 

Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 

e-mail: raquel.grazina@dq.fct.unl.pt 

Ferredoxins are simple iron-sulphur proteins that contain prosthetic groups composed of iron and sulphur atoms 

and they play a functional role in electron transfer processes relevant for sulphate-reducing bacteria (SRB) 

metabolism. In particular, Desulfovibrio gigas ferredoxin II (DgFdII) is a small tetrameric protein of 58 amino 

acids which contains a single [3Fe-4S] cluster and a redox active internal disulfide bridge [1, 2]. Electrochemical 

tools can provide important information for the understanding of the redox and mechanistic/structural role of Fe- 

S clusters and to the interconvertion process occurring between 3 Fe and 4 Fe clusters, as well as to the effect of 

the addition of an extra metal to form heterometal clusters of the type [M3Fe-4S] ) [3]. From direct 

electrochemistry (cyclic voltammetry and differential pulse voltammetry) the DgFdII presents two distinct 

electrochemical signals correspondent to the following transitions: [3Fe-4S] +1/0 and [3Fe-4S] 0/-2 . Concerning the 

electronic properties of the [3Fe-4S] cluster these have been also explored by EPR spectroscopy and besides the 

two sates already assigned in the literature for the oxidized state [3Fe-4S] +1 (S = 1/2 and g = 2.02) [4] and for the 

reduced state [3Fe-4S] 0 (S = 2 and EPR transition around g = 12) [5, 6], a new reduced state was also achieved 

using titanium(III) citrate, as reductant, and EPR signals at very low magnetic fields were observed. This further 

reduced state is also being attempted to be generated by bulk electrochemical methods. 

Acknowledgments: We thank FCT-MCTES for financial support. 

References: 

[1] Moura et al., Methods in Enzymology, Vol. 243, 166-188 (1994), Peck, HD and LeGall, J, Ed., AP. 

[2] Moura et al., in Methods in Enzymology, J.H.D. Peck and J. LeGall Editors (1994). 

[3] Moreno et al., J. Inorg. Biochem., 53, 219 (1994). 

[4] Huynh et al., J. Biol. Chem., 255, 3243 (1980). 

[5] Papaefthymiou et al., J. Am. Chem. Soc., 109, 4703 (1987). 

[6] Bush et al., Biochem. J., 323, 95 (1997). 

_____________________________________________________________________ 

187


P69. Adsorption Studies of Famotidine on the Sodium Starch Glycolate – in 

vitro and DSC 

B. Grimling a , A. Górniak b , J. Pluta a , J. Świątek-Kozłowska b 

a 

Department of Drug Form Technology, Wroclaw Medical University, Szewska 38, 50-139 Wroclaw, Poland 

email: agata.gorniak@interia.pl 

b 


Drug excipient interactions are among the most important factors that should be considered in any 

preformulation study. Many reports in the last few decades showed that excipients can physically or chemically 

interact with drug substances either in the solid state or liquid state. Adsorption is one of the most important 

mechanisms of interaction between drugs and excipients [1]. The forces involved in this type of interaction can 

be physical or chemical in nature, or a combination of both. Physical interaction occurs to some extent in all 

systems and is primarily due to weak van der Waals` attraction forces. In some systems, however, stronger 

electrostatic attractions can be involved. Chemical interaction is more specific and is primarily attributed to 

covalent bond formation and occurs only when chemical interaction between the drug and excipient is possible. 

Sodium starch glycolate (SSG) is a cross-linked substituted potato starch - sodium salt of carboxymethyl ether of 

starch which is widely used in oral pharmaceuticals as a disintegrant in capsule and tablet formulations [2]. 

Famotidine (Fig.1) is an active compound of the pharmaceutical formulation. It competitively inhibits the action 

of histamine on the H2-receptors of parietal cells and reduces gastric acid secretion under daytime and nocturnal 

basal conditions. 

Fig.1 Structural formula of famotidine (isopropyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropionate) 

In the present study we have decided to investigate the character of the interactions between sodium starch 

glycolate - excipient and famotidine used in the treatment of peptic ulcers and related disorders, taking into 

account certain physicochemical factors. 

Adsorption of drug on polymer was investigated by means of a static method. The findings of the amount of the 

drug bound on SSG were used to determine Langmuir adsorption isotherms [3]. The chemical analysis of drug - 

polymer interactions was determined by means of qualitative interpretation of thermograms obtained during 

thermochemical assessment by means of DSC. The findings indicate that famotidine is adsorbed on polymer in 

all applied pH ranges, and the capability of binding depends on polymer its swelling properties of the polymer, 

which increase with increasing alkaline pH of the environment. 

The incompatibilities were detected by appearance, shift or disappearance of peaks in the DSC thermograms. 

The DSC thermogram of pure famotidine showed a sharp endotherm maximum melting point at 166°C. The 

excipient sodium starch glycolate exhibited broad endothermal peak with maximum temperature at 127°C. The 

preliminary DSC curves showed that while the thermal effects of either individual components (or eutectic 

mixture?) were present for the physical mixture, on the DSC thermograms of complexes these melting 

endotherms were absent. For both precipitates, prepared at pH=1.5 and pH=7.6, on the DSC curves only one 

shallow broad exothermic peak or broad endothermal "step" respectively was observed. The presence of these 

single thermal effects may point to significant interactions between the components of the mixture and it may 

prove formation of complexes bound between them. These interactions probably result from ion-dipol reactions 

between the substances and formation of hydrogen bonds of internal salt between the carboxyl groups of the 

polymer and amine groups of the drug. 

References: 

[1] W. X. Huang, Int. J. Pharm., 33, 311 (2006) 

[2] S. Edge, A. M. Belu, U. J. Potter, D. F. Steele, P. M. Young, R. Price, J. N. Staniforth, Int. J. Pharm., 240, 67 

(2002) 

[3] B. Grimling, J. Pluta, Macromol. Symp., 253, 186 (2007) 

_____________________________________________________________________ 

188


P70. Compatibility Study Between Sulfasalazin and Sucralfat in vivo Using 

HPLC 

B. Grimling, J. Meler, J. Pluta 

Dispensing Pharmacy of Wroclaw Medical University, Szewska 38/39, 54-139, Wrocław, Poland, 

The need of parallel treatment of two or more affections and the resulting necessity of using a few drugs from 

different pharmacological groups creates the danger of interactions witch may decrease effectiveness of the 

applied therapy. Sucralfat is a complex salt of saccharose octosulfate with apparent adsorption qualities. The 

proximity of a weak acid such as sulfasalazin makes it possible to assume an interaction based on adsorption of 

this medicine on the surface of sucralfat polyanions. In the first study, four healthy male volunteers received a 

single, oral 500-mg dose of salicylazosulfapyridine three times a day. In the second study, six patients receive 

oral 500 mg oral dose salicylazosulphapirydine three times a day and Sucralfate 1g one once a day. 

In study 1, urine was collected just before dosing and in 24-hour blocks for at least 1 day after administration of 

Sulfasalazin. In study 2, all urine collected for 24 hours after Sulfasalazin and Sucralfate[1]. The concentration 

of sulfasalazin (salicylazosulfapyridine, SSA) and its metabolites: 5-aminosalicylic acid (5-ASA), acetyl-5 - 

aminosalicylic acid (Ac-5-ASA), sulphapyridine (SP), acetylsulphapyridine Ac-SP) was determined by a 

reverse-phase HPLC (Gilson) using Zorbax ODS C-18 (250mm×4.6mm, 5µm) column. The mobile phase 

consisted of 25% methanol in 5mM phosphate buffer (pH 6.0) containing 0.5mM tetrabutylammonium chloride, 

which was filtered through 0.45 µm membrane filter before use. Samples (20 µl) were injected and eluted with 

the mobile phase at a flow rate of 2, 0 ml/min. The eluate was monitored at 257nm at sensitivity of AUFS 0.001. 

The retention times of 5-ASA was, SP and SP was 8, 49, SAS was 12, 01, Ac-SP was 15.03 and Ac-5-ASA was 

15.75 min, respectively.The observed differences of concentrations in dependence from applied therapy were 

extremely essential statistic, on level of significance and = 0, 001(the p < 0, 001). This analysis, that the fall of 

concentration of higher described metabolite in urine during applying the therapy associated it is not the random 

phenomenon treat with rule. It the analysis of introduced data was affirmed was, that presence sucralfat in 

associated from sulfasalasine therapy reduces concentration sulfasalasine as well as her two metabolite in studied 

patients' urine[2]. 

References: 

[1]. Jung, Y.J., Kim, H.H., Kong, H.S., Kim, Y.M., 2003. Synthesis and properties of 5-aminosalicyl-taurine as a 

colon-specific prodrug of 5-aminosalicylic acid. Arch. Pharm. Res. 26, 264-269. 

[2]. Schroder, H., Campbell, D.E., 1972. Absorption, metabolism, and excretion of salicylazosulfapyridine in 

man. Clin. Pharmacol. Ther. 13, 539-551. 

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189


P71. Interaction of Ruthenium and Gold Derivatives of H2TMPyP 4+ 

(Ru(II)TMPyP 4+ and Au(III)TMPyP 5+ ) with DNA 

M.D. A. Habib a , M. Tabata b 

a 

Department of Chemistry, University of Dhaka, Dhaka, 1000, Dhaka, Bangladesh 

e-mail: mahabibbit@yahoo.com 

b 

Chemistry, Saga University, Saga, Saga, Japan 

We investigated the interaction of free base porphyrin, tetrakis(1-methylpyridium-4-yl)porphyrin (H2TMPyP 4+ ), 

and its metallo-derivatives of ruthenium(II) and gold(III) with DNA using UV-vis, fluorescence and circular 

dichroism (CD) spectroscopy at 0.1 M NaCl, 7.5 pH and 25 °C. The results indicated that Ru(II)TMPyP 4+ 

interacted with DNA via outside binding with self-stacking manner. This is because the UV-vis data indicated 

that a small red shift (∆λ = 3 nm) and a minimal hypochromicity (10%) were observed upon addition of DNA. 

Moreover, the CD spectra of DNA indicated that a new peak was developed at the Soret region upon the addition 

of the Ru(II)TMPyP 4+ . However, in the case of Au(III)TMPyP 5+ , a significant hypochromicity (55%) was 

observed at high concentration (5.0x10-4 M bp) of DNA but a small red shift (∆λ = 4.5 nm) was observed. 

Moreover, the CD results indicated the development of positive and negative peaks at the Soret region during the 

titration of DNA with Au(III)porphyrin. Therefore, both the spectroscopy results indicated that Au(III)TMPyP 5+ 

interacted with DNA as outside binding with a partial intercalation. On the other hand, the free base porphyrin, 

H2TMPyP 4+ , interacted with DNA as intercalation because the UV-vis results indicated a significant 

hypochromicity (30%) and a large red shift (∆λ = 20 nm). In addition, the CD results also indicated only a 

negative peak was developed at the Soret region during the titration of DNA with the free base porphyrin. 

Fluorescence spectroscopy results indicated that initially the aggregated porphyrins de-aggregate and then 

interacted with DNA upon addition of DNA. This phenomenon has been confirmed with the fluorescence 

experiments of the porphyrins in the presence of different concentrations of NaCl and ethanol. 

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190


P72. Functionalized Cytotoxic Ruthenium(II) Arene Complexes 

A. Habtemariam a , P.C.A. Bruijnincx b , P.J. Sadler b 

a Chemistry, University of Warwick, Gibett Hill Road, CV4 7AL, Coventry, United Kingdom 

e-mail: a.habtemariam@warwick.ac.uk 

b Chemistry, University of Warwick, Gibett Hill Road, CV4 7AL, Coventry, United Kingom 

Organometallic half-sandwich Ruthenium(II) arenes of the type [(η6- arene)Ru(YZ)(X)]+ where YZ is 

typically a chelating diamine ligand (e.g. ethylenediamine, en) and X is a halide (e.g. Cl) exhibit anticancer 

activity in vitro and in vivo.[1, 2] 

A number of design features are inherent in these classes of compounds. Systematic variations of the 

coordinated arene, and the mono- and bidentate ligands can result in complexes having preselected, desirable 

features. A number of organometallic Ru(II) arene complexes bearing versatile functional groups, are being 

synthesised in our laboratory with a view to generate bimetallic and hetero-bimetallic complexes. In addition 

these transformations may enable us to explore the possibility of tuning the selectivity of the compounds in their 

interaction with biomolecules by attaching an appropriate probe. The inclusion of specific markers such as 

fluorescent dyes may aid in studies of the biodistribution of complexes. The results of these studies will be 

discussed in this presentation. 

Acknowledgement: We thank NWO (fellowship for PCAB for support and members of COST Action D39 for 

discussion. 

References: 

[1] Habtemariam, A., Melchart, M., Fernandez, R., Parsons, S., Oswald, I. D. H., Parkin, A., Fabbiani, F. P. A., 

Davidson, J. E., Dawson, A., Aird, R. E., Jodrell, D. I., Sadler, P. J. (2006) J. Med. Chem. 49, 6858-6868. 

[2] Yan, Y. K., Melchart, M., Habtemariam, A., Sadler, P. J. (2005) Chem. Commun., 4764-4776. 

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191


P73. A Near-atomic Resolution Crystal Structure of Melanocarpus 

Albomyces Laccase 

N. Hakulinen a , J. Kallio a , M. Andberg b , A. Koivula b , K. Kruus b , J. Rouvinen 

a 

Dept. of Chem. , University of Joensuu, Yliopistokatu 7, 80100, Joensuu, Finland 

e-mail: nina.hakulinen@joensuu.fi 

b 

VTT Technical Research Centre of Finland, PO Box 1000, 02044 , Espoo, Finland 

Laccases (E.C. 1.10.3.2, p-diphenol dioxygen oxidoreductases) are redox enzymes that use molecular oxygen to 

oxidize various phenolic compounds. They share the arrangement of the catalytic sites with other blue multicopper 

oxidases including ascorbate oxidase, ceruloplasmin, CueO, and Fet3p. For catalytic activity, four copper 

atoms are needed: one type-1 (T1) copper forming a mononuclear site, one type-2 (T2) copper and two type-3 

(T3 and T3´) coppers forming a trinuclear site. Reducing substrates are oxidized near the mononuclear site and 

then electrons are transferred to the trinuclear site, where dioxygen is reduced to water. 

Melanocarpus albomyces is an ascomycete fungus expressing a thermostable laccase with a neutral pH optimum. 

The three-dimensional structure of M. albomyces laccase (MaL) at 2.4 Å was solved among first complete 

laccase structures [1]. In MaL structure, dioxygen was observed at the first time inside the trinuclear site. At the 

present, laccase structures show wide variety of trinuclear site geometries having one or two oxygen atoms. Our 

recent studies have shown that the trinuclear site of laccase is sensitive to X-rays and the observed structure may 

depend on the data collection strategy or the intensity of the beam [2]. 

We have recently solved the three-dimensional structure of recombinant M. albomyces laccase (rMaL) at 1.3 Å 

resolution [3]. At the moment, this is the highest resolution that has been attained for any multicopper oxidase. 

This structure confirmed our earlier proposal regarding the dynamic behaviour of the trinuclear site and it 

allowed us to describe important solvent cavities of the enzyme. T2 and T3 solvent cavities, and a putative SDSgate, 

formed by Ser142, Ser510 and the C-terminal Asp556 of rMaL, were found. We also observed a 

2-oxohistidine, an oxidized histidine, possibly caused by a metal-catalysed oxidation by the trinuclear site 

coppers. To our knowledge, this is the first time that 2-oxohistidine has been observed in a protein crystal 

structure. 

References: 

[1] Hakulinen, N., Kiiskinen, L.-L., Kruus, K., Saloheimo, M., Paananen, A., Koivula, A. and Rouvinen J. 

(2002) Nature Structural Biology 9, 601 

[2] Hakulinen, N., Kruus, K., Koivula, A. and Rouvinen, J. (2006) Biochem. Biophys. Res. Comm. 350, 929 

[3] Hakulinen, N., Andberg, M., Kallio, J., Kruus, K., Koivula, A. and Rouvinen, J. (2008) J. Struct. Biol. 

162, 29 

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192


P74. Cytotoxic Properties, DNA and Glutathion Interactions of New 

Lipophilic trans-platinum Complexes Tethered to 1-adamantylamine 

A. Halamikova a , P. Heringova a , J. Kasparkova a , V. Brabec a , G. Natile b 

a 

Institute of biophysics ASCR, Kralovopolska 135, CZ-612-65, Brno, Czech Republic 

e-mail: halamikova@ibp.cz 

b 

Department of Pharmaceutical Chemistry, University of Bari, , I-70125, Bari, Italy 

Platinum-based anticancer compounds are a clinically successful group of therapeutic agents. In spite of the fact 

that they belong to the world’s best selling anticancer drugs, their use is constrained by dose-limiting side-effects 

and the problem of acquired resistance. To avoid these problems, for over three decades, continuous efforts have 

been made with a primary focus on the development of new platinum drugs and mechanisms underlying their 

antitumor effects. 

Cytotoxicity and mutagenicity of new platinum complexes trans, trans, trans-[PtCl2(CH3COO)2(NH3)(1adamantylamine)] 

and its reduced analog trans-[PtCl2(NH3)(1-adamantylamine)] were examined. In addition, 

several factors underlying biological effects of these trans-platinum complexes, such as drug accumulation in 

cells, DNA binding and deactivation of complexes by sulfur-containing compounds, using various biochemical 

methods were investigated. 

A notable feature of the growth inhibition assays using human cancer cell lines was the remarkable 

circumvention of both acquired and intrinsic resistance of conventional cisplatin by the two new lipophilic transplatinum 

compounds. The results also suggest that the pharmacological factors, such as enhanced accumulation 

of the drug in cells, altered DNA binding mode and deactivation of the trans-[PtCl2(NH3)(1-adamantylamine)] 

by glutathione, are likely to play a significant role in the mechanism of the biological action of these new transplatinum 

complexes. 

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193


P75. Structural Evidence for the Catalytic Mechanism of Nitrile Hydratase 

Proposed by Time-resolved X-ray Crystallography Using a Novel 

Substrate, tert-butylisonitrile 

K. Hashimoto a , H. Suzuki b , K. Taniguchi c , M. Yohda a , T. Noguchi b , M. Odaka a 

a 

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 

Naka-cho, 184 8588, Koganei, Tokyo, Japan 

e-mail: kounda@bel.bio.tuat.ac.jp 

b 

Institute of Materials Science, University of Tsukuba, 1-1-1 Tennohdai, 305 8573, Tsukuba, Ibaraki, Japan 

c 

Eco-Soft Material Research Units, RIKEN, Wako, Saitama, 2-1 Hirosawa, 351 0198, Wako, Saitama, Japan 

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides. NHase of Rhodococcus sp. N771 has a 

non-heme Fe catalytic center with two oxidized Cys ligands, αCys112-SO2H and αCys114-SOH. The metal is 

proposed to function as a Lewis acid, but the detailed mechanism remains unclear. Recently, we found that 

NHase catalyzes the conversion of tert-butylisonitrile (tBuNC) to tert-butylamine (tBuNH2). Here we present the 

first structural evidence for the catalytic mechanism of NHase. When the reaction was monitored by ATR-FTIR 

only tBuNH2 was observed, suggesting that the product from the isonitrile carbon escaped as a gas. The product 

was identified as CO by being trapped with reduced hemoglobin. Thus, NHase catalyze the reaction of both 

nitriles and isonitriles with one equivalent molar amount of water molecules. Crystals of the nitrosylated inactive 

NHase were soaked with tBuNC. The catalytic reaction was initiated by photo-induced denitrosylation and 

stopped by flash-cooling with nitrogen gas at elapsed times. tBuNC was first trapped at the hydrophobic pocket 

above the Fe center and then coordinated to the Fe ion at 120 min. From 340 to 440 mins, the shapes of the 

electron densities of tBuNC were significantly changed and a new electron density observed near the isonitrile 

carbon as well as the sulfenate oxygen of αCys114. These results demonstrate that the substrate was coordinated 

to the iron and then attacked by a water molecule activated by αCys114-SOH. 

_____________________________________________________________________ 

194


P76. Molecular Intelligence: Flexible Response of Zinc Fingers on Metal 

Ion Supply 

U. Heinz, L. Hemmingsen, H. W. Adolph 

Department of Natural Sciences, University of Copenhagen, 1871, Frederiksberg C, Denmark, 

e-mail: u.heinz@gmx.de, hwa@life.ku.dk 

Zinc fingers are considered as small, independently folded protein domains that require coordinative binding of 

zinc ions for structure stabilization, and serve in molecular recognition processes involving DNA, RNA, other 

proteins or membranes. The present study demonstrates that both nature of and supply with metal ions determine 

whether zinc fingers fold into the well-known, fully loaded structures or alternatively form complexes with 

ligands for one metal ion contributed by different binding sites. The identification and characterization of such 

adaptive and intelligent system changes in terms of alternative structural states of single proteins according to the 

element-specific requirements of metal ions contributes to the understanding of zinc homeostasis, trafficking, 

signalling and heavy metal toxicity. The prevailing view on molecular regulation in terms of "on and off control" 

might be replaced by a more differentiated process - oriented view on complex adaptive systems. 

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195


P77. A Novel Technique to Probe the Inorganic and Bioinorganic 

Chemistry of Lead: 204m Pb-PAC Spectroscopy 

L. Hemmingsen a , J. Vibenholt b , M. Magnussen b , M. Stachura a , M. J. Bjerrum a , 

P. W. Thulstrup a 

a 

Department of Natural Sciences, University of Copenhagen, Denmark 

e-mail: lhe@life.ku.dk 

b 

Department of Chemistry, University of Copenhagen, Denmark 

Perturbed angular correlation of γ-rays (PAC) spectroscopy has been applied routinely in solid state physics over 

the past 5 decades, and to a lesser extent in coordination chemistry and biochemistry using a variety of PAC 

probes, providing information on the molecular and electronic structure in the vicinity of the PAC probe [1]. 

204m Pb-PAC spectroscopy was first applied in 1973 by Haas and Shirley [2], in investigations of inorganic 

compounds such as PbCl2, PbFCl, PbI2, PbO, PbSO4, PbCrO4, PbC2O4, and Pb(SCN)2. Only recently the 

technique was “revived” in an effort by the PAC-group in Leipzig, Germany, and the ISOLDE collaboration at 

CERN [3], where additional inorganic compounds (PbCO3, Pb2O(CO3), Pb3(PO4)2, Pb(CN)2, PbO, Pb3O4, 

Pb(IO3)2, PbBr2, PbTiO3) were investigated. No applications to coordination compounds nor applications in 

biochemistry have been published yet in peer reviewed journals, although a convincing attempt to determine the 

NQI in Pb(II)-substituted azurin was carried out in the Ph.D. work by Frank Heinrich [4]. 

In this work [5] we present the first application of 204m Pb-PAC spectroscopy to a coordination compound, in the 

sense that it possesses a full molecular entity in the unit cell, see Figure 1, rather than the periodic crystalline 

structure of the inorganic compounds investigated previously. 

Figure 1 Left: The structure of [Pb(II) iso-maleonitriledithiolate] 4- ([6], counterions not shown). Right: the 

Fourier tranform of the perturbation function recorded by 204m Pb-PAC spectroscopy (Thin line: data; boldfaced 

line: fit). 

Acknowledgements: The Danish Natural Science Research Council is acknowledged for funding, and 

ISOLDE/CERN for beam time 

References: 

[1] Hemmingsen L., Sas K.N., Danielsen E. Chem. Rev. 2004, 104, 4027. 

[2] Haas H., Shirley D. J. Chem. Phys. 1973, 58, 3339. 

[3] Tröger W., Dietrich M., Araujo J.P., Correia J.G., Haas H., and the ISOLDE collaboration Z. Naturforsch. 

2002, 57A, 586. 

[4] Heinrich F., Ph.D. thesis, 2005, Faculty of Physics and Geosciences, University of Leipzig, Germany 

[5] Vibenholt J., Magnussen M., Stachura M., Bjerrum M.J., Thulstrup P.W. and Hemmingsen L. in prep. 

[6] Magyar J.S., Weng, T.-C., Stern C.M., Dye D.F., Rous B.W., Payne J.C., Bridgewater B.M., Mijovilovich 

A., Parkin G., Zaleski J.M., Penner-Hahn J.E., and Godwin H.A. J. Am. Chem. Soc. 2005, 127, 9495 

_____________________________________________________________________ 

196


P78. Structural Studies of the Intermediates in the Reaction Between 

Myoglobin and Peroxides 

H. P. Hersleth a , Y. W. Hsiao b , C. H. Görbitz c , U. Ryde b , K.K. Andersson a 

a 

Department of Molecular Biosciences, University of Oslo, P.O.Box 1041 Blindern, N-0316, Oslo, Norway 

e-mail: h.p.hersleth@imbv.uio.no 

b 

Department of Theoretical Chemistry, Lund Univeristy, P.O.Box 124, S-221 00, Lund, Sweden 

c 

Department of Chemistry, University of Oslo, P.O.Box 1033 Blindern, N-0315, Oslo, Norway 

The intermediates generated in the reaction between myoglobin and peroxides mimic the intermediates found in 

many peroxidases, oxygenases and catalases [1, 2]. These myoglobin intermediates are also relevant because 

myoglobin is proposed to take part as scavenger of reactive oxygen species during oxidative stress. We have in 

this study combined crystallography and single-crystal light absorption spectroscopy (microspectrophotometry). 

Radiation-induced changes of the different intermediates in this reaction cycle have been observed and followed 

by microspectrophotometry [2, 3, 4] . We have been able by cryoradiolytic reduction of an oxymyoglobin 

equivalent (compound III) to generate and trap the so-called peroxymyoglobin intermediate, a Fe(II)-superoxide 

form indicated by quantum refinement analysis [2, 4]. By annealing of this compound the oxygen-oxygen bond is 

broken and the reaction propagates to the compound II intermediate [3, 4]. The structures have further been 

refined with quantum refinement [3, 4, 5]. 

References: 

[1] Hersleth, H.-P., Ryde, U., Rydberg, P., Görbitz, C.H. & Andersson, K.K. (2006). J. Inorg. Biochem. 100, 

460-476. 

[2] Hersleth, H.-P. Varnier, A., Harbitz, E, Røhr, Å. K., Schmidt, P. P., Sørlie, , M., Cederkvist, F. H., Marchal, 

S., Gorren, A. C. F., Mayer, B., Uchida, T., Schünemann, V., Kitagawa, T., Trautwein, A. X., Shimizu, T., 

Lange, R., Görbitz, C. H. & Andersson, K. K. (2008) Reactive Complexes in Myoglobin and Nitric Oxide 

Synthase. Inorg. Chim. Acta 361, 831-843. 

[3] Hersleth, H.-P., Uchida, T., Røhr, Å.K., Teschner, T., Schünemann, V., Kitagawa, T., Trautwein, A.X., 

Görbitz, C.H. & Andersson, K.K. (2007) Crystallographic and spectroscopical studies of peroxide-derived 

myoglobin compound II and Occurence of protonated FeIV-O. J. Biol. Chem. 282, 23372-23386. 

[4] Hersleth, H.-P., Hsiao, Y.-W., Ryde, U., Görbitz, C.H. & Andersson, K.K. (2008) The crystal structure of 

peroxymyoglobin generated through cryoradiolytic reduction of myoglobin compound III during data collection. 

Biochem. J. 412, 257-264. 

[5] Nilsson, K., Hersleth, H.-P., Rod, T.H., Andersson, K.K. & Ryde, U. (2004) The Protonation Status of 

Compound II in Myoglobin, Studied by a Combination of Experimental Data and Quantum Chemical 

Calculations: Quantum Refinement. Biophys. J. 87, 3437-3447. 

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197


P79. Indenyl Iron Carbonyls as CO-releasing Molecules 

L. Hewison a , B. Mann a , P. Sawle b , R. Motterlini b 

a 

Department of Chemistry, University of Sheffield, Brook Hill, S3 7HF, Sheffield, United Kingdom 

e-mail: chp05lh@sheffield.ac.uk 

b 

Vascular Biology Unit, Department of Surgical Rese, Northwick Park Institute for Medical Research, HA1 

3UJ, Harrow, Middlesex, United Kingom 

Heme oxygenase is an important enzyme within our bodies, converting heme to carbon monoxide (CO), Fe II , and 

initially biliverdin that is subsequently reduced to bilirubin.[1] Carbon monoxide plays a vital role in the 

cardiovascular system and provides protection against reperfusion injury and inflammation. The use of metal 

carbonyls to deliver CO in a solid form was first described in 2002, [2] and a range of viable metal carbonyls 

have been described and recently reviewed.[3] 

The use of [CpFe(CO)3] + and its derivatives as CO releasing molecules has recently been reported.[4] As 

replacement of cyclopentadienyl by indenyl frequently increases rates, as has been reported for [CpFe(CO)3] + 

and [(indenyl)Fe(CO)3] + , [5] a selection of derivatives of [(indenyl)Fe(CO)3] + have been examined as CO 

releasing molecules. The rate of CO release to myoglobin and macrophages in culture was used to examine the 

effect of the compounds on cell viability, toxicity and suppression of nitrite formation. While t1/2 is 69 min for 

[CpFe(CO)3] + , it reduces to


P80. Synthesis and Characterization of N3-type Copper(II) Complex with 

Calix[6]arene Upper Rim 

T. Higa, T. Fujii, Y. Kajita, T. Inomata, Y. Funahashi, T. Ozawa, H. Masuda 

Department of Materials Science and Engineering, Graduate School of Engineering, Nagoya Institute of 

Technology, Showa-ku, Nagoya 466-8555, Japan 

Dopamine β-monooxygenase (DβM) and peptidylglycine-α-hydroxylating monooxygenase (PHM) are two 

mononuclear uncoupled copper-containing enzymes which catalyze essential hydroxylation reactions of 

substrates by activating dioxygen. We previously synthesized their biomimetic model copper(II) complexes with 

dipyridylamine-type ligands.[1] Addition of hydrogen peroxide to the solution containing the copper(II) 

complex, [Cu(bpba)(MeOH)](ClO4)2 (bpba = bis(2-pyridylmethyl)tert-butylamine) generates copperhydroperoxide 

species.[1] The reaction of the hydroperoxo species with dimethyl sulfide has exhibited catalytic 

oxidation of dimethyl sulfide to dimethyl sulfoxide.[1] We introduce a calixarene group into the dipyridylaminetype 

ligand as a cavity of encapsulating organic substrates nearby the reaction center, because increase in the 

reaction probability is expected. In this study, we desgined a novel calixarene derivative, CA[6]BPA(Figure 1), 

and synthesized the novel copper(II) and palladium(II) complexes. We will also discuss characterization of these 

complexes and encapsulation of substrates by 1 H NMR measurements. 

Acknowledgment: We gratefully acknowledge the support of this work by grants for overseas investigation 

research from Tatematsu Foundation. 

References: 

[1] T. Fujii, A. Naito, S. Yamaguchi, A. Wada, Y. Funahashi, K. Jitsukawa, S. Nagatomo, T. Kitagawa, and 

H. Masuda, Chem. Commun. 2003, 2700-2701. 

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199


P81. Fe L-edge XAS Determination of Differential Orbital Covalency of 

Siderophore Model Compounds: Electronic Structure Contributions to 

High Stability Constants 

R.K. Hocking a, b , J. Xu c , E. Dertz c , S. DeBeer George d , K.N. Raymond c , K.O. Hodgson d , 

B. Hedman d a, d 

and E.I. Solomon 

a Department of Chemistry, Stanford University, Stanford, California 94305, USA 

b Monash Centre for Synchrotron Science and School of Chemistry, Monash University. 3800, Australia 

c Department of Chemistry, University of California, Berkeley, USA 

d Stanford Synchrotron Radiation Laboratory, SLAC, Stanford University, Stanford California, 94309, USA 

Many micro-organisms produce low-molecular weight iron-chelators called siderophores. Although many 

different siderophore structures have been characterised, the iron bonding moieties generally contain 

carboxylate, hydroxamate, or catecholate binding groups. Siderophores function because of their extraordinarily 

high stability constants. Recently we have developed a methodology that enables the interpretation of Fe L-edges 

in terms of differential orbital covalency (i.e. the differences in the mixing of the metal d orbitals with ligand 

valence orbitals) using a valence bond configuration interaction model. We have now applied this methodology 

to a series of model siderophores understand the electronic structure contributions to these stability constants in 

terms of σ and π covalent as well as ionic contributions to bonding. 

Acknowledgement: This work was in part performed at SSRL, which is funded by the DOE Office of Basic 

Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the NIH National Center for 

Research Resources, Biomedical Technology Program and by the DOE Office of Biological and Environmental 

Research. 

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200


P82. Coordination abilities of alloferon peptides towards copper ions 

A. Janicka-Kłos a , A. Bonna b , A. Prahl c , H. Kozłowski d , J. Świątek-Kozłowska a 

a 

Department of Inorganic Chemistry, Faculty of Pharmacy Silesian Piast University of Medicine in Wroclaw, 

Szewska 38, 50-139 Wroclaw, Poland 

e-mail: anna_janicka@yahoo.pl 

b 

Department of Biophysics, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Pawinskiego 

5a, 02-106 Warszawa, Poland 

c 

Department of Organic Synthesis, Faculty of Chemistry, University of Gdansk, Sobieskiego 18/19, 80-952 

Gdansk, Poland 

d 


Two peptides isolated from bacteria-challenged larvae of the blow fly Calliphora vicina: HGVSGHGQHGVHG 

(alloferon 1) and GVSGHGQHGVHG (alloferon 2) posses potent antiviral and antitumoral activity [1]. Both 

peptides are rich in histidine and thus are supposed to be able to effectively bind Cu 2+ ions. It is proved that 

copper is required by several key proteins involved in the stimulation of angiogenesis as it acts as an important 

cofactor for their angiogenic activity [2]. Several reports show that reduction of copper level at tumor sites 

interrupts angiogenesis what results in prevention of tumor growth and its metastasis. An essential step in 

tumor proliferation, expansion and metastasis is angiogenesis. It is believed that a switch of angiogenic 

phenotype in a tissue is dependent upon the local balance between angiogenic factors and inhibitors. It is also 

speculated that most endogenous angiogenesis inhibitors in a vascular tissues are protein molecules and their 

fragments [1, 3, 4]. 

The aim of this work was to experimentally check coordination abilities of both alloferon peptides towards 

copper ions. Potentiometric and spectroscopic techniques (CD, UV-Vis and EPR) were performed and provide 

evidence of Cu 2+ /peptide complexes. At the physiological pH (pH 7-8) three species with different protonation 

state are present in equilibrium where metal ion is bound via the imidazole nitrogen of His and amide nitrogens. 

References: 

[1] S. Chernysh, S. I. Kim, G. Bekker, V. A. Pleskach, N. A. Filatova, V. B. Anilin, V. G. Platonov, P. Bulet, 

PNAS, 99, 12628, (2002) 

[2] S. Brem, Cancer Control, 6, 436, (1999) 

[3] M.A. Moses, R. Langer, J. Cell. Biochem., 47, 230, (1991) 

[4] V. L. Goodman, G. J. Brewer, S. D. Merajver, Endocrine-Related Cancer, 11, 255, (2004) 

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201


P83. Influence of Physiological Anions on Spectral Properties of 

Lanthanide(III) Complexes with 

Ethylenediamine(tetramethylenephosphonic acid) H8EDTMP 

R. Janicki, K. Rubka, A. Walkowiak, A. Mondry 

Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland, 

e-mail: anm@wchuwr.pl 

Thermodynamically stable lanthanide(III) complexes with organophosphonate ligands have found a variety of 

applications, particularly in the radiopharmaceutical and biomedical NMR fields. Though the complex of 

radioactive isotope of 153 Sm(III) with EDTMP, known clinically as Quadramet ® , is in widespread use to relieve 

pain from metastatic bone cancer, the mechanism by which it achieves is still unknown. Whereas the uptake by 

metastatic tissue in bones of the Sm(III)–EDTMP complex is good, in the case of the Ho(III)–EDTMP is rather 

poor. The latter complex also stays longer in blood plasma. To understand these significant differences between 

both Ln(III) complexes, we have undertaken studies on spectral properties of light and heavy ions with EDTMP 

ligand in the presence of physiological anions. 

Previously it was shown that replacement of inner-sphere water molecules and/or hydroxy anions by a carbonate 

anion in the Eu(III)–EDTMP complex at physiological pH results in the formation of [Eu(EDTMP)(CO3)] 7– 

species [2]. A very good fitting of a carbonate anion into the coordination space vacated by water molecules 

and/or hydroxy groups in [Eu(EDTMP)(H2O)2] 5– and [Eu(EDTMP)(H2O)(OH)] 6– species which are in 

equilibrium at physiological pH, is a reason that kinetically labile species become inert. 

Molecular structure of [Eu(EDTMP)CO3] 7– anion [2]. 

Therefore in the present work the absorption and/or emission spectra of the mixed Ln(III)–EDTMP–L 

complexes (where Ln are Nd, Sm, Eu, Tb, Er, Yb ions and L are carbonate, phosphonate and acetate anions) 

have been analyzed and association constants of the Ln(III)–EDTMP–carbonate systems at physiological pH 

were determined. The relative stability of various chelates has been discussed. 

References: 

[1] N.V. Jarvis, J.M. Wagener, G.E. Jackson, J. Chem Soc. Dalton Trans., 1411 (1995). 

[2] A. Mondry, R. Janicki, Dalton Trans., 4702 (2006). 

_____________________________________________________________________ 

202


P84. Vibrational Properties of the NO-carrier Protein Nitrophorin 

A. Janoschka a , J. Wolny a , B. Hewener a , H. Paulsen b , I. Filipov c , A.I. Chumakov d , 

F.A. Walker c , V. Schünemann a 

a 

Department of Physics, University of Kaiserslautern, Erwin-Schrödinger-Str. 46, 67663 Kaiserslautern, 

Germany 

e-mail: schuene@physik.uni-kl.de 

b 

Institute of Physics, University of Lübeck, Ratzeburger Allee 160, 23556 Lübeck, Germany 

c 

Department of Chemistry, University of Arizona, Tucson AZ 85721-0041, USA 

d 

Nuclear Resonance Group, ESRF, 6 rue Jules Horowitz, BP220, 38043 Grenoble Cedex, France 

The protein nitrophorin (NP) is found in the salivary glands of the Amazon river-based kissing bug Rhodnius 

prolixus. For obtaining a sufficient blood-meal, these proteins are injected into the victim by the insect prior to 

feeding. When the pH value suddenly rises from 5 in the salivary glands of the kissing bug to pH 7.5 in the 

tissues of the victim, the binding affinity is changed and thus the signaling molecule nitric oxide (NO), which is 

bound to the iron in the active heme-center of the protein, is released. The NO can migrate through the victim’s 

tissue to the capillaries to dilate them to allow more blood to flow to the site of the bite. 

The Rhodnius NP’s have a molecular weight of 20 kDa. Crystallographic data show that the tertiary structure of 

the NP’s exhibit a β-barrel with a histidine residue (His-59) that serves as the proximal ligand to the heme [1]. 

The NP’s represent the first examples of proteins with stable Fe(III)-NO complexes, where the NO can be stored 

for a long period of time. 

Figure 1: Density of states (DOS) obtained 

from NIS spectra obtained from NP-2 under 

four different conditions, from top to 

bottom: ligand-free (NP2-HS), NO-bound 

(NP2-NO), CN- bound (NP2-CN). 

Histamine bound (NP2-Histamine). The 

arrows denote the iron-ligand stretching 

modes. 

0 10 20 30 40 50 60 70 80 

We have performed nuclear inelastic scattering of synchroton radiation (NIS) at the ESRF to detect iron centered 

molecular vibrations of NO-ligated nitrophorins. Due to the high protein concentration of 10mM as well as the 

extraordinary beam stability at ID-18 during our experiment we have been able to measure the isoform 

nitrophorin 2 under four different conditions: (i) ligand-free (NP2-HS), (ii) NO-bound (NP2-NO), (iii) histamine 

bound (NP2-HIS), and (iv) CN- bound (NP2-CN) (see Figure 1). A striking feature of the vibrational spectrum 

obtained from NP2-NO is the well resolved and intense vibration at 73.5 meV (594 cm -1 ) with a shoulder at 72 

meV (581 cm -1 energy (meV) 

). The former mode has been assigned to a NO-Fe stretching mode and the latter to a Fe-NO 

bending mode by Resonance Raman spectroscopy [2]. However these bands are only hardly visible in the 

Resonance Raman spectra and isotope labelling was used for the assignments. Currently theoretical QM/MMcalculations 

are undertaken to understand and assign all iron related stretching modes visible in Fig. 1. Already it 

can be said that the Fe-axial ligand interaction is strongest for NO, decreases for CN- and decreases even more in 

the case of Fe-Histamine binding in nitrophorin. We consider this study as a textbook example of how NIS can 

be used to study the interaction of an active iron site in a protein with different ligands without the need of 

isotope labelling experiments. 

Acknowledgement: This work has been supported by the state Rhine-Palatine, by the BMBF and by the ESRF 

via experiment No. SC 2122. 

References: 

[1] J.F. Andersen, W.R. Montfort, J. Biol. Chem., 275, 30496 (2000). 

[2] E.M. Maes, F.A.Walker, W.R. Montfort, R.S. Czernuszewicz, J. Am. Chem. Soc., 123, 11664 (2001). 

iron density of states (DOS) 

0,3 

0,2 

0,1 

0,0 

NP2-HS 

NP2-NO 

NP2-CN 

NP2-Histamine 

_____________________________________________________________________ 

203


P85. Homo- and Heterodinuclear Metal Complexes of Symmetric and 

Asymmetric Ligands for Modeling of Active Sites in Metallohydrolases 

M. Jarenmark, a M. Haukka b and E. Nordlander a 

a Inorganic Chemistry Research Group, Department of Chemical Physics, Centre for Chemistry and Chemical 

Engineering, Lund University, Box 124, SE-221 00, Sweden 

e-mail: martin.jarenmark@chemphys.lu.se 


Several hydrolytic enzymes containing dinuclear active sites catalyse the cleavage of phosphoester bonds with 

high efficiency. Prominent examples are zinc phosphotriesterase that contain two hydroxido bridged Zn(II) ions 

and purple acid phosphatases that contain a Fe(III) and a divalent metal ion (Fe, Zn or Mn) bridged by a 

hydroxido or oxido group in the active site. 

To model these sites several dinuclear metal complexes have been synthesized using the ligands IPCPMP and 

BCPMP.[1] Zinc complexes with these ligands catalyse the transesterfication of 2-hydroxypropyl-p-nitrophenol 

phosphate (HPNP) yielding a considerable higher rate with the asymmetric ligand IPCPMP and a strong pH 

dependence. The solid-state structures indicate that a tetranuclear complex may be responsible for the higher 

activity. 

With IPCPMP a mononuclear Fe(III) complex can be synthesized which can be used to selectively form several 

heterodinuclear metal complexes where the structure of the FeZn, FeCo, FeNi and FeCu derivatives have been 

determined by X-ray crystallography. The FeZn, FeCo and FeNi complexes also catalyses the transesterfication 

of HPNP. 

Acknowledgements: The authors would like to thank the Swedish research council, the International research 

training group ‘Metal sites in biomolecules: Structures, regulation and mechanisms’ and the faculty of natural 

sciences at Lund University for financial support. 

References: 

[1]M. Jarenmark, S. Kappen, M. Haukka E. Nordlander, Dalton Trans., 2008, 993 - 996 

_____________________________________________________________________ 

204


P86. Equilibrium Studies of Phytic Acid Interactions with Spermine 

R. Jastrzab a , A. Odani b , L. Lomozik a , R. Bregier-Jarzebowska a 

a 

Faculty of Chemistry, A.Mickiewicz University, Grunwaldzka 6, 60-780, Poznan, Poland 

e-mail: renatad@amu.edu.pl 

b 

Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, 920-1192, 

Kanazawa, Japan 

The in vivo as well as in vitro studies have shown that phytic acid (IP6) is an important antioxidant and the 

presence of IP6 in living cells has beneficial effects, such as protection against cancer, heart diseases and renal 

calculosis [1, 2]. A large number of phosphate groups (Figure) provide strong multidonor interaction sites in the 

ligand that take part in reactions with metals or organic cations, e.g. polyamines [3]. 

Polyamines are present in the physiological fluids as protonated species and in this form interact with negative 

fragments of other bioligands including IP6. The mechanism of such an action at the molecular level has not 

been recognised yet. 

This presentation shows results of the potentiometric equilibrium study of the interactions of phytic acid with 

spermine and other polyamines. To characterise the mode of interactions, the systems studied have been 

investigated by 31 P NMR. The noncovalent interactions between partly protonated spermine and partly 

deprotonated phosphate groups of phytic acid lead to the formation of molecular complexes of the type 

(IP6)Hx(Spm). The IP6 protonation constants, stability constants of the adducts and equilibrium constants of 

their formation have been determined. The interactions between phytic acid and polyamines have ion-ion 

character and the charge is the main factor determining their effectiveness, although the structure of polyamines 

should be also taken into account. Unexpectedly, it has been found that the effectiveness of the noncovalent 

interactions with phytic acid is greater for the derivatives of biogenic amines than for the amines occurring in 

living organisms. In contradistinction to molecular complexes of polyamines with nucleotides, the stability of the 

complexes formed with phytic acid decreases with increasing length of amine chain. 

References: 

[1] B.F. Harland, G. Narula, Nutr. Res., 19 (1999) 633 

[2] G. Urbano, M. Lopez-Jurado, P. Aranda, C. Vidal-Valverde, E. Tenorio, J. Porres, J. Physiol. Biochem., 56 

(2000) 283 

[3] C.W. Tabor, H. Tabor, Ann. Rev. Biochem., 53 (1984) 749 

_____________________________________________________________________ 

205


P87. Solid-State NMR Study of Anticancer Cisplatin Interaction with 

Phospholipid Bilayers 

M. Jensen, W. Nerdal 

Chemistry Department, University of Bergen, Allegaten 41, N-5007, Bergen, Norway 

e-mail: magnus.jensen@kj.uib.no 

Cisplatin has been used for many years in cancer chemotherapy of testicular cancer with a cure rate better than 

90%[1]. Chemotherapy with use of cisplatin in treatment of other tumor types such as cervical cancers has also 

been done for many years [2-4]. It is well established that cisplatin forms platinum-DNA adducts that initiate 

tumor cell death [5-8]. Despite the widespread use of cisplatin in chemotherapy and its high cure rate of 

testicular tumors, drawbacks are side effects such as neurotoxicity and cellular cisplatin resistance. 

It is likely that the molecular mechanisms that take cisplatin across the cellular membrane are vital in the 

development of cisplatin resistance with reduced intracellular accumulation. Unfortunately, these biochemical 

mechanisms are not fully understood. 

It is therefore important to find the molecular mechanisms of how cisplatin gets across the cellular membrane 

and enters the cytosol, as well as establishing to what extent cisplatin interacts with the phospholipid bialyer. A 

consequence of cisplatin binding to phospholipids of the cellular membrane is that this can change the fluidity of 

the membrane and alter its function. 

Results of cisplatin interaction with phospholipid bilayers studied by different NMR techniques using 1H, 13C, 

31P and 15N , both magic angle spinning (MAS) and static spectra, will be presented. 

References: 

[1] Bosl, G. J., Bajorin, D. F. and Sheinfeld, J. Cancer of the Testis (eds, DeVita, V. T. J., Hellman, S. and 

Rosenberg, S. A.) (Lippincott, Williams and Wilkins, Philadelphia, 2001). 

[2] Morris, M., Eifel, P. J., Lu, J., Grigsby, P. W., Levenback, C., Stevens, R. E., Rotman, M., Gershenson, D. 

M. and Mutch, D. G., N. Engl. J. Med. 340, 1137, (1999) 

[3] Rose, P. G., Bundy, B. N., Watkins, E. B., Thigpen, J. T., Deppe, G., Maimann, M. A., Clarke-Pearson, D. L. 

and Insalaco, S., N. Engl. J. Med. 340, 1144, (1999) 

[4] Keys, H. M., Bundy, B. N., Stehman, F. B., Muderspach, L. I., Chafe, W. E., Suggs, C. L., Walker, J. L. and 

Gersell, D., N. Engl. J. Med. 340, 1154, (1999) 

[5] Jamieson, E. R. and Lippard, S. J., Chem. Rev. 99, 2467, (1999) 

[6] Kartalou, M. and Essigman, J. M., Mut. Res. 478, 23, (2001) 

[7] Brabec, V. and Kasparkova, J., Drug Resist. Updates 5, 147, (2002) 

[8] Wang, D. and Lippard, S. J., Nat. Rev. 4, 307, (2005) 

_____________________________________________________________________ 

206


P88. The Influence of pH on the Loop-structure of an Oligonucleotide 

Containing Artificial Nucleobases 

S. Johannsen a , D. Böhme b , N. Düpre b , J. Müller b , R.K.O. Sigel a 

a 

Institute of Inorganic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland 

e-mail: silkej@aci.uzh.ch 

b 

Faculty of Chemistry, Dortmund University of Technology, Otto-Hahn-Strasse 6, 44227, Dortmund, Germany 

We synthesised a 17 nt long oligonucleotide including three imidazole moieties as nucleobase surrogates (see 

Figure).[1] In the absence of coordinating metal ions the oligonucleotide adopts a hairpin structure and 

constitutes a model system for reaction centres in ribozymes exhibiting acid-base catalysis. 

For a detailed analysis of the acid-base properties of each imidazole moiety we used pD-dependent 1H-NMR 

spectroscopy of the aromatic protons to determine the intrinsic pKa values. Starting in acidic solution, with 

increasing pD all imidazole protons shift to higher field, broaden out or even disappear around pD=7 and 

become sharper again at higher pD values. Compared to the pKa of imidazole nucleoside 5'-monophosphate 

(ImMP), Im8 and Im10 show an increase in basicity, of up to 0.4 log units. We further calculated the NMR 

solution structures of the hairpin at three different pD values: pD=4.7, 7.2 and 10.2. Two different, but welldefined 

loop structures are observed at low and high pD. In contrast, at neutral pD the loop is rather 

unstructured. This may result from a structural intermediate between the two loop structures, i.e., the fully 

protonated and completely unprotonated form. 

Acknowledgement: Financial support by the European ERAnet-Chemistry, the Swiss National Science 

Foundation (20EC21-112708 and SNF-Förderungsprofessur PP002-114759 to R.K.O.S.) and the DFG 

(JM1750/2-1 and Emmy Noether programme JM1750/1-3, J.M.) is gratefully acknowledged. 

References: 

[1] J. Müller D. Böhme, P. Lax, M. Morell Cerdà, M. Roitzsch, Chem. Eur. J. 2005, 11, 6246-6253. 

_____________________________________________________________________ 

207


P89. Coordination Modes and Structural Aspects of Mixed-ligand 

Copper(II) Complexes Containing Some Polyamines and Amino Acids 

A. Kamecka a , K. Bogusz a , J. Jezierska . b , A. Woźna. a , B. Kurzak a 

a Department of Chemistry, University of Podlasie, 3 Maj 54, 08-110, Siedlce, Poland 

b Faculty of Chemistry, University of Wrocław, F. Joliot Curie 14, 50-383, Wrocław, Poland 

Amino acids and peptides, which are the basic structural units of proteins, are potential chelating reagents for 

copper(II) ion [1, 2]. There is continuing interest in coordination compounds of copper(II) with various 

combinations of heterocyclic nitrogen, thiolate and thioether donors because of the coordination of copper to two 

histidine nitrogen atoms and cysteine and methionine sulfur atoms in the electron transfer blue copper proteins 

[3, 4]. Furthermore, in a number of biochemical processes copper(II) is involved in mixed-ligand complex 

formation and ligand catalysed complex formation reactions [5]. Due to the fact that polyamines are found at a 

significant concentration in young cells and, particularly, in cancer tumor tissues [6], it seems to be interesting to 

investigate the ternary copper(II)-polyamine-histidine/methionine systems (the polyamines such as 

ethylenediamine (en), diethylenetriamine (dien) or N, N, N’, N’’, N’’-pentamethyldiethylenetriamine 

(Me5dien)). The stoichiometries, stability constants and bonding modes of the species formed at aqueous 

solution in the copper(II) ternary systems have been established. Our spectroscopic results indicate the tetragonal 

geometry for the mixed-ligand complexes of en, the geometry slightly deviated from square pyramidal for the 

species of dien and strongly deviated from square pyramidal towards trigonal bipyramidal for the complexes of 

Me5dien. 

References: 

[1] P. Deschamps, P.P. Kulkarni, M. Gautam-Basak, B. Sarkar, Coord. Chem. Rev., 249 (2005) 895. 

[2] B.G. Malstrom, J. Leckner, Current Opinion in Chemical Biology, 2 (1998) 286. 

[3] E.I. Solomon, M.J. Baldwin, M.D. Lowery, Chem. Rev. 92 (1992) 521. 

[4] E.I. Solomon, R.K. Szilagyi, S.D. Georgie, L. Basumallick, Chem. Rev. 104 (2004) 419. 

[5] H. Sigel, Metal Ions in Biological Systems, Vol.3, Marcel Dekker Inc., New York, 1974. 

[6] C.W. Tabor, H. Tabor, Ann. Rev. Biochem. 53 (1984) 749. 

_____________________________________________________________________ 

208


P90. Mechanism for Autoxidation of Nitric Oxide Adduct of Manganese(II) 

Porphyrin Placed in Microscopically Hydrophobic Environments in 

Aqueous Solution 

K. Kano, Y. Itoh, H. Kitagishi 

a Molecular Chemistry and Biochemistry, Doshisha University, Tatara, 610-0321, Kyotanabe, Japan 

e-mail:kkano@mail.doshisha.ac.jp 

Nitric oxide (NO) is bound to various hemoproteins to inhibit or activate protein functions. Since NO induces 

oxidation of oxyMb yielding inactive metMb, the mechanism for oxidation of oxyMb by NO has been well 

studied and established. Meanwhile, Mb binds NO and (NO)Mb is gradually oxidized to metMb and NO3 - . 

However, the mechanism for oxidation of (NO)Mb by dioxygen has not been clarified. Recently, we 

demonstrated the mechanism for oxidation of the NO adduct of a Mb model [1]. In this work, we studied the 

mechanism for oxidation of the NO adduct of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinato manganese(II) 

(Mn(II)TPPS) complexed with a model protein to confirm our previous mechanism. The model system is 

composed of a per-O-methylated beta-cyclodextrin dimer (Py2CD) having a pyridine linker and Mn(II)TPPS. 

Py2CD formed an extremely stable 1:1 inclusion complex (Mn(II)PCD) of Mn(II)TPPS in aqueous phosphate 

buffer solution at pH 7.0. Mn(II)PCD bound NO and (NO)Mn(II)PCD was gradually oxidized to Mn(III)PCD 

and NO3 - under aerobic conditions, the half-life time being ~35 h. The oxidation obeyed the zero-order kinetics, 

suggesting that NO gradually dissociates from its adduct (rate-determining step) and Mn(II)TPPS formed is 

autoxidized by dioxygen affording Mn(III)PCD and superoxide. The superoxide anion immediately reacts with 

NO to yield NO3 - . The present study supports the previous mechanism for (NO)Fe(II)PCD [1]. 

References: 

[1]K. Kano, Y. Itoh, H. Kitagishi, T. Hayashi, S. Hirota, J. Am. Chem. Soc., in press 

_____________________________________________________________________ 

209


P91. Antimicrobial Activity of Amino Acid and Dipeptide Based 

Amphiphiles 

N. Kayal a , S. Roy b 

a 

Department of Biotechnology, Vellore Institute of Technology, University (VITU, 1/1 thakurpukur road, kol- 

63, 700063, kolkata, India 

e-mail: nilanjan_kayal@hotmail.com 

b 

Department of Biological Chemistry, Indian Association for the Cultivation of Science, , 1/1 thakurpukur 

road, kol-63, 700063, kolkata, India 

Cationic surfactants bear anti-bacterial activity. Surfactants are usually organic compounds which are 

amphiphilic in nature; they contain both hydrophobic groups (their “tails”) and hydrophilic groups (their 

“heads”). Thus they are soluble in both organic solvents and water. Quaternary ammonium compounds are 

found to be quite effective against both Gram’s positive and Gram’s negative bacteria, but they are also toxic. 

Their toxicity is related primarily to the various biological effects of the quaternary ammonium head and its 

metabolism (such as oxidative dealkylation), but it is also believed that the surfactant characteristics of the 

molecules, particularly in liver, causes additional alterations in a number of chemical, biological and transport 

phenomena. The mechanism of action of cationic surfactants on bacteria is understood to be purely 

electrostatic interaction and physical disruption. The cationic site of the agent is able to bind to the anionic 

sites of the cell wall surface. With a significant lipophilic component present, it is then able to diffuse through 

the cell wall and bind to membrane. As a surfactant it is able to disrupt the membrane and permit the release of 

electrolytes and nucleic materials, leading to cell death. Amino acid (Tryptophan) based cationic surfactants 

having carbon lengths C14 and C16 were tested with Klebshiella aerogens (Gram -ve) and Bacillus substilis 

(Gram +ve) and also give rise to semi-solid materials i.e. Gels, which can in turn have antimicrobial properties 

and can have a variety of uses in terms of antibiotics. 

References: 

[1] Salton, M. R. J. J. Gen. Physiol. 1968, 52, 227S-252S. 

[2] Hugo, W. B.; Frier, M. Appl. Microbiol. 1969, 17, 118-127. 

[3] Tomlinson, E.; Brown, M. R; Davis, S. S. J. Med. Chem. 1977, 20, 1277- 1282. 

[4] Denyer, S. P. Int. Biodeterior. Biodegrad. 1995, 36, 227-245. 5. McDonnel, G.; Russell, A. D. Clin. 

Microbiol. Rev. 1999, 12, 147-179. 

[6] (a) Das, D.; Roy, S.; Dasgupta, A.; Mitra, R. N.; Debnath, S.; Das, P. K. Chem. Eur. J. 2006, 12, 5068- 

5074, (b) Das, D.; Roy, S.; Das, P. K. Org. Lett. 2004, 6, 4133-4136 

[7] Willemen, H. M.; de Smet, L. C.P.M.; Koudijis, A.; Stuart, M.C.A.; Heicamp de-Jong, I.G.A.M.; Marcelis, 

A.T.M.; Sudholter, E.J.R. Angew. Chem. Int. Ed. 2002, 41, 4275-4277 . 

_____________________________________________________________________ 



P92. Study on the Interaction Between DNA and a New Uranyl Schiff Base 

Complex 

M. Khorasani-Motlagh, M. Noroozifar, A. Heydari 

Department of Chemistry, University of Sistan & Baluchestan (USB), Zahedan, Iran, 

e-mail: mkhorasani@hamoon.usb.ac.ir 

Uranyl ion (UO2 2+ ) having favorable photophysical properties and high bond affinity toward phosphate 

backbone across the minor groove of DNA, is worth pursuing as a possible photonuclease. Besides, this ion was 

found to be potentially important in many biochemical applications such as probing local DNA structure and 

metal ion-binding sites in a DNA [1-2]. In earlier investigations, UO2(CH3COO)2.2H2O and UO2(NO3)2.6H2O 

salts have been used in photoinduced DNA scission and other biochemical applications. However, one of the 

major disadvantages of working with simple UO2 2+ salts for a wide range of biochemical applications is that, the 

pH must be maintained as neutral or highly acidic to prevent the uranyl ion from forming insoluble 

aggregates [3]. 

Here, to circumvent this problem, we used a new UO2 2+ - Schiff base complex, [UO2(L)(H2O)] (1), 

L= C13H20N2O2 with good solubility in water within the physiological pH range. Complex 1 have been 

synthesized and characterized by different spectroscopes method. Interaction between [UO2(L)(H2O)] (1) and 

DNA has been studied with Uv-Vis spectroscopy as well as cyclic voltammetry within the physiological pH 

range (pH 6-8). Upon addition of DNA to an aqueous solution of complex 5 (in Tris buffer), intensity of 

absorption band at λ =311 nm (or current in CV) decreases which is due to the interaction of the Schiff base 

complex with DNA. Kb, binding constant of this interaction found out to be 1.8×10 6 M -1 . 

References: 

[1] V. Balzani, F. Bollette, M.T. Gandolfi, Top. Curr. Chem., 75 (1978) 1. 

[2] P.E. Nielsen, C. Hiort, S. H. Sonnichesen, O. Buchardt, J. Am. Chem. Soc., 114 (1992) 4967. 

[3] P. E.Nielsen, C.Jeppesen, O. Buchardt, FEBs Lett. 235 (1988) 122. 

_____________________________________________________________________ 

211


P93. Linkage Isomerism in Complexes of Uracil with cis-(NH3)2Pt 2+ 

A. Khutia and B. Lippert 

Fakultät Chemie, Technische Universität Dortmund, 44227 Dortmund, Germany 

e-mail: akhutia@gmail.com 

The coordination chemistry of Pt II with the simple nucleobase uracil provides a wealth of binding patterns. 

Interest in these patterns relates, among others, to Pt antitumor agents (“platinum pyrimidine blues”[1]) and 

supramolecular chemistry (“metallacalix[n]arenes”[2, 3]). The existence of bis(ligand) complexes of a metal 

containing two identical ligands bonded to the metal through different sites (“linkage isomers”) is a relatively 

rare case in coordination chemistry and relates to basic questions such as their formation, chemical properties of 

the individual isomers and their mutual interference respectively. 

NH 

3 

3 1 

N NH 

_____________________________________________________________________ 

212 

O 

H 3N 

1 

N 

Pt 

H 3N 

1 

O 

Here we report on cis-(NH3)2Pt(UH-N1)(UH-N3), 1.5H2O (1), a neutral complex containing the two tautomers of 

the uracil anion (UH) bonded simultaneously. The compound was synthesized in a stepwise manner from cis- 

(NH3)2Pt(UH-N1)Cl and neutral uracil (UH2) at pH = 4-5 in water. The compound has been characterized by 1 H- 

NMR spectroscopy (1-D; pD dependence) and elemental analysis. A unique feature of the title compound is the 

isotopic exchange of the proton at C5 for deuterium when kept in D2O. 

Acknowledgement: A. Khutia thanks the International Max-Planck Research School in Chemical Biology 

(IMPRS-CB); Dortmund for a fellowship. 

References: 

[1] J.P. Davidson, P.J. Faber, R.G. Fischer, Jr., S. Mansy, H.J. Peresie, B. Rosenberg, L. VanCamp, Cancer 

Chemother. Rep. Part I, 59, 287 (1975). 

[2] H. Rauter, E.C. Hillgeris, A. Erxleben, B. Lippert, J. Am. Chem. Soc., 116, 616 (1994). 

[3] E.G. Bardaji, E. Freisinger, B. Costisella, C.A. Schalley, W. Brüning, M. Sabat, B. Lippert, Chem. Eur. J., 

13, 6019 (2007). 

O 

O


P94. ESI-MS Investigation of Interactions of Peptide-derived Amadori 

Products with Borate 

M. Kijewska, P. Stefanowicz, K. Kapczyńska, Z. Szewczuk 

Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland, 

e-mail: monikabr@eto.wchuwr.pl 

Non-enzymatic glyaction products are complex and heterogeneous group of compounds which accumulate in 

plasma and tissues and may play a role in the pathogenesis of diabetes, rethinopathy, cataracta, atherosclerosis, 

nephropathy and neurological diseases such Alzheimer’s disease [1, 2]. 

We have obtained a series of glycated peptides derived from the fragments of bovine serum albumin using two 

independent methods. First method involved reductive alkylation of the ε-amino groups of lysine with 2, 3:4, 5di-O-isopropylidene-β-arabino-hexos-2-ulo-2, 

6-pyranose in the presence of sodium cyanoborohydride on a 

solid support [3]. The secend approach is based on the synthesis of a suitable building block containing protected 

fructose residue attached to the ε-amino group of lysine. The novel lysine derivative: 

Fmoc-Lys(Fru, Boc)-OH is compatible with the Fmoc protocol of solid phase peptide synthesis [4]. 

According to literature data borate is well known for its ability to form complex with hydroxyl groups and has 

been used as a complexing buffer in CE for the separation of sugars [5]. Moreover, CE is fast and convenient 

method for separation of glycated peptides which is also benefits from complexation with borate [6]. 

In the present study we investigated the formation of borate complex of the peptide-derived Amadori products 

by MS experiment. The fragmentation pathways of obtained complexes were also analyzed in order to find the 

characteristic fragments ions. 

References: 

[1] A. Lapolla, P.Traldi, D. Fedele, Clin. Biochem., 38, 103 (2005). 

[2] N. Ahmed, Diabetes Res. Clin. Pr., 67, 3 (2005). 

[3] P. Stefanowicz, K. Kapczyńska, A. Kluczyk, Z. Szewczuk, Tetrahedron Lett., 48, 967 (2007). 

[4] K. Kapczyńska, P. Stefanowicz, M. Brunatna, Z. Szewczuk, 19. Polskie Sympozjum Peptydowe, 23-27 IX 

2007, Pułtusk, Polska 

[5] S. Hoffstetter-Khun, A. Paulus, E. Gassmann, Anal. Chem., 63, 1541 (1991). 

[6] L. Royle, R.G. Bailey, J.M. Ames, Food Chem. 62, 425 (1998). 

_____________________________________________________________________ 

213


P95. UV Resonance Raman Spectroscopic Studies of Azurin I and Azurin II 

from Alcaligenes Xylosoxidans NCIM 

S. Kimura, R.F. Abdelhamid, T. Kohzuma 

Applyed Beam Science, Ibaraki University, 310-8512, Mito, Japan 

e-mail: 07nd603y@mcs.ibaraki.ac.jp 

Azurin is a blue copper protein, which functions as an electron donor to nitrite reductase. A denitrifying bacteria, 

Alcaligenes xylosoxidans NCIMB 11015 has two different types of azurins, azurin I (AzI) and azurin II (AzII) 

[1]. Resonance Raman spectroscopic technique is strong methods to obtain the structure of a certain 

chromophore having specific electronic absorption in the visible region. Oxidized azurin has a intense blue color 

due to the SCys→Cu(II) ligand to metal charge transfer (LMCT), but the reduced form of azurin does not have 

any electronic absorption in the visible region. UV resonance Raman (UVRR) spectroscopc technique is a 

powerful technique to elucidate the structure and dynamics of protein molecules, which does not have electronic 

absorption in the visible region. Most of all protein molecules has electronic absorption band in the UV region 

due to the electronic absorption of aromatic amino acids. UVRR spectra of AzI and AzII were measured to 

elucidate the protein structural differences between the oxidized and reduced forms under the various conditions. 

UVRR of AzI and AzII were measured by the excitation at 244 nm. UVRR of AzI and AzII were readily to be 

assigned according to the previous report [2]. Raman bands at 1628cm -1 (Y8a, ring stretch mode), 1207cm -1 

(Y7a, ring-C stretch mode), and 1173cm -1 (Y9a, CH in-plane bending mode) are contributed from tyrosine 

residues. Raman bands at 1360 cm -1 and 1340 cm -1 are assigned to W7 Fermi doublet for N1C8 stretching mode 

of tryptophane indole. The intensity ratio of the W7 Fermi doublet (I1360/I1340) is known to reflect the 

environment of tryptophane indole [2, 3]. The intensity ratio of the W7 Raman bands of AzI and AzII were 

calculated to be 1.0 and 0.8, respectively. The environment of tryptophane residue in AzI and AzII are estimated 

to be hydrophobic and hydrophilic, respectively, from the intensity ratio of the W7 Fermi doublet Raman bands 

of AzI and AzII. Detailed UVRR spectroscpic studies of those oxidized and reduced azurins from Alcaligenes 

xylosoxidans NCIMB 11015 under the various pH conditons will be disscussed. 

Acknowledgement: A part of this work is supported by Research Promotion Bureau, Ministry of Education, 

Culture, Sports, Science and Technology (MEXT), Japan to TK, a Grant-in-Aid for Scientific Research from 

JSPS (No. 18550147), Japan to TK, and the Project of Development of Basic Technologies for Advanced 

Production Methods Using Microorganism Functions by the New Energy and Industrial Technology 

Development Organization (NEDO) to TK. 

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214


P96. Application of High Resolution Mass Spectrometry to the Analysis of 

Complexes of Substituted 3-(benzoxazol-5-yl)alanines with Pt(II) 

A. Kluczyk, H. Bartosz-Bechowski, J. Kierzenkowska, P. Stefanowicz, K. Guzow, W. Wiczk, 

Z. Szewczuk 

Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland, 

e-mail: hbb@wchuwr.pl 

The complexing properties of heterocycles are widely investigated because of the broad range of applications 

found for their metal complexes, from dyes and pigments to DNA intercalators. The heterocyclic side-chains of 

natural amino acids, as well as the delocalized amides in peptide backbone are responsible for the affinity of 

peptides and proteins to metal ions, creating the specific activity of metalloenzymes and structural properties of 

zinc fingers. Therefore the introduction of novel nonproteinaceous heterocyclic amino acids into peptides may 

result in new compounds with desired structural, physicochemical and biological properties [1]. 

A series of 2-substituted 3-(benzoxazol-5-yl)alanines were investigated for their photochemical and biological 

activity [2, 3]. It was also found that such benzoxazole derivatives substituted with 8-quinolinyl group could be 

used as effective chemosensors for Zn(II), Tb(III) and Eu(III) ions [4]. It is known that 2-pyrid-2-ylbenzoxazole 

forms a chelating didentate complex with Pt(II), where coordination to the metal occurs via the pyridine and ring 

imine nitrogens [5]. Taking all this into account we investigated the affinity of 3-(benzoxazol-5-yl)alanines 

towards Pt(II) ions, using high resolution electrospray mass spectrometry. 

C 

H 3 

O 

O 

O 

R 

N 

NH 

O 

O 

CH 3 

CH 3 

CH 3 

where R is: 

Structures of 2-substituted 3-(benzoxazol-5-yl)alanines investigated for their affinity to Pt (II). 

O 

S 

The mass spectra of investigated complexes revealed the differences between variously substituted benzoxazoles 

in respect to their affinity to Pt(II) ions; especially the three quinoline isomers show a diversity in their relation 

to the metal ion. The fragmentation experiments indicate that the binding occurs in the heterocyclic part of the 

protected amino acid derivatives. The gradual changes in complex composition, as well as the differences in 

stability in gas phase could be investigated and analyzed using mass spectrometry, which seems to be a method 

of choice for selecting promising ligands from libraries of organic compounds. 

References: 

[1] A. Staszewska, P. Stefanowicz, Z. Szewczuk, Tetrahedron Lett., 46, 5525 (2005). 

[2] M. Szabelski, M. Rogiewicz, W. Wiczk, Anal. Biochem. 342, 20 (2005). 

[3] K. Guzow, D. Szmigiel, D. Wroblewski, M. Milewska, J. Karolczak, W. Wiczk, J. Photochem. Photobiol. A: 

Chem. 187, 87 (2007). 

[4] K. Guzow, M. Milewska, D. Wróblewski, A. Giełdoń, W. Wiczk, Tetrahedron, 60, 11889 (2004). 

[5] X.F. He, Ch.M. Vogels, A. Decken, S.A. Westcott, Polyhedron, 23, 155 (2004). 

CH 3 

CH 3 

N 

N 

H 

N 

_____________________________________________________________________ 

215 

N 

N 

N


P97. Peptides Conjugated with Quinoxalines and Their Affinity to Metal 

Ions Analysed by High Resolution Mass Spectrometry 

A. Kluczyk, B. Predko, A. Staszewska, P. Stefanowicz, M. Jeżowska-Bojczuk, Z. Szewczuk 


e-mail: kluczyk@wchuwr.pl 

The search for new bioactive peptides directed our attention to new methods for introducing heterocyclic motifs 

into peptide side chains [1]. Considering the biological activity and complexing abilities of quinoxalines we 

developed a direct solid-phase synthesis of quinoxaline-peptide conjugates, expecting these new compounds to 

express novel biological properties [2]. 

We investigated two solid phase protocols of synthesis of quinoxaline-containing peptides. The first method uses 

the commercially available 4-amino-3-nitrobenzoic acid, which could be attached to the ε-amino group of lysine 

or the N-terminal α-amino group of peptide. For the second procedure we developed a special phenylalanine 

derivative, Fmoc-Phe(4’-NH2-3’-NO2)-OH, which can be used as a building block for standard Fmoc protocol 

for peptide synthesis [2]. In both these methods the quinoxaline formation involves reduction of the aromatic 

nitro group and the subsequent condensation of the o-phenylenediamine intermediate with 

α-dicarbonyl reagents. 

N 

N 

N 

N 

N 

N 

_____________________________________________________________________ 

216 

A 

N 

N 

O 

Aaa 

O 

NH 

NH 

Peptides containing quinoxaline motifs: A - as a modification of amino groups, B - in the form of a novel amino 

acid residue. Aaa represents amino acid residue or peptide fragment. 

Using these procedures we obtained a series of peptides conjugated with quinoxalines containing various 

substituents. We concentrated our attention on compounds with two 2-pyridil moieties attached to the 

quinoxaline skeletons. Such heterocyclic systems are known for their affinity to metal ions [3], whereas 

bipyridine-peptide conjugates complexed with ruthenium ion and additional quinoxaline derivative were 

investigated as metallointercalators [4]. 

We investigated the binding of copper (II) ions to both types of quinoxaline-peptide conjugates using high 

resolution electrospray mass spectrometry. The method is ideal for initial screening of potential high affinity 

ligands because of minimal sample consumption and the insight into the structure of the complex through the 

fragmentation analysis and the experiments involving proton-deuter exchange. 

Acknowledgements: Supported in part by Grant No. N N204 1616 33 from the MSHE (Poland). 

References: 

[1] A. Kluczyk, A. Staszewska, P. Stefanowicz et al., J. Peptide Sci., 12, 111 (2006). 


[3] A.A. Abdel-Shafi, M.M.H. Khalil, H.H. Abdalla, R.M. Ramadan, Transition Metal Chemistry, 27, 69 (2002). 

[4] K.D. Copeland, A.M.K. Lueras, E.D.A. Stemp, J.K. Barton, Biochemistry, 41, 12785 (2002). 

O 

Aaa 

N 

Aaa 

B 

N 

N 

NH 

O 

N 

Aaa


P98. Dinuclear Metal Complexes of a bis-intercalater Ligand as a Strong 

DNA-binder 

M. Kodera, K. Hamada, T. Nakamura, Y. Hitomi, T. Funabiki, K. Kano 

Molecular Chemistry and Biochemistry, Doshisha University, Tatara Miyakotani, 610-0321, Kyotanabe, Japan 

e-mail: mkodera@mail.doshisha.ac.jp 

In order to explore a strong DNA-binder we have synthesized dinuclear metal complexes with a new 

bis-intercalater ligand (H2L1) that has two phenanthrene groups. The ligand reacts with Zn(OAc)2 and Cu(OAc)2 

in MeOH to form dinuclear complexes [Zn2(AcO)2(L1)] and [Cu2(AcO)2(L1)], respectively. The dicopper 

complex is stable in an aqueous solution at pH 7.1, but the dizinc complex decomposes to mononuclear complex 

under aqueous conditions. The crystal structure of the dicopper complex was determined by X-ray analysis. 

DNA-binding studies are carried out with both the ligand and the dicopper complex. The dicopper complex 

binds DNA much more strongly than the ligand. DNA-binding affinity of the dicopper complex may be 

enhanced by synergistic intercalation of the phenanthrene groups with binding of phosphate group of DNA to the 

dicopper center. 

_____________________________________________________________________ 

217


P99. NMR Investigations of Cobalt(III)-hexammine Coordination to 

Domain 6 of a Group II Intron Ribozyme 

M.M.T. Korth a , M.C. Erat b , R.K.O. Sigel a 

a 

Institute of Inorganic Chemistry, University of Zurich, Winterthurerstr. 190, 8057, Zurich, Switzerland 

e-mail: maximiliane.korth@aci.uzh.ch 

b 

Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, Oxford, United Kingdom 

Group II intron ribozymes are large self-splicing RNA molecules that have a highly conserved secondary 

structure of six individual domains projecting from a central wheel (see Figure). Domain 6 (D6) contains a 

conserved adenosine (the branch point) whose 2'-OH acts as the nucleophile in the first step of splicing. Mg 2+ 

plays an essential role for the folding into the three-dimensional structure and the catalytic activity of 

ribozymes. [1] The NMR solution structure of a 27 nucleotide long D6 construct (D6-27) that supports catalysis 

was recently elucidated [2] and its intrinsic Mg 2+ binding properties were determined. [3] One Mg 2+ thereby binds 

at the branch region. 

Here we investigate possible outersphere coordination of this Mg 2+ ion in the branch region of D6-27. 

Cobalt(III)-hexammine is a commonly used mimic for the spectroscopically silent [Mg(H2O)6] 2+ in NMR studies 

and can even show NOE contacts with RNA protons in favorable cases. [Co(NH3)6] 3+ titration experiments were 

performed in H2O and D2O observing the chemical shift changes of the RNA protons. Chemical shift mapping 

shows distinct binding of [Co(NH3)6] 3+ to the branch region of D6-27. Furthermore direct NOE contacts between 

the ammine protons of [Co(NH3)6] 3+ and RNA protons are detected, providing proton-proton distances for 

structural calculations to elucidate the geometry of [Mg(H2O)6] 2+ coordination to the branchpoint. 

Acknowledgement: Financial support by the Swiss National Science Foundation (SNF-Förderungsprofessur 

PP002-114759 to R.K.O.S.) is gratefully acknowledged 

References: 

[1] R. K. O. Sigel, Eur. J. Inorg. Chem., 2005, 12, 2281-2292. 

[2] M. C. Erat, O. Zerbe, T. Fox, R. K. O. Sigel, ChemBioChem, 2007, 8, 306-314. 

[3] M. C. Erat, R. K. O. Sigel, Inorg. Chem., 2007, 46, 11224-11234. 

_____________________________________________________________________ 

218


P100. Binding of MMR Protein MutS to Mispaired DNA Adducts of 

Intercalating Ru(II) Arene Complexes 

H. Kostrhunova a , V. Marini a , J. Kasparkova a , P.J. Sadler b , J.-M. Malinge c , V. Brabec a 

a 

Institute of Biophysics, AS CR, v.v.i., Kralovopolska 135, 612 65, Brno, Czech Republic, 

e-mail: kostrhunova@ibp.cz 

b 

Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom 

c 

Centre de Biophysique Moleculaire, CNRS, Rue Charles Sadron, 45071, Orleans, France 

We examined the binding properties of Escherichia coli mismatch repair protein MutS with various DNA 

substrates containing a single centrally located adduct of Ru(II) arene complexes [(eta 6 -arene)Ru(II)(en)Cl][PF6] 

[arene=tetrahydroanthracene (Ru-THA) or p-cymene (Ru-Cym), en=ethylenediamine]. These complexes were 

chosen as representatives of two different classes of monofunctional Ru(II)-arene compounds which differ in 

DNA binding modes: one that involves combined coordination to G N7 along with noncovalent, arene 

intercalation (tricyclic-ring Ru-THA) and the other that binds to DNA only via coordination to G N7 and does 

not interact with DNA by intercalation (mono-ring Ru-Cym). Using electrophoretic mobility shift assay, the 

binding properties of MutS protein with various DNA duplexes (homo- or mismatched duplexes) containing a 

single centrally-located adduct of Ru(II)-arene compounds were examined. We have shown that presence of 

Ru(II)-arene adducts decreases the affinity of MutS for ruthenated duplexes that either have a regular sequence 

or contain a mismatch and that intercalation of the arene considerably contributes to this inhibition effect. Since 

MutS initiates mismatch repair by recognizing DNA lesions the results of the present work support the view tha 

DNA damage due to intercalation is removed from DNA by mechanism(s) other than mismatch repair. 

_____________________________________________________________________ 

219


P101. Complexation of Boric Acid with Vitamin C 

D. A. Köse and B. Zümreoğlu-Karan 

Hacettepe University Science Faculty Department of Chemistry Beytepe/Ankara/TURKEY 06800 

The exact biological function of boron in animals and humans remains unknown. The current hypothesis is that 

it stabilizes biological molecules by linking them through ester bridges. An exciting article suggests that borate 

minerals could play a crucial role in the early world by stabilizing the cyclic ribose during RNA synthesis [1]. At 

intracellular pH, nearly all boron exists as boric acid which behaves as a Lewis acid and forms molecular 

addition compounds with amino- and hydroxy acids, carbohydrates, nucleotides and vitamins through electron 

donor-acceptor interactions. The complexation with sugars is particularly important in understanding the role of 

boron as carrier for nucleotides and carbohydrates and for the appropriate design of boron compounds in terms 

of biomimetic aspects. 

(a) (b) 

Vitamin C (Ascorbic acid, H2A) is a sugar acid with a cis-enediol group on the sugar ring and adjacent alcoholic 

hydroxy groups on the side chain, available for complex formation with boron. Little is known about the 

interaction of ascorbic acid with boric acid. We present here the complexation of H2A and iso-propylidene-H2A 

with boric acid, characterization of the isolated complexes by 13 C CP- & 11 B-MAS NMR and the relative 

stabilities of 1:1 (a) and 1:2 (b) complexes in aqueous phase. 

Acknowledgement: Support from Hacettepe University through project 06D021002 is acknowledged. 

References: 

[1] A. Ricardo, M.A. Carrigan, A.N. Olcott, S.A. Benner, Science, 303 (2004) 196. 

_____________________________________________________________________ 

220


P102. Electron Transfer Dynamics of Cytochrome C at Interfaces 

A. Kranich a , K. H. Ly a , P. Hildebrandt a , D. H. Murgida. b 

a Institut für Chemie, TU Berlin, Str. des 17. Juni 135, 10623, Berlin, Germany 

e-mail: anja.kranich@tu-berlin.de 

b INQUIMAE, Universidad de Buenos Aires, , C1428EH, Buenos Aires, Argentina 

Elucidating the mechanism and dynamics of electron transfer processes between redox proteins is one of the 

fundamental challenges in molecular biophysics. Upon coating Ag electrodes with self-assembled monolayers 

of amphiphiles, it is possible to mimic biological interfaces where most of the electron transfer processes of 

redox proteins take place. This approach has the specific advantage of facilitating the determination of ET rate 

constants as a function of distance by simply varying the chain length of the thiols. In most of the studies 

reported so far, “unusual” distance-dependences of the non-adiabatic electron transfer rate constants have been 

found. The origin for this behavior has been elusive and discussed controversially. Using a two-colour timeresolved 

surface enhanced resonance Raman spectroelectrochemical approach we have been able to monitor 

simultaneously and in real time the structure, electron transfer kinetics and configurational fluctuations of 

cytochrome c electrostatically adsorbed to SAM-coated electrodes. It is shown that the overall electron transfer 

kinetics is determined by protein dynamics rather than by tunnelling probabilities and, in turn, is controlled by 

the interfacial electric field. Implications for inter-protein electron transfer at biological membranes are 

discussed. 

_____________________________________________________________________ 

221


P103. Square-Planar Metallointercalators as Potential Anticancer Agents 

A. Krause-Heuer a , N. Orkey a , B. Singh a , J. Aldrich-Wright a 

a School of Biomedical and Health Sciences, University of Western Sydney, Narellen Road, 2560, Campbelltown, 

Australia 

e-mail: j.Aldrich-Wright@uws.edu.au 

The majority of clinically used platinum-based anticancer agents (eg cisplatin and carboplatin) effect their 

cytotoxic action by coordinating to specific atoms in the base-pairs of the DNA helix. However, unfavourable 

side effects such as nephrotoxicity and neurotoxicity are associated with these drugs. In an attempt to overcome 

the potential side effects with such drugs, a series of platinum(II) compounds have been innovatively designed 

which interact via a completely different mechanism – intercalation. These square-planar metallointercalators are 

composed of two bidentate ligands positioned around the platinum(II) metal centre: an intercalating moiety and a 

non-intercalating (ancillary) group. The planar intercalating segment is comprised of a minimum of three 

aromatic rings fused together and both chiral and achiral ancillary ligands have been utilised. The first compound 

in our series of platinum(II) metallointercalators was 1S, 2S diaminocyclohexane-1, 10-phenanthroline 

platinum(II). This compound displays a higher level of activity than cisplatin in all cell lines tested and is up to 

twenty times more soluble in water. These features suggest that this and similar compounds may demonstrate 

higher clinical effectiveness and lower toxicity than platinum-based compounds in clinical use. In vitro 

experiments have shown these complexes to be biologically active. The binding affinities of these compounds 

have also been investigated through UV-vis and CD spectroscopy. 

References: 

[1] Fenton, R. R.; Aldrich-Wright, J. R. In PCT Int. Appl.: Australia, 2002, pp 1-26. 

_____________________________________________________________________ 

222


P104. N-H⋅⋅⋅S and N-H⋅⋅⋅O Hydrogen Bonds as a Structure Forming Factor 

in Metal Silanethiolate Complexes with Additional N-donors 

A. Kropidłowska a , I. Turowska-Tyrk b , B. Becker a 

a 

Chemical Faculty, Gdańsk University of Technology, 11/12 Narutowicza Str., 80-952 Gdańsk, Poland 

e-mail: anna@urethan.chem.pg.gda.pl 

b 

Chemical Faculty, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland 

The N–H···S interactions are known for their important role in biological systems since the sulfur and nitrogen 

atoms of the side-chain of cysteine and histidine residues are two of the most common donors in biochemistry. 

They can bind to metal ions and form the active centers of numerous metalloenzymes and metalloproteins. Two 

Cys and two His residues bind e.g. to the zinc ion in the zinc finger proteins (transcription factor IIIA, enhancer 

binding protein in the human immunodeficiency virus type 1 (HIV-EPI) [1] and related nucleic acid binding 

proteins) [2]. Also a copper protein, Alcaligenes azurin, possesses SSN2 binding in the active site [3], although 

the role of N-H⋅⋅⋅S interaction in this system remains unclear. It was the reason which has stimulated scientists to 

search for the structural models of the S2N2 binding sites (see e.g. [4]). Since zinc is a spectroscopically silent 

metal, therefore cadmium and cobalt have been substituted in the native proteins to further aid in the study of the 

spectroscopic features of zinc centers [5]. For this reason, the study of Cd(II) coordination by protein-related 

ligands has attained renewed interest and attention. 

Our research efforts are also prompted towards the goal of generating complexes supported by a mixed 

nitrogen/sulfur coordination environment. Recently we reported on silanethiolate complexes containing 

aminopyridines [6] and diamines [7] as coligands with a sulfur atom serving as the N-H···S hydrogen bond 

acceptor. Now, we turned our attention to other N-donors capable of providing the NH donor group, reasoning 

that hydrogen bonding interactions might prove useful towards the stabilization and isolation of a nitrogen/sulfur 

ligated species.Using [Cd{SSi(OBu t )3}2]2 [8] as a substrate and cyclic aliphatic amines as coligands we 

synthesized new heteroleptic silanethiolate complexes with S2N2 binding sites. Thus, we have obtained four 

tetrahedral complexes i.e. [Cd{SSi(OBu t )3}2(NC4H9)2] 1 with pyrrolidine, [Cd{SSi(OBu t )3}2(NC5H11)2] with 

piperidine 2, [Cd{SSi(OBu t )3}2(NC4H9O)2] with morpholine 3 and [Cd{SSi(OBu t )3}2(N2C4H10)2] with piperazine 

4. The X-ray structural analysis revealed basic forms of supramolecular packing mediated by inter- (see 3 in Fig. 

1) and intramolecular hydrogen bonds of the N-H···S and N-H···O types. 

Fig.1. 

Acknowledgement: The research was supported by the grant of the Polish Ministry of Education and Science 

(grant No. 1 T09A 117 30). A. Kropidłowska thanks The Foundation for Polish Science for the fellowship. 

References: 

[1] A. J. van Wijnen, K. L. Wright, J. B. Lian, J. L. Stein, G. S. Stein, J . Biol. Chem., 264 (1989) 15034. 

[2] R. M. Evans and S. M. Hollenberg, Cell, 52 (1988) 1. 

[3] E. N. Baker, J. Mol. Biol., 203 (1988) 1071. 

[4] C. E. Forde, A. J. Lough, R. H. Morris, R. Ramachandran, Inorg. Chem., 35 (1996) 2747. 

[5] B. L. Vallee, in Zinc Proteins (T. G. Spiro, ed.) Wiley-lnterscience, New York, 1983. 

[6] A. Kropidłowska, I. Turowska-Tyrk, B. Becker, 18 ICPOC, Warsaw (2006) Book of Abstracts, 72. 

[7] A. Kropidłowska, I. Turowska-Tyrk, B. Becker, XVth Winter School on Coordination Chemistry, Karpacz 

(2006) Book of Abstracts, 61. 

[8] W. Wojnowski, B. Becker, L. Walz, K. Peters, E.-M. Peters, H.G. von Schnering, Polyhedron 11 (1992) 607. 

_____________________________________________________________________ 

223


P105. Structural Studies of Copper(II) Binding to the Novel Peptidyl 

Derivative of Quinoxaline: N-(3-(2, 3-di(pyridin-2-yl)quinoxalin-6yl)alanyl)glycine 

M. Kucharczyk a , W. Szczepanik a , P. Młynarz b , N. D’Amelio c , A. Staszewska a , 

P. Stefanowicz a , Z. Szewczuk a , A. Olbert-Majkut a and M. Jeżowska-Bojczuk a 

a 

Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland 

e-mail: marzenak@eto.wchuwr.pl 

b 

Faculty of Chemistry, Wrocław University of Technology, Wyspiańskiego 27, 50-373 Wrocław, Poland 

c 

Department of Chemistry, University of Siena, Via Aldo Moro, 53100 Siena, Italy 

Studies of interactions between organic compounds and DNA, especially the characteristics of structural aspects 

of DNA interaction with small molecules, are of particular interest as they can result in effective in highly 

targeted therapeutic applications. It may be expected that peptides conjugated with quinoxaline analogs may 

deliver novel probes of DNA structure and may help to design new DNA targeting agents. Attachment of the 

peptidic chain to the substance of potential biological activity may enable its cellular recognition and subsequent 

transport. 

Transition metal complexes capable of cleaving DNA are of importance for their potential use as new structural 

probes in nucleic acids chemistry and as therapeutic agents. They also can be used to studies of the polymorphic 

nature of nucleic acid conformation. In this context, several metal ions were tested for their ability to form stable 

complexes with 2, 3-di(pyridin-2-yl)quinoxaline. Cupric complexes appeared efficient to generate oxidative 

nicks within DNA [1], while the species that contained platinum [2], palladium [3] and ruthenium ions [4] 

focused attention as highly effective metal chelators that may show significant chemotherapeutic activity. 

Our previous study on the DNA cleavage in the presence of the copper(II) and iron(II) complexes of N-(3-(2, 3di(pyridin-2-yl)quinoxalin-6-yl)alanyl)glycine 

(DPQa-Gly) showed that this ligand may greatly enhance the 

yield of DNA degradation by metal ions, especially copper(II) [5]. 

N 

H 2 

_____________________________________________________________________ 

224 

O 

N 

NH 

Herein we report the coordination characteristics of the Cu(II)-DPQa-Gly system. By means of potentiometric 

titrations, NMR, EPR, CD and UV-Vis spectroscopic methods, mass spectrometry as well as molecular 

modeling it was resolved, which residue in the studied substance possesses stronger chelating properties: 2, 3di(pyridin-2-yl)quinoxaline 

or Ala-Gly dipeptides. 

Acknowledgement: This work is financially supported by the Polish State Committee for Scientific Research 

(KBN), grant no. N N204 1616 33. 

N 

N 

N 

COOH 

References: 

[1] B.K. Santra, P.A.N. Reddy, G. Neelakanta, S. Mahadevan, M. Nethaji, A.R.J. Chakravarty, J. Inorg. 

Biochem. 89, 191 (2002) 

[2] J. Granifo, M.E. Vargas, M.T. Garland, R. Baggio, Inorg. Chim. Acta 305, 143 (2000) 

[3] J. Granifo, M.T. Garland, R. Baggio, Inorg. Chim. Acta 348, 263 (2003) 

[4] R.L. Williams, H.N. Toft, B. Winkel, K.J. Brewer, Inorg. Chem. 42, 4394 (2003) 

[5] W. Szczepanik, M. Kucharczyk, A. Staszewska, P. Stefanowicz, Z. Szewczuk, J. Skała, A. Mysiak, M. 

Jeżowska-Bojczuk, submitted


P106. In Silico Approaching to Cisplatin Toxicity Quantum Chemical 

Studies on Platinum(II) – Cysteine Systems 

J. Kuduk-Jaworska a , I. Jaroszewicz a , J. Jański a , H. Chojnacki b 

a Faculty of Chemistry Wroclaw University, F. Joliot-Curie 14, 50-383 Wroclaw, Poland 

b Institute of Physical and Theoretical Chemistry, Wrocław University of Technology, Wyb. Wyspiańskiego 27, 

50-370 Wrocław, Poland 

The differences in therapeutic ability of antitumor drug cisplatin (cis- diamminedichloroplatinum(II)) and its 

trans-isomer, are still intriguing for investigators who would like to learn in more detail about the molecular 

bases of such behavior [1]. It is well documented that cisplatin has high antitumor activity and strong toxicity, 

especially towards kidneys, whereas the trans isomer is therapeutically inefficient and non nephrotoxic. 

However, the studies on mechanisms of biological activity of both isomers, performed so far, were focused on 

interaction of platinum(II) complexes with DNA as critical target molecule. The problems of cisplatin and its 

congeners toxicity, though clinically very important, were far less studied [1]. Similar preferences can be 

observed in theoretical approaches [2]. 

Contrary to this tendency, our interest has been concentrated on the toxicity of cisplatin in comparison with nontoxicity 

of its trans isomer. Therefore we evaluated the reactions responsible for harmful biological effects, and 

consequently, the impact of platinum(II) complexes on molecules containing sulphur donors became the object 

of the presented study. 

Our approach relied on applying quantum chemical in silico methodology for the evaluation of "platinum(II) - 

cysteine" and its model systems. There were investigated the following systems: (1) a/cisplatin (b/transplatin) 

with CH3SH; (2) a/cisplatin (b/transplatin) with cysteine. 

The electronic structure for molecular systems has been studied at non-empirical all-electron level by using 

density functional (DFT) or Moeller-Plesset (MP2) methods within the correlation consistent cc-pVTZ [3] basis 

set. In the case of platinum the widest Huzinaga basis set with polarization functions has been used. In order to 

avoid the long optimization process, at the first stage, the optimization was performed at the all-valence 

MOPAC-PM6 method [4] following the B3LYP density functional or MP2 formalism [5] in the next step. The 

B3LYP [6] density functional was applied using GAUSSIAN-03 program package [7]. 

Acknowledgements: The numerical calculations have been performed in part at Wrocław Networking and 

Supercomputing Center. Wrocław University of Technology support is also acknowledged. 

References: 

[1] Reedijk, J.; Tauben, J. M. in: Cisplatin. Chemistry and Biochemistry of a Leading Anticancer Drug, Lippert, 

B. (Ed.), Wiley-WCH, 1999, 339-362. 

[2] Kozelka, J.; ibid, 537-556. 

[3] Huzinaga, S.; (Ed.), Gaussian Basis Sets for Molecular Calculations, Elsevier, Amsterdam 1984. 

[4] . 

[5] Szabó, A.; Ostlund, N.S. Modern Quantum Chemistry, Dover, Mineola 1996. 

[6] Becke, A.D.; J Chem Phys, 1993, 98, 5648- 5653. 

[7] GAUSSIAN-03, Rev. D-01, Pople, J.A. Gaussian Inc., Pittsburgh, PA, 2004. 

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225


P107. Cys-His Motifs as a Target for Ni(II) Ions in Peptides 

K. Kulon a , D. Valensin b , W. Kamysz c , R. Nadolny c , E. Gaggelli b , G. Valensin b , 

H. Kozłowski a 

a 

Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383, Wroclaw, Poland 

e-mail: kulon@eto.wchuwr.pl 

b 

Department of Chemistry, University of Siena, via Aldo Moro, 53-100, Siena, Italy 

c 

Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdansk, Al. Gen. Hallera 

107, 80-416, Gdansk, Poland 

Waglerin I was isolated from venom of Wagler’s pit viper (Trimeresurus wagleri), a small venomous arboreal 

snake occurring in Malaysia, the Philippines, Thailand, Indonesia and the Indo-Australian archipelago [1, 2, 3]. 

It is a peptide toxin composed of 22 amino acid residues with one disulfide bond. Characteristic for this sequence 

are seven proline residues and a high content of basic amino acids, which beside intramolecular disulfide bond 

and His-10 are believed to be essential for biological activity of the toxin [3, 4]. 

Three fragments of the toxin (GGKPDLRPCHP-NH2, PCHYIPRPKPR-NH2, PCHPPCHYIPR-NH2), due to the 

presence of two Cys and His residues, are potentially very attractive ligands for transition metal ions. The main 

aim was to establish the impact of these two adjacent residues on Ni 2+ ion binding, especially because this kind 

of motif is very common in nature. Waglerin’s fragments and their N-protected analogues were studied with 

Ni2+ ions using combined potentiometric and spectroscopic measurements (UV-Vis, CD, EPR and NMR). In all 

peptides, except PCHPPCHYIPR-NH2 with disulfide bridge, Cys-His motif was found to be crucial for the 

coordination of Ni 2+ ions. In the case of the N-unprotected analogues, N-terminal amino group participates in the 

coordination as well [5]. 

Acknowledgment 

This work was supported by Polish Ministry of Science and Higher Education (MEiN 1 T09A 008 30 and N204 

2608 33). 

References: 

[1] L. Chuang, H. Yu, C. Chen, T. Huang, S. Wu, K. Wang, Biochim. Biophys. Acta, 1996, 1292, 145-155 

[2] J. J. Schmidt, S. A. Weinstein, Toxicon., 1995, 33, 1043-1049 

[3] Y. Hsiao, C. Chuang, L. Chuang, H. Yu, K. Wang, S. Chiou, S. Wu, Biochem. Biophys. Res. Commun., 

1996, 227, 59-63 

[4] L. C. Sellin, K. Mattila, A. Annila, J. J. Schmidt, J. J. McArdle. M. Hyvönen, T. T. Rantala, T. Kivistö, 

Biopchys. J., 1996, 70, 3-13 

[5] K. Kulon, D. Valensin, W. Kamysz, R. Nadolny, E. Gaggelli, G. Valensin, H. Kozłowski J. Chem. Soc. 

Dalton. Trans, accepted 

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226


P108. Oxidation of Nitrite by a trans-Dioxoruthenium(VI) Complex. 

Direct Evidence for Reversible Oxygen Atom Transfer 

T. Ch. Lau 

Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Hong Kong, China 

e-mail:bhtclau@cityu.edu.hk 

The inter-conversion between nitrite and nitrate is of fundamental interest and of biological importance. Reaction 

of trans-[RuVI(L)(O)2] 2+ (1, L = 1, 12-dimethyl-3, 4:9, 10-dibenzo-1, 12-diaza-5, 8- dioxacyclopentadecane, a 

tetradentate macrocyclic ligand with N2O2 donor atoms) with nitrite in aqueous solution or in H 2 O/CH 3 CN 

produces the corresponding (nitrato)oxoruthenium(IV) species, trans-[RuIV(L)(O)(ONO2)] + (2), which then 

undergoes relatively slow aquation to give trans-[RuIV(L)(O)(OH2)] 2+ . These processes have been monitored by 

both ESI/MS and UV/Vis spectrophotometry. The structure of trans-[RuIV(L)(O)(ONO2)] + (2) has been 

determined by X-ray crystallography. The ruthenium center adopts a distorted octahedral geometry with the oxo 

and the nitrato ligands trans to each other. The Ru=O distance is 1.735(3) Å, the Ru-ONO2 distance is 2.163(4) 

Å and the Ru-O-NO2 angle is 138.46(35)°. Reaction of trans-[RuVI(L)(18O)2] 2+ (1-18O2) with N16O2- in 

H2O/CH3CN produces the 18O-enriched (nitrato)oxoruthenium(IV) species 2-18O2. Analysis of the ESI/MS 

spectrum of 2-18O2 suggests that scrambling of the 18O atoms has occurred. A mechanism that involves linkage 

isomerization of the nitrato ligand and reversible oxygen atom transfer is proposed.Financial support by the 

Research Grants Council of Hong Kong (CityU 1105/02P and CityU 2/06C) is gratefully acknowledged. 

References: 

[1] W. L. Man, W. W. Y. Lam, W. Y. Wong, T. C. Lau J. Am. Chem. Soc. 2006, 128, 14669-14675. 

_____________________________________________________________________ 

227


P109. Characterization and Reactivity of [Ru(β)(L)Cl2]ClO4 ( L= Tpea, 

Me6tpea ) 

J. E. Lee , J. H. Cho , H. I. Lee 

Department of Chemistry, Kyungpook National University, 1370 SangKyeok-Dong, 702-701, Deagu, Republic of 

Korea 

e-mail: jjuni32@hanmail.net 

We prepared Ru(III) complexes with tripodal, pyrazoyl ligand [Ru(β)(L)Cl2]ClO4 (L= Tpea 1, Me6tpea 2)]. 

Single crystal study identified octahedral geometry with tetradentate Tpea ligand and two chloride ions. 

Reactivity toward the oxidation of d-glucose and d-fructose by the complexes and hydrogen peroxide was 

investigated using UV-VIS and EPR, which showed that only 1 could oxidize the hexoses at pH 7 (0.01M 

phosphate). We describe the stabilization of high oxidation state and other properties. 

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228


P110. Oxygen Sensibility of Ni-Fe Hydrogenases with Modified Gas 

Channels 

F. Leroux a , S. Dementin a , B. Burlat a , A. Volbeda b , J. Fontecilla-Camps b , M. Rousset a , 

P. Bertrand a , B. Guigliarelli a , Ch. Léger a 

a 

Laboratoire de Bioénergétique et Ingénierie des, CNRS - Université de Provence, 31 chemin Jospeh Aiguier, 

13402, Marseille Cedex 20, France, 

b 

Laboratoire de Cristallographie et Cristallogenès, CEA, Grenoble, France 

e-mail: fanny.leroux@ibsm.cnrs-mrs.fr 

Hydrogenases catalyze the conversion between H2 and H+ as part of the bioenergetic metabolism of most 

bacteria. The main obstacle for their use as catalysts in biofuel cells is the fact that they are inhibited by 

molecular oxygen [1]. The active site of the so-called Ni-Fe hydrogenase is buried in the protein. Structural and 

molecular dynamic studies suggest that a hydrophobic channel guides hydrogen and inhibitors towards the active 

site and recent results suggest that the shape of this tunnel may determine the sensitivity to oxygen [2]. 

We introduce a quantitative experimental method for probing the rates of intra-molecular diffusion within 

hydrogenases based on using Protein Film Voltammetry, a technique where enzymes molecules are adsorbed on 

an electrode, a potential is applied and the resulting current is proportional to enzyme's activity [3-4]. We use it 

to resolve the kinetics of binding and release of the competitive inhibitor CO and of the reaction with O2 [5-6]. 

We study how the structure of the channel affects the diffusion of CO and the rate of O2 inhibition in several 

mutants whose structures have been determined. We will show that certain mutations slow down gas diffusion 

[7] and affect the sensitivity of the enzyme. However, CO always reacts with the enzyme much faster than O2 

does, this implies that intramolecular transport does not limit the rate of oxygen inhibition, at least in the WT 

enzyme. 

References: 

[1] De Lacey, Fernandez, Rousset, Cammack, Chem. Rev. 2007, 107 (10), 4304-30. 

[2] Buhrke, Lenz, Krauss, Friedrich, J. Biol. Chem. 2005, 23791. 

[3] Léger, Elliott, Hoke, Jeuken, Jones, Armstrong, Biochemistry, 2003, 42, 8653. 

[4] Léger, Bertrand, Chem Rev, in press (july 2008). 

[5] Léger, Dementin, Bertrand, Rousset, Guigliarelli, J. Am. Chem. Soc., 126, 2004, 38. 

[6] Baffert, Demuez, Cournac, Burlat, Guigliarelli, Bertrand, Girbal, Léger, Angew. Chem. Int. Ed. 47, 2008, 

2052. 

[7] Leroux, Dementin, Burlat, Cournac, Volbeda, Champ, Martin, Guigliarelli, Bertrand, Fontecilla-Camps, 

Rousset, Léger, submitted. 

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229


P111. Correlating Properties, Structure and Function in the EcI/II 

Metallothionein from Wheat Embryos 

O. Leszczyszyn a , E. Peroza b , E. Freisinger b , C. Blindauer a 

a Department of Chemistry, University of Warwick, Gibbett Hill Road, CV4 7AL, Coventry, United Kingdom, 

b Institute of Inorganic Chemistry, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, 

Switzerland 

e-mail: o.i.leszczyszyn@warwick.ac.uk 

Over the last decade, the advancement of genome sequencing, microarray and high-throughput protein 

identification techniques has resulted in an exponential increase in the number of plant metallothionein (pMT) 

sequences in protein and translated nucleotide databases. More and more evidence shows that the four pMT 

subfamilies are differentially expressed during various developmental stages and in different organs [1], and 

that they display significant variation in the number and arrangement of CXC motifs [2]. Therefore, it is 

reasonable to suggest that pMTs carry out compartmentalised roles for which they possess specific properties. 

However, the relative paucity of structural and biochemical information for pMTs severely limits the scope for 

the correlation of properties with structure and function. Therefore, further research focussing on both structure 

and metal binding dynamics is required to advance our understanding in this field. 

Given the critical need for structural and biochemical information on pMTs, our research focuses on the 

elucidation of the solution structure of wheat EcI/II; the prototype type 4 pMT. In addition, we have probed 

both the kinetics and thermodynamics of metal binding using a range of techniques, including multinuclear 

NMR, mass spectrometry and molecular biology. These studies show that EcI/II binds six zinc ions in two 

distinct domains with stoichiometries of Zn2Cys6 and Zn4Cys11His2 [3-4]. Structure calculations reveal that the 

individual EcI/II domains possess unique structural features not previously reported in MT literature. These 

novel structural features confer distinct backbone and metal dynamic properties to each domain, and are likely 

to have a functional significance. 

References: 

[1] N.J. Robinson, A.M. Tommey, C. Kuske, P.J. Jackson, Biochem J, 295, 1-10 (1993) 

[2] C. Cobbett, P. Goldsbrough, Annu Rev Plant Biol, 53, 159-168 (2002) 

[3] O.I. Leszczyszyn, R. Schmidt, C.A. Blindauer, Proteins: Struc Func Bioinf, 68, 922-935 (2007) 

[4] E.A. Peroza, E. Freisinger, J Biol Chem, 12(3), 377-391 (2007) 

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230

L. Lista 


P112. Heme-Protein Models: Toward Artificial Enzymes 

Chemistry, University of Naples, via Cynthia, 80126, Naples, Italy 

e-mail: lilista@unina.it 

Metalloproteins are involved in fundamental biological processes and utilize a relatively small number of metal 

based prosthetic groups to serve numerous chemical functions [1]. Heme-proteins represent a fascinating 

example in this respect; a single prosthetic group, the heme, promotes a variety of functions such as dioxygen 

transport and storage, electron transfer and catalysis of redox reactions [2]. In the last decades, numerous studies 

have been devoted to the structure-activity relationships in hemoproteins: model compounds, able to reproduce 

the structural and functional features of heme-proteins, represent a useful tools to elucidate the mechanism of 

action of natural systems. A new class of heme-peptide conjugates have been developed in our laboratory [3], 

with the aim of investigating the effects of peptide chain composition and folding in modulating the properties of 

the metal ion inserted into the porphyrin ring. The main features are the covalent structure and a well defined 

helical conformation of the peptide chains linked to the deuteroporphyrin ring [4]. Here, we present a 

pentacoordinated metal ion porphyrin miniprotein, developed to allow the binding of exogenous ligands at the 

sixth vacant position. This model is stable and water soluble and exhibits peroxidase activity. Structural and 

functional data, compared to other model systems and natural peroxidases, will be presented. 

References: 

[1] a) S. J. Lippard, J.M. Berg, Principles of Bioinorganic Chemistry, University Science Books, Mill Valley, 

CA, 1994; b) R. H: Holm, P. Kennepohl, E. I. Solomon, Chem. Rev. 1996, 96, 2239-2314. 

[2] The Porphyrins, (Ed.: D. Dolphin), Academic Press, New York, 1979. 

[3] A. Lombardi, F. Nastri, D. Marasco, O. Maglio, G. De Sanctis, F. Sinibaldi, R. Santucci, M. Coletta, V. 

Pavone, Chem. Eur. J. 2003, 9, 5643-5654. 

[4] A. Lombardi, F. Nastri, V. Pavone Chem. Rev. 2001, 101, 3165-3189. 

_____________________________________________________________________ 

231


P113. Labeling Thiele’s Acid and its Derivatives with [ 99m Tc(CO)3(H2O)3] + 

Through Retro-Diels-Alder Reaction in Aqueous Media 

Y. Liu, P. Schmutz, B. Spingler, R. Alberto 

Institute of Inorganic Chemistry, University of Zürich, Winterthurerstr. 190, CH-8057, Zürich, Switzerland 

e-mail: liuyu@aci.uzh.ch 

The amino acids coupled with tripodal Dap (Diamino propionic acid) has been introduced in our group to 

prepare Re(I)/Tc(I) tri-carbonyl complexes, which can be recognized and transported by LAT1 transporter[1]. 

Based upon this observation, Cp (Cyclopentadiene), with its smaller and more compact size than Dap, should be 

a good chelator candidate for Re(I)/Tc(I) tri-carbonyl chemistry. Contrary to the conventional view of moisture 

sensitive chemistry of Cp, C5H5COOH can react with Re(I)/Tc(I) triscarbonyl to produce (CO)3MC5H4COOH 

(M=Re(I), 99m Tc(I)) in aqueous media (Scheme). What is more, the Diels-Alder dimer of C5H5COOH, Thiele’s 

acid (C5H5COOH)2, reacting with Re(I)/Tc(I) triscarbonyl as well in aqueous media, results in the formation of 

(CO)3MC5H4COOH (Scheme), which gives a first example of retro-Diels-Alder complexation of triscarbonyl 

compound at low temperature (


P114. Complexes of Copper(II) with Nicotinamide Adenine Dinucleotide 

(NAD + ) in Binary and Ternary Systems Including Spermine (Spm) 

L. Lomozik, A. Gasowska, K. Basinski 

Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland 

e-mail: basinski@amu.edu.pl 

Nicotinamide adenine dinucleotide (NAD + ) is a coenzyme found in all living cells which consists of two 

nucleotides linked through their phosphate groups. In metabolism, NAD + is involved in redox reactions of 

cellular respiration, carrying electrons from one reaction to another [1, 2]. The bioligand makes an interesting 

object of research because of its biochemical reactivity with the enzymes from the group of dehydrogenases [3]. 

Unfortunately not much literature on the coordination of NAD + through metals present in living organism has 

been published as yet [4]. 

Interaction of nicotinamide adenine dinucleotide with copper(II) ions in binary and ternary systems including 

spermine has been observed. Computer analysis of potentiometric titration data in the Cu(II)-NAD + system has 

shown the formation of CuH2(NAD + ), CuH(NAD + ), Cu(NAD + )(OH) and Cu(NAD + )(OH)2 complexes and in the 

ternary system including spermine (1, 12-diamino-4, 9-diazadodecane) – the formation of Cu(NAD + )H4(Spm) 

and Cu(NAD + )H5(Spm) complexes. To determine the coordination centres, Vis spectra of the complexes were 

made. For example, the λmax values obtained were 808.0, 793.7 and 665.0 nm for CuH2(NAD + ), CuH(NAD + ) and 

Cu(NAD + )(OH)2, respectively, which corresponds to the {O} type of metallation in CuH2(NAD + ) and 

CuH(NAD + ) and the {N, O} type of metallation in Cu(NAD + )(OH)2 complex [5]. Analysis of the 13 C and 31 P 

NMR spectra has confirmed these conclusions. 

Full recognition of the character of copper-NAD + and copper-NAD + -spermine interactions in model systems is 

expected to give information on their probable participation in biological processes, in which nicotinamide 

adenine dinucleotide takes part. The results of these observations should yield better insight into intracellular 

mechanisms and emphasise the role of metals in living organisms. 

References: 

[1] S. Sivaranam et al., Biochem., 42, 4406-4413 (2003) 

[2] J. M. Berg, J. L. Tymoczko, L. Stryer, Biochemistry, 465-514 (2005) 

[3] G. R. Stockwell, J. M. Thornton, J. Mol. Biol., 356, 928-944 (2006) 

[4] L. A. Herrero et al., J. Biol. Inorg. Chem., 7, 313-317 (2002) 

[5] A. Gasowska, J. Inorg. Biochem., 99, 1698-1707 (2005) 

_____________________________________________________________________ 

233


I. P. Lorenz 

P115. Aziridines: Coordination Chemistry And Biological Activity 

Ludwig-Maximilians-University of Munich, Department of Chemistry and Biochemistry, Butenandtstr. 9-13, 

81377 Munich, Germany 

e-mail: ipl@cup.uni-muenchen.de 

Aziridines, the three-membered N-heterocycles, are important not only as synthetic tools in organic chemistry. 

Compounds with aziridine mojeties show cytostatic, cytotoxic and mutagene activity. The family of 

mitomycines, for instance, are used in the chemotherapy of cancer deseases. Their mode of action is assumed to 

the strained ring structure which is opened enzymatically resulting in the methylation of DNA-tumor cells. 

Aziridine complexes may also possess therapeutic applications, which is the reason we investigate in detail the 

coordination chemistry of aziridines and their metal-induced or metal-mediated reactions. 

Our results demonstrate that aziridines are not only useful as ligands for transition metals yielding mono-, bis, 

tris- and tetrakis-aziridine complexes. 

Aziridines are also versatile and active ligands which lead to compounds with different functionalities. In special 

cases aziridines react via single C-N-opening reaction with co-ligands (like CO or itself) to give e. g. 

β-aminoacyl complexes, heterocyclic carbene complexes or β-aminoaziridine complexes. The last hitherto 

unknown N, N’-chelate is the formal dimer of aziridine and can be separated from the metal and isolated; 

especially its Cu(II) complex is highly cytotoxic. Rh(I) and Ir(I) complexes with this N, N’-chelate undergo an 

oxidative addition reaction after a second C-N-opening reaction to yield the new tridentate ligand 

(N-aminoethyl)-2-amidoethyl with two M-N and one M-C bonds. 

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234


P116. Vanadate, a Nonlinear Competetive Inhibitor of Human Prostatic 

Acid Phosphatase, Exhibiting Positive Cooperativity in Ligand Binding 

E. Luchter-Wasylewska, N. Hutyra, M. Górny 

Department of Medical Biochemistry, Jagiellonian University, Collegium Medicum, Kopernika 7, 31-034 

Krakow, Krakow, Poland 

e-mail: mbwasyle@cyf-kr.edu.pl 

Human prostatic acid phosphatase (PAP) catalyses hydrolysis of phosphoesters; it removes phosphate moiety 

from phosphoserine (Pser), phosphothreonine (Pthr) and phosphotyrosine (Ptyr) residues. PAP belongs to 

regulatory enzymes: it exhibits positive cooperativity in substrate binding; degree of cooperativity grows when 

PAP concentration is increased. 

PAP was found to be a prostate tumor suppressor. Receptor cErbB2 of the prostate cell was identified as PAP 

substrate in vivo: PAP dephosphorylates cErbB2 at tyrosine residues what results in the reduction of its kinase 

activity. In advanced prostate cancer cells, the level of PAP is decreased; thus hyperphosphorylation of cErbB2 

at tyrosine residues and activation of the downstream extracellular signal-regulated kinase (ERK)/mitogen 

activated protein kinase (MAPK) signaling is observed, which results in prostate cancer progresssion. 

Studies on inhibition of PAP catalytic activity by vanadate were performed after the cooperative properties of 

PAP were stated by us. Vanadate was found to be a nonlinear competitive inhibitor of phenyl phosphate PAPcatalysed 

hydrolysis. When inhibitor concentration was increased, the half saturation constant rose, but the 

turnover number and the Hill cooperation coefficient remained constant. Thus, sodium vanadate at growing 

concentration did not change the cooperativity in substrate bindng exhibited by PAP. 

_____________________________________________________________________ 

235


P117. Structural Characterization of a Series of Novel Pt(II) Complex with 

1,2,4-triazolo[1,5-a]pyrimidines as Nonleaving Group 

I. Łakomska 

Faculty of Chemistry, Nicolaus Copernicus University, Gagarina 7, 87-100 Toruń, Poland 

e-mail: dziubek@umk.pl 

Clinical inconveniences in cisplatin chemotherapy prompted the design and synthesis of more effective and less 

toxic platinum based anticancer drugs. 

Following this research line, a series of platinum(II) complexes with 1, 2, 4-triazolo[1, 5-a]pyrimidine (tp) (1), 5methyl-1, 

2, 4-triazolo[1, 5-a]pyrimidin-7(4H)-one (HmtpO) (2) and 5, 7-dimethyl-1, 2, 4-triazolo[1, 5a]pyrimidine 

(dmtp) (3) has been prepared and studied by spectroscopic methods, especially by multinuclear 

NMR ( 1 H, 13 C, 15 N, 195 Pt) and IR spectroscopy. Analysis of 1 H NMR spectra revealed, that binding of 

triazolopyrimidine to Pt(II) ions results in the deshielding of H(2) and H(6) resonances (∆coord=0.28-0.74 ppm). 

However, those changes do not indicate unambiguously which of the heterocyclic nitrogen atoms is engaged in 

formation of the platinum-nitrogen bond. This problem was solved by means of 15 N- 1 H HETCOR spectra 

analysis. After coordination all signals corresponding to nitrogen atoms in heterocycle ligand are shielding (0.2- 

91 ppm). However the biggest coordination shift (∆coord =� 80–91 ppm) was observed for the N(3) signal. Such a 

significant shielding of N(3) resonances signal indicates the monodentate coordination to Pt(II) in solution, what 

is very important for in vitro test and their application as antitumor prodrugs. Addition, in 195 Pt NMR, the cisdiiodo 

compounds were observed between -3143 and -3263 ppm. 

The complexes were tested for antitumor activity against three human cells. The results showed that the activities 

of these complexes are significantly dependent on the nature of the alkyl group in heterocyclic ligands. The 

compounds indicate low in vitro cytotoxic activity of against tested human cancer lines. 

_____________________________________________________________________ 

236


P118. Dizinc(II) Pyrazolate Complexes as Functional Models of Enzyme 

Active Sites 

A. Maciąg a , L. V. Penkova b , F. Meyer c , I. O. Fritsky b and H. Kozłowski a 

a 

University of Wroclaw, Faculty of Chemistry, 14 F. Joliot-Curie, 50-383 Wroclaw, Poland 

e-mail: annam@wcheto.chem.uni.wroc.pl 

b 

National Taras Shevchenko University, Department of Inorganic Chemistry, Volodymyrska str. 64, Kyiv 01033, 

Ukraine; 

c 

Georg-August-Universität Göttingen, Institut für Anorganische Chemie, Tammannstr. 4, 37077 Göttingen, 

Germany; 

Various hydrolytic enzymes that mediate the cleavage of biologically important phosphate esther bond are 

known to contain two proximal zinc ions within their active sites. Examples include some metallo-β lactamases, 

several aminopeptidases, phosphotriesterase, and alkaline phosphatase. Despite intensive investigation, still not 

much is known about the mechanism of enzyme action. Metal complexes with small organic molecules that 

resemble the active sites of the enzymes may provide some insight into the basic principles that govern the 

enzymatic activity. 

Two factors are crucial in hydrolytic activity of dizinc complexes. One is the zinc – zinc separation distance, and 

the other is the ability to generate strongly nucleophilic hydroxide group by lowering the pKa of bound water. 

Pyrazolate-based ligands with chelating side arms in the 3- and 5-positions of the heterocyclic ring have a high 

tendency to span two metal ions, and the metal-metal separations can be adjusted by modification of chelating 

substituents [1]. 

In this work, the RNA model substrate, 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) was exploited to 

study the hydrolytic behavior of dizinc pyrazolate complexes. The increase in product concentration (pnitrophenolate 

anion) was followed by UV-Vis spectroscopy. The obtained results were correlated by 

potentiometric and spectroscopic methods to reveal the active compounds. 

References: 

[1] B. Bauer-Siebenlist, F. Meyer, E. Farkas, D. Vidovic, S. Dechert, Chem. Eur. J., 11, 4349 (2005) 

_____________________________________________________________________ 

237


P119. Generation of NO by Xanthine Oxidase Family Enzymes 

L. Maia, R. Duarte, J. Moura 

REQUIMTE, Fac. Ciencias e Tecnologia, Univer. Nova de Lisboa, Depart. Química, C. Quimica Fina 

e Biotecnologia, 2829-516, Caparica, Portugal 

e-mail: lbmaia@dq.fct.unl.pt 

Xanthine oxidase (XO) is a homodimer, containing one molybdopterin, one FAD and two different [2Fe-2S] 

centres [1]. XO has a broad specificity for both reducing and oxidizing substrates. In addition to the well-known 

oxidation of hypoxanthine and xanthine, XO also catalyses the oxidation of a wide variety of aldehydes and 

nitrogen-containing heterocycles [2]. Much less known are nitrate and nitrite reductase activities related to XO 

[3-4], an reaction somewhat surprising due to the structural differences/similarities found between XO and 

bacterial nitrate reductases [5]. We investigate, in detail, the reduction of nitrate and nitrite to • NO by the 

bacterial (Desulfovibrio species) aldehyde oxidoreductase (AOR), one mononuclear molybdenum enzyme 

containing two different [2Fe-2S] centres, an enzyme of the XO family [6]. The present study examines the 

kinetics of • NO formation catalyzed by XO and by AOR, in the presence of dihydroxybenzaldehyde and 

benzaldehyde as reducing substrates. Anaerobic • NO formation was directly demonstrated using a • NO meter 

(ISO-NO TM ) and the • NO trap iron-N-methyl-D-glucamine dithiocarbamate, which in the presence of • NO gives 

rise to the characteristic EPR signal with g=2.04 and a N =12.7G. Additional EPR studies were performed to 

provide evidence for the sites of action of the substrates and the characteristic axial signals of the nitrosyl-iron 

complex were observed. 

Acknowledgement: L. Maia (SFRH/BPD/39036/2007) wishes to acknowledge to Fundacao para a Ciencia 

e a Tecnologia for financial support. 

References: 

[1] Hille, R., Nishino, T. (1995), FASEB J, 9, 995-1003. 

[2] Krenitsky, T.A., Neil, S.M., Elion, G.M., Hitchings, G.H. (1972), Arch. Biochem. Biophys., 150, 585-599. 

[3] Westerfield, W. W., Richert, D. A., Higgins, E. S. (1959), J. Biol. Chem., 234, 1897-1900. 

[4] Millar, T. M., Stevens, C. R., Benjamin, N., Eisenthal, R., Harrison, R., Blake, D. R. (1998), FEBS Lett., 

427, 225-228. 

[5] González, P.J., Correia, C., Moura, I., Brondino, C.D., Moura, J.J.G. (2006), J. Inorg. Biochem., 100, 1015- 

1023. 

[6] Brondino, C.D., Romao, M.J., Moura, I., Moura, J.J.G. (2006), Curr. Opin. Chem. Biol., 10, 109-114. 

_____________________________________________________________________ 

238


P120. Effect of DNA Modification by Platinum Complexes on 

Topoisomerase I Cleavage Activity 

J. Malina, V. Brabec 

Institute of Biophysics, AS CR, v.v.i., Kralovopolska 135, 612 65, Brno, Czech Republic 

e-mail: malina@ibp.cz 

Topoisomerase I (top 1) is ubiquitous and vital enzyme that participates in nearly all events related to DNA 

metabolism including replication, transcription and recombination. It is now viewed as important therapeutic 

target for cancer therapy and top 1 inhibitors, such as camptothecin or topotecan, are considered promising 

anticancer agents. In vitro experiments combining anticancer platinum drugs with top 1 poisons demonstrated 

synergistic activity in various human tumor cell lines. While the molecular mechanism of each of these agents is 

relatively well understood, the mode of action of these anticancer agents in combination is not clear. We studied 

influence of the DNA modification by selected platinum complexes on the activity of top 1 by gel 

electrophoresis on globally modified supercoiled plasmid DNA and short linear DNA fragment (161 bp). Further 

experiments were performed on DNA oligonucleotide (30 bp) containing specific adducts of platinum complexes 

in the close proximity to the cleavage site of top 1. The results show that the activity of top1 is inhibited by the 

presence of platinum adducts close to its cleavage site and that severe alteration in DNA structure is not 

necessary for inhibition to occur. It suggests that platinum adducts prevent top1 from recognizing its binding site 

and formation of top1-DNA cleavable complex. 

Acknowledgement: This research was supported by the Academy of Sciences of the Czech Republic (Grant 

B400040601). 

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239


P121. Novel Anticancer-active Fluorescent Platinum(II) Complexes 

Containing Anthracene Derivatives as Carrier Ligands 

P. Marques-Gallego, a H. den Dulk, a J. Brouwer, a H. Kooijman, b A. L. Spek, b S. J. Teat, c 

J. Reedijk a * 

a 

Coordination and Bioinorganic Chemistry, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC, Leiden, 

Netherlands 

e-mail: p.marques@chem.leidenuniv.nl 

b 

Bijvoet Center for Biomolecular Research, Utrecht University 

c 

ALS, Berkeley Lab, 1 Cyclotron Rd 

Since the introduction of cisplatin into oncology practice, several studies dealing with molecular mechanisms of 

action have provided considerable and significant information about how cisplatin induces its antitumor effects. 

However, cellular processing of anticancer agents remains largely unknown. Several efforts have been 

undertaken to elucidate these mechanisms; one is the attachment of a fluorescent label to a cisplatin-derivative 

complex [1]. Another approach, also from our laboratory, is the use of fluorescent ligands for the synthesis of 

fluorescent platinum complexes [2]. Biological studies of novel fluorescence platinum(II) complexes have 

shown high activity against several human tumor cell lines, as compared to cisplatin. In addition, a different 

cellular response was found for the free ligands, as compared to their corresponding platinum(II) complexes. 

Therefore, these compounds are of interest for molecular and optical studies. The present contribution describes 

the cytotoxic activity against several human tumor cell lines and their cellular processing. 

References: 

[1] Molenaar, C., Teuben, J. M., Heetebrij, R. J., Tanke, H. J., and Reedijk, J. (2000) J. Biol. Inorg. Chem. 5, 

655-665 

[2] Jansen, B. A. J., Wielaard, P., Kalayda, G. V., Ferrari, M., Molenaar, C., Tanke, H. J., Brouwer, J., and 

Reedijk, J. (2004) J. Biol. Inorg. Chem. 9, 403-413 

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240


P122. Novel Pt (II) Complexes with 4-nitropyrazole Derivatives Ligands 

A. Mastalarz a , M. Kubiak a , T. Lis a , J. Kuduk-Jaworska a , A. Regiec b , H. Mastalarz b 

a Faculty of Chemistry, Wroclaw University, 14 F. Joliot-Curie str., 50-383 Wroclaw, Poland, 

b Faculty of Pharmacy , Wroclaw Medical University, 8 Grodzka str., 50-137 Wrocław, Poland 

The results of screenings done so far on thousands of platinum complexes towards their cytotoxicity, and on 

some of them towards radiosensitizing activity, are not very satisfied. The reason of the therapeutic usefulness of 

many screened candidates seems to be caused by inappropriate choice of the ligands, in particular N-donors 

which strongly modulate the biological properties of complexed compounds. 

Our goal was to synthesize a series of potentially radiosensitizing compounds which contain platinum (II) centre 

able to interact with DNA and to stabilize its damages caused by radiotherapy in the tumor cells. Amongst 

synthesized Pt(II) complexes of general formula PtL2Cl2 and KPtLCl3 we paid special attention on complexes 

with N-methyl-4-nitropyrazole, N-methyl-4-nitro-3- or –5- carbomethoxypyrazole. These ligands were chosen 

due to their potential electron-affinic properties [1]. In this way it might be achieved a goal of this work: to 

obtain complexes with dual mode of action, binding to DNA and implying electron accepting mechanism. 

Presented poster summarizes the results of chemical and structural investigations of new compounds by using 

the NMR, IR, MS spectroscopic methods and X-ray crystallography. The preliminary results of biological tests 

on antiproliferative properties of presented compounds are promising. 

References: 

[1]. Anna Zięba-Mizgała, Aniela Puszko, Andrzej Regiec, Janina Kuduk-Jaworska 

Electrophilic properties of nitroheterocyclic compounds.Potential hypoxic cells radiosensitizers. 

Bioelectrochemistry, 65, 113 - 119 (2005). 

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241


P123. Aminomethane-1, 1-diphosphonic Acids with N-2-pyridyl, N-2thiazolyl, 

and N-2-benzothiazolyl Side Chains. Structure − Properties − 

Complex-forming Abilities Relationships 

E. Matczak-Jon a , T. Kowalik-Jankowska b , P. Kafarski a , J. Jezierska b 

a 

Department of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, 

Poland 

e-mail: ewa.matczak-jon@pwr.wroc.pl 

b 

Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland. 

Bisphosphonates are now the most widely used drugs for diseases associated with increased bone resorption or 

turnover such as osteoporosis, Paget’s disease or cancer induced osteolysis. The reason for that is their 

exceptional affinity for bone mineral, most of which is in the form of hydroxyapatite [Ca10(PO4)4(OH)2] with 

some impurities, including magnesium. In addition, bisphosphonates act at cellular level inhibiting the 

magnesium-dependent enzyme of the mevalonate pathway, the farnesyl pyrophosphate (FPP) synthase. It is thus 

apparent that complexation of calcium and magnesium is of vital importance for their biological activity. 

Compounds 1 ÷ 6 (scheme 1), recognized as potent inhibitors of the FPPS, represent a class of nitrogen 

containing bisphosphonates with a nitrogen atom directly attached to the α-carbon. Continuing our systematic 

studies on the relations between the structure and properties of this special class of acids [1, 2] herein we report 

solution speciation, stability constants and possible coordination modes of the Ca(II), Mg(II), Zn(II) and Cu(II) 

complexes with 1 ÷ 6. 

Scheme 1 

The complexation features of studied ligands are discussed in the context of intramolecular dynamics and ligands 

predispositions for the formation of hydrogen-bonded aggregates. The main complex species proven to exist in 

solution are compared with those found in the gas phase. The role of N−H…O versus O−H…O hydrogen bonds 

on the formation of multinuclear metal−bisphosphonate complexes is demonstrated as well. 

Acknowledgement: The financial support from the Polish Ministry of Higher Education and Science (project 

R05 034 03) is thankfully acknowledged. 

References: 

[1] E. Matczak-Jon, V. Videnova-Adrabińska, Coord. Chem. Rev., 249, 2458 (2005). 

[2] E. Matczak-Jon, B. Kurzak, P. Kafarski, A. Woźna, J. Inorg. Biochem., 100, 1155 (2006) 

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242


P124. The Impact the α-COOH Group on the Binding Abilities of the 

Simple Tetrapeptides with βAsp-His-Gly-His Sequence 

A. Matera-Witkiewicz a , J. Brasuń a , M. Cebrat b , J. Świątek-Kozłowska a 

a) Department of Inorganic Chemistry, Wroclaw Medical University, Szewska 38, 50-139 Wroclaw, Poland; 

b)Faculty of Chemistry, Wroclaw University, F. Joliot-Curie 14, 50-383 Wroclaw, Poland; 

e-mail:amatera@chnorg.am.wroc.pl 

Histidine has significant role in many interactions of peptides and proteins with metal ions. The multi-His 

sequence is basic for Cu 2+ binding motifs in amyloid precursor protein (APP) [1] or secreted protein, acidic and 

rich in cysteine (SPARC) [2, 3]. The recent studies show that prions from many species in their function may 

involve copper binding motif with multi-His region [3]. 

Moreover, Armstrong et al. have shown that in the blood stream a low molecular fractions of Cu 2+ complexes 

have been detected with the fraction bound to macromolecular ligand such as serum albumin with Asp-Ala-His- 

system [4]. 

The investigations for the simple tetrapeptides including two His residues in the peptide sequence and β-aspartic 

acid or β-alanine are presented. The analysis of the potentiometric as well as spectroscopic results shows that the 

presence of the β–aspartic acid in the first position in the peptide chain makes the imidazole rings binding (and 

formation of the CuHL and CuL complexes) much more difficult (Fig.1). Furthermore, the binding of the first 

amide nitrogen occurs at lower pH in comparison to its α-analogue. 

%Cu 2+ 


80 

60 

40 

20 

CuHL 

CuH 2 L 

Cu 2+ 

CuL 

CuH -1 L 

0 

2 4 6 8 10 

pH 

CuH -2 L 

CuH -3 L 

Figure 1. Distribution diagram of complexed species formed as a function of pH in 

the β-DHGH -Cu 2+ (solid line) and DHGH-Cu 2+ data from [5] (dashed line) systems 

References: 

[1] M. Łuczkowski, K. Wiśniewska, L. Łankiewicz, H. Kozłowski, J. Chem. Soc. Dalton Trans., , 2266 (2002) 

[2] H. Kozłowski, D.R Brown, G. Valensin, Royal Society of Chemistry in ”Metallochemistry of 

Neurodegeneration; biological, chemical and genetic aspects”, (2006) 

[3] E. Gagelli, H. Kozłowski, D. Valensin, G. Valensin, Chem. Rev., 106, 1995, (2006), 

[4] P.W. Jones, D.M. Taylor, D.R. Williams, J. Inorg. Biochem., 81, 1, (2000), 

[5] A.Matera-Witkiewicz, JBrasuń, J.Świątek-Kozłowska, A.Pratesi, M.Ginnaneschi, L.Messori, data unpulished 

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243


P125. Effect of the Dihedral Angles of Two CuS2 Planes of µ-η 2 :η 2 - 

Disulfidodicopper(II) Complexes on Their Reactivities 

J. Matsumoto a , Y. Kajita a , Y. Wasada-Tsutsui b , I. Takahashi c , S. Hirota c , Y. Funahashi a , 

T. Ozawa a , H. Masuda a 

a 

Materials Science and Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya, 466-8555, Aichi, Japan 

e-mail: 19415166@stn.nitech.ac.jp 

b 

Graduate School of Natural Sciences, Nagoya City University, Mizuho-ku, Nagoya, 467-8501, Aichi, Japan 

c 

Graduate School of Materials Science, Nara Institute of Science and Technology, Takayama-cho, Ikoma, 630-0192, 

Nara, Japan 

A new focus of interest in copper-sulfur coordination chemistry is derived from the recent discovery of 

a tetracopper-sulfur cluster at the CuZ active sites of nitrous oxide reductase that catalyzes the terminal step in 

bacterial denitrification. 

We newly synthesized three µ-η 2 :η 2 -disulfidodicopper(II) complexes with cis, cis-1, 3, 5-triaminocyclohexane 

derivatives (Figure 1), which were characterized in detail by elemental, electrochemical, and X-ray structure 

analyses, and electronic absorption, IR, ESI mass, and resonance Raman spectroscopic methods. In this study, 

we found that their reactivities with exogenous substrates are related to the dihedral angles defined by two CuS2 

planes.[1] This finding was also examined from the DFT calculation of the two complexes with bent and planar 

Cu2S2 structures. The atomic charges on Cu and S atom were localized when the dihedral angles were decreased; 

1.137, -0.470 for the bent one and 1.107, -0.450 for the planar one, respectively. These results indicate that the 

reactivities of disulfidodicopper(II) complexes depend upon the nucleophilicities of S atoms affected by the 

localized charge. 

References: 

[1] Y. Kajita, J. Matsumoto, I. Takahashi, S. Hirota, Y. Funahashi, T. Ozawa, and H. Masuda, Eur. J. Inorg. 

Chem. accepted. 

_____________________________________________________________________ 

244


P126. Preparation and Characterization of Manganese Schiff Base 

Complexes as Models for PSII 

T. Matsushita, H. Imagawa, H. Asada, M. Kasuno, and M. Fujiwara 

Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, 

Seta, Otsu 520-2194 Japan 

e-mail: matusita@chem.ryukoku.ac.jp 

Manganese ions are believed to function dioxygen generation from water in PSII of green plants. So far many 

model manganese compounds have been prepared and characterized to mimic this process. However, a few 

artificial photosynthetic dioxygen generation using higher-valent manganese complexes has been reported. 

Previously we have reported on the preparation of a series of dichloromanganese(IV) Schiff base complexes, 

their molecular structures and reactions with water, which can generate dioxygen [1-4]. 

In this study we have prepared and characterized dinucleating Schiff base ligands, which were obtained by the 

reaction of triethylenetetramine and salicylaldehyde derivatives, and their manganese(III) complexes(Fig.1). 

Moreover, novel dinucleating ligands, which were derived from 5, 5’-methylene-bis-salicylaldehyde and 

alkylesters of 1 : 1 condensation product of 5-carboxysalicylaldehyde and ethylenediamine, and their 

manganese(III) complexes have been prepared. All the manganese complexes were identified by several 

physico-chemical measurements. These manganese(III) Schiff base complexes have been allowed to react with 

chlorine to form the corresponding manganese(IV) complexes and then with water molecules. The reactions of 

the manganese(III) complexes with Cl2 and then water have been monitored by measuring the changes in UVvisible 

spectra and cyclic voltammograms. In the visible spectra, new intense bands around 600 nm were 

observed by the addition of Cl2 to the solutions of manganese(III) complexes, which can be assigned to charge 

transfer transitions from Cl - to Mn. These bands decreased in intensity by the addition of water. In addition, in 

the CV, new cathodic waves appeared near -0.9 V vs. SCE by the addition of water after addition of Cl2 to the 

solutions of manganese(III) complexes, which disappeared by passing through argon. These results indicate that 

the present manganese(III) complexes can be oxidized by Cl2 to yield dichloromanganese(IV) complexes, which 

can react with water to generate dioxygen. Moreover, amounts of dioxygen generated by the reactions of the 

manganese(IV) complexes with water have been monitored by using an oxygen electrode. We have confirmed 

dioxygen evolved from water, but their yields are low: about 3 to 5% based on the manganese complexes, which 

may be caused by some decomposition of the complexes. In addition, heterodinuclear complexes which include 

both manganese(III) and copper(II) ions have been prepared and characterized. 

X X 

N N 

X 

N N 

Y OH 

OH HO 

Y 

Y 

Mn(OAc)3･2H2O 

or 

X = H, CH3, Y = H, 5-Cl, 5-Br, 5-NO2, 5-COOEt, 5-SO3Na, Z = Cl - , OAc - Fig. 1. Dinuclear manganese(III) complexes 

studied. 

References: 

[1] T. Matsushita, M. Fujiwara, and T. Shono, Chem. Lett., 1981(5), 631. 

[2] T. Matsushita, H. Kono, and T. Shono, Bull. Chem. Soc. Jpn., 54(9), 2646 (1981). 

[3] M. Fujiwara, T. Matsushita, and T. Shono, Polyhedron, 4(11), 1895 (1985). 

[4] H. Asada, M. Fujiwara, and T. Matsushita, Polyhedron, 19 (18), 2039 (2000). 

X X 

N Z N 

X 

N 

Z 

N 

Y O Z 

O O 

Y 

_____________________________________________________________________ 

245 

Mn 

Y 

Mn


P127. Metal-ion Mediated Hoogsteen-type Base Pairs Between the Natural 

Pyrimidine Bases and Artificial 1-deaza and 1, 3-dideazapurine Bases 

D.A. Megger, F.A. Polonius, J. Müller 

Inorganic Chemistry, Dortmund University of Technology, Otto-Hahn-Str. 6, 44227, Dortmund, Germany 

e-mail: dominik.megger@tu-dortmund.de 

In the past few years many new base pairs containing artificial nucleobases have been reported. Those base pairs 

are mediated by hydrogen bonding, metal-ion binding, hydrophobic interactions or a combination of these 

interactions. As had been reported earlier, the incorporation of 19 metal ions in a row was achieved in our group 

using 1-deazaadenine D as base complementary to a deprotonated thymine T.[1] In this case, Hoogsteen-type 

base pairs are formed by one hydrogen bond and two coordinative bonds (Fig. A). 

The aim of our current work is the investigation of the metal-ion binding properties of oligonucleotides 

containing the artificial nucleobases 1, 3-dideazaadenine dD and 1, 3-dideaza-6-nitropurine dN. The reason for 

choosing dD as an artificial nucleobase is on the one hand the easier accessibility of dD compared to D and on 

the other hand the formal substitution of the N3 atom by a CH-group. This defunctionalisation of the nucleobase 

allows metal-ion binding only via the Hoogsteen edge. In case of dN we intend to develop an artificial 

nucleobase that is able to form metal-ion mediated Hoogsteen-type base pairs with cytosine C (Fig. B). 

The results of UV- and CD-spectroscopic experiments with oligonucleotides containing the above mentioned 

nucleobases in absence and presence of several metal ions will be discussed. Additionally the pKa values of the 

nucleosides of D, dD and dN will be presented. 

References: 

[1] F.-A. Polonius, J. Müller, Angew. Chem. Int. Ed., 2007, 46, 5602-5604. 

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246


P128. Antimicrobial Activity of the Co(II), Zn(II) and Cd(II) Complexes 

with N-benzyloxycarbonyl-S-phenylalanine 

D. Mitić a , M. Milenković b , S. Milosavljević a , Z. Miodragović a , K. Anđelković a and 

Dj. Miodragović a 

a 

Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia 

e-mail: dmiodrag@chem.bg.ac.yu 

b 

Department of Microbiology and Immunology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 

450, Serbia 

There is a pressing need for new antifungal agents because of the fast development of resistance of 

microorganisms to the state-of-the-art drugs currently used to treat different fungal infections. For this reason, the 

elaboration of new types of antifungal agents is presently a very real task. A promising field for this search is 

metal-based drugs. Metal-based drugs have a different mode of action compared to the commonly used 

commercial polyene and azole antifungal drugs. Treatment of fungal cells with, for example, Cu(II) and Ag(I) 

complexes [1] resulted in a reduced amount of ergosterol in the cell membrane and a subsequent increase in its 

permeability. 

In spite of interesting biological activities, only a few complexes with N-Boc amino acids have been described. As 

N-benzyloxycarbonylglycine has favorable membrane penetration properties [2], in our previous paper the 

preparation of neutral complexes of this ligand with various metal ions was described [3]. The antimicrobial 

activity of the obtained metal complexes was also determined and it was established that among the investigated 

strains, the Zn(II) and Co(II) complexes were selective, acting only against the yeast Candida albicans. In a 

further investigation, the complex of Zn(II) with N-benzyloxycarbonyl-S-alanine was synthesized and was shown 

to possess the same selectivity against Candida albicans [4]. 

In this study, new complex compounds of Zn(II), Cd(II) and Co(II) with N-benzyloxycarbonyl-S-phenylalanine 

(1-3) were synthesized and characterized. As N-benzyloxycarbonyl-S-phenylalanine is more hydrophobic (and 

more lipophilic) than N-Boc-glycine and N-Boc-S-alanine, it was supposed that these complexes could have 

better antimicrobial activities than the previously investigated ones. 

MIC values obtained for complexes are lower than MIC values obtained for ligand and simple metal salts. The 

comparison of MIC value of complex 2 with MIC value of complex with N-Boc-gly indicates that substitution of 

N-Boc-gly with N-Boc-S-phe ligand resulted in a more than twelve fold increase in the anti-Candida activity, 

from 1.11 to 0.09 mM. The increase in activity was also observed for complexes 1 and 3 [5]. The increase in the 

lipophilicity of N-benzyloxycarbonyl–S-phenylalaninato ligand is probably the reason for the better penetration 

of the complexes with this ligand in comparison to the complexes with N-Boc-glycine or N-Boc-S-alanine. 

It is interesting to note that Hitherto investigated complexes with N-benzyloxycarbonyl-amino acids exhibited 

the best activity against the yeast Candida albicans of the until now investigated bacterial and fungal strains. 

Complex 2 has MIC value almost the same as that of the standard drug nystatin in the case of Candida albicans 

ATCC 24433. 

Acknowledgement: This investigation was supported by the Ministry of Science of the Republic of Serbia, 

Grant No. 142010. 

References: 

[1] D.M. Lambert, G.K.E. Scriba, J.H. Poupaert, P. Dumont, Eur. J. Pharm. Sci. 4, 159 (1996). 

[2] B.S. Creaven, D. A. Egan, D. Karcz, K. Kavanagh, M. McCann, M. Mahon, A. Noble, B. Thati, M. Walsh, J. 

Inorg. Biochem. 101, 1108 (2007). 

[3] D.U. Miodragović, D.M. Mitić, Z.M. Miodragović, G.A. Bogdanović, Ž.J. Vitnik, M. D. Vitorović, M.Đ. 

Radulović, B.J. Nastasijević, I.O. Juranić, K.K. Anđelković, Inorg. Chim. Acta 361, 86 (2008). 

[4] D.M. Mitić, Đ.U. Miodragović, D.M. Sladić, Ž.J. Vitnik, Z.M. Miodragović, K.K. 

Anđelković, M.Đ. Radulović, N.O. Juranić, J. Serb. Chem. Soc. (2008), in press. 

[5] D. Mitić, M. Milenković, S. Milosavljević, D. Gođevac, Z. Miodragović, K. Anđelković, Dj. Miodragović, 

submited for Eur. J. Med. Chem. 

_____________________________________________________________________ 

247


P129. Selenium, Tellurium and Transition Metals as Chemical Ingredients 

of Intelligent Antioxidants 

H. Mohammed a , S. Mecklenburg a , M. Doering a , S. Shaaban a , T. Burkholz a , C. Collins b , 

M. Abbas a , A. Anwar a , C. Jacob Claus a 

a Bioorganic Chemistry, University of Saarland, University Campus, 66123, Saarbruecken, Germany, 

b School of Biological and Chemical Sciences, University of Exeter, Stocker Road, Exeter, UK 

e-mail: h.mohammed@mx.uni-saarland.de 

Elements such as selenium and tellurium are generally not too popular in Bioinorganic Chemistry. Nonetheless, 

the combination of selenium, tellurium and various transition metal ions in chemically simple molecules 

provides a successful recipe for powerful pro- and antioxidants, which may be further ‘spiced' by the addition of 

organic redox centres [1, 2]. Antioxidants are used against oxidative stress (OS), a disturbance in redox 

homeostasis associated with numerous human diseases. Importantly, OS is not a single molecule event, but is 

associated with the increase in intracellular concentrations of various reactive, oxidizing species, and loss of 

antioxidant defence. Since reactive oxygen species (ROS), iron and copper ions are among the major culprits 

causing and propagating OS, we have considered the design of multifunctional molecules which combine the 

ability to remove ROS and bind (and disarm) adventitious metal ions [2]. These molecules are synthesised using 

various synthetic techniques, including multicomponent reactions and integrate one or more sulfur, selenium and 

tellurium redox centres with macrocycles, phenolic and quinone-based redox sites [3]. They exhibit an exciting 

electrochemical behaviour and bind/exchange zinc, copper and iron ions [1]. They are catalytically active and, 

when tested in skin cell culture, show strong antioxidant effects, probably due to catalytic destruction of ROS 

and exchange of beneficial zinc ions for toxic copper/iron ions. 

References: 

[1] Mecklenburg, S.; Collins, C. A.; Doring, M.; Burkholz, T.; Abbas, M.; Fry, F. H.; Pourzand, C.; Jacob, 

C. Phosphorus Sulfur and Silicon and the Related Elements 2008, 183 (4), 863-88. 

[2] Collins, C. A.; Fry, F. H.; Holme, A. L.; Yiakouvaki, A.; Al-Qenaei, A.; Pourzand, C.; Jacob, C. Org. 

Biomol. Chem. 2005, 3 (8), 1541-1546. 

[3] Shabaan, S.; Abbas, M.; Jacob, C. 2008, Manuscript in preparation. 

_____________________________________________________________________ 

248


P130. Mo and W Containing Formate Dehydrogenases: 

Structural and Biochemical Characterization 

C. Mota a , M.G. Rivas a , P.J. Gonzalez a , C.D. Brondino b , J.J.G. Moura a , I. Moura a 

a 

REQUIMTE/CQFB - Departamento de Química, Faculdade de Ciências e Tecnologia - UNL, Quinta da Torre, 

2829-516, Caparica, Portugal, 

b 

Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas -UN, Barrio El Pozo, 3000, Santa Fe, 

Argentina 

e-mail: cristianomota@dq.fct.unl.pt 

Formate dehydrogenases (Fdh) belong to the DMSO reductase family and catalyze the two electron oxidation of 

formate to carbon dioxide via a cleavage of the C-H bond [1]. The oxidized active site of these enzymes can 

contain a hexacoordinated molybdenum or tungsten atom [2, 3, 4]. The reaction mechanism of these enzymes 

seems to involve pentacoordinated or hexacoordinated species in presence or absence of inhibitors, respectively 

[5, 6]. The present work reports biochemical and EPR studies of a Mo- and a W-Fdh isolated from Desulfovibrio 

desulfuricans ATCC 27774 and Desulfovibrio gigas, respectively. The main aim is to compare the catalytic 

properties of these enzymes to evaluate the relevance of the metal in the active site. 

References: 

[1] Khangulov S. V., Gladyshev V. N., Dismukes G. C., Stadtman T. C. Biochemistry 37 (1998), 3518-3528. 

[2] H. Raaijmakers, S. Macieira, J.M. Dias, S. Teixeira, S. Bursakov, R. Huber, J.J. Moura, I. Moura, M.J. 

Romao. Structure 10 (2002) 1261-1272. 

[3] M. Jormakka, S. Tornroth, B. Byrne, S. Iwata. Science 295 (2002) 1863-1868. 

[4] M. Jormakka, S. Tornroth, J. Abramson, B. Byrne, S. Iwata. Acta Crystallogr. D. Biol. Crystallogr. 58 

(2002) 160-162. 

[5] J.C. Boyington, V.N. Gladyshev, S.V. Khangulov, T.C. Stadtman, P.D. Sun. Science 275 (1997) 1305-1308. 

[6] M.G. Rivas, P.J. González, C.D. Brondino, J.J.G. Moura, I. Moura. J. Inorg. Biochem. 101 (2007) 

(11-12):1617-1622. 

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249


P131. Acid-Base Properties of 2'-Deoxyribose versus Ribose Nucleotides 

A. Mucha a , B. Knobloch a , M. Jeżowska-Bojczuk b , H. Kozłowski b , R.K.O. Sigel a 

a Institute of Inorganic Chemistry, University of Zürich, Winterthurerstrasse 190, 8057, Zürich, Switzerland, 

b Faculty of Chemistry, University of Wrocław, F. Joliot Curie 14, 50-383, Wrocław, Poland 

e-mail: ariel@eto.wchuwr.pl 

The extent of the influence of the exchange of a ribose by a 2'-deoxyribose on the acid-base properties of 

nucleotides has so far not yet been determined in detail. In this study, we have measured by potentiometric pH 

titrations in aqueous solution the acidity constants of the 5'-mono-, 5'-di- and 5'-triphosphates of 2'deoxyadenosine 

[i.e., of H2(dAMP) ± , H2(dADP) – and H2(dATP) 2– ] as well as of the 5'-di- and 5'-triphosphates of 

2'-deoxyguanosine [i.e., of H2(dGDP) – and H2(dGTP) 2– ] (see Fig.1). These in total 12 acidity constants are 

compared with the corresponding ribose derivatives, which have been measured under the same experimental 

conditions (published data). The results show that all protonation sites in the 2'-deoxynucleotides are more basic 

than in their ribose counterparts. The influence of the 2'-OH group is thereby dependent on the number of 5'phosphate 

groups as well as on the nature of the purine nucleobase. The basicity of N7 in guanine nucleotides is 

most significantly enhanced (about 0.2 pK units), the effect on the phosphate groups and the N1H or (N1H) + 

sites is less pronounced but clearly present. In addition, for the dAMP, dADP and dATP systems 1 H-NMR 

chemical shift change studies in D2O in dependence on pD were carried out confirming the results from the 

potentiometric pH titrations and showing that the nucleotides are in their anti conformation. [1] Overall, our 

results are not only of relevance for metal ion binding to nucleotides or nucleic acids, but also constitute an exact 

basis for the calculation, determination, and understanding of perturbed pKa values in DNAzymes and ribozymes 

needed for an acid-base mechanism in catalysis. 

Figure 1. The chemical structures of the investigated nucleotides. 

Acknowledgements: Financial support from the Swiss National Science Foundation (SNF-Förderungsprofessur 

to R.K.O.S., PP002-114759/1), the Polish State Committee for Scientific Research (KBN Grant No. N.204 029 

32/0791), the Universities of Zürich and Wrocław, and within the COST D39 programme from the Swiss State 

Secretariat for Education and Research is gratefully acknowledged, as are the International Relations Office of 

the University of Zürich (fellowship to A.M.) and helpful hints by Prof. Dr. H. Sigel (University of Basel). 

References: 

[1] A. Mucha, B. Knobloch, M. Jeżowska-Bojczuk, H. Kozłowski, R.K.O. Sigel, Comparison of the Acid-Base 

Properties of Ribose and 2'-Deoxyribose Nucleotides, Chem. Eur. J., 2008, DOI: 10.1002/chem.200800496 

_____________________________________________________________________ 

250


P132. Transferrin Fibrillation and Iron Nanomineralization 

A. Mukherjee a , S. Ghosh b , M. A. Barnett c , J. P. Willaims a , N. Wilson d , P. J. Sadler a , 

S. Verma b 

a 

Department of Chemistry, University of Warwick, Gibbett Hill Road, CV47AL, Coventry, United Kingdom, 

b 

Chemistry, Indian Institute of technology Kanpur, Kanpur, India 

c 

Research Support Services, University of Warwick, Department of Chemistry, CV47AL, Coventry, United 

Kingdom 

d 

Physics, University of Warwick, Gibbett Hill Road, CV47AL, Coventry, United Kingdom 

e-mail: arindam.mukherjee@warwick.ac.uk 

Aggregation of extracelluar proteins and peptides such as prion protein, alpha-synuclein, insulin, beta2microglobulin 

and amyloid beta- peptide is found in patients with various neurodegenerative diseases, e.g. 

Alzheimer’s, Parkinson’s and Halloverden-spatz disease [1]. Fe, Mn, Cu are known to induce oxidative stress, 

and in addition abnormal iron deposits are found in the brains of dementia patients. We are investigating the 

possibility that human serum transferrin (hTf), an extracellular Fe(III)-transporting glycoprotein [2] present in 

blood and in brain could play a role in iron deposition. Using various types of microscopy (TEM, AFM, SEM), 

we have found that human serum transferrin readily forms fibres, typically 200-300 nm wide, on various 

surfaces (e.g. carbon, formvar, mica)[3]. Fibrillation is observed with apo-, holo-, Mn2III-hTf, Bi2III-hTf and 

holo deglycosylated hTf. Thus the glycan chains and the metal appear to have little or no role in fibril formation. 

Periodic iron nanomineralization was observed in fibrils of holo-hTf. TEM experiments show that fibrils can 

form under physiologically relevant conditions. Mass spectrometry shows that transferrin can form dimers and 

trimers in the gas phase. Other proteins from the same family such as lactoferrin and ovo-transferrin also 

undergo fibrillation on carbon-coated formvar surfaces. 

References: 

[1] A. Khan, J.P. Dobson, C. Exley, Free Rad. Biol. Med. 2006, 40, 557. 

[2] H. Sun, H. Li and P.J. Sadler, Chem. Rev. 1999, 99, 2817. 

[3] S.Ghosh, A. Mukherjee, P.J. Sadler, S. Verma, Angew. Chem. Int. Ed. 2008, 47, 2217. 

_____________________________________________________________________ 

251


P133. Crystal structures of Cytochrome C Peroxidase from Pseudomonas 

stutzeri in Active and Inactive Forms 

A. Mukhopadhyay, J. Trincão, C. Trimoteo, C. Bonifacio, I. Moura, M. J. Romão 

REQUIMTE/CQFB, Departamento de Química, FCT, Universidade Nova de Lisboa, 2829-516 Caparica, 

Portugal 

Bacterial di-heme Cytochrome c Peroxidase (CCP) is essential to maintain H2O2 below toxic levels by catalyzing 

its reduction to H2O. Bacterial CCP consists of two heme domains, one harboring the electron transfer heme 

(E heme) and the other the peroxidatic heme (P heme). The crystal structure of the CCP from Pseudomonas 

stutzeri was obtained in two different redox states. The oxidized form (inactive) crystal structure was refined to 

1.6 Å resolution[1]. In this structure the peroxidatic heme is coordinated to six ligands. The reduced form, in the 

active mixed valence state, was refined to 2.02 Å resolution and has a water molecule bound to the peroxidatic 

heme. These two structures have significant conformational differences in some regions, in particular around the 

P heme and the interface between the two domains. Previous studies indicate that CCP from P. stutzeri has a 

very high affinity for calcium [2]. This property has been addressed from a structural point of view. Structural 

data will also be obtained for the CCP from the same organism in the calcium free state in the oxidized form. 

These structures, along with the IN and OUT forms of P. Nautica [3] will help to obtain a better understanding 

of the electron transfer mechanism within di-heme CCP and also of the role of the calcium in its activation and 

mechanism. 

Acknowledgement: This work was supported by the post-doctoral grant SFRH/BPD/30142/2006 

References: 

[1] C. Bonifácio, C. A. Cunha, A. Müller, C. G. Timóteo, J. M. Dias, I. Moura M. J. Romão, Acta Cryst. D59, 

345, (2003). 

[2] C. G. Timóteo, P. Tavares, C. F. Goodhew, L. C. Duarte, K. Jumel, F. M. Gírio, S. Harding, G. W. Pettigrew, 

I. Moura, J. Biol. Inorg. Chem., 8, 29, (2003) 

[3] J. M. Dias, T. Alves, C. Bonifacio, A. S. Pereira, J. Trincao, D. Bourgeois, I. Moura, M. J. Romao, Structure, 

12, 961, (2004) 

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252


P134. Electrocatalytic Aldehyde Oxidation and Acid Reduction by 

Hyperthermophilic Tungsten-containing Oxidoreductase on Ferredoxin- 

Modified Gold 

M. Nahid Hasan a , S. de Vries a , W.R. Hagen a , H.A. Heering b 

a. 

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The 

Netherlands. 

b. 

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands. 

The hyperthermophilic aldehyde:ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate 

(GAP):ferredoxin oxidoreductase (GAPOR) from Pyrococcus furiosus form complexes with their native redox 

partner ferredoxin (Fd), chemisorbed on a gold electrode. With GAPOR, a well-developed non-turnover 

voltammetric response is observed at room temperature and pH 7.4, assigned to the [4Fe 4S] clusters of the 

enzyme and Fd, at 368mV and 383 mV, respectively. At 60ºC a catalytic oxidation wave is observed upon 

addition of the substrate GAP. With AOR, a broad, reversible non-turnover voltammetric response is observed at 

room temperature due to overlapping potentials of the Fd and AOR [4Fe-4S] clusters, in addition to 

tungstopterin species. The AOR / Fd complex formation on the electrode is corroborated by ellipsometric 

detection of Fd-labeled gold on immobilized AOR. At 80ºC and in the presence of the substrate crotonaldehyde, 

three distinct catalytic oxidation responses are observed. Remarkably, these responses are peak-shaped due to a 

rapid switch-off at high overpotentials, and are each accompanied by a peak due to the catalytic reduction of the 

produced acid. The averages of the oxidation and reduction peaks are found at 0.38 V, 0.27 V and 0.10 V. The 

bi-directional AOR activity and potential-dependent switching are confirmed by colorimetric aldehyde analysis 

and dye mediated optical activity assays. A minimal mechanism is proposed, involving reversible productinduced 

switching between an aldehyde oxidizing form and an acid reducing form of the enzyme. The present 

work opens the way for the application of Fd electrodes in achieving controlled reduction of carboxylic acids on 

a preparative scale. 

References: 

[1] Hasan MN, Kwakernaak C, Sloof WG, Hagen WR, Heering HA (2006) J Biol Inorg Chem 11:651-662 

[2] Koehler BP, Mukund S, Conover RC, Dhawan I K, Roy R, Adams MWW, Johnson MK (1996) J Am Chem 

Soc 118:12391-12405 

[3] Mukund S, Adams MWW (1991) J Biol Chem 266:14208-14216 

[4] Hagedoorn P-L, Freije JR, Hagen WR (1999) FEBS Lett 462:66-70 

[5] van den Ban ECD, Willemen HM, Wassink H, Laane C, Haaker H (1999) Enzyme Microb Tech 25:251-257 

[6] Bernhardt PV (2006) Aust J Chem 59:233-256 

_____________________________________________________________________ 

253


P135. New Crystallographic Data Provide New Insights in the Catalytic 

Mechanism of Nitrate Reduction: Mo or Ligand-based Redox Chemistry? 

S. Najmudin a , C. Coelho a , J. Trincão a , P.J. González a , C. Brondino c , I. Moura a , J.J.G. Moura a , 

C.C. Romão b and M. J. Romão a 

a 

REQUIMTE/CQFB, Departamento de Química, FCT-UNL, 2829-516 Monte de Caparica, Portugal. 

b 

Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, Av da República, EAN, 2780- 

157, Oeiras, Portugal 

c 

Departamento de Física, Universidad Nacional del Litoral, 3000ZAA Santa Fe, Argentina. 

e-mail: mromao@dq.fct.unl.pt 

Nitrate reductases are key enzymes present in the biological cycle of nitrogen and catalyze the conversion of 

nitrate to nitrite. We have performed extensive crystallographic studies on two periplasmic nitrate reductases: the 

enzyme from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harbouring a 

bis-molybdopterin guanine dinucleotide (bis-MGD) active site and a [4Fe-4S] cluster and its crystal structure was 

solved to 1.9Å resolution [1, 2] while Nap from Cupriavidus necator (CnNapAB) comprises a 91 kDa catalytic 

subunit (NapA) and a di-haem c-type cytochrome 17 kDa subunit (NapB) involved in electron transfer. The 

CnNapAB crystal structure was solved to atomic resolution (1.5 Å) [3]. For both Naps, crystals were prepared in 

different conditions when reacted with reducing agents, substrate or inhibitors and the corresponding structures 

were solved. 

The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on 

that basis, the sixth Mo ligand, originally proposed to be an OH ligand, was clearly assigned as a sulfur atom after 

refinement and analysis of the B-factors of all the structures. This unexpected result was confirmed by 

independent means. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the 

Sγ atom of the Mo ligand CysA140 was substantially shorter than the van der Waals contact distance and varies 

between 2.2 Å and 2.85 Å, indicating a partial disulfide bond: 

Mo S 

This new and unexpected coordination sphere of Mo derived from our studies led us to revise the currently 

accepted reaction mechanism for nitrate reductases. Proposals for a new mechanism are discussed taking into 

account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry 

based solely on the Mo atom. We propose that reduction of nitrate to nitrite involves a combined Mo and ligandbased 

redox chemistry of the active site probably via a sulfoxide-ligated Mo-S=O species, instead of the currently 

accepted redox chemistry based solely on the Mo atom in the redox cycle of the enzyme. These results show that 

distinct aspects attributed to the chemistry of Mo inorganic complexes to date, can also occur in mononuclear Moenzymes 

of the DMSO reductase family, opening new research directions in the study of these proteins. 

Acknowledgement:. 

This work was supported by projectsPOCI/QUI/57641/2004 and PTDC/QUI/64733/2006 financed by the program 

POCI2010 and co-financed by FEDER. 

References: 

[1] Dias et al. Structure, 1999, 7, 65-79. 

[2] Coelho et al. Acta Cryst. 2007, F63, 516-519. 

[3] Najmudin et al, J Biol Inorg Chem. 2008 Jun;13(5):737-53 

_____________________________________________________________________ 

254 

S 

S 

RS SR 

Mo VI 

S 

Cys 

S 

S 

RS SR 

Mo V 

RS SR 

Mo IV


P136. An Ethanol/Dioxygen Biofuel Cell Based on Alcohol Dehydrogenase- 

and Bilirubin Oxidase-immobilized Electrodes 

N. Nakamura , K. Murata, K. Kajiya, M. Masuda, and H. Ohno 

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, 

Tokyo 184-8588, Japan 

e-mail: nobu1@cc.tuat.ac.jp 

Biofuel cells using enzymes as a catalyst have attracted considerable attention because biomass products such as 

ethanol, glucose, and fructose, which are easy for handling, may be used as a fuel. The achevement of the fast 

electron transfer between enzymes and electrodes on both electrodes (an anode and a cathode) is one of the most 

immportant themes to construct a highly efficient biofuel cell. 

Anode: When nicotinamide adenine dinucleotide (NAD + /NADH)-dependent enzymes are used as an anode 

catalyst, the electrochemical regeneration of NAD + at high rate constants and low over potential is an important 

issue. Many compounds have been investigated as potential redox-mediators for electrocatalytic oxidation of 

NADH so far. In this study, the novel bioanode using NAD(H)-dependent alcohol dehydrogenase was 

constructed. The electrochemical polymerization of ruthenium(II) tris(5-amino-1, 10-phenanthroline) ([Ru(5phenNH2)3] 

2+ ) in nonaqueous solution (0.1M TBAP acetonitrile) was performed by the continuous cycling of the 

potential of the working electrode according to a similar method reported previously [1]. The cyclic 

voltammogram shows the typical Ru(II/III) redox couple with E = +1.2 V when the potential of a glassy carbon 

electrode was cycled between −1.0 and +1.5 V (vs. Ag/AgCl). After the electrode is transferred to a pH 7.0 

phosphate buffer solution, the redox couple was observed at E = −25 mV (vs. Ag/AgCl). The plot of the redox 

potential versus pH was linear with the slope −55 mV/pH, indicating that protons take part in the redox reaction 

of the polymer. It was found that the poly-[Ru(5-phenNH2)3] 2+ formed onto a carbon black-modified carbon 

paper electrode was an effective catalyst for electrochemical oxidation of NADH. A high current densitybioanode 

was fabricated from the poly-[Ru(5-phenNH2)3] 2+ modified electrode that was coated with a layer of 

poly(diallyldimethylammonium chloride) and NAD(H)-dependent alcohol dehydrogenase (ADH). In the 

presence of 10 mM NAD + and 1 M ethanol, well-defined faradaic currents were observed and the current density 

reached a value as large as 2.5 mA/cm 2 at 800 rpm rotation rate. 

Cathode: A direct electron transfer type 

biocathode was fabricated using bilirubin 

oxidase which is a one of multi-copper 

oxidases and catalyzes reduction of 

dioxygen to water. Bilirubin oxidase was 

adsorbed onto a carbon black-modified 

carbon paper electrode whose surface had 

been electrochemically oxidized. The 

observed current density was about 0.7 

mA/cm 2 in an air-saturated buffer solution 

(pH 7.0) at 800 rpm rotation rate. 

Biofuel cell: The alcohol dehydrogenasemodified 

electrode and the bilirubin 

oxidase-modified electrode were combined 

to prepare an alcohol/dioxygen biofuel cell. 

The prepared biofuel cell without a 

separator showed the open circuit potential 

of 0.45 V, the short circuit current of 0.6 

mA/cm 2 , and the maximum power density 

of 0.08 mW/cm 2 at the cell voltage of 0.25 

V with stirring solution at 800 rpm under air-saturated condition (figure 1). 

0 

0 0.2 0.4 0.6 

Current density/ mA cm -2 

Reference 

[1] C.D. Ellis, L.D. Margerum, R.W. Murray, T.J. Meyer, Inorg. Chem., 22, 1283 (1983). 

E cell / V 

0.5 

0.4 

0.3 

0.2 

0.1 


Figure 1. The dependences of cell potential (dotted lines) of 

the biofuel cell and of the power output (solid lines) on the 

current density. The measurements were performed in 

phosphate buffer pH 7.0 without stirring (open triangle) and 

with stirring at 800 rpm (closed circle) under air-saturated 

conditions. 

_____________________________________________________________________ 

255 

50 

0 

Power density / µW cm -2


P137. Synthesis of New Oxo-vanadium(IV) Coordination Compounds and 

Evaluation of Their Insulin Mimetic Actvity 

J. Nilsson, a M. Haukka, b E. Degerman, c D. Rehder, d E. Nordlander a 

a Inorganic Chemistry Research Group, Chemical Physics, Center for Chemistry and Chemical Engineering, 

Lund University, Getingevägen 6, SE-22100 Lund, Sweden 

email: jessica.nilsson@chemphys.lu.se 


c Department of Experimental Medical Science, Biomedical Center, Lund University, SE-221 48 Lund, Sweden 

d Institut für Anorganische und Angewandte Chemie, Universität Hamburg, Martin-Luther-King-Platz 6, D- 

20146 Hamburg, Germany 

With the intention of preparing new insulin mimetic compounds, two new V(IV) oxo complexes of the 

tetradentate ligand N-(2-hydroxybenzyl)-N, N-bis(2-pyridylmethyl)amine, L, (Figure 1a) have been prepared and 

characterized by NMR and IR spectroscopy, mass spectrometry, elemental analysis and X-ray diffraction. In 

vitro effects on the insulin signaling pathways of the new complexes [V IV O(HSO4)(L)] (Figure 1b) and 

[V IV O(Cl2)(L)] . MeOH as well as of a related V(IV) oxo complex of trispyridylmethylamine [1] have been 

investigated. Neither of the complexes did, however, show the insulin mimetic effect observed for VOSO4 or 

Na3VO4 salts. In fact this type of tetradentate coordinating ligands seems to inhibit the inherent insulin mimetic 

effect of vanadium. To investigate this further we are now in the process of developing complexes containing 

derivates of the original ligand, altering the coordination mode as well as the hydro/lipophilicity. 

N 

a) b) 

N 

Figure 1. a) The ligand, N-(2-hydroxybenzyl)-N, N-bis(2-pyridylmethyl)amine, used for synthesis of the new 

complexes, one of which is b) [V IV O(HSO4)(L)]. 

Acknowledgements: The authors would like to thank The Research School in Pharmaceutical Science (FLÄK), 

The Swedish Foundation for International Cooperation in Research and Higher education (STINT), The 

European Cooperation in the Field of Scientific and Technical Research (COST Action D21) and The Royal 

Physiographic Society in Lund for financial support. 

Reference: 

[1] Y. Tajika, K. Tsuge and Y. Sasaki, Dalton Trans., 1438 (2005) 

_____________________________________________________________________ 

256 

OH 

N


P138. Electrocatalytic Epoxidation of Olefins Using Iron(III) 

Tetraphenylporphyrin Chloride as a Model of Cytochrome P-450 

M. Noroozifar, M. Khorasani-Motlagh, A. Naghi Torabi 

Department of Chemistry, University of Sistan & Baluchestan, Zahedan, Iran, P.O.Box: 98155-147 

e-mail: mnoroozifar@chem.usb.ac.ir 

The cytochromes P-450 are a superfamily of cysteine thiolate ligated heme iron enzymes that activate dioxygen 

for the insertion or addition of a single oxygen atom into a wide variety of substrates, including alkanes to form 

alcohols, alkenes to form epoxides, sulfides to form sulfoxides, etc. [1-2]. The chemical oxidation of alkene 

catalyzed metalloporphyrins in mimicking cytochrome P-450 has been studied extensively. Literature oxidations 

have been carried out almost exclusively by chemical methods in the catalytic reactions. The oxidants, which are 

also oxygen atom sources, include organic alkyl peroxides, peracids, iodosyl benzene as well as inorganic 

compounds. Almost two decades ago, however, Groves and Gilbert demonstrated the olefin epoxidation could be 

effected electrochemically [3]. They used an iron porphyrin as the catalyst in solution and water as the oxygen 

source. 

We describe here an electrocatalytic cycle (Scheme I) in which Fe(III)-porphyrin undergoes two-electron 

electro-oxidation. This cycle is of special interest since it contains the essential elements of the cytochrome P- 

450 catalytic cycle: metalloporphyrin catalyst, substrate, reducing equivalents. FeTPPCl, (TPP = 

tetraphenylporphyrin) in an acetone aqueous Na2SO4 two phase system media, have been used for 

electrocatalytic epoxidation of olefins. The epoxidation reactions have been monitored by gas chromatographic 

analysis. Optimization of the electrolysis conditions and estimation of the reaction mechanism are discussed. 

OH 

1.1 V 1.5 V 

P - Fe(III) P- Fe(IV)= O P - Fe(V) = O 

O 

Scheme I 

or 

P .+ -Fe(IV) = O 

References: 

[1] M. Sono, M. P. Roach, E. D. Coulter, J. H. Dawson, Chem. Rev., 96 (1996) 2841. 

[2] S. G. Sligar, Essays Biochem., 34 (1999) 71. 

[3] J. T.Groves, J. A. Gilbert, Inorg. Chem., 25 (1986) 123. 

_____________________________________________________________________ 

257


P139. The Methylation of Monofunctional Dienplatin Affects Conformation 

of DNA Adducts and Their Processing by DNA Polymerases 

O. Novakova a , J. Malina a , G. Natile b , V. Brabec a 

a 

Institute of Biophysics AS CR v.v.i., Kralovopolska 135, 612 65, Brno, Czech Republic 

e-mail: olga@ibp.cz 

b 

Department Farmaco-Chimico, University of Bari, Via E. Orabona 4, I-70125, Bari, Italy 

To learn more about structure-function relationship of platinum complexes and factors affecting inhibition by 

these compounds of DNA replication, we investigated DNA polymerization using DNA templates sitespecifically 

modified by the adducts of monofunctional [PtCl(dien)]+, dien = diethylenetriamine (dienplatin, 

dienPt) and its methylated derivatives in three sequence contexts. These adducts were characterized structurally 

and analyzed for their ability to affect DNA polymerization using two DNA polymerases which differed in 

processivity and fidelity. We found that character of DNA distortions induced by the adducts of the 

monofunctional complexes was dependent on the sequence context and extent of the methylation. The adducts of 

the methylated complexes strongly inhibited DNA polymerases in a sequence dependent manner whereas those 

of nonmethylated dienPt did not. Also interestingly, the polymerases discriminated between Me3dienPt and 

Me5dienPt adducts and the adducts of Me5dienPt strongly inhibited a single nucleotide misincorporation 

opposite the platinated nucleotide. The results indicate that the bulkiness of DNA adducts of monofunctional 

platinum complexes is a key factor in mechanism of the blockage of DNA polymerases. 

Acknowledgement: This research was supported by Grant Agency of the Czech Republic (Grant 203/06/1239) 

_____________________________________________________________________ 

258


P140. Metallothioneins – What Are Their Physiological Functions in 

Barley? 

J. Nymark Hegelund , M. Schiller , S. Husted , J.K. Schjoerring 

Agricultural Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark 

e-mail: jnh@life.ku.dk 

Metallothioneins (MTs) are a family of small cysteine rich metalloproteins (Mr < 10 kDa). In vivo MTs have 

been found to coordinate Zn, Cu and Cd. Metallothioneins in mammals have been associated with numerous 

physiological functions such as heavy metal detoxification, scavenging of reactive oxygen species[1], and metal 

loading of transcription factors[2]. Transcripts of plant MTs are induced by metal ions, hormones, wounding and 

oxidative stress[3, 4, 5]. However at the protein level, plant MTs are not well characterized. 

The entire MT family of barley have been cloned to characterize their physiological functions and redundancies. 

We are particularly focusing on the importance of MTs during ear development in monocots. During grain 

development, Zn, Cu and possibly Cd are mobilized from roots and leaves and stored in the aleurone, embryo 

and endosperm tissues of the grain. Using the stabile Zn67 isotope as a tool, we have managed to relate Zn 

remobilization to the developing ear to the expression of barley MTs. Transcripts of 6 barley MTs are abundant 

in developing grains. However, the protein levels arising from the MT transcripts are uncertain. We chemically 

analyze the composition of Zn, Cu and Cd binding compounds in barley seeds using HPLC-ICP-MS and LC- 

ESI-QTOF-MS. These methods detect barley MTs expressed in and purified from E. coli. Ultimately, the 

identification of barley MTs in vivo will provide valuable new knowledge to the physiological functions of plant 

MTs. 

References: 

[1] Coyle P. et.al. (2002) Cell. Mol. Life Sci. 59 (2002) 627-647 

Metallothionein: The multipurpose protein 

[2] Huang M. et.al. (2004) J. Inorg. Biochem. 98 639-648 

Interprotein metal exchange between transcription factor IIIa and apo-metallothionein 

[3] Lü S. et.al. (2007) Transgenic Res 16:177-191 

The GUS reporter-aided analysis of the promoter activities of a rice metallothionein gene reveals different 

regulatory regions responsible for tissue-specific and inducible expression in transgenic Arabidopsis 

[4] Yuan J. et.al. (2008) Plant Physiol, April 2008, Vol. 146, pp. 1637-1650, 

Characteristic and Expression Analysis of a Metallothionein Gene, OsMT2b, Down-Regulated by Cytokinin 

Suggests Functions in Root Development and Seed Embryo Germination of Rice 

[5] Obertello M. et. al. (2007) MPMI Vol. 20: 10, 1231-1240. 

Functional Analysis of the Metallothionein Gene cgMT1 Isolated from the Actinorhizal Tree Casuarina glauca 

_____________________________________________________________________ 

259


P141. Effect of Weak Interaction on the Electronic Structure and 

Electrochemical Properties of Pseudoazurin Met16X Mutants 

Y. Obara a , R. F. Abdelhamid a , K. Fujita b , D. E. Brown b , D. M. Dooley b , H. Tanaka c , 

I. Kitagawa c , M. Okada c , and T. Kohzuma a 

a 

Institute of Applied Beam Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan 

e-mail: 06nd603s@hcs.ibaraki.ac.jp 

b 

Department of Chemistry, Montana State University, Bozeman, Montana 59717, U. S. A. 

c 

Hitachi Ltd , Omika-cho 7-1-1, Hitachi, Ibaraki 319-1292, Japan 

Pseudoazurin (PAz) is a blue copper protein, which functions as an electron carrier in several denitrifying 

bacteria. Very recently, we have reported the spectroscopic and electrochemical properties of Met16Phe, 

Met16Tyr, and Met16Trp mutants to elucidate the effects of the π-π interaction at the active site of blue copper 

protein [1, 2]. The introduction of aromatic amino acid in the vicinity of the blue copper active site shows 

a drastic spectroscopic changing and significantly higher redox potential as compared to the wild-type protein. 

Two different types of PAz mutants, Met16Lys and Met16Glu were constructed and studied to further explore 

the effects of weak interactions involving electrostatic effects on the structure and function of the blue copper 

active site. 

Wild-type PAz exhibits three intense absorption bands at 454 (ε = 1700 M -1 cm -1 ) and 594 (ε = 3700 M -1 cm -1 ) nm 

due to the SCys→Cu(II) ligand to metal charge transfer (LMCT) and a d-d transition at 753 nm (ε = 1700 M -1 cm - 

1 ). These assignments are based on the electronic structure analysis by Solomon and co-workers [3]. Perturbation 

of the ratios of the LMCT bands (A~460/A~600) by the substitution at Met16 reflects the structural changes around 

the active site [4]. The ratio of the absorption at 460 and 600 nm, A~460/A~600 of Met16Lys and Met16Glu 

pseudoazurin were estimated to be 0.54 and 0.64, respectively. These values are larger than that of wild-type 

PAz, and the larger A~460/A~600 values of Met16Lys and Met16Glu suggest the larger population of rhombic 

structure. X-band EPR spectra of the Met16Lys and Met16Glu variants showed almost identical spectra to the 

spectrum of Met16Val variant, which shows rhombic EPR spectral pattern. The redox potentials of Met16Lys 

and Met16Glu mutants were evaluated to be 306 and 245 mV vs NHE (pH 7.0), respectively. The redox 

potentials of the Met16Lys and Met16Glu are reasonably explained by the electrostatic effect of the side chain 

groups of Met16Lys and Met16Glu variants. 

Acknowledgement: A part of this work is supported by Research Promotion Bureau, Ministry of Education, 

Culture, Sports, Science and Technology (MEXT), Japan to TK, a Grant-in-Aid for Scientific Research from 

JSPS (No. 18550147), Japan to TK. 

References: 

[1] R. F. Abdelhamid, Y. Obara, Y. Uchida, T. Kohzuma, D. M. Dooley, D. E. Brown, H. Hori, J. Biol. Inorg. 

Chem, 12, 165 (2007). 

[2] R. F. Abdelhamid, Y. Obara, T. Kohzuma, J. Inorg. Biochem, 102, 1373 (2008). 

[3] E. I. Solomon and M. D. Lowery, Science, 259, 1575 (1993). 

[4] A. A. Gewirth and E. I. Solomon, J. Am. Chem. Soc., 110, 3811 (1988). 

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260


P142. Maturation Mechanism of a New Nitrile Hydratase Family Protein, 

Thiocyanate Hydrolase 

M. Odaka a , S. Hori a , T. Arakawa a , H. Nakayama b , N. Dohmae b , H. Mino c , 

Y. Katayama d , M. Yohda a 

a 

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 

Naka-cho, 184-8588, Koganei, Japan, 

b 

Biomolecular Characterization Team, RIKEN, 2-1 Hirosawa, 351-0198, Wako, Japan 

c 

Division.of Material Science (Physics), Nagoya University, Chikusa, 464-8602, Nagoya, Japan 

d 

Department of Environmental and Natural Resource S, Tokyo University of Agriculture and Technology, 3-5-8 

Saiwaicho, 183-8509, Fuchu, Japan 

e-mail: modaka@cc.tuat.ac.jp 

Nitrile hydratase (NHase) family proteins are Co- or Fe-containing enzymes having two post-translationally 

modified Cys ligands, Cys-SO2H and Cys-SOH. NHase family proteins require their specific activator proteins 

for the functional expression. Here, we studied the function of P15K, the activator protein of a new Co-type 

NHase family protein, thiocyanate hydrolase (SCNase). SCNase catalyzes the hydrolysis of thiocyanate to 

carbonyl sulfide and ammonia. It consists of α, β and γ subunits and has a hetero-dodecamer structure, (αβγ)4. 

When P15K was co-expressed with each SCNase subunit in E. coli, only γsubunit which had the Co-binding site 

formed a stable 1: 1 complex (γP15K). In contrast, the isolated γ subunit (γ(+Co)) as well as γP15K 

(γ(+Co)P15K) was obtained when they were co-expressed in the Co-enriched medium. γ(+Co) as well as 

γ(+Co)P15K incorporated stoichiometric amounts of Co ions and possessed the Cys-SO2H modification like 

native SCNase, suggesting that these complexes are intermediate species in the maturation systems of SCNase. 

Then, we expressed SCNase (A) α, (B) β or (C) γ subunits in the presence of γ(+Co) using a cell free protein 

synthesis system. In (A) and (C), the αβ or αβγ complexes were obtained, respectively, while no βγ complex was 

detected in (B). Interestingly, only the reaction mixture of (C) exhibited the SCNase activity. Thus, we 

concluded that γ(+Co) assembles with the α subunit subsequently with the β subunit, to form mature SCNase. 

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261


P143. High Selective Epoxidation of Cyclohexene by Non-heme Ruthenium 

Complexes Incorporated into Mesoporous Silicate 

K. Okumura, K. Jitsukawa, T. Ozawa, Y. Funahashi, and H. Masuda 

Department of Applied Chemistry, Graduate School of Engineering, Nagoya Institute of Technology, 

Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan 

e-mail: masuda.hideki@nitech.ac.jp 

Binding and activation of oxygen species in biological metallo-enzymes are regulated by the metal coordination 

structures and the non-covalent interaction groups surrounding them, such as hydrophobic and hydrogen bonding 

interaction ones. On the basis of the active site structures in biological systems, we have designed and 

synthesized some new artificial metallo-enzymes containing transition metal ions. The objective of our research 

project is to construct the artificial metallo-enzymes using these transition metal complexes and to develop 

environmentally-benign catalysts. We recently prepared the biomimetic materials consisted of metal complexes 

as an active site and nanoporous silicate as a reaction field, and the oxidation reaction with some substrates were 

carried out.[1][2][3] In this paper, we newly designed and synthesized new ruthenium(II) complex of 6,6’bis(benzoylamido)-2,2’-bipyridine 

(BABP) as an oxidation catalyst. This catalyst exhibited high efficient 

oxidations for cyclohexene when tert-butyl hydroperoxide was employed as an oxidant, but it does not show any 

selectivity. So we tried the immobilization of the ruthenium(II) complex into the nanoporous silicate FSM-16 

that is often used as the substrate-specific reaction field in biological enzyme systems. Interestingly, it showed a 

higher epoxidation selectivity for cyclohexene. The material after the oxidation reaction was ESR active, 

indicating that the starting material for the catalytic reaction is Ru(III) species and the oxidation active species is 

Ru(V)=O. So we can propose that the oxidation reaction was carried out with cycle of Ru(III) and Ru(V)=O. 

Interestingly, this Ru(III) species was repeatedly used for epoxidation reaction. These findings indicate the 

nano-silicate FSM-16 can stabilize the active species and promote the effective capturing of the substrates. 

Acknowledgment: We gratefully acknowledge the support of this work from a Grant-in-Aid for Scientific 

Research from the Ministry of Education, Science, Sports and Culture, and in part by a grant from the NITECH 

21st Century COE program. 

References: 

[1] T. Okumura, H. Takagi, Y. Funahashi, T. Ozawa, Y. Fukushima, K. Jitsukawa, and H. Masuda, Chem. Lett., 

36, 122-123 (2007) 

[2] Y. Honda, H. Arii, T. Okumura, A. Wada, Y. Funahashi, T. Ozawa, K. Jitsukawa, and H. Masuda, Bull. 

Chem. Soc. Jpn. (Accounts, Invited), 80, 1288-1295 (2007). 

[3] T. Okumura, Y. Morishima, H. Shiozaki, T. Yagyu, Y. Funahashi, T. Ozawa, K. Jitsukawa, and H. Masuda, 

Bull. Chem. Soc. Jpn., 80, 507-517 (2007). 

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262


P144. New Complexes with N, N-dimethylbiguanide Displaying Low 

Cytotoxicity as Potential Large Spectrum Antimicrobial Agents 

R. Olar a , M. Badea a , D. Marinescu a , G. Vasile b , V. Lazar c , C. Chifiriuc Balotescu c 

a Faculty of Chemistry University of Bucharest Panduri, 050663, Bucharest, Romania, 

b Agrochemistry, University of Agronomical Sciences and Veterinary, Marasti, , Bucharest, Romania 

c Faculty of Biology, University of Bucharest, Aleea portocalelor, 060101, Bucharest, Romania 

e-mail: rodica_m_olar@yahoo.com 

Selective and effective antimicrobial activities against Gram-positive and Gram-negative bacteria were found for 

a series of new complexes with N, N-dimethylbiguanide (DMBG). The complexes with general formula 

M(DMBG)2(ClO4)2 ((1) M:Mn; (2) M:Ni, (3) M:Cu and (4) M:Zn) have been synthesized. The methods used in 

order to characterize the bonding and the stereochemistry of the complexes were IR, EPR, 1 H NMR and 

electronic spectroscopy. Recrystallization from DMF of (2) afforded crystals of type 

[Ni(DMBG)2](ClO4)2·2CHON(CH3)2 (2a) in the monoclinic P2(1)/c space group. The antimicrobial activities of 

complexes was investigated by qualitative (disk diffusion) and quantitative (liquid medium serial microdillution) 

methods on planktonic and biofilm embedded bacterial and fungal strains. Metal-free N, N-dimethylbiguanide 

derivatives and the complexes exhibited specific antiinfective properties as demonstrated by the low MIC values, 

the large antimicrobial spectrum and by the inhibition of the microbial ability to colonize the inert surfaces. At 

the same time, excepting the complex (2), the other metal complexes exhibited low citotoxicity levels on HeLa 

cells, this representing a great advantage for the in vivo use of the tested complexes as antimicrobial agents. 

Acknowledgement: This work was partially supported by the grants PNII nr. 61-48/2007 and VIASAN 

142/2006 of the Romanian Ministry of Education and Research. 

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263


P145. The Crystal Structure of the DsrAB Dissimilatory Sulfite Reductase 

from D. Vulgaris Bound to DsrC Provides Novel Insights into the 

Mechanism of Sulfate Reduction 

T.F. Oliveira a , C. Vonrhein b , P.M. Matias a , S.S. Venceslau a , M. Archer a , 

I.A. Cardoso Pereira a 

a 

Instituto de Tecnologia Química e Biológica/UNL, Av. da República - EAN, 2780-157, Oeiras, Portugal 

e-mail: ipereira@itqb.unl.pt 

b 

Global Phasing Ltd.,Sheraton House, Castle Park, CB3 0AX, Cambridge, United Kingdom 

Sulfate reduction is one of the earliest energy metabolisms detected on Earth, at ~3.5 billion years ago [1]. 

Despite extensive studies, many questions remain about the way respiratory sulfate reduction is associated with 

energy conservation [2]. A crucial enzyme in this process is the dissimilatory sulfite reductase (dSiR; DsrAB), 

which contains a unique siroheme-[4Fe4S] coupled cofactor, and was present in one of the earliest life forms on 

Earth. The number of cofactors of dSiRs is not clear with studies reporting from two to four sirohemes and 10 to 

32 non-heme irons per α2β2 module. In Desulfovibrio spp. this protein (desulfoviridin) is particularly intriguing 

since it is reported that up to 80% of its siroheme is not metallated but is in the form of sirohydrochlorin, and it 

forms a stable complex with DsrC. DsrC is one of the few proteins of the dsr operon to be conserved in both 

sulfate/sulfite reducers and sulfur oxidisers. In D. vulgaris DsrC is very highly expressed, at a level twice of 

DsrAB [3], pointing to an important role in cellular metabolism. 

Here, we report the structure of desulfoviridin from D. vulgaris, in which the dSiR DsrAB subunits form a 

complex with DsrC [4]. This structure elucidates several pending questions about desulfoviridin and points to an 

essentail role of DsrC in sulfite reduction. We propose a novel mechanism for this reduction that changes our 

understanding of sulfate respiration and has important implications for models used to date ancient sulfur 

metabolism based on sulfur isotope fractionations. 

References: 

[1] Y. Shen, R. Buick, D.E. Canfield, Nature, 410, 77 (2001). 

[2] P.M. Matias, I.A.C. Pereira, C.M. Soares, M.A. Carrondo, Prog. Biophys. Mol. Biol., 89, 292 (2005). 

[3] S.A. Haveman, V. Brunelle, J.K. Voordouw, G. Voordouw, J.F. Heidelberg, R. Rabus, J. Bacteriol., 185, 

4345 (2003). 

[4] T.F. Oliveira, C. Vonrhein, P.M. Matias, S.S. Venceslau, I.A.C. Pereira, M. Archer, (2008) submitted. 

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264


P146. Key Role of Val567 on L-Arginine Analogues and Heme Ligands 

Binding to nNOS. 

I. K. Olsbu a , M. Lombard b , H.P. Hersleth a , K.K. Andersson a , J.L. Boucher b 

a Department of Molecular Biosciences, University of Oslo, Blindernv. 31, 0371, Oslo, Norway, 

b Université Paris Descartes UMR8601 CNRS, Rue st Peres, 75270, Paris, France 

e-mail: i.k.Olsbu@imbv.uio.no 

Nitric Oxide (NO) is a key inter and intracellular messenger involved in maintenance of vascular tone, neuronal 

signalling and immune response in mammals. The biosynthesis of NO involves a two step oxidation of 

L-Arginine (L-Arg) to Citrulline and NO by heme thiolate proteins called Nitric Oxide SYntases (NOSs). The 

crystallographic studies have revealed a highly conserved hydrophobic pocket among NOSs isoforms, containing 

Val, Pro and Phe residues [1]. In bacterial NOSs, this Val residue is change for an Ile residue [2]. THis Val/Ile 

swich significantly reduses the rate of NO release due to increased shielding of the heme pocket [3]. and could 

alter the reactivity of the heme. Based on these findings, the importanse of this Val567(rat nNOSoxy) residue on 

substrtate recognition, heme ligand binding and NO formation was investigated. 

Two mutants of nNOSoxy were constructed by point mutation, namely V567S and V567Y and the proteins were 

characterised in respect to their binding capability of natural substrates of NOS and substrate analogues, as well 

as common heme ligands such as alkylisocyanides and CO 

References: 

[1] Li H, Shimizu H, Flinspach M, Jamal J, Yang W, Xian M, Cai T, Wen EZ, Jia Q, Wang PG, Poulos TL. 

Biochemistry 41 (2002) 13868-13875 

[2] Pant K, Bilwes AM, Adak S, Stuehr DJ, Crane BR. Biochemistry 41 (2002) 11071-11079 

[3] Wang ZQ, Wei CC, Sharma M, Pant K, Crane BR, Stuehr DJ. J.Biol.Chem. 279 (2004) 19018-19025 

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265


P147. How Does CUP1 Cope with Cd(II) or Zn(II) Instead of Cu(I)? 

R. Orihuela a , F. Monteiro b , S. Atrian b , M. Capdevila a 

a Departament de Química, Universitat Autònoma de Barcelona, , 08193, Bellaterra, Spain, 

b Departament de Genètica, Universitat de Barcelona, , 08028, Barcelona, Spain 

e-mail: ruben.orihuela@uab.es 

Metallothioneins (MTs) are ubiquitous, small cysteine-rich proteins that bind d 10 heavy metal ions such as the 

essential Cu(I) and Zn(II) or the toxic Cd(II) and Hg(II). 

CUP1 is the paradigmatic copper-thionein from the yeast Saccharomyces cerevisiae, whose gene responds to 

copper but not to cadmium overload. 

In this work we have used spectroscopic and spectrometric techniques to characterize the copper, zinc, and 

cadmium complexes formed by CUP1 recombinantly synthesized in E. coli. 

Furthermore, we have studied native Cd-CUP1 complexes produced by the mutant strain 301N of S. cerevisiae, 

where a promoter mutation enables a high cadmium-induced CUP1 expression [1, 2]. 

Preliminary results corroborate the literature reporting the coordination of 8 copper ions by CUP1, and the 

features of the Cu-CUP1 species [3]. The metalated protein recovered from zinc supplemented cultures was 

found to contain 4 metal ions. The cadmium complexes from enriched cadmium cultures contain a variable 

number of Cd(II) ions together with a considerable amount of sulphide ligands. 

Owing to the fact that CUP1 is the Cu-thionein par excellence, the comparison of the recombinant (E.coli) Cd- 

CUP1 with the native Cd-CUP1 preparations will reveal if the native metal-protein complexes have the ability to 

harbour non proteic ligands, as we have previously described happening when recombinant metallothioneins are 

forced to coordinate their “non-preferred” metal ions [4]. 

References: 

[1] A.K. Sewell, F. Yokoya, W. Fu, T. Miyagawa, T. Murayama, D.R. Winge, The Journal of Biological 

Chemistry, 1995, 270, 25079-25086. 

[2] M. Inouhe, M. Hiyama, H. Tohoyama, M. Joho, T. Murayama, Bioquimica et Biophysica Acta, 1989, 993, 

51-55. 

[3] D.R. Winge, K.B. Nielson, W.R. Gray, D.H. Hamer, The Journal of Biological Chemistry, 1985, 260, 14464- 

14470. 

[4] M. Capdevila, J. Domenech, A. Pagani, L. Tío, L. Villareal, S. Atrian, Angew. Chem. Int. Ed., 2005, 44, 

4618-4622. 

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266


P148. High-Frequency EPR and Magnetic Studies on a Complex Useful in 

Modelling Cu(II) Transport through Biological Membranes 

A. Ozarowski a , K. Wojciechowski b M. Brynda c , J. Jezierska d 

a 

National High Magnetic Field Laboratory, Florida State University, 1800 E. Paul Dirac Drive, Tallahassee, 

FL 32310, USA 

e-mail: ozarowsk@magnet.fsu.edu 

b 

Warsaw University of Technology, Department of Analytical Chemistry, Noakowskiego 3, 00-664 Warsaw, 

Poland, 

c 

Department of Chemistry University of California, Davis One Shields Avenue, Davis, CA 95616, USA, 

d 

Department of Chemistry, Wroclaw University, F. Joliot-Curie 14, Wroclaw 50-383, Poland 

Permeation Liquid Membranes (PLM), capable of selective transport of metal ions, are used for biomimetic 

purposes to study heavy metal ions speciation. Membranes containing long-chain azacrown ethers and 

carboxylic acids transport transition metal ions with the selectivity order Cu(II)> Pb(II) >> Cd(II), and were used 

as models of biological membranes of algae [1]. 

In the course of our mechanistic studies on Cu(II) transport through PLM we have characterized a model of the 

active complex formed in membranes during the transport of Cu(II), which is an adduct of dimeric Cu(II)hexanoate 

and Cu(II)-1, 10-diaza-18-crown-6 ether complex [2]. The X-Ray structure (Fig. 1) could be only 

partially resolved due to high disorder in the carboxylic acid chains. IR, UV/Vis as well as magnetic and highfield 

EPR studies presented here support the 1D polymer structure in which Cu-hexanoate dimers alternate with 

Cu-azacrown monomers. Exchange integral J of 333 cm -1 (H=JS1S2) was determined from the magnetic 

susceptibility measurements. 

Magnetic Induction, Tesla 

12.0 12.5 13.0 13.5 14.0 14.5 

Figure 1. Left: Fragment of the alternating chain of [Cu(C 5H 11COO) 2] 2[Cu(C 12H 26N 2O 4)(C 5H 11COO) 2]. For clarity, all 

hydrogen atoms were omitted and only the carboxyl groups of hexanoates are shown. 

Right: bottom: EPR spectrum measured at 287K and 412.8 GHz. Asterisks indicate resonances due to the copper-azacrown 

complex (g x=2.055 g y=2.066 g z=2.288). Top: Triplet spectrum of the binuclear copper hexanoate simulated with parameters 

given in text. 

In HF EPR, signals of carboxylato-bridged dimer were observed in addition to the monomeric Cu-azacrown 

entities. The spectra indicate that symmetry of the dimer being part of the polymeric chain is higher (axial, 

gx=gy=2.072 gz=2.375, D=0.348 cm -1 , E=0) than the symmetry of a separate copper hexanoate dimer (rhombic, 

gx=2.050 gy=2.075, gz=2.348, D=0.336cm -1 , E=0.0121cm -1 ). No dimer-monomer exchange interactions were 

observed, in agreement with our DFT calculations. 

Acknowledgement: This work was supported by the NHMFL. The NHMFL is funded by the NSF through the 

Cooperative Agreement No. DMR-0654118 and by the State of Florida. KW acknowledges the financial support 

from Warsaw University of Technology. 

References: 

[1] Zhang, Z.; Buffle, J.; van Leeuwen, H. P.; Wojciechowski, K. Anal. Chem., 78, 5693 (2006) 

[2] Wojciechowski, K.; Kucharek, M.; Buffle, J. J. Membr. Sci., 314, 152 (2008) 

* 

_____________________________________________________________________ 

267 

* 

*


P149. Approach to the Metal Specificity of the Terrestrial Snail 

Metallothionein Isoforms through the Analysis of Their Recombinant 

Complexes 

O. Palacios a , A. Pagani b , M. Egg c , M. Hockner c , R. Dallinger c , M. Capdevila a , 

S. Atrian b 

a Química, Universitat Autònoma de Barcelona, Campus de Bellaterra, 08193, Cerdanyola del Vallés, Spain, 

b Genètica, Universitat de Barcelona, Av. Diagonal, 08028, Barcelona, Spain 

c Zoology and Limnology, University of Innsbruck, , 6020, Innsbruck, Austria 

e-mail: oscar.palacios@uab.cat 

Many organisms contain several metallothionein (MT) isoforms able to play different physiologic roles. A clear 

example is found in the snail H.pomatia [1], harboring two MT isoforms. The HpCdMT gene is induced by Cd 2+ 

and expressed in the midgut gland. HpCuMT is expressed constitutively only in the rogocites, yielding 

homometallic Cu + complexes, so that a role in Cu homeostasis is hypothesized. In other invertebrates, such as 

D.melanogaster, the existence of several MT isoforms, all of them with similar metal binding preferences, is 

known [2]. In order to shed light on the metal binding behavior of the two H.pomatia MTs, HpCuMT and 

HpCdMT, they were recombinantly produced in E.coli cultures supplemented with either Zn 2+ , Cd 2+ or Cu 2+ 

ions. The metal-MT complexes obtained in vivo from the recombinant bacteria, as well as those obtained in vitro 

after Zn/M (M= Cd or Cu) replacement, were analysed by ICP-AES, UV and CD spectroscopy and ESI-TOF 

MS.Our results confirm the high specificity of HpCuMT for copper, since it is able to fold into single Cu12-MT 

species under low O2 conditions, unlike the HpCdMT isoform. Also, the high specificity of the HpCdMT 

isoform for divalent metal ions (Zn 2+ and Cd 2+ ) is highlighted, as it yields Zn6- and Cd6-MT species, 

respectively. These data are in perfect concordance with those available about the metal specificity of both native 

isoforms, and thus their specific functions, an scenario that has so far not been described for other organisms. 

References: 

[1] R. Dallinger, B. Berger, P.E. Hunziker, J.H.R. Kägi, Nature, 1997, 388, 237-238 

[2] D. Egli, J. Domenech, A. Selvaraj, K. Balamurugan, H. Hua, M. Capdevila, O. Georgiev, W. Schaffner, S. 

Atrian, Genes to Cells, 2006, 11, 647-658 

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268


P150. Enzymatic Electron Transfer in Respiratory Escherichia coli Nitrate 

Reductase 

A. Parkin a , C. F. Blanford a , J. Weiner b , F. A. Armstrong a 

a Inorganic Chemistry, University of Oxford, South Parks Road, OX1 3QR, United Kingdom 

b Membrane Protein Research Group, Dept. of Biochemistry, Univ. of Alberta, Edmonton AB T6J 2C2, Canada 

We have examined the electrocatalytic properties of mutants of Escherichia coli respiratory nitrate reductase in 

order to explore the role of an irregular sequence of reduction potentials in enzymatic electron transfer relays. In 

the wild type enzyme, one [4Fe4S] cluster (FS2) located halfway along the electron ‘wire’ in nitrate reductase 

has a reduction potential (-420 mV) that is more than 300 mV more negative than its neighbouring clusters 

(electron-acceptor FS1 and electron-donor FS3). The electron-transport relay’s potential barriers were 

“smoothed out” in two mutants. 

By simulating the protein film voltammetry activity of the wild type and mutant nitrate reductases we have been 

able to explore the relative importance of distance and potential barriers in limiting the rate of electron transfer in 

proteins. We conclude that low potential [4Fe4S] clusters which are also found in DMSO reductase, 

quinol:fumarate oxidoreductase and succinate:quinone oxidoreductase (SQR), may be an irreplaceable element 

in biological catalysis. 

Acknowledgement: UK Engineering and Physical Sciences Research Council (Supergen V). 

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269


P151. Artificial Ribonucleic Acids as Scaffolds for Mercury(II) Ions 

S. Paulus a , S. Johannsen a , N. Düpre b , J. Müller b , R.K.O. Sigel a 

a Institute of Inorganic Chemistry, University of Zurich, Winterthurstr. 190, 8057, Zurich, Switzerland, 

b Faculty of Chemistry, Dortmund University of Technology, Otto-Hahn-Str. 6, 44227, Dortmund, Germany 

e-mail: supaulus@aci.uzh.ch 

Metal-modified nucleic acids have become increasingly important as they provide scaffolds that can potentially 

be used for molecular wires. A DNA duplex with mismatched T-T pairs can bind Hg 2+ by forming T-Hg 2+ -T 

base pairs, which are as stable as a Watson-Crick base pair.[1] We are interested in the formation of an 

analogous array of Hg 2+ by using RNA instead.[2] The required single-stranded RNA sequences were 

synthesized by in vitro transcription, which yields the RNA in amounts sufficient for a detailed characterization 

by NMR. Indeed, T7 RNA polymerase successfully incorporates two to twenty consecutive uracil residues into 

RNA. In the presence of Hg 2+ , a structural rearrangement from hairpin to regular duplex by forming Hg 2+ - 

mediated base pairs has been characterized in detail for one of the sequences (see Figure). This rearrangement 

was verified by several techniques, e.g. NMR, DLS, UV and CD spectroscopy.[2] Ongoing investigations now 

focus on the incorporation of stretches of the chemically more stable thymine into RNA. For this purpose we are 

using the T7 RNA polymerase mutant Y639F, which does not discriminate deoxyribose against ribose sugars.[3] 

Indeed, we were able to construct RNAs containing poly T-sequences, which are presently subject to structural 

studies. 

Acknowledgement: Financial support by the European ERAnet-Chemistry, the Swiss National Science 

Foundation (20EC21-112708 and SNF-Förderungsprofessur PP002-114759 to R.K.O.S.) and the DFG 

(JM1750/2-1 and Emmy Noether programme JM1750/1-3 to J.M.) is gratefully acknowledged. 

References: 

[1] Y. Tanaka, S. Oda, H. Yamaguchi, Y. Kondo, C. Kojima, A. Ono, J. Am. Chem. Soc. 2007, 129, 244-245. 

[2] S. Johannsen, S. Paulus, N. Düpre, J. Müller, R.K.O. Sigel, J. Inorg. Biochem. 2008, 102, 1141-1151. 

[3] R. Sousa, R. Padilla, EMBO J. 1995, 14, 4609-4621. 

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270


P152. Quantitative Electrospray Mass Spectrometry of Zinc Finger 

Oxidation 

K. Piątek a , J. Smirnova b , L. Zhukova a , A. Witkiewicz-Kucharczyk a , E. Kopera a , 

J. Olędzki a , A. Wysłouch-Cieszyńska a , T. Schwerdtle c , A. Hartwig c , P. Paluma b , 

W. Bal a 

a 

Department of Biophysics, Institute of Biochemistry and Biophysics, PAS, Pawińskiego 5a, 02-106, Warsawa, 

Poland, 

b 

Department of Gene Technology, Tallinn Technical University, Akadeemia tee 15, 12618 Tallinn, Tallinn, 

Estonia 

c 

Institute of Food Technology and Food Chemistry, Technical University Berlin, Gustav-Meyer-Allee 25, 

D-13355, Berlin, Germany 

e-mail: kpiatek@ibb.waw.pl 

Oxidation is a crucial inhibitor of zinc fingers (ZF) [1]. Using the Zn(II) complex of 37-peptide acetyl- 

DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (ZnXPAzf), the Cys4 ZF of human DNA 

repair protein XPA, we developed an ESI MS approach for quantitative study of ZF oxidation kinetics. For this 

purpose we studied oxidation of ZnXPAzf by H2O2 using HPLC of covalent reaction products, PAR-based zinc 

release assay, and ESI MS. All yielded independently the same reaction rate, thus demonstrating quantitative 

applicability of ESI MS [2]. We then used this approach to study aerobic reactions of ZnXPAzf with Snitrosoglutathione 

(GSNO), arsenite and monomethylarsonous acid (MMA). GSNO initially formed a ZnXPAzf- 

GSNO complex, followed by thiol transnitrosylations and finally disulfide formation. These results showed that 

at low exposures GSNO may reversibly regulate the ZF, while transnitrosylation by GSNO, occurring at 

prolonged exposures, is damaging. For XPA this may lead to DNA repair inhibition [3]. MMA, but not arsenite, 

released Zn(II) from ZnXPAzf easily, forming arsenical thiol esters of XPAzf. Unprotected thiol groups were 

oxidized to disulfides. The estimated affinity of MMA to XPAzf is 30-fold higher than the values typical for 

arsenite binding to thiols, indicating a particular susceptibility of ZFs to MMA, and providing a novel molecular 

pathway in arsenic carcinogenesis [4]. In summary, ESI MS is a valuable tool for quantitative bioinorganic 

studies. 

References: 

[1] A Witkiewicz-Kucharczyk, W Bal, Damage of zinc fingers in DNA repair proteins, a novel molecular 

mechanism in carcinogenesis. Toxicol Lett 162, 29-42, 2006. 

[2] J Smirnova, L Zhukova, A Witkiewicz-Kucharczyk, E Kopera, J Olędzki, A Wysłouch-Cieszyńska, 

P Palumaa, A Hartwig, W Bal, Quantitative electrospray mass spectrometry of zinc finger oxidation: the reaction 

of XPA zinc finger with H2O2, Anal Biochem 369, 226-231, 2007. 

[3] J Smirnova, L Zhukova, A Witkiewicz-Kucharczyk, E Kopera, J Olędzki, A Wysłouch-Cieszyńska, 

P Palumaa, A Hartwig, W Bal, Reaction of the XPA zinc finger with GSNO, Chem Res Toxicol 21, 386-392, 

2008. 

[4] K Piątek, T Schwerdtle, A Hartwig, W Bal, Monomethylarsonous acid destroys a tetrathiolate zinc finger 

much more efficiently than inorganic arsenite. Mechanistic considerations and consequences for DNA repair 

inhibition. Chem Res Toxicol 21, 600-606, 2008. 

_____________________________________________________________________ 

271


P153. Kojic Acid Derivatives as Iron (III) and Aluminium (III) Chelators 

T. Pivetta a , V. M. Nurchi a , G. Crisponi a , J. Lachowicz a , M. Remelli b , J. M. Gonzalez-Perez c , 

J. Niclós-Gutiérrez c , A. Castiñeiras d 

a 

Dip. di Scienze Chimiche, Università di Cagliari, 09042 Monserrato-Cagliari, IT 

e-mail: lachowicz@unica.it 

b 

Dip. di Chimica, Università di Ferrara, Via L.Borsari 46, 44100 Ferrara, IT 

c 

Dpt. of Inorganic Chemistry, Campus Cartuja, University of Granada, E-18071 Granada, ES 

d 

Dpt. of Inorganic Chemistry, University of Santiago de Compostela, E-15782 Santiago de Compostela, ES 

Kojic acid (HKj, 1) is a 3-hydroxy-4-pyrone derivative produced by Aspergillus oryzae with antibacterial and 

antifungal activities, also used by its lighten skin properties. HKj metal complexes have received large attention 

for long time to nowadays [1-3]. A binuclear Cu-1 complex inhibits the catecholase activity and metal complexes 

of HKj have an interesting medicinal inorganic chemistry [4, 5]. Because of the hard nature of its O-donor atoms 

and the strategic site of its phenol group, HKj is a suitable ligand for iron(III) ions, with excellent chelating 

properties. In this connection, spectrophotometric studies carried out by Murakami [6] proved that in HKjiron(III) 

solutions complexes of different stoichiometries form, also if the pFe 3+ value ~14 prevents its use as 

iron chelator. Nevertheless, Fox and Taylor [7] synthesized the methylene-bis-6-kojic acid (H2MbK, 2) and 

checked its ability in the intracellular mobilization of ferritin-bound iron(III). A behaviour similar to that of 

Desferal and Deferiprone was found. 

HO 

O 

O 

OH 

HO 

_____________________________________________________________________ 

272 

O 

O 

OH 

HO 

O 

O 

(1) (2) (3) 

Since no work is reported on iron(III)-2 complexes, we studied the complex formation of H2MbK with iron(III) 

and aluminium(III) using potentiometric, spectrophotometric, 1 H-NMR and crystallographic methods (Figure 1). 

Furthermore, to increase the lipophilicity of compound 2, we synthesized a new bis-kojic-like acid with a 

bridging o-vanillin moiety (H3VbK, 3). 

absorbance 

1.2 

0.8 

0.4 

0 

pH = 3.5 

pH = 10.9 

240 280 320 360 400 

wavelength (nm) 

Figure 1. (A) pH-spectrophotometric titration of H2MbK; (B) Crystal structure of Fe(III)-HKj3; (C) Crystal 

structure of H3VbK. 

Acknowledgement: JL thanks a recent stay in the Research Group FQM283 (Junta de Andalucía, Spain) of 

Prof. J. Niclós-Gutiérrez 

References: 

[1] B. E. Bryants, W.C. Fernalius, J. Am. Chem. Soc. 76, 5351 (1954). 

[2] S.M. Reilly, A. Stenson, 235 th ACS National Meeting, 2008, New Orleans, USA. 

[3] A.C. Stenson, E.A. Cioffi, Rapid Comm. Mass Spectrom., 21, 2594 (2007). 

[4] G. Battaini, E. Monzani, L. Casella, L. Santagostini, R. Pagliarin, J. Biol. Inorg. Chem., 5, 262 (2000). 

[5] K.H. Thompson, C.A. Batrta, C. Orving, Chem. Soc. Rev., 35, 545 (2006). 

[6] Y. Murakami, J. Inorg. Nucl. Chem., 24, 679 (1962). 

[7] R.C. Fox, P.D. Taylor, Bioorg. Med. Chem. Lett., 8, 443 (1998) 

OH 

HO 

H3C 

O 

O 

HO 

O 

OH 

HO 

O 

O 

OH 

C

H. Podsiadły 


P154. Interaction of Vanadium(III) Ions with Small Biomolecules 


Vanadium is an important trace element for different organisms . Among others, it is present in vanadium 

dependent enzymes and is accumulated in high concentrations in certain sea squirts and mushrooms Amanita 

muscaria. Vanadium compounds have also therapeutic effect as insulin-mimetics, antitumor and antileukemic 

agents [1]. 

For a deeper understanding of the biological role of vanadium necessarily is to the study of model compounds 

e.g. interaction of vanadium with amino acids and with smaller peptides. The complexing behaviour of 

vanadium(III) with small biomolecules is still not well understood. In recent papers we reported the speciation 

and structure of vanadium(III) with amino acids and their derivatives [2, 3, 4] 

In this communication I have investigated the stability, speciation and structures of vanadium(III) complexes 

with L-carnosine, which is the first report on the speciation of the vanadium(III) complexes with peptides. 

L-carnosine is a dipeptide composed of covalently bonded amino acids alanine and histidine and is found in 

brain, heart, skin, muscles and stomach. The exact biological role of carnosine is not well understood, but many 

studies indicate that carnosine has antioxidant potential and decreases the intracellular level of reactive oxygen 

species. Carnosine may also act as a neurotransmitter. Biochemical behavior and biological activities of this 

dipeptide depend on the participation of metal cations. The presence of metal cations is also important for the 

stabilization and activation of the enzyme of carnosinase [5]. 

N 

N 

H 

NH 

O OH O 

Carnosine is a polydentate ligand offering six potential binding sites: two imidazole nitrogens, one carboxylate 

and one amino group, and the peptide linkage. The type of complexes formed strongly depends on metal cation, 

ligand – metal ratios, and pH range of the solution. 

The speciation in the aqueous V(III) – carnosine system has been determined from the potentiometric and 

spectroscopic (UV-Vis absorption and CD) data. The application of the Hyperquad program to the experimental 

potentiometric data indicates that in experimental conditions (I=0.5 M NaClO4, pH range 2-6.5 and L/M>5) only 

ML2H4, ML2H3, ML2H2 and ML2H species existed in solution. Potentiometric results proved formation of stable 

complexes and with the use of spectroscopic methods the identification of the binding sites was made. 

References: 

[1] H. Sigel, A. Sigel ‘Metal Ions in Biological Systems” vol. 31 (1995) 

[2] K. Bukietyńska, H. Podsiadły, Z. Karwecka, J.Inorg.Biochem. 94, 317 (2003) 

[3] I. Osińska-Królicka, H. Podsiadły, K. Bukietyńska, M. Zemanek-Zboch, D. Nowak, K.Suchoszek-Łukaniuk, 

M. Malicka-Błaszkiewicz, J. Inorg. Biochem.98, 2087 (2004) 

[4] H. Podsiadły, Polyhedron 27, 1563 (2008) 

[5] E.J. Baran, Biochemistry 65(7) 789 (2000) 

NH 2 

_____________________________________________________________________ 

273


P155. Structural Properties of L-X-L-Met-L-Ala Phosphono Tripeptides: 

A Combined FT-IR, FT-RS, and SERS Spectroscopy Studies and DFT 

Calculations 

E. Podstawka, a* P. Kafarski, b a, c 

L. M. Proniewicz 

a 

Laser Raman Laboratory, Regional Laboratory of Physicochemical Analysis and Structural Research, 

Jagiellonian University, ul. Ingardena 3, 30-060 Krakow, Poland 

b 

Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw Technical University, ul. Wybrzeże 

Wyspiańskiego 27, 50-370 Wroclaw, Poland 

c 

Chemical Physics Division, Faculty of Chemistry, Jagiellonian University, ul. Ingardena 3, 30-060 Krakow, 

Poland; 

e-mail: proniewi@chemia.uj.edu.pl 

FT-IR and FT-RS spectra of three phosphonate tripeptides containing P-terminal L-Met-L-Ala (L-Gly-L-Me-L- 

Ala-PO3H2 (GMA), L-Leu-L-Met-L-Ala-PO3H2 (LMA), and L-Phe-L-Met-L-Ala-PO3H2 (PMA)) were recorded 

and analysed. Vibrational wawenumbers and intensities were calculated by density functional theory (DFT) at 

the B3LYP/6-311++G** level of theory and compared to these molecules in solid form. Based on this 

comparison, conclusions were drawn about the molecular structures. At the same time, the experimental data 

served as a test for the computational results. 

SERS spectra were recorded in a silver colloidal dispersion. Silver colloidal dispersions prepared by simple 

borohydride reduction of silver nitrate were used as substrates. A comparison is made between the SERS spectra 

and the spectra of the solid sample. Also, the capability of SERS for spectral fingerprinting of analytes with 

close structural properties using easily prepared substrates and relatively simple instrumentation is illustrated. By 

careful analysis, we obtained information on the orientation of these tripeptides and specific-competitive 

interactions of their functional groups with the silver surface. For example, all molecules are thought to adsorb 

on a silver surface via a P=O bond and a sulfur atom. In addition, the amide bond of GMA assists in the 

adsorption process, adopting a tilted orientation on the surface, with the N-H unit being closer to the surface than 

the C=O moiety. Conversely, the C=O unit of the LMA -CONH- bond lies closer to the silver surface than the 

N-H moiety. The -CH3 group and P-O bond of LMA additionally interact with the silver surface, whereas for 

PMA the L-Phe lies almost flat on the colloidal silver surface. 

GMA 

LMA 

PMA 

O CH3 OH 

S 

H3C NH P 

O 

O NH 

HO 

Acknowledgements: 

This work was supported by the Polish State Committee for Scientific Research (Grant No. 1 T09A 112 30 to 

E.P.). The authors are grateful to the Academic Computer Center “Cyfronet” in Krakow for allowing us to 

conduct all calculations presented in this work. 

References: 

E. Podstawka, P. Kafarski, L.M. Proniewicz, J. Phys. Chem. A., (2008) submitted. 

_____________________________________________________________________ 

274 

N 

H 2 

N 

H 2 

C 

H 3 

C 

H 3 

S 

O 

S 

O 

NH 2 

NH 2 

NH 

NH 

O 

NH 

O 

HO 

NH 

HO 

CH 3 

CH 3 

OH 

P 

O 

OH 

P 

O


P156. Heme Coordination in Porphyromonas gingivalis HmuY Protein 

H. Połata , M. Olczak , T. Olczak 

Faculty of Biotechnology, University of Wrocław, Tamka 2, 50-137, Wrocław, Poland 

e-mail: hpolata@tlen.pl 

Periodontitis is an infectious disease to which genetic, microbial, immunological, and environmental factors 

combine to influence disease risk and progression, resulting in the destruction of tooth-supporting tissues. 

Porphyromonas gingivalis, Gram-negative, anaerobic bacterium implicated in the development and progression 

of chronic periodontitis, requires iron and heme for growth. One of the mechanisms of heme uptake in this 

bacterium comprises the outer-membrane heme transporter HmuR and a putative heme-binding lipoprotein 

HmuY. We propose that heme derived from host sources binds to HmuY and is then delivered to HmuR, which 

further transports the heme molecule into the bacterial cell. The aim of this study was to characterize the nature 

of heme binding to HmuY. The protein was expressed, purified and detailed spectroscopic investigations (UV- 

Vis absorption spectroscopy, spectrofluorimetry, CD, and MCD) were performed. The spectrophotometric 

titrations showed a 1:1 molar ratio of heme binding to HmuY with a Kd of ~3 uM. Ferric heme bound to HmuY 

may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. The heme complexed to HmuY 

is in a low-spin Fe(III) hexa-coordinate environment and the heme binding does not change the protein's 

structure. Using site-directed mutagenesis, several single and double HmuY mutants were constructed and the 

ability of the mutated proteins to bind heme was analyzed. This analysis identified histidines 134 and 166 as 

potential heme ligands. It is also likely that a reversible coordination by a histidine chain may occur, allowing 

heme transfer from hemoglobin to HmuY and subsequently to HmuR. 

_____________________________________________________________________ 

275


P157. Mo and W Complexes of Pyrane Dithiolene Derivatives as Synthetic 

Analogues of the Cofactors in Oxotransferases 

P. S. Prinson, C. Schulzke 

Institute for Inorganic Chemistry, Georg-August-University of Goettingen, Tammanstrasse 4., 37077, 

Goettingen, Germany, 

e-mail: Prinson.Samuel@ chemie.uni-goettingen.de 

Dithiolene complexes of Mo and W have been well documented since early 1960s, but the role of this chemistry 

in biology was revealed only after the structure of molybdopterin, a dithiolene ligand coordinated to the Mo 

centres in the cofactors of oxotransferases, was proposed by Rajagopalan in 1982 [1, 2]. Since this time, 

considerable efforts have been made by the inorganic chemists to synthesize the dithiolene complexes of Mo and 

W as the structural analogues of these cofactors [3]. But adequate analogues in terms of the chemical structure of 

the dithiolene ligands are rare in the literature either due to the difficulty to prepare or due to the overwhelming 

tendency to achieve the analogous metal centers using conventional type ligands. Our approach to this problem 

utilizes dithiolene ligands of derivatives of pyrane which is already present in the molybdopterin structure. Our 

present study involves the preparation of Monooxobis(chromanedithiolene) Mo(IV) complexes as well as its 

W(IV)analogue (Fig.). In addition to the basic characterizations, cyclic voltametry or differential pulse 

voltametry of the complexes have been carried out to investigate the response of the redox potentials to 

temperature and the nature of the substituents in the dithiolene ligand. The results have been compared with 

dithiolene complexes with non-pyrane substituents [4]. Finally, the catalytic activities of the new complexes 

have been explored in the model oxotransfer reaction between PPh3 and DMSO. 

References: 

[1]. Johnson J.L., Rajagopalan K.V., Proc. Natl. Acad. Sci. USA, 1982, 79, 6856. 

[2]. Rajagopalan K.V., Adv.Enzymol. Relat. Areas. Mol. Biol., 1991, 64, 215. 

[3]. Enemark J.H., Cooney J.A., Wang J.J., Holm R.H., Chem. Rev., 2004, 104, 1175. 

[4]. Schulzke C., Dalton. Trans., 2005, 713. 

_____________________________________________________________________ 

276


P158. Zn Complexes of Oxolinic Acid: Structure and DNA-binding 

Properties 

G. Psomas a , A. Tarushi a , C. Raptopoulou b , D. P. Kessissoglou a 

a 

Department of General and Inorganic Chemistry, Fac, Aristotle University of Thessaloniki, P.O. Box 135, 

GR-54124, Thessaloniki, Greece, 

b 

Institute of Materials Science, NCSR “Demokritos”, , GR-15310, Aghia Paraskevi Attikis, Greece 

e-mail: gepsomas@chem.auth.gr 

Zinc is an element of great biological interest. It is critical for numerous cell processes and is a major regulatory 

ion in the metabolism of cells. In the literature, diverse zinc complexes with antidiabetic, antifungal, 

antimicrobial, anti-inflammatory, antitumor and antiulcer activity are reported. 

Quinolones (quinolonecarboxylic acids or 4–quinolones) are a group of synthetic antibacterial agents that 

effectively inhibit DNA replication and are commonly used as treatment for many infections[1]. Oxolinic acid 

(=Hoxo), a first–generation quinolone antimicrobial drug, is known for the treatment of urinary tract infections. 

Although its pharmaceutical role is known for the last four decades, only one crystal structure of its complexes 

has been reported [2]. 

Here we report the synthesis, the structure and DNA binding properties of the neutral mononuclear zinc 

complexes with oxolinic acid in the absence or presence of the N-donor heterocyclic ligands pyridine (=py), 2, 

2’-bipyridine (=bipy) and 1, 10-phenanthroline (=phen). The complexes Zn(oxo)2(H2O)2, Zn(oxo)2(py)2, 

Zn(oxo)(bipy)Cl, Zn(oxo)(phen)Cl, Zn(oxo)2(bipy) and Zn(oxo)2(phen) (Figure 1) have been isolated and 

spectroscopically and structurally characterized. The interaction of the complexes with CT DNA has been 

investigated with UV and fluorescence spectroscopies in order to determine the intrinsic binding constants of the 

complexes with DNA, the mode of binding and its correlation with the structure of the complexes. 

References: 

[1] I. Turel, Coord. Chem. Rev. 232 (2002) 27–47. 

[2] G. Psomas, A. Tarushi, E.K. Efthimiadou, Y. Sanakis, C.P. Raptopoulou, N. Katsaros, J. Inorg. Biochem. 

100 (2006) 1764–1773. 

_____________________________________________________________________ 

277


P159. Copper(II) Complexes with Allantoin and Hydantoin. Synthesis, 

Spectroscopic and Pharmacokinetic Characterization 

M. Puszyńska-Tuszkanow, a M. Cieślak-Golonka, a G. Maciejewska, a T. Grabowski b 

a Faculty of Chemistry, Wrocław University of Technology, Smoluchowskiego str. 23, 50-372 Wrocław, Poland 

b Pharmacokinetic Testing Centre Filab-Ravimed, Polna str. 54, 05-119 Łajski k/Legionowa, Poland 

Allantoin and hydantoin (Fig 1) are natural species playing a crucial role e.g. in the purine metabolisms [1]. 

Besides, allantoin is widely used in dermatology as a drug [2, 3]. In contrast to hydantoin, no data that cover the 

topic of the coordination properties of allantoin was found in the literature. 

In this work the interaction of allantoin and hydantoin with copper(II) ion in ammonia solution was investigated. 

As a result, four new Cu(II) complexes were isolated and spectroscopically (UV/Vis, FIR, IR) characterized. 

For a preliminary evaluation of the potential pharmacokinetic activity of the species, the calculation of 

characteristic physiochemical parameters like e.g. the magnitude of polar surfaces of a molecule, lipophilicity 

and hydrogen bonding were performed and discussed. 

N(3) 

O 

O 

HN3 

2 

1NH 

4 

5 

NH 

Fig 1 allantoin hydantoin 

References: 

[1]. An encyclopedia of chemicals, drugs, and biologicals (2001); 

[2]. V. Shevstopolov, P. Guskov, et. al., Biology Bulletin, 33, (2006), 437-440; 

[3]. P. Guskov, N. Procofev, et. al., Dokl. Biochem. Biophys., 389, (2004), 320-324; 

_____________________________________________________________________ 

278 

O 

NH 2 

N(1) 

N(3) 

O 

HN 

2 

3 

1NH 

4 

5 

O 

N(1)


P160. The Peptide-heterocycle Conjugates: Derivatives of 3-(benzimidazol- 

5-yl)alanine in the Presence of Selected Metal Ions 

M. Ratajska, A. Kluczyk, P. Stefanowicz, H. Bartosz-Bechowski, M. Cebrat, Z. Szewczuk 

Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland, 

e-mail: mkoprow@eto.wchuwr.pl 

In our search for new bioactive peptides we investigate methods for introducing heterocyclic motifs into peptide 

side chains [1]. Considering the biological activity and complexing abilities of benzimidazoles we developed a 

direct solid-phase synthesis of benzimidazole-peptide conjugates, expecting these new compounds to express 

novel biological properties [2]. 

The peptide-heterocycle conjugates are obtained by the on-resin reaction between aldehydes and peptides 

containing a specially designed β-(3, 4-diaminophenyl)alanine residue [3]. The stoichiometric amount of 

aldehyde leads to 2’-substituted 3-(1H-benzimidazol-5-yl)alanine–containing peptides, whereas the increase in 

aldehyde content results in 1’, 2’-disubstituted derivatives. The reaction with dialdehydes produces novel amino 

acid residues containing tricyclic systems, in the case of o-phthalic aldehyde - the pyrido[1, 2-a]benzimidazole. 

The compatibility of our method with the Fmoc solid-phase peptide synthesis protocols was further proven by 

the synthesis of analogues of immunosuppressory fragments of ubiquitin and HLA-DQ [4, 5]. 

NH 

O 

N 

N 

H 

1. 2 M SnCl 2 * 2H 2 O 

2. 1.2 eq aldehyde, DMF 

3. piperidine 

4. TFA 

1. 2 M SnCl 2 * 2H 2 O 

2. 2 eq dialdehyde, DMF 

3. piperidine 

4. TFA 

Fig. 1. On-resin synthesis of substituted 3-(benzimidazol-5-yl)alanines. 

R 

NH 

NH 

O 

O 

NH 2 

NO 2 

NH 

1. 2 M SnCl 2 * 2H 2 O 

2. 2.1 eq aldehyde, DMF 

3. piperidine 

4. TFA 

Taking into account the known affinity of benzimidazoles to metal ions [6] we investigated the complexing 

abilities of peptides containing substituted 3-(benzimidazol-5-yl)alanines using high resolution mass 

spectrometry. We observed the formation of the complex of HLA-DQ fragment containing (2-(pyridin-2yl)benzimidazol-5-yl)alanine 

with copper ion. The collision-induced dissociation of the investigated complex 

indicates the part of the peptide-heterocycle conjugate that is involved in binding of the metal ion. 

Our results demonstrate that the high resolution electrospray mass spectrometry is an ideal tool for studying 

interactions of peptides with metal ions. The sub-femtomolar amount of sample required for measurement and 

the possibility of analyzing mixtures, as well as the richness of information brought by the analysis of isotopic 

patterns and the fragmentation pathways, make mass spectrometry an interesting alternative for investigating 

bioinorganic compounds. 

Acknowledgements: Supported by Grant No. N N 204 249934 from the MSHE (Poland). 

References: 

[1] A. Kluczyk, A. Staszewska, P. Stefanowicz et al., J. Peptide Sci., 12, 111 (2006). 

[2] M. Koprowska-Ratajska, A. Kluczyk, P. Stefanowicz et al., Amino Acids, in press. 


[4] Z. Szewczuk, P. Stefanowicz, A. Wilczynski et al., Biopolymers, 40, 571 (1996). 

[5] Z. Szewczuk, P. Stefanowicz, A. Wilczynski et al., Biopolymers, 74, 352 (2004). 

[6] M. Devereux, D. O’Shea, A. Kellett, J. Inorg. Biochem., 101, 881 (2007). 

N 

N 

_____________________________________________________________________ 

279 

O 

N 

N 

R 

R


P161. Oxidation of L-Tryptophan in Biology 

E. Raven a , J. Basran b , S. Rafice a , N. Chauhan a , I. Efimov a , M. Cheesman c 

a Chemistry, University of Leicester, University Road, LE1 7RH, Leicester, United Kingdom, 

b Biochemistry, University of Leicester, Lancaster Road, le1 9hn, Leicester, United Kingdom 

c Chemical Sciences and Pharmacy, University of East Anglia, Earlham Road, NR4 7TJ, Norwich, United 

Kingdom 

e-mail: emma.raven@le.ac.uk 

The L-kynurenine pathway – which leads to the formation of NAD – is the major catabolic route of L-tryptophan 

metabolism in biology. The initial step in this pathway is oxidation of L-tryptophan to N-formylkynurenine, 

Scheme 1. In all biological systems examined to date, this is catalysed by one of two heme enzymes, tryptophan 

2, 3-dioxygenase (TDO) or indoleamine 2, 3 dioxygenase (IDO), in a reaction mechanism that involves binding 

of dioxygen to ferrous heme. Although they catalyse the same reaction, TDO and IDO are otherwise distinct and 

we know little about their structure and mechanism. 

There is essentially nothing known about human TDO. Here, we describe spectroscopic, kinetic and redox 

analyses on recombinant human TDO [1]. We find unexpected differences between human TDO and the closely 

related human IDO [2] in terms of both substrate binding and the catalytic reaction intermediates. These data 

widen the scope of information available on these new heme dioxygenase enzymes and we use it to make 

functional comparisons both with human IDO and more generally across the heme dioxygenase family. 

References: 

[1] Basran, J.; Rafice, S.; Chauhan, N.; Efimov, I.; Cheesman, M. R.; Ghamsari, L.; Raven, E. L. Biochemistry 

2008, 47, 4752-4760. 

[2] Chauhan, N.; Basran, J.; Efimov, I.; Svistunenko, D. A.; Seward, H. E.; Peter C. E. Moody, Raven, E. L. 

Biochemistry 2008, 47, 4761-4769. 

_____________________________________________________________________ 

280


P162. What Controls the Reactivity of High Valent Oxoiron(IV) 

Complexes? 

Steric vs Thermodynamic Considerations 

K. Ray, and L. Que Jr. 

Department of Chemistry and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, 

Minnesota, 55455, USA 

High-valent oxoiron species are often invoked as the oxidants in the catalytic cycles of dioxygen activating 

mononuclear nonheme iron enzymes. To date, such iron(IV) intermediates have been characterized for four 

enzymes, lending strong support for this notion. Within the same time frame, synthetic nonheme complexes 

containing oxoiron(IV) units have also been described that serve as models for such reactive intermediates. 

Herein, we compare the reactivities of a series of oxoiron(IV) complexes involving aminic nitrogen and/or 

pyridine donor ligands and show that the spatial orientation of the donor groups plays a vital role in determining 

the oxidizing capabilities of this important class of biologically relevant oxoiron(IV) complexes. The 

thermodynamic driving force for reaction, the reduction potential, was found to be the controlling factor in 

determining the relative reactivity of the complexes; whereas steric factors had little noticeable impact. 

_____________________________________________________________________ 

281


P163. Towards Photo-catalytic Hydrogen Production with 

Desulfomicrobium baculatum [NiFeSe]-hydrogenase Adsorbed to Titanium 

Dioxide Nanoparticles 

E. Reisner and F.A. Armstrong 

Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, UK 

e-mail: erwin.reisner@chem.ox.ac.uk 

Many consider a hydrogen economy as the most promising solution to replace today’s carbon-based fuel driven 

energy system. However, efficient and inexpensive H2 production, storage, and combination with O2 in 

affordable fuel cells are major obstacles, which need to be solved to sustain such an economy. The solar-driven 

conversion of water into H2 would be an ideal solution for hydrogen fuel production.[1,2] Given the enormity of 

scale up, investigations at the atomic and molecular level seem a far cry from reality: Yet Biology has managed 

to handle the complicated task of water reduction by using enzymes known as hydrogenases to catalyze the 

reversible reduction of protons into H2 at high turnover rates. Direct electron transfer to hydrogenases attached to 

a photo-catalytic center allows for the generation of H2 upon irradiation. [NiFeSe]-Hydrogenases from 

Desulfomicrobium baculatum are both very efficient H2 oxidation and production catalysts under anaerobic 

conditions and show high catalytic activity for H2 evolution in the presence of H2 or traces (at least 1 %) of 

O2.[3] Herein, we report on our preliminary findings of direct electron transfer between the [NiFeSe]hydrogenase 

and titanium dioxide nanoparticles and its consequences for photo-catalytic H2 production. In 

particular, protein film voltammetry of [NiFeSe]-hydrogenase on a photo-catalytic titanium dioxide nanoparticle 

electrode shows direct electron transfer and high stability compared to graphite. 

Acknowledgement: This work is supported by BBSRC (BB/D52222X/1). 

[1] Balzani, V.; Credi, A.; Venturi, M. ChemSusChem 2008, 1, 26–58. 

[2] Esswein, A. J.; Nocera, D. G. Chem. Rev. 2007, 107, 4022–4047. 

[3] Parkin, A.; Goldet, G.; Cavazza, C.; Fontecilla-Camps, J. C.; Armstrong, F. A. submitted. 

_____________________________________________________________________ 

282


P164. New Mo-Fe Cluster in a Protein that Responds to Molybdenum 

Isolated from Desulfovibrio alaskensis 

M. G. Rivas a , M. S. Carepo a , C. S. Mota a , M. Korbas b , M. C. Durand c , A. T. Lopes d , 

C. D. Brondino e , A. Pereira a , G.N. George b , A. Dolla f , J.J.G. Moura a , I. Moura a 

a 

Chemistry, Requimte, FCT-UNL, Quinta da Torre, 2829-516, Monte de Caparica, Portugal, 

b 

Anatomy and Cell Biology, University of Saskatchewan, , S7N5E5, Saskatoon, Canada 

c 

Unité Interactions et Modulateurs de Réponses, BSM – CNRS, 31 chemin Joseph Aiguier, 13402, Marseille, 

France 

d 

Chemisty, Requimte, FCT-UNL, Quinta da Torre, 2829-366, Monte de Caparica, Portugal 

e 

Physics, FBCB-UNL, Barrio El Pozo S/N, 3000, Santa Fe, Argentina, 

f 

Unité Interactions et Modulateurs de Réponses, BSM – CNRS, 31 chemin Joseph Aiguier, 13402, Marseille, 

France 

e-mail: gabriela.rivas@dq.fct.unl.pt 

This work reports the characterization of a novel Mo-Fe protein isolated from Desulfovibrio alaskensis. The 

protein, which is located at the periplasmic face of the cytoplasmic membrane, is a homomultimer of high 

molecular weight (260 ± 13 kDa) consisting of 16-18 monomers of 15321.1 ± 0.5 Da. The UV/Visible 

absorption spectrum of the protein shows absorption peaks around 280, 320, and 570 nm with extinction 

coefficients of 18700, 12800, and 5000 M-1 cm-1, respectively. XAS and biochemical data shows that the Mo- 

Fe protein contains a Mo-2S-[2Fe-2S]-2S-Mo cluster shared per each two subunits. The expression of the protein 

responds to the Mo concentration in the media and was designated as MorP (Molybdenum response associated 

protein). Interestingly, the morP encoding gene is located downstream of a two component system. This kind of 

gene arrangement is used by the cell to regulate diverse physiological processes in response to changes in 

environmental conditions. 

Acknowledgements. We thank Fundação para a Ciência e a Tecnologia (MCTES) for financial support (POCI 

POCI/QUI/55350/2004) 

_____________________________________________________________________ 



P165. Electron Paramagnetic Resonance as a Tool for Investigating how 

Redox Active Enzymes Attach to the Surfaces of Electrodes 

Maxie M. Roessler, a Jeffrey Harmer, a Fraser A. Armstrong a 

a Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford 

OX1 3QR, United Kingdom 

Electron paramagnetic resonance (EPR) is being applied to redox-enzymes bound to conductive surfaces to 

probe the interaction between them. Such surfaces can be thought of as replacing the physiological redox partner 

of the enzyme and are the basis of the technique called protein film voltammetry (PFV) for studying enzymes 

[1]. PFV has been used extensively to gain fundamental information about the active sites of enzymes, to control 

their catalytic action, to measure absolute turn-over frequencies in the absence of substrate-diffusion limitations, 

and to quantify the rate of electron transfer within the redox-active protein [2,3]. 

Pyrolytic graphite ‘edge’ has been shown to be a versatile electrode surface for binding enzymes. After abrasive 

pre-treatment it is very rough and rich in C-O functionalities. When measured with a small molecule (N2), the 

actual surface area of a typical electrode is in fact 10 4 times greater than its visible geometric surface area [4]. 

However, to date little is known about the way an enzyme interacts with such a surface, which is important for 

further development of the technique. For instance, it may be that only a fraction of the total number of adsorbed 

enzymes on the surface are electrochemically active, that is, only a fraction of the enzyme molecules are in the 

‘correct’ orientation for electrons from the electrode to tunnel through the protein to the active site. 

Enzyme-modified micro- and nano-particles of the electrode material, in particular graphite, have been produced 

and explored with EPR. First investigations have been carried out on laccase from Trametes versicolor, which 

possesses four copper centres, two of which are paramagnetic and can be probed directly. A schematic 

illustration is given in the figure below. 

Cartoon of an enzyme attached to an electrode as studied with PFV and its extension to ‘electrode particles’ 

modified with enzyme that is a suitable arrangement for EPR experiments. 

Acknowledgements: The authors acknowledge the EPSRC (EP/D048559/1) for support of this research and St. 

John’s College Oxford for a travel grant. 

References: 

[1] Leger, C.; Elliott, S. J.; Hoke, K. R.; Jeuken, L. J. C.; Jones, A. K.; Armstrong, F. A. Biochemistry 2003, 42, 

8653-8662. 

[2] Vincent, K. A.; Armstrong, F. A. Inorg. Chem. 2005, 44, 798-809. 

[3] Vincent, K. A.; Parkin, A.; Armstrong, F. A. Chem. Rev. (Washington, DC, U. S.) 2007, 107, 4366-4413. 

[4] Blanford, C. F.; Armstrong, F. A. J. Solid State Electrochem. 2006, 10, 826-832. 

_____________________________________________________________________ 

284


P166. Gold(III)-based Anticancer Agents: Peptide Derivatives of Sulfur 

Donor Ligands as Improved Intracellular Drug Transfer and Delivery 

Systems Supported by Transporter Proteins 

L. Ronconi a , M. Negom Kouodom a , V. Milacic b , Q.P. Dou b , F. Formaggio a , D. Fregona a 

a Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131, Padova, Italy 

b Ann Karmanos Cancer Institute, School of Medicine, Wayne State University, 640.1 HWCRC, 4100 John R. 

Road, MI 48201, Detroit, United States 

e-mail: luca.ronconi@unipd.it 

Only a few Au(III) compounds are currently under evaluation for their extremely promising antitumor 

properties. Recently, we have reported on some Au(III)-dithiocarbamato derivatives which have proved to be 

much more cytotoxic in vitro than clinically established platinum-based drugs, and showed encouraging results 

in terms of both high in vivo effectiveness and lack of nephrotoxic side-effects [1,2]. Moreover, for the first 

time, we have identified the ubiquitin-proteasome system as a major in vitro and in vivo target for these 

compounds [3], and we have also extended the evaluation of their interaction with mitochondria [4], thus 

supporting the hypothesis of a different mechanism of action compared to cisplatin. We have now extended our 

research towards new Au(III) derivatives of peptides as improved intracellular drug transfer and delivery 

systems supported by transporter proteins, that mediate the cellular uptake of di/tripeptides. As their substratebinding 

site can accommodate a wide range of different molecules, they represent excellent targets for the 

delivery of pharmacologically active compounds [5]. The rationale behind our research was to design Au(III) 

complexes of the type [AuX2L] (X = Cl, Br; L = di/tripeptide derivatives) which can be able to both maintain the 

antitumor properties and the lack of nephrotoxicity of the previously reported Au(III) analogues, together with 

an enhanced bioavailability through the di/tripeptide-mediated cellular internalization. 

References: 

[1] L. Ronconi, C. Marzano, P. Zanello, M. Corsini, G. Miolo, C. Maccà, A. Trevisan, D. Fregona, J. Med. 

Chem. 2006, 49, 1648. 

[2] V. Milacic, D. Fregona, Q.P. Dou, Histol. Histopathol. 2008, 23, 101. 

[3] V. Milacic, D. Chen, L. Ronconi, K.R. Landis-Piwowar, D. Fregona, Q.P. Dou, Cancer Res. 2006, 66, 10478. 

[4] D. Saggioro, M.P. Rigobello, L. Paloschi, A. Folda, S.A. Moggach, S. Parsons, L. Ronconi, D. Fregona, A. 

Bindoli, Chem. Biol. 2007, 14, 1128. 

[5] I. Rubio-Aliaga, H. Daniel, Trends Pharmacol. Sci. 2002, 23, 434. 

_____________________________________________________________________ 

285


P167. DNA-based Catalysis: Mechanism and Applications 

F. Rosati, A.J. Boersma, J. Klijn, B.L. Feringa and G. Roelfes 

Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The 

Netherlands 

e-mail: F.Rosati@rug.nl, http:// roelfes.fmns.rug.nl 

DNA’s characteristic right-handed helical structure is one of the most elegant examples of chirality in nature. 

Due to its unique chiral architecture, it is an ideal scaffold for asymmetric catalysis. Previously, we have 

demonstrated that DNA can induce enantiomeric excess in the product of a reaction, i.e. a Diels-Alder reaction 

between an aza-chalcone and cyclopentadiene, by non-covalent binding of a catalytically active metal complex 

[1, 2]. 

A key aim is to gain insight into the structure of the active site generated by the DNA-bound complexes. Our 

current research efforts are directed towards the development of a tentative stereochemical model of this active 

site and a mechanistic study was performed with the aid of CD spectroscopy, in combination with other 

spectroscopic techniques (UV, EPR).The effect of DNA on the reaction rate was investigated, as well as the 

effect of different DNA sequences on the enantioselectivity. 

Additionally we are currently exploring other reactions using this novel catalytic concept. For example, 

incorporation of DNA-based catalysts into micellar aggregates has been investigated. 

References: 

[1] G. Roelfes & B.L. Feringa Angew. Chem. Int. Ed. 2005, 44, 3230-3232. 

[2] G. Roelfes, A.J. Boersma, B.L. Feringa Chem. Commun. 2006, 635-637. 

_____________________________________________________________________ 

286


P168. A Versatile Electronic Hole in One-electron Oxidized Ni II bissalicylidene 

Phenylenediamine Complexes 

O. Rotthaus a , O. Jarjayes a , C. Perez Del Valle b , C. Philouze a , F. Thomas a 

a 

Département de Chimie Moléculaire - Chimie Inorganique Redox Biomimétique (CIRE) – UMR-5250, 

Université Joseph Fourier, Grenoble, France 

e-mail: Fabrice.Thomas@ujf-grenoble.fr; Olivier.Jarjayes@ujf-grenoble.fr 

b 

Département de Chimie Moléculaire - Chimie Théorique – UMR-5250, Université Joseph Fourier, Grenoble, 

France. 

Phenoxyl radicals coordinated to divalent or trivalent metal ions are the focus of a considerable interest since the 

discovery of a Cu II -tyrosyl radical entity in the active site of Galactose Oxidase. Several Cu II -coordinated 

phenoxyl radicals have been characterized during the last decade in order to mimic the enzyme active site. 

Recently, nickel complexes of pro-phenoxyl ligands (the phenolate moiety is substituted by electron-donating 

groups) have emerged in literature. Compared to Cu II -phenoxyl complexes, they exhibit ligand and metal redox 

active orbitals closer in energy. Consequently, either Ni II -phenoxyl or Ni III -phenolate redox state are expected to 

be reached upon one-electron oxidation, making these compounds particularly interesting. We present in this 

poster a series of one-electron oxidized nickel salen complexes that exhibit a radical character with partial 

delocalization of the spin density onto the orbital of the nickel ion. We show that the degree of delocalization 

could be modulated by the electron-donating properties of phenolate para substituent R (Fig. 1-3). In addition, 

we found that it greatly influences the affinity of exogenous ligands for the metal, and thus the ability of the 

complexes to exhibit valence tautomerism properties (tetracoordinated Ni II -phenoxyl to octahedral Ni III - 

phenolate transformation in the presence of exogenous ligands). 

N 

Ni 

R O O 

R 

N 

R = t-Bu: 1 

R = OMe: 2 

R = NMe 2: 3 

(R = NHMe 2 + : 3H2 2+ ) 

Figure 1 Formula of the neutral phenolate-nickel 

complexes 

dX'' 

dB 

1 + 

2 + 

3 + 

g = 2.034 

g = 2.017 

g = 2.006 

320 325 330 335 340 345 

B / mT 

Figure 3 X-Band EPR spectra of CH2Cl2 solutions 

(anhydrous + 0.1 M TBAP) of 1 + (1mM), 2 + (0.5 mM) 

and 3 + (1 mM) at 233 K. Microwave Freq : 9.42 GHz, 

power: 20 mW, Mod. Freq: 100 KHz, Amp. 0.4 mT 

(1 + ), 0.05 mT (2 + , 3 + ). 

Figure 2 Optimized structures and calculated SOMO 

for 1 + , 2 + and 3 + ; The Mulliken contribution of the 

nickel orbitals (mainly the dyz) to the total spin density 

is indicated. 

_____________________________________________________________________ 

287


P169. Quinolinate Synthase, an Iron-sulfur Enzyme as a Key Target for 

the Design of Antibacterial Agents 

C. Rousset a , S. Ollagnier de Choudens a , M. Thérisod b , O. Hamelin a , M. Fontecave a 

a LCBM, iRTSV, CEA Grenoble, 17 Avenue des Martyrs, 38054, Grenoble, France 

b UMR 8182, ICMMO, UMR 8182, Université Paris-Sud XI, , 15, Rue Georges Clémenceau, 91405, Orsay 

Cedex, France 

e-mail: carine.rousset@cea.fr 

Nicotinamide adenine dinucleotide (NAD) plays a crucial role as a cofactor in numerous essential redox 

biological reactions. In fact, in all living organisms, NAD derives from quinolinic acid (QA), the biosynthetic 

pathway of which differs among organisms. In most eukaryotes, QA is produced via the degradation of 

tryptophan, whereas in E.coli and most prokaryotes organisms it is synthetized from L-Aspartate and 

dihydroxyacetone phosphate (DHAP) as the result of the concerted action of two enzymes, L-Aspartate oxidase, 

a flavin adenine dinucleotide (FAD)-dependent flavoenzyme encoded by nadB gene, and the quinolinate 

synthase, encoded by the nadA gene. 

Besides the de novo synthesis of NAD, a salvage pathway exists that enables NAD to be recycled. The presence 

of distinctly different pathway in most prokaryotes and eukaryotes for the biosynthesis of QA and the absence of 

the salvage pathway for some (M.tuberculosis and H.pylori) suggests that NadA might prove to be a key target 

for the design of antibacterial agents. 

Despite the importance of the process, there has been little characterization of NadA. Recently, NadA from E. 

coli was characterized as an Fe-S enzyme with a [4Fe-4S] cluster essential for its activity [1, 2]. The [4Fe-4S] 

cluster proved to be very sensitive to oxygen, in agreement with previous in vivo observations that de novo NAD 

biosynthesis is a pathway sensitive to hyperbaric oxygen and that NadA is specifically the oxygen-sensitive site 

[3]. Design of molecules required first a complete biochemical, spectroscopic and enzymatic characterization of 

the NadA enzyme. Using in vivo and in vitro approaches with cysteine to alanine mutants we recently identified 

the ligands of the cluster. 

As potential inhibitors, we tested different molecules like substrate analogues or analogues of the reaction 

intermediates. Three of them proved to be active in vitro toward the enzyme and we are actually investigating 

their inhibition mechanism. It is worth noting that such molecules might be interested at a fundamental level to 

elucidate molecular mechanism of the reaction catalyse by NadA. 

References: 

[1]. Ollagnier-de Choudens S., FEBS, 2005. 

[2]. Cicchillo R., JACS, 2005. 

[3]. Gardner P.R., Arch. Biochem. Biophys. 1991. 

_____________________________________________________________________ 

288


P170. Copper (II)-aminohydroxamate Ternary Complexes Evidenced by 

Mass Spectrometry 

M. Rowińska-Żyrek a E. Gumienna-Kontecka a , Z. Szewczuk a , I.O. Fritsky b , H. Kozłowski a 

a 


e-mail: magdalena.zyrek@gmail.com 

b 

Department of Chemistry, National Taras Shevchenko University, 01033 Kiev, Ukraine 

The formation of the pentameric 12-MC-4 Cu(II) complexes of α- and β-aminohydroxamic acids is well 

described in literature with a four species model: [CuL] + , [Cu5L4H-4] 2+ , [CuL2] and [CuL2H-1] - . [1-3] 

Since only binary, and not ternary complexes have been studied before, there is not much evidence on 

hydroxamic acid mixed pentanuclear metallaorganic complexes. Therefore, in order to learn more about the 

process of metallacrown formation we have investigated the Cu(II) : glycinehydroxamate : 

phenylalaninehydroxamate system by means of electrospray mass spectrometry, potentiometry and UV-Vis 

spectroscopy, looking for the formation of ternary species. 

Mass spectrometry proves the existence of a ternary complex consisting of glycine and phenylalanine 

hydroxamic acids [Cu5GlyhaPheha3H-4] 2+ . MS/MS results show the extreme stability of the metallacrown. 

Mass spectrometric data together with potentiometric results are complementary to each other and give an idea 

about the true nuclearity of the complexes and about the dominant forms in the solution. In the calculations the 

ternary [Cu5Glyha2Pheha2H-4] 2+ species have first been taken into account, however it’s presence did not lead to 

a satisfactory fitting of the titration curves. The fitting was much improved when the [Cu5GlyhaPheha3H-4] 2+ 

complex was introduced into the model and a stability constant of logK= 44.89(7) was calculated. The 

[Cu5GlyhaPheha3H-4] 2+ complex seems to dominate over others at pH 5. 

Fig. 1. The ternary [Cu5GlyhaPheha3H-4] 2 + complex. 

Because of the incredibly broad field of applications for both metal chelators and metallacrowns, other transition 

metals have been reinvestigated to prove their potential ability to form 12-MC-4 complexes with hydroxamic 

acids. The experimental findings are in perfect agreement with the literature data. [4, 5] Copper seems to be the 

only known metal capable of forming stable 12-metallacrown-4 complexes. 

References: 

[1] F. Dallavalle, M. Tegoni, Polyhedron 20, 2697 (2001). 

[2] M. Careri, F. Dallavalle, M. Tegoni, I. Zagnoni, J. of Inorg. Biochem., 93, 174 (2003). 

[3] M. Tegoni M, M. Remelli, D. Bacco, L. Marchio, F. Dallavalle, Dalton Trans., 2693 (2008). 

[4] A. Dobosz, N. M. Dudarenko, I. Fritsky, T. Głowniak, A. Karaczan, H. Kozłowski, T. Yu. Silva, J. Swiątek- 

Kozłowska, J. Chem. Soc. Dalton Trans., 743 (1999). 

[5] E. Farkas, P. Buglyó, J. Chem. Soc. Dalton Trans., 1549 (1990). 

_____________________________________________________________________ 

289


P171. NMR and PAC Properties of Heavy Metal Ions in Proteins: A 

Theoretical Study 

K. Rud-Petersen a , K.V. Mikkelsen b , S.P.A. Sauer b , L. Hemmingsen a 

a 

Natural Sciences,University of Copenhagen,Bülowsvej 17, 1870, Frederiksberg C,Denmark 

e-mail: kristian@rud-petersen.dk 

b 

Chemistry,University of Copenhagen, Denmark 

In order to interpret the NMR and perturbed angular correlation (PAC) spectra, we apply ab initio and DFT 

calculations to acquire the parameters from model complexes containing the heavy metal ions Cd(II), Hg(II) and 

Pb(II). NMR parameters being 2. order properties are known to be very sensitive to conformational changes and 

since the atoms are heavy, it is essential to include relativistic effects. The systems to be investigated are both 

naturally occurring proteins, de novo designed helix bundles (collaboration with professor V.L. Pecoraro, 

University of Michigan), and coordination compounds. PAC and NMR spectroscopy provide complementary 

information about both structure and dynamics at the metal ion binding sites, but interpretation of NMR (207Pb, 

199Hg and 113Cd) and PAC (204mPb, 199mHg, and 111mCd) spectroscopic data in most cases depend on 

empirical methods. Thus, we carry out calculations of electric field gradients (which via the nuclear quadrupole 

interaction is the property measured in PAC spectroscopy), chemical shifts and spin-spin coupling constants for 

Pb(II), Hg(II) and Cd(II) a series of model systems. 

The groups involved have previously, with success, applied both ab initio and DFT methods for calculations of 

electric field gradients and chemical shifts in Cd(II) containing compounds [1,2], and recently NMR properties 

of mercury containing systems have been investigated in detail [3], providing an excellent basis for the current 

investigation. 

References: 

1. Hemmingsen L., Ryde U. Ab initio calculations of electric field gradients in cadmium complexes J. Phys. 

Chem. 1996, 100, 4803-4809. 

2. Hemmingsen L., Olsen L., Antony J., Sauer S.P.A. First principle calculations of Cd-113 chemical shifts in 

proteins and model systems J. Biol. Inorg. Chem. 2004, 9, 591-599. 

3. Autschbach J., Sterzel M. Molecular Dynamics Computational Study of the 199Hg-199Hg NMR Spin-Spin 

Coupling Constants of [Hg-Hg-Hg]2+ in SO2 Solution J.Am.Chem.Soc. 2007, 129, 11093-11099. 

4. Figure: HgHAH1 metal binding site, made by Monika Stachura 

_____________________________________________________________________ 

290


P172. New Photoactivable Ruthenium Complexes 

L. Salassa a , A.M. Pizarro a , P.J. Sadler a , C. Garino b , R. Gobetto b 

a Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom 

b Dipartimento di Chimica IFM, Università di Torino, via P. Giuria 7, 10125, Torino, Italy 

Metal complexes able to dissociate coordinated ligands when irradiated by light of appropriate wavelength have 

stimulated much recent research. Such attention is motivated by the possibility of phototriggering specific and 

desired interactions between the metal complexes and biological targets, like DNA,[1] but also by the 

opportunity to deliver biologically-active small molecules.[2] 

We have studied photoactivable ruthenium complexes of formula [Ru(N�N)(4AP)4] 2+ , where N�N = bpy (1), phen 

(2), 4-methyl-phen (3) and dppz (4) and 4AP = 4-aminopyridine. Complexes 1�4 are stable in the dark, but 

undergo double ligand substitution when irradiated with visible light. The case of complex 1 is discussed in 

detail. 1 shows two photoreactions leading to the formation of the photoproducts mer-[Ru(bpy)(4AP)3(H2O)] 2+ 

and trans-(4AP)-[Ru(bpy)(4AP)2(H2O)2] 2+ . The photodissociation yields are ϕ1 = (6.1±1.0) 10 �3 and 

ϕ2 = (1.7±0.1) 10 �4 , respectively. Insights on the ligand photodissociation mechanism were obtained by 

combining a mixed experimental and theoretical approach. DFT and TDDFT were used to characterize the 

photophysical properties of the complex as well as to determine the relevant singlet and triplet excited states for 

the dissociation mechanism.[3] Finally, in vitro cytotoxicity to cancer cells of complexes 1�4 will be discussed 

with the aim of designing new photoactivable anticancer agents. 

References: 

[1] F. S. Mackay, J. A. Woods, P. Heringová, J. Kašpárková, A. M. Pizarro, S. A. Moggach, S. Parsons, V. 

Brabec, P. J. Sadler, PNAS, 104, 20743 (2007). 

[2] L. Zayat, C. Calero, P. Albores, L. Baraldo, R. Etchenique, J. Am. Chem. Soc., 125, 882 (2003). 

[3] L. Salassa, C. Garino, G. Salassa, R. Gobetto, C. Nervi, J. Am. Chem. Soc., In Press (2008). 

_____________________________________________________________________ 

291


P173. Synthetic Approaches in the Aqueous Structural Speciation of Binary 

Cr(III)-hydroxycarboxylate Systems. Relevance to Chromium Toxicity 

A. Salifoglou, C. Gabriel 

Department of Chemical Engineering, Aristotle University of Thessaloniki, Corner of Egnatia and 3rd 

September St, 54124, Thessaloniki, Greece 

e-mail: salif@auth.gr 

Chromium has since long been considered as a metal with widespread applications in industry [1]. Its biological 

role [2], albeit conjectured to a great extent, has been the subject of considerable research efforts due to its 

proclivity to induce toxic effects in lower and higher organisms [3, 4, 5]. Poised to comprehend chromium 

toxicity at the molecular level, synthetic efforts were launched targeting the aqueous binary interactions of 

Cr(III) with citric acid. The latter substrate is abundantly present in biological fluids and exhibits a diverse 

chemical reactivity toward metal ions. To that end, synthetic efforts in the specific binary system led, in a pHdependent 

fashion, to the isolation of discrete Cr-citrate species, such as (NH4)4[Cr(C6H4O7)(C6H5O7)]•3H2O (1), 

in the solid state [6]. The structure of such species reveals a mononuclear octahedral Cr(III) complex with two 

citrate ligands bound to it. Detailed aqueous speciation studies in the Cr(III)-citrate system suggest the presence 

of a number of species, among which is the mononuclear [Cr(C6H4O7)(C6H5O7)] 4- complex at pH ~5.5. The 

collective physicochemical data of these species in the solid state and in solution a) emphasize the importance of 

concerted synthetic and aqueous speciation studies in the binary Cr(III)-citrate system, and b) denote the 

ramifications of well-defined soluble Cr(III)-species in comprehending the arisen chemical reactivity that could 

be linked to toxicity at the cellular level. 

References: 

[1] Bae, W.-C.; Lee, H.-K.; Choe, Y.-C.; Jahng, D.-J.; Lee, S.-H.; Kim, S.-J.; Lee, J.-H.; Jeong, B.-C. J. 

Microbiology 2005, 43, 21-27. 

[2] Ramos, R. L.; Martinez, A. J.; Coronado, R. M. G. Water Sci. Technol. 1994, 30, 191. 

[3] Pettrilli, F. L.; Miller, W. Appl. Environ. Microbiol. 1977, 33, 805-809. 

[4] Levis, A. G.; Bianchi, V. Biological and environmental aspects of chromium, S. Langard, Ed.; Elsevier 

Science, Amsterdam, 1982, p. 171-208. 

[5] Walsh, A. R.; O’Halloran, J.; Gower, A. M. Ecotoxicol. Environ. Safety 1994, 27, 168. 

[6] Gabriel, C.; Raptopoulou, C. P.; Terzis, A.; Tangoulis, V.; Mateescu, C.; Salifoglou, A. Inorg. Chem. 2007, 

46, 2998-3009 

_____________________________________________________________________ 

292


P174. Non-Covalent Metallo-Drugs Coupled to Sex Hormone Steroids 

C. Sanchez Cano a , A. Gómez Quiroga b , C. Navarro Ranninguer b , M. Hannon a 

a School of Chemistry, University of Birmingham, , B15 2TT, Birmingham, United Kingdom 

b Departamento de Química Inorganica, Universidad Autonoma de Madrid, , 28049, Madrid, Spain 

e-mail: cxs527@bham.ac.uk 

Different biomolecules have been used in the last years as delivery vectors with varying degrees of succes. Sex 

hormones such as estrogens and testoteron are of interest as vectors because of their importance in the 

development and treatment of reproductive system cancers (a high number show over-expression of steroid 

receptors). Conjugates of metallo-drug units and bioactive steroids have therefore been created with the aim of 

localizing cytotoxic drugs[1, 2]. Almost all of these previous bioconjugates have focused on DNA alkylating 

agents that form a direct covalent or coordinate bond to the bases like cisplatin. 

Non-covalent DNA-binding metallo-drugs have been extensively studied, and showing a broad spectrum of 

direct activities[3, 4, 5] they represent an interesting alternative to covalent DNA-binders, because their different 

mode of action can circunvent some problems, such as cross-resistance or side effects. 

For this reason we have explored conjugating non-covalent metallo-drug units to steroids. Our approach is to 

attach an intercalating Pt(II) terpyridine moeity to estradiol and testosterone units. Synthetic techniques and 

approaches that allow very easy accests to such conjugates will be described. We show that these 

metallointercalator-steroids conjugates are potent cytotoxic agents. They also possess interesting fluorescence 

properties, demostrating fluorescent enhancement on interaction with DNA. 

References: 

[1] A. Jackson, J. Davis, R.J. Pither, A. Rodger and M.J. Hannon, Inorg. Chem., 2001, 40, 3964-73. 

[2] M.J. Hannon, P.S. Green, D.M. Fisher, P.J. Derrick, J.L. Beck, S.J. Watt, M.M. Sheil, P.R. Barker, N.W. 

Alcock, R.J. Price, K.J. Sanders, R. Pither, J. Davis and A. Rodger, Chem. - Eur. J., 2006, 12, 8000-8013. 


Intl. Ed., 2006, 45, 1227-1231. 

[4] G. I. Pascu, A. C. G. Hotze, C. Sanchez Cano, B. M. Kariuki, M. J. Hannon, Angew. Chem., Intl. Ed., 2007, 

46, 4374-4378. 

[5] D. Ma, T. Y. Shum, F. Zhan, C. Che, M. Yang, Chem. Commun., 2005, 4675-4677. 

_____________________________________________________________________ 

293


P175. A Novel Paddlewheel Dicopper(II) Molecule 

with Four µ2-N3,N7- [N1,N6-dideaza-adeninate(1-)] Bridges 

C. Sánchez de Medina-Revilla a , D. Choquesillo-Lazarte b , A. Domínguez-Martín a , 

J.M. González-Pérez a , A. Castiñeiras c , J. Niclós-Gutiérrez a 

a 



e-mail: celiasm@ugr.es 

b 



c 



The adeninate(1-) ion has probed to act as and µ-N3,N7,N9-bridging ligand, in an hexanuclear molecule [1] 

and in a polymer [2]. Such a role would be displayed by 4-azabencimidazolate(1-) [also N1,N6-dideazaadeninate(1-), 

4abim - ], but without a N6-H···A interaction reinforcing a metal-N7 bond. Reedijk et al. [3] have 

reported the structure of [Cu2(µ2-N3,N7-4abim)4X2]X2·solvated salts (X = Cl or Br) and studied their strongly 

anti-ferromagnetic coupling behaviour. Now we inform of the closely related paddlewheel centro-symmetric 

molecule [Cu2(µ2-N3,N7-4abim)4(SO4)2]·12H2O (293 K, monoclinic, space group C2/c, final R 0.056,see 

figure). Relevant inter-atomic distances (Å): Cu···Cu 2.960(1), Cu-N3 2.019(3), Cu-N9 2.016(3), Cu-O(sulphate) 

2.113(14). In the crystal, pairs of H-bonding interactions N7-H···O(sulphate) give chains. 

References: 

[1] J.M. González-Pérez, C. Alarcón-Payer, A. Castiñeiras, T. Pivetta, L. Lezama, D. Choquesillo-Lazarte, 

G. Crisponi, J. Niclós-Gutiérrez, Inorg. Chem., 45, 877 (2006). 

[2] J.P. García Terán, O. Castillo, A. Luque, U. García Cruceiro, P. Román, Inorg. Chem., 43, 4549 (2004). 

[3] G.A. van Albada, I. Mutikainen, U. Turpeinen, J. Reedijk, Polyhedron, 25, 3278 (2006). 

_____________________________________________________________________ 

294


P176. Structural Studies on Desulfoviridin from Desulfovibrio desulfuricans 

ATCC 27774 

A.S. Serra a , M. Carepo a , S.L.A. Andrade b , I. Moura a , J.J.G. Moura a and M.G. Almeida 

a REQUIMTE / CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de 

Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal 

b Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität 

Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany 

c Escola Superior de Saúde Egas Moniz, Quinta da Granja, Monte de Caparica, 2829-511 Caparica, Portugal 

Desulfoviridin (Dsv) is a dissimilatory sulfite reductase capable of reducing sulfite to sulfide during anaerobic 

respiration. A detailed knowledge of the enzyme’s multimeric (α2β2γ2) structure from Desulfovibrio genus is 

not available, and it is our goal to carry out such characterization using Dsv from Desulfovibrio desulfuricans 

ATCC 27774 cells. The molecular mass of the native protein as obtained from gel filtration chromatography, 

compared with the molecular mass and intensity of the corresponding bands identified by SDS-PAGE (9, 35, 

49 kDa), indicates that Dsv is purified as a α2β2γ2 complex. The total and free iron content is in agreement with 

the existence of two sirohaems and four [Fe4S4] clusters. For obtaining the complete sequence of Dsv subunits 

(α, β and γ), primers were designed for DNA amplification by PCR, based on multiple sequence alignments of 

dsr genes from several Desulfovibrio species and N-terminal chemical sequencing of the subunits. The 

sequences obtained were compared with deposited homologous sequences as well as with D. desulfuricans 

internal peptide sequences obtained after enzymatic digestion. In order to use X-ray diffraction techniques to 

solve the crystallographic structure of D. desulfuricans Desulfoviridin, preliminary screenings were made 

yielding small protein crystals. The refinement of the crystallization conditions is currently under optimization. 

References: 

[1] Steuber, J. et al; Eur.J.Biochem.; 1995; 233; 873-879 

[2] Marritt, S. J.; Hagen, W. R.; Eur.J.Biochem.; 1996; 238; 724-727 

[3] Mander, G. J. et al; FEBS Let.; 2005, 579, 4600-4604 

[4] Schiffer, A. et al; Journal of Molecular Biology; 2008; in press 

_____________________________________________________________________ 

295 

a, c


P177. 1-Deazapurine as a Potential Artificial Nucleobase for Metal- 

Mediated Base Pairing 

K. Seubert , J. Müller 

Inorganic Chemistry, TU Dortmund, Otto-Hahn-Str. 6, 44227, Dortmund, Germany 

e-mail: kristof.seubert@tu-dortmund.de 

The growing area of research of developing and analysing oligonucleotides containing artificial nucleobases 

provides a way towards the generation of functionalized nanoarchitectures. Metal-mediated base pairs are of 

special interest since one expects interesting magnetic or electric properties.[1, 2] 

We report the synthesis of the artificial 1-deazapurine nucleoside as well as the characterisation of its metal-ion 

binding properties in oligonucleotides. The addition of silver(I) ions results in a concentration-dependend 

melting behaviour with various oligonucleotides (d(X14A), d(X16A), d(X19A) (X = 1-deazapurine)). This can 

be explained by the formation of a double helix with up to 19 consecutive metal-mediated artificial base pairs 

(Scheme). 

References: 

[1] Müller, J., Eur. J. Inorg. Chem. 2008, in press (doi: 10.1002/ejic.200800301). 

[2] Carell, T., Clever, G.H.; Kaul, C., Angew. Chem. Int. Ed., 2007, 6226-6236. 

_____________________________________________________________________ 

296


P178. Reactivity for Isocyanide and Nitrile by Using Cobalt(III) Complexes 

Directed to an Active Site Model of Nitrile Hydratase 

T. Shibayama, T. Yano, Y. Funahashi, T. Ozawa, H. Masuda 

Department of Materials Science and Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, 

Nagoya 466-8555, Japan, 

e-mail: 19415083@stn.nitech.ac.jp 

Nitrile Hydratase (NHase) is an enzyme which hydrates a nitrile compound to the corresponding amide. A noncorrin 

Co(III) or a non-heme Fe(III) ion is included in its active site. Crystal structural analyses of their proteins 

demonstrated that both metal centers were coordinated by two amide nitrogens from the peptide backbone and 

two oxidized cystein sulfurs (Cys-SO and Cys-SO2) in the equatorial plane and a cystein sulfur and H2O (Cotype) 

[1] or NO (Fe-type) [2] in the axial positions. (Fig. 1) Recently, Odaka and co-workers reported that Fetype 

NHase also hydrolyzed t-butyl isocyanide (tBuNC) to t-butyl amine (tBuNH2) [3]. 

In order to understand the reaction mechanism, we have prepared and characterized six Co(III) complexes with 

N2S2- [4] or N2S3-type ligands [5]; [Co III (L1)(tBuNC)2] - (1) (L1: N, N'-bis((2-mercapto-2-methyl)propioyl)-1, 3propanediamine) 

with two thiolate sulfurs, [Co III (L1(SO)2)(tBuNC)2] - (2) with two sulfenate sulfurs and 

[Co III (L1(SO2)2)(tBuNC)2]- (3) with two sulfinate sulfurs, [Co III (L2)] - (4) (L2: 3, 3'-bis(2-mercapto-2methylpropionylamino)-dipropylsulfide) 

with two thiolate sulfurs, [Co III (L2(SO)(SO2))] - (5) with a sulfenate 

sulfur and a sulfinate sulfur, and [Co III (L2(SO2)2)] - (6) with two sufinate sulfurs. In this study, we tried the 

hydrolysis reaction of tBuNC and acetonitrile using their complexes under of a large excess of the substrate 

concentrate. 

References: 

[1] A. Miyanaga, S. Fushinobu, K. Ito, T. Wakagi, Biochem. Biophys. Res. Commun., 288, 1169 (2001). 

[2] S. Nagashima, M. Nakasako, N. Dohmae, M. Tsujimura, K. Takio, M. Odaka, M. Yohda, N. Kamiya, I. 

Endo, Nat. Struct. Biol., 5, 347 (1998). 

[3] K. Hashimoto, M. Yohda, M. Odaka, 13th International Conference on Biological Inorganic Chemistry, 

Vienna, Austria, Abstr., P133 (July, 2007). 

[4] T. Yano, Y. Wasada-Tsutsui, H. Arii, S. Yamaguchi, Y. Funahashi, T. Ozawa, H. Masuda, Inorg. Chem., 46, 

10345 (2007). 

[5] T. Yano, T. Ikeda, Y. Funanhashi, T. Ozawa, H. Masuda, Adv. Mater. Res., 11-12, 347 (2006). 

_____________________________________________________________________ 

297


Y. Shimazaki 

P179. Oxidation Behavior of Metal-Phenolate Complexes and 

Characterization of Their Phenoxyl Radical Species 

College of Science, Ibaraki University, Bunkyo, 310-8512, Mito, Japan, 

e-mail: yshima@mx.ibaraki.ac.jp 

The Cu(II)-phenoxyl radical formed during the catalytic cycle of galactose oxidase (GO) attracted much 

attention, and the structures and properties of a number of metal-phenoxyl radical complexes have been studied. 

As a functional model system of GO, Stack et al. reported that the Cu complex of a distorted salen 

(di(salicylidene)ethylenediamine) derivatives showed the catalytic oxidation of primary alcohols to aldehydes, 

and formation of the Cu(II)-phenoxyl radical species was revealed in the catalytic cycle. 

As an extension of the studies on Metal-phenoxyl radical species, we synthesized Co(II), Co(III), Ni(II), Cu(II), 

and Zn(II) complexes of the N3O tripodal ligands with a 2, 4-di(tert-butylphenolate moiety and characterized the 

one- and two-electron oxidized complexes. The structures of these phenolate complexes depended on the central 

metal ion. Co(II) and Zn(II) complexes were very similar structures while Cu(II) and Ni(II) complexes were 

slightly different . Upon one-electron oxidation the Co(II) complexes were converted to the Co(III)-phenolate 

species, while the one-electron oxidized species of the Ni(II) complexes were the Ni(II)-phenoxyl radical in the 

ground state. The stability of he Ni(II)-phenoxyl radical depends on the N-donor properties; the half-life 

increased with the increase of the N-donor ability of the ligands. This characteristic was also observed in the case 

of the Zn(II)-phenoxyl radical complexes. Further, the results of the oxidized Co(III) complexes revealed that the 

oxidation center is dependent on the properties of the pyridine nitrogen donors. These results illustrate the 

control of the oxidation locus that can be reached by modulating the ligand field. 

_____________________________________________________________________ 

298


P180. Antioxidant Reaction of Mononuclear Mn(III) Complex, 

Mn(III)BEBP (BEBP = Benzenamine, 2, 2'-(1, 2-ethanediyl) bis[N-(2pyridiymethylene 

)]) 

M. J. Shin , S. Sarkar , H. I. Lee 

Department of Chemistry, Kyungpook National University, 1370 SangKyeok-Dong, 702-701, Deagu, Republic of 

Korea 

e-mail: kaps25@hanmail.net 

Recently, mononclear manganese complexes with Schiff-base ligands have been reported to have peroxidase and 

catalase activities.[1, 2, 3, 4] They suggested that highly symmetric environments of the metal center could 

dimerize by itself or via exogenous axial ligand during the catalytic process, fullfiling the requirements of the 

peroxidase activity. The authors proposed two possible reaction pathways; one to form high valent Mn(IV)=O 

intermediate and the other to form dimer bridged by hydrogen peroxide for the catalytic process. In an attempt to 

explore this issue, we synthesized manganese complex with a new Schiff-base ligand, BEBP (Benzenamine, 

2, 2'-(1, 2-ethanediyl) bis [N-(2-pyridine methylene)] ) which was produced by condensation reaction of 

2, 2'-ethylendianiline and 2-pyridinecarboxaldehyde. We tested its catalytic activity using H2O2 as an oxidant 

and the reaction was monitored by UV-vis, EPR and FAB-Mass. 

References: 

[1] M. Maneiro, M. R. Bermejo, M. I. Fernandez, E. G. Forneas, A. M. G. Noya, A. M. Tyryshkin, New J. 

Chem., 2003, 27, 727-733 

[2] M. R. Bermejo, M. I. Fernandez, A. M. G. Noya, M. Maneiro, R. Pedrido, M. J.Rodrıgue, J.C. G. 

Monteagudo, B. Donnadieu, Journal of Inorganic Biochemistry 100 (2006) 1470-1478 

[3] M. Maneiro, M. R. Bermejo, A. Sousa, M. Fondo, A. M. Gonzalez, A. S. Pedrares, C. A. McAuliffe, 

Polyhedron 19 (2000) 47-54 

[4] L. R. Guilherme, S. M. Drechsel, F. Tavares, C. J. da Cunha, S. T. Castaman, S. Nakagaki, I. Vencato, A. J. 

Bortoluzzi, Journal of Molecular Catalysis A: Chemical 269 (2007) 22-29 

_____________________________________________________________________ 

299


H. Sigel 

P181. Metal Ion-Binding Properties of Two Isomeric 

Uracilmethylphosphonates 

Department of Chemistry, Inorganic Chemistry, University of Basel, Spitalstrasse 51, CH-4056, BASEL, 

Switzerland 

e-mail: helmut.sigel@unibas.ch 

The uracilmethylphosphonates, 5Umpa 2– = uracil(5)-CH2-PO3 2– and 6Umpa 2– = uracil(6)-CH2-PO3 2– , show 

interesting pharmacological properties [1] and they fit thus into a series of artificial acyclic nucleotide analogues, 

a wellknown member in use in hepatitis B therapy is Adefovir = PMEA 2– = adenine(9)-CH2CH2-O-CH2-PO3 2– 

[2]. Since the action of such phosphonates involves metal ions [2], we studied the M 2+ -binding properties of 

5/6Umpa 2– in aq. solution [3]. In both cases mainly the phosphonate group determines the stability of the 

M(Umpa) complexes (M 2+ = Mg 2+ , Ca 2+ , Cu 2+ , Zn 2+ , Cd 2+ ). However, for M(5Umpa) with Cu 2+ , Zn 2+ or Cd 2+ an 

increased stability is observed, which, based on steric considerations, must be attributed to a 7-membered chelate 

formed by the phosphonate-coordinated M 2+ with the oxygen of (C4)O. The formation degree of the M(5Umpa) 

chelates varies with the metal ion involved; e.g., for Mg 2+ , Cu 2+ and Zn 2+ it is ca. 0, 46 and 26%, respectively. Of 

course, the (C4)O interaction facilitates deprotonation of (N3)H giving thus also rise to a larger formation degree 

of the chelates (up to 90%) in the M(5Umpa – H) – species. For uracil the (N3) – /(N1) – ratio is about 80/20 [4], 

showing that (N1)H is only a bit less acidic than (N3)H. Indeed, the M(6Umpa) complexes undergo (N1)H 

deprotonation at physiological pH forming 6-membered M(6Umpa – H) – chelates with high formation 

degrees [3]. 

Acknowledgements:Supported by the Department of Chemistry, University of Basel, Switzerland. 

References: 

[1] J. Ochocki, J. Graczyk, Pharmazie 53 (1998) 884–885. 

[2] H. Sigel, Chem. Soc. Rev. 33 (2004) 191–200. 

[3] E. Freisinger, R. Griesser, B. Lippert, C.F. Moreno-Luque, J. Niclós-Gutiérrez, J. Ochocki, B.P. Operschall, 

H. Sigel, submitted. 

[4] C.F. Moreno-Luque, E. Freisinger, B. Costisella, R. Griesser, J. Ochocki, B. Lippert, H. Sigel, J. Chem. Soc., 

Perkin Trans 2 (2001) 2005–2011. 

_____________________________________________________________________ 

300


P182. Kinetic Studies on the Multihemic Nitrite Reductase from 

Desulfovibrio desulfuricans ATCC 27774 

C.M. Silveira a , S. Besson a, b , J.J.G. Moura a , M. Gabriela Almeida 

a REQUIMTE - Departamento de Química, CQFB, Faculdade de Ciências e Tecnologia, 

Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 

b UIBD, Faculdade de Engenharia e Ciências Naturais, Universidade Lusófona de Humanidades e Tecnologias, 

Campo Grande 376, 1749-024 Lisboa, Portugal 

c Escola Superior de Saúde Egas Moniz, Monte de Caparica, 2829-511 Caparica, Portugal 

e-mail: celia.silveira@dq.fct.unl.pt 

The multiheme nitrite reductase (ccNiR) from the δ-proteobacterium Desulfovibrio desulfuricans ATCC 27774 

is able to reduce nitrite to ammonia in a six-electron transfer reaction. Though, low activities have also been 

reported for the reduction of hydroxylamine, nitric oxide and sulphite [1]. ccNiR is a membrane associated 

complex composed of two subunits NrfA and NrfH, apparently following a α4β2 architecture [2]. Although 

extensively characterized from the spectroscopic, biochemical and structural points of view, most of ccNiR’s 

kinetic characteristics are still unknown. Due to its high specific activity towards nitrite, ccNiR has been targeted 

for biosensor applications using different electronic mediators for signal transduction. As a result, a set of kinetic 

data concerning the immobilized protein was obtained [3-5]. 

In this work the homogeneous kinetic behaviour of ccNiR is being evaluated in the presence of several redox 

mediators (methyl viologen, diquat, phenosafranine, anthraquinone-sulfonate). Enzyme activities were measured 

by a continuous method following the mediator reoxidation, and by a discrete manner, titrating nitrite and 

ammonia at regular time intervals. Regardless the redox potential of the electron donor, ammonium was always 

the main product. Among the tested mediators, methyl viologen showed the highest turnover number. The kcat 

and KM values for both nitrite and mediator were influenced by the incubation temperature. 

Acknowledgements: The authors thank the financial support funded by REQUIMTE and Fundação para 

a Ciência e Tecnologia (POCI/QUI/58026/2004 and SFRH/BD/28921/2006). 

References: 

[1] Stach P. et al, J. Inorg. Biochem., 2000, 79(1-4), 381-385. 

[2] Rodrigues M.L. et al, EMBO J., 2006, 25(24), 5951–5960. 

[3] Strehlitz B. et al, Anal. Chem., 1996, 68, 807-816. 

[4] Almeida M.G. et al, Biosens. Bioelectron., 2007, 22(11), 2485-2492. 

[5] Chen H. et al, Electrochem. Comm., 2007, 9, 2241–2246. 

_____________________________________________________________________ 

301 

a, c


P183. New Rhodium Water-soluble Complexes with 

1,3,5-triaza-7-phosphaadamantane 

P. Smoleński a, b , M. Fátima C. Guedes da Silva a, c , F. P. Pruchnik b , A. J. L. Pombeiro a 

a Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, TU Lisbon, 

Av. Rovisco Pais, 1049–001 Lisbon, Portugal 

b Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland 

c Universidade Lusófona de Humanidades e Tecnologias, ULHT Lisbon, Av. do Campo Grande, 376, 1749-024, 

Lisbon, Portugal 

Water-soluble phosphines are playing an important role in studies on chemical diagnostic and therapy. Indeed, 

following their adaptable capacity to coordinate metals ions and their radioisotopes, they allow the preparation of 

new water-soluble radiopharmaceutical compounds. The strong metal-phosphorus bond promotes the stability of 

the complexes even under less favourable conditions as those encountered in vivo. In order to promote the 

stability of Rh I systems in aqueous phase, we selected the chemically stable and water-soluble 1, 3, 5-triaza-7phosphaadamantane 

(PTA) ligand. Its coordination chemistry has experienced in the last years a rapid 

development mainly justified by the search for water soluble transition metal phosphine complexes which could 

find several applications e.g. as catalysts in aqueous phase or water-soluble antitumor agents. 

In this contribution we report the syntheses, structural and spectroscopic characterization of some new rhodium 

complexes: [RhCl2(PTA-H)(PTA)] (1), [RhCl2(PTA)4]Cl (2), [Rh(CO)(PTA)4]Cl (3) and [RhH(PTA)4] (4). All 

of them were characterized by IR, 1 H, 13 C and 31 P NMR spectroscopies, elemental analyses and (for 1 - 3) also 

by X-ray structural analysis. 

P 

PTA = PTA-H = 

N N 

N H 

N 

N 

+ 

N 

Acknowledgements: This work has been partially supported by the Foundation for Science and Technology 

(FCT), grants and BPD/20869/04, and its POCI 2010 programme (FEDER funded). 

_____________________________________________________________________ 

302 

P


P184. The Bioinspired Direct Synthesis of New Copper(II) Complex with 

alfa – alaninehydroxamic Acid. Magnetic and Spectroscopic Properties 

J. Sowińska, W. Wojciechowski, U. Oleksińska 

Department of Chemistry, Wroclaw University of Technology, Wybrzeże Wyspinskiego 27, 50 - 370, Wroclaw, 

Poland 

e-mail: jolanta.sowinska@pwr.wroc.pl 

Construction of chloride-bridged transition metal complexes is a productive research field, allowing to obtain 

compounds with a quite wide range of applications and attractive frameworks, for example as chelating resin. 

Hydroxamates and corresponding transition-metal complexes have been widely reported to exhibit antitumor, 

antifungal and antibacterial properties – amongst several other biological activities[1]. The new polynuclear 

copper(II) cluster with alfa-alaninehydroxamic acid of formula [Cu6(alfa-alaha)5Cl2.5]2 * 2HCl * 18H2O, where 

alfa - alaha stands for alfa - alaninehydroxamate (C3H6N2O2 -2 ), was obtained. Crystal powder of the complex 

was growing up in water solution. The cluster is composed of two hexanuclear subunits connected by five 

chloride bridges . The characterization of this complex was based on elementary analysis, atomic absorption, 

IR- and EPR-spectroscopy and magnetic measurements. Magnetic studies allowed to describe the shown spin 

correlation within hexanuclear clusters and interaction between them. Spectroscopic behavior was studied at the 

range of 4 – 300 K. Basing on computational calculations we try to predict antitumor properties. 

References: 

[1] H. Kehl, Chemistry and Biology of Hydroxamic Acids, Karger, New York, 1982. 

_____________________________________________________________________ 

303


P185. Coordination Ability of Hydroxamic Acid Analogues Towards 

Biometal Ions 

M. Sowińska a , J. Gałęzowska a , I. Fritsky b , J. Świątek-Kozłowska a 

a 

Department of Inorganic Chemistry, Wrocław Medical University, Szewska 38, 50139, Wrocław, Poland, 

malgorzata.sowinska@chnorg.am.wroc.pl 

b 

Department of Chemistry, National Taras Shevchenko University, , 01033, Kiev, Ukraine 

The trials with metal chelators for neurodegenerative diseases treatment prompted renewed interest in assessing 

whether their therapeutic action is related to the coordination of neurotoxic trace metals, such as Cu(II), Fe(III) 

and Zn(II). 

Neurodegenerative disorders are characterized by the evidence implicating the central role of these metals. 

Numerous studies have established the roles of redox-active transition metals in the pathogenesis of 

neurodegenerative diseases [1, 2]. Iron, copper, zinc and other metals are usually found in essential normal 

biological processes and are also involved in enzymatic activities. However, in appropriate accumulation of 

excessive metal deposits can also be cytotoxic. Evidences of alteration in metal ions metabolism have been 

reported in various diseases like Alzheimer's, Wilson, Menkes, Prion, Pick, Huntington disease, epilepsy and 

other pathological events. Thus, metal ions play a important role in neurodegenerative phenomena. 

Hydroxamic acids are very effective chelators for different metal ions [3-7]. Basically, trivalent metal ions i.g. 

Fe(III) or Al(III) complexation by hydroxymates takes place via the two oxygens of the hydroxamic group 

through the formation of a stable 5-membered chelate ring [3]. 

Cyclic voltammetry offers a convenient route for the determination of ligand protonation/deprotonation 

constants and also for metal-ligand complex stability constants in aqueous media. The obtained results allow us 

to calculate the stability constants of the studied ligands towards the: Cu(II), Ni(II), Fe(III) and Zn(II) metal ions. 

Conditional stability constants (Kc) are the key for quantifying and therefore understanding reactions that may 

be relevant to biology and therapeutics of drugs. Spectroscopic and electrochemical methods were used in the 

present studies. 

References: 

[1]. A.I.Bush, Neurobiol Aging, 23, 1031 (2002) 

[2]. E.Ferrada, V.Arancibia, NeuroToxicology, 28, 445 (2007) 

[3]. E.Gumienna-Kontecka, J. Chem. Soc. Dalton Trans., 4201 (2000) 

[4]. E.Farkas, E.A.Enyedy, J. Inorg. Biochem., 79, 205 (2000) 

[5]. H.Kurzak, H.Kozłowski, Coord. Chem. Rev., 114, 169 (1992) 

[6]. A.Dobosz, N.M.Dudarenko, J. Chem. Soc. Dalton Trans., 743 (1999) 

[7]. T.W. Failes, T.W.Hambley, J. Inorg. Biochem., 101, 396 (2007) 

_____________________________________________________________________ 

304


P186. Metal Site Dynamics on the Nanosecond Time Scale 

M. Stachura a , N.J. Christensen b , L. Olsen c , S. Chakraborty d , V.L. Pecoraro d , 

L. Hemmingsen a 

a 

Department of Natural Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, 

Denmark 

e-mail: msta@life.ku.dk 

b 

Department of Food Science/Quality and Technology, University of Copenhagen, Rolighedsvej 30, 1958, 

Frederiksberg C, Denmark 

c 

Department of Medicinal Chemistry, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, 

Denmark 

d 

Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109 - 1055, United States 

Perturbed angular correlation of γ-rays (PAC) spectroscopy is a well establihed technique suitable for 

investigation metalloprotein structure [1]. Here we demonstrate that it is also a valuable tool for monitoring the 

dynamics occuring at the metal ion binding site on the nanosecond time scale. In the present work the nuclear 

quadrupole interactions (NQI) of the 111m Cd derivatives of the de novo designed α-helical peptide TRI 

L16CL23A have been investigated in order to monitor the exchange dynamics between and . Only one signal 

was observed in Nuclear Magnetic Resonance (NMR) spectroscopy, corresponding to a weighted average of the 

two complexes [2]. This suggested that these two structures interconvert rapidly on the NMR timescale (0.01 - 

10 ms), whereas in fact they are slow enough to be observed on PAC timescale (0.1 - 100 ns). By carrying out 

measurements at several different temperatures, ranging from -196 ºC to 50 ºC, see the figure, the dynamics 

changed from slow exchange (at -20 ºC) to intermediate/fast (at 50 ºC) exchange on the PAC time scale. 

Exchange rates for the water molecule, and thermodynamic parameters for the reaction have been estimated. 

PAC spectra at different temperatures – from slow exchange at -20ºC to intermediate/fast exchange at 50ºC. 

References: 

[1] Hemmingsen, L., Sas, K. & Danielsen, E. Biological applications of perturbed angular correlations of 

gamma-ray spectroscopy. Chem. Rev. 104, 4027-4061(2004). 

[2] Matzapetakis M., Farrer B.T., Weng T-C., Hemmingsen L., Penner-Hahn J.E., P ecoraro V.L., Heavy metal 

complexation by de novo designed peptides: Comparison of peptid e aggregation preferences in the presence of 

cadmium(II), mercury(II) and arsenic(III) J. Am. Chem. Soc. 2002, 124, 8042-8054. 

_____________________________________________________________________ 

305


P187. Copper(I) Iodide Complexes with Aromatic Diimines and Aliphatic 

Aminophosphines: Characterization, Structures, Antibacterial Properties 

and Study of Interaction with pUC18 Plasmid and Bovine Serum Albumin. 

R. Starosta a , M. Florek b , J. Król b , W. Barszczewski c , M. Puchalska a , A. Kochel a 

a 

Faculty of Chemistry, University of Wroclaw, ul. F. Joliot-Curie 14, 50-383 Wroclaw, Poland 

e-mail: sta@wchuwr.pl 

b 

Department of Veterinary Microbiology, Wroclaw University of Environmental and Life Sciences, ul. Norwida 

31, 50-375 Wroclaw, Poland 

c 

Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life 

Sciences, ul. Norwida 25, 50-375 Wroclaw, Poland 

A large number of copper(I) complexes with tertiary phosphines and tertiary diphosphines have been studied for 

their tumoricidal properties [1]. Phosphane copper(I) complexes with 1, 10-phenanthroline and its derivatives, 

have been extensively studied mainly for their photophysical properties [2], but have not been investigated for 

their potential biological activity to the best of our knowledge. 

In this work we present novel copper(I) iodide complexes with aliphatic aminophosphines of general formula: 

CuI(NN)P(CH2R)3, where NN = 2, 2’-bpy or 1, 10-phen and P(CH2R)3 = P(CH2-N(CH2CH2)2O)3, P(CH2- 

N(CH2CH2)2N-CH3)3 or P(CH2-N(CH2CH2)2N-CH2CH3)3. 

All CuI(NN)P(CH2R)3 complexes were prepared in direct 

reactions of CuI with diimine and phosphine in 1:1:1 molar ratios 

at room temperature. The obtained compounds were characterized 

using spectroscopic and crystallographic methods. 

Cu(I) has distorted tetrahedral coordination in all investigated 

complexes. NMR data indicate that imine ligands are bound to 

copper atom symmetrically. The influence of phosphines 

coordination to Cu(I) atom on the phosphine part of the 

CuI(1, 10-phen)P(CH2-N(CH2CH2)2O)3 

1 H NMR 

spectra suggest that the phosphorus atoms in phosphine ligands 

coordinate more strongly in the complexes with 1, 10-phen than in 

the complexes with 2, 2’-bpy. 

Presented complexes were screened for their in vitro antibacterial 

activity against Gram-negative Escherichia coli and Pseudomonas 

aeruginosa and Gram-positive Staphylococcus aureus strains, and 

for in vitro antifungal activity against Candida albicans. All the 

compounds show significant antibacterial activity against 

Staphylococcus aureus. 

Investigations of interactions of CuI(1, 10-phen)PR3 and CuI(2, 2’-bpy)PR3 complexes with bovine serum 

albumin showed that the diminution of BSA fluorescence signal is stronger for 1, 10-phenanthroline than for 2, 

2’-bipyridine derivatives. A circular dichroism study of interactions of the presented complexes with BSA gave 

similar results: the decrease of the negative bands characteristic for α-helical structure caused by interactions 

with 1, 10-phenanthroline derivatives is bigger than with 2, 2’-bipyridine derivatives. 

The agarose gel electrophoresis studies have been carried out and it was found that the investigated complexes 

interact with pUC18 plasmid and break the DNA supercoiled to nicked and linear DNA forms. 

References: 

[1] C. Marzano, M. Pellei, D. Colavito, S. Alidori, G.G. Lobbia, V. Gandin, F. Tisato, C. Santini J. Med. Chem. 

49 (2006) 7317; C. Marzano, M. Pellei, S. Alidori, A. Brossa, G.G. Lobbia, F. Tisato, C. Santini J. Inorg. 

Biochem. 100 (2006) 299; N.J. Sanghamitra, P. Phatak, S. Das, A.G. Samuelson, K. Somasusundaram J. Med. 

Chem. 48 (2005) 977; M.J. McKeage, P. Papathanasiou, G. Salem, A. Sjaarda, G.F. Swiegers, P. Waring, 

S.B.Wild Metal-Based Drugs 5 (1998) 217; V. Scarcia, A. Furlani, G. Pilloni, B. Longato and B. Corain Inorg. 

Chim. Acta 254 (1997) 199; S.J. Berners-Price, R.A. Johnson, C.K. Mirabelli, L.F. Faucette, F.L. McCabe, P.J. 

Sadler Inorg. Chem. 26 (1987) 3383 

[2] see for example: X. Gan, W.F. Fu, Y.Y. Lin, M. Yuan, C.M. Che, S.M. Chi, H.F.J. Li, J.H. Chen, Z.Y. Zhou 

Polyhedron 27 (2008) 2202; W.F. Fu, X. Gan, J. Jiao, Y. chen, M. Yuan, S.M. Chi, M.M. Yu, S.X. Xiong 

Inorg. Chim. Acta 360 (2007) 2758; N. Armaroli, G. Accorsi, G. Bergamini, P. Ceroni, M. Holler, O. Moudam, 

C. Duhayon, B. Delavoux-Nicot, J.F. Nierengarten Inorg. Chim. Acta 360 (2007) 1032 

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306


P188. Cytochrome C Peroxidase and Its “Mimics” as 

Fret-Based NO Biosensors 

M. Strianese a , F. De Martino a , G.W. Canters b , V. Pavone c , C. Pellecchia a , A. Lombardi c 

a Dipartimento di Chimica, Università di Salerno, via S. Allende, I-84081 Baronissi, Salerno, Italy, 

b Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands 

c Dipartimento di Chimica, Università Federico II of Napoli, Via Cintia 45, I-80126 Napoli, Italy 

e-mail: mstriane@unisa.it 

Nitric oxide (NO) has a number of significant roles in physiology and microbiology. For example, NO regulates 

vasodilatation in the circulatory system and long-term potentiation in the brain. Furthermore, micromolar 

concentrations of NO can cause carcinogenesis and neurodegenerative disorders [1]. Therefore, detection of NO 

is an especially challenging problem [2]. The techniques commonly used for NO detection, due to the limitations 

of low sensitivity or expensive instrumentation, are not generally useful, especially in biological settings [2]. 

Recently, a novel use of FRET has been proposed to monitor the activity of a donor-acceptor pair on a protein, 

opening the doors to a new generation of fluorescence based biosensors [3]. This method translates the changes 

in absorption into a change of fluorescence intensity of a label covalently attached to the protein [3]. 

The overall properties of Cytochrome C peroxidase (CcP) make it an attractive candidate for developing 

a FRET-based NO biosensor. Here, we present data demonstrating that CcP can be successfully used for NO 

detection. 

Artificially and properly tailored CcP model compounds are also shown to be ideally suited for being used as 

FRET-based NO sensors. 

References: 

[1] M. H. Lim and S. J. Lippard Acc. Chem. Res. 2007, 40, 41 

[2] E. M. Boon and M. A. Marletta J. Am. Chem. Soc. 2006, 128, 10022 

[3] G. Zauner, M. Strianese, L. Bubacco, T. J. Aartsma, A. W.J.W. Tepper and G. W. Canters Inorg. Chim. Acta, 

2008, 361, 1116 

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307


P189. Fluorescently Labeled Hemocyanin as Oxygen Biosensor for Cell 

Viability 

M. Strianese a , G. Zauner a , A.W.J.W. Tepper a , L. Bubacco b , E. Breukink c , 

T.J. Aartsma d , G.W. Canters a , L.C. Tabares a 

a 

Leiden Institute of Chemistry, University of Leiden, Einsteinweg 55, 2300 RA, Leiden, Netherlands 

b 

Department of Biology, University of Padua, Via Trieste 75, 30121, Padua, Italy 

c 

Department of Chemical Biology and Organic Chemist, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, 

Netherlands 

d 

Leiden Institute of Phyics, University of Leiden, Niels Bohrweg 2, 2300 RA, Leiden, Netherlands 

e-mail: lctabares@chem.leidenuniv.nl 

Hemocyanin acts as oxygen carrier in molluscs and arthropods by reversible binding of oxygen into its binuclear 

Tipe-3 copper site. Deoxygenated Hemocyanin has no strong absorption in the visible spectrum but upon 

oxygenation two strong bands centered at 340 nm and 570 nm appear. As it has been previously shown attaching 

a fluorescent label to the N-terminus of Hemocyanin it is possible to translate this change in absorption into 

a change in Fluorescence by means of Forster Resonance Energy Transfer. This effect results in a dramatic 

increase of the limit of detection making practical the use of Hemocyanin as an oxygen sensor. In this work we 

explore the efficacy of this biosensor for monitoring the biological oxygen consumption by bacteria and its use 

in bacterial cell growth and viability tests. By using a microwell plate, the time courses for the complete 

deoxygenation of samples with different initial concentrations of cells were obtained and the doubling times 

could be extracted. The applicability of our fluorescence-based cell growth assay as antibacterial drugs screening 

method was also explored. The results provide proof of principle for a simple, quantitative, sensitive and costeffective 

method for high-throughput monitoring of prokaryotic cell growth and antibiotic susceptibility 

screening. 

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308


P190. Interaction of Zebrafisch “Prion Related Protein” (Prp-Rel-2) with 

Zn 2+ Ions 

Ł. Szyrwiel a , E. Jankowska b , A. Marcinkowska b , Z. Szewczuk a , H. Kozłowski a 

a Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wroclaw, Poland 

b Faculty of Chemistry, University of Gdańsk, Sobieskiego 18, 80-952 Gdańsk, Poland 

e-mail: lukszyr@wp.pl 

Mammalian prion protein (PrP) seems to play two basic biological functions: i) transport of metal ions (Cu) and 

ii) SOD-like activity. 1, 2 The very effective binding of Cu 2+ to PrP derive from the fact that protein contains an 

octapeptide repeat region with 4 His residues and 2 His in the neurotoxic peptide fragment. 

His are very effective binding sites by using their imidazole moieties to anchor and coordinate metal ions and the 

multi-His sites are even more efficient. 2 However, the fact that the His residues are separated by seven other 

amino acid residues the multi-His site in mammalian protein is only effective for Cu 2+ ions, while Zn 2+ binds 

very poorly. Fish proteins which could be included into the prion family contain also His rich domains with His 

residues being much closer to each other. 2 In these cases proteins were found to be very effective to bind also 

Zn 2+ ions. In this work we have investigate by potentiometric, NMR and ESI method the interactions of zebrafish 

protein, zPrP63-87 fragments with Zn 2+ ions showing that this protein is not only effective to bind Zn 2+ ions, 

but ZnL species coexists in cooperative mode with Zn2L complex. 

Aknowledgements 

This work was supported by Polish Ministry of Higher Education and Science 

(1 T09A 008 30 and 1 T09A 149 30). 

1. Stańczak, P., Kozlowski, H., Biochem Biophys Res Commun., 2007, 1, 352: 198-202. 

2. Kozłowski, H., Brown, D., Valensin G., Metalochemistry of Neurodegeneration, The Royal Society of 

Chemistry 2006. 

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309


P191. Molecular Dynamics Study On Human Prion Protein 

M. Taraszkiewicz a , E. Molteni b , G. Valensin b , H. Kozłowski a 

a Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wroclaw, Poland, 

b Department of Chemistry, University of Siena, Via Aldo Moro, 53100 Siena, Italy. 

e-mail: lena@wcheto.chem.uni.wroc.pl 

Prion diseases belong to the diseases of 21 st century. These disorders are thought to be caused by conformational 

instability of normal prion protein (PrP C ). 

Prion protein is a cell membrane anchored glycoprotein expressed in many cell types, but mainly expressed in 

the brain of many animal species. These proteins may play a crucial role in copper homeostasis and the copperbased 

antioxidant enzymatic activity in the brain. [1, 2] 

Human prion protein consist of two domains. The N-terminal region is flexible and the C-terminal region 

containing the globular domain is rigid. The strucutred part contains three helices and two antiparaller β-sheets. 

It’s postulated that four Cu 2+ ions being able to bind with PrP C within tandem repeat region in the N-terminal 

domain consisting of octarepeat peptide repeats. Also it’s possible to coordinate one or two Cu (II) ions within 

neurotoxic peptide fragment. [3] 

Classical molecular dynamics is widely spread technique used to investigation of biological molecules. This kind 

of calculations allow to monitor behaviour of protein/peptide at atomic level hence one could obtain insight into 

conformational changes. The aim of performed simulations was to see if there are some structural effects on 

prion protein upon copper binding. Calculations are based on the sequence, which contains the octarepeat region 

and the fifth metal binding site. The aminoacid sequence of the octarepeat fragment (PHGGGWGQ) contains 

imidazole nitrogens and carboxylate groups, which are efficient donors in copper cooradination. 

From the series of simulations it emerges that only two types of metal coordination mode could be significant for 

the biologocal effect, the intra-repeat and inter-repeat case. The obtained results encourages us to investigate also 

diffrent copper coordination patterns within neurotoxic fragment of the protein with two further histidines His96 

and His111 which are thought to be the fifth copper binding site. 

References: 

[1] E. Gaggelli, H. Kozlowski, D. Valensin, G. Valensin, Chem. Rev. 106 (2006) 1995. 

[2] H. Kozlowski, D.R. Brown, G. Valensin, Metallochemistry of Neurodegenaration, RSC Publishing, 

Cambridge, 2006. 

[3] H. Kozlowski, A. Janicka-Klos, P. Stanczak, D. Valensin, K. Kulon, Coord. Chem. Rev. 252 (2008) 1069. 

_____________________________________________________________________ 



P192. A Binding Site of Metal Complexes on Serum Albumin: 

Computational Binding Energies and Calorimetric Binding Constants 

T. Taura, K. Suyama 

Group of Chemistry, Graduate School of Information Science and Technology, University of Aichi Prefecture, 

Nagakute, 480-1198 Aichi, Japan 

Human serum albumin is the main transport protein in the blood. This protein comprises 60% of the total plasma 

protein. Albumin plays an essential role in the transport and delivery of metal ions, fatty acids and other small 

molecules or ions [1, 2]. It seems that anionic compounds of these materials strongly bind to the amino acids, 

Lys, Arg and His which provide a three dimensional space around cationic side chains in subdomain IIA of the 

albumin structure (Site I). Although albumin is thought to be the major transport protein for inorganic and 

organic compounds in the blood, precise images of these binding sites are not clear. 

Therefore, we tried to detect the sites of albumin to which metal complexes bind by computational simulations. 

Binding energies of the metal complex anions to albumins including human, bovine, pig and sheep were 

calculated for docking poses after structural optimizations. By contrast, binding constants of these complex 

anions with albumins were obtained by the calorimetric measurement. The large values of binding constants 

correspond to the large binding energies obtained by the computational methods. Good correlation between 

computed binding energies and measured binding constants suggests that the images of binding sites could be 

correct. 

References: 

[1] T. Peters, Jr., All about Albumin, 1996, Academic Press. 

[2] D. C. Carter, J. X. Ho, Adv. Protein Chem., 1994, 45, 153-204. 

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311


P193. A Unique Synchrotron-Radiation Flow Linear Dichroism 

Spectroscopy Facility for the Study of Oriented Macromolecules 

P.W. Thulstrup a , S.V. Hoffmann b 

a 

Department of Natural Sciences, LIFE, University of Copenhagen, Thorvaldsensvej 40, DK-1871 

Frederiksberg, Denmark, e-mail: pwt@life.ku.dk 

b 

Institute for Storage Ring Facilities, University of Aarhus, Ny Munkegade, Bldg. 1520, DK-8000 Århus, 

Denmark. 

Spectroscopic studies of ordered samples constitute an underutilized, yet highly useful range of techniques, 

particularly for studies of biological systems, which often possess an inherent orientation. One prominent 

technique is Linear Dichroism (LD) spectroscopy, where a sample with a partial molecular orientation is probed 

with linearly polarized light. The LD signal is defined as the difference in absorption between the polarization 

oriented parallel and perpendicular to the sample orientation axis: LD = ∆A = A⊥ - A|| 

Naturally oriented samples include crystals, fibres and membranes, but sample orientation may also be induced, 

as for example in Flow LD (see e.g. ref. [1]). This technique (illustrated in the figure) can be applied to any rigid, 

elongated sample molecule. The most notable examples are DNA and fibrous proteins. They are oriented in a 

laminar flow due to their rigidity and their extended molecular shape. Since the sample is in aqueous solution the 

structural properties can be studied as a function of temperature and chemical composition (e.g. salts, denaturing 

agents, and other small molecules), where intra- and intermolecular reactions can be studied and physical 

changes can be monitored. 

_____________________________________________________________________ 

312 

Figure 1: Long molecules like e.g. DNA can 

be oriented in a flow field of a Couette cell. 

Small molecules (e.g. a potential drug) remain 

randomly oriented until they bind to the DNA. 

The plane polarized light passes through the 

central part of the cell and thus only probes the 

molecules aligned perpendicular to the direction 

of the light. 

Small molecules are not oriented by the laminar flow gradient in the sample. Flow-LD studies can, therefore, 

reveal information both on molecular structure of the oriented sample (i.e. changes in molecular shape / protein 

secondary structure for fibrous proteins) as well as information on the binding of small molecules to the oriented 

sample. In the latter case information can both be obtained with regard to kinetics, equilibrium binding constants 

as well as structural information. Thus, linear dichroism spectroscopy can provide many different types of 

information; both with regard to the sample constitution and with regard to molecular shape, symmetry and 

structure. 

The flow LD facility has been designed specifically for the study of: 

• The interaction between membranes and membrane-binding peptides and proteins. 

• The interaction between DNA and DNA-binding molecules, including proteins and coordination compounds. 

• The structure and structural dynamics of fibre-forming peptides and proteins. 

The use of flow oriented LD spectroscopy has until very recently only been realised on commercial CD 

instruments modified into LD spectrometers. This despite the fact that the same advantages in spectral range and 

intensity can be realised with synchrotron radiation based LD (SRLD) as has been obtained for SRCD. A 

Couette Flow LD facility has been implemented at the existing SRCD facilities on the UV1 and CD1 beamlines 

at the synchrotron radiation source ASTRID at Aarhus University in Denmark. This SRLD facility is the first of 

its kind world wide. The implementation has been very successful, and it has shown that SRLD is a drastically 

improvement over the commercial LD spectrometers: The dynamic range of molecular concentrations is higher, 

the spectral quality is far better, and lower wavelengths can be measured enabling the studies of protein-lipid 

membrane interactions/insertions. 

Acknowledgement: A grant from The Danish Natural Science Research Council is gratefully acknowledged 

(no. 272-07-0240) 

References: [1] R. Marrington, T.R. Dafforn, D.J. Halsall, J.I. MacDonald, M. Hicks, A. Rodger, Analyst, 130, 

1608 (2005).


P194. CD and MCD Studies of the Reduced Binuclear Iron Site of 

Ribonucleotide Reductase from B. cereus 

A.B. Tomter a , C.B. Bell b , A.K. Røhr a , E.I. Solomon b , K.K. Andersson a 

a 

Department of Molecular Biosciences, University of Oslo, P.O box 1041 Blindern, 0316, Oslo, Norway 

e-mail: a.b.tomter@imbv.uio.no 

b 

Department of Chemistry, Stanford University, 94305, Stanford California, United States 

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis deoxyribonucleotides from the 

corresponding ribonucleotides needed for DNA synthesis and repair in all living organisms. Class I RNR is 

divided into three different classes and they all consist of two non-identical subunits called R1 and R2. The R1 

subunit contains the active site, and the R2 subunit a tyrosyl radical and a diiron-oxygen cofactor which are 

essential for initiation of the nucleotide reduction process in R1. The R2 subunit of the enzyme complex reacts 

with ferrous iron and dioxygen to generate a diferric iron-oxygen cluster and a tyrosyl radical that is essential for 

enzymatic activity [1]. The reduced form of the class Ib enzyme, Bacillus cereus R2, has been studied using 

a combination of circular dichroism (CD), magnetic circular dichroism (MCD) and variable-temperature 

variable-field (VTVH) MCD spectroscopies. Spectral features of individual iron sites have been analyzed to 

obtain detailed geometric and electronic structural information. VTVH MCD data have been collected and 

analyzed using two complementary models to obtain detailed ground state information including the zero-field 

splitting (ZFS) of both ferrous centers and the exchange coupling (J) between the two sites [2]. The results have 

been compared to the studies of Escherichia coli R2 [3], mouse R2 [4] and p53R2 [5] which all are of RNR 

class 1a. 

References: 

[1] Kolberg M., Strand K.R., Graff P., Andersson K.K. Biochim. Biophys. Acta- Proteins and Proteomics, 2004, 

1699; 1-34. 

[2] Solomon, E. I., Brunold, T. C., Davis, M. I., Kemsley, J. N., Lee, S. K., Lehnert, N., Neese, F., Skulan, A. J., 

Yang, Y. S., Zhou, J. Chem. Rev., 2000, 100; 235-349. 

[3] Yang, Y.-S., Baldwin, J., Ley, B. A., Bollinger, J. M., Solomon, E. I. J.Am. Chem. Soc. 2000, 122; 8495- 

8510. 

[4] Strand, K.R., Yang, Y.-S., Andersson, K.K., Solomon, E.I. Biochemistry, 2003, 42; 12223-12234. 

[5] Wei P.P, Tomter A.B, Røhr, Å.K. Andersson K.K., Solomon, E.I. Biochemistry, 2006, 45; 14043-14051. 

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313


P195. Solvent-Free Synthesis of 

N-Dodecyl-2-Aminocyclopentene-1-Dithiocarboxylic Acid under 

Microwave Irradiation and Complexes With Cu(II) and Ni(II) 

L. Torres, R.R. Contreras, B. Fontal, F. Bellandi, I. Romero, M. Reyes, T. Suárez 

and P. Cancines 

Laboratorio de Organometálicos, Departamento de Química, Facultad de Ciencias, Universidad de Los Andes, 

Mérida, Venezuela. 

e-mail: laurat@ula.ve 

In this work we employ the solvent-free conditions to synthetized N-dodecyl-2-aminocyclopentene-1dithiocarboxylic 

acid (L) from 2-aminocyclopentene-1-dithiocarboxylic acid (acda) and n-dodecylamine on 

silice using a domestic microwave, we explorer if this method is acording to green chemistry. The synthesis of 

this compound was reported with 49.7% yield in 24h by classic method [1] , by microwave irradiation in 4minutes 

we obtained 36.97% yield, characterized by IR and RMN 1 H. We prepare two metal complexes of L using 

nickel(II) [Ni(L)2] and copper(II) [Cu(L)2]. They were characterized by conductimetry, UV-Vis, and IR (and 

RMN 1 H only for [Ni(L)2] complex). Both complexes are electrically neutral. The IR and RMN 1 H spectrum 

showed the same signals of L, but displaced respect this. The IR spectrum showed one band at 3400cm -1 (vN-H), 

the absence of one at 2546 cm -1 (vS-H) in both complexes and the asymetrically split bands due to vCSS at 

990cm -1 indicates the unequal involvement of coordination mode (S-C-S). We hope that both complexes shows 

biological activity by imitation of Cu(II) and Ni(II) enviroment in proteins or biological molecules according to 

biomimetic inorganic. 

C 12H 25 

NH 

S 

SH 

Figure 1. Complexes reaction 

NH 

+ M (Ac)2 (reflujo en MeOH 24h) + 


Laboratorio química orgánica (ULA) - Lic. Iris Santos 

Laboratorio de Organometálicos (ULA) 

Laboratorio de Resonancia Magnética Nuclear – Dr. Alí Bhasas 

References: 

[1] T. Abbas, A. Ashrafolmolouk, Journal of Inorganic Chemistry, 2002, 645, 102 

_____________________________________________________________________ 

314 

C 12H25 

S 

S 

M 

S 

S 

HN 

C 12H25 

S 

HN 

S 

C 12H25 M 

S 

HN 

S 

C 12H25


P196. Interaction of Eu(III) Derivatives with Human Serum Albumin 

L. Trynda-Lemiesz, R. Janicki, A. Mondry 


e-mail: ltl@wchuwr.pl 

Serum albumins are the most abundant proteins in blood plasma, accounting for about 60% of the total protein. 

Human serum albumin (HSA) binds and transports many exogenous and endogenous ligands, including fatty 

acids, metal ions, and pharmaceuticals HSA consists of three structurally homologous domains (I, II, and III) 

that assemble to form a heartshaped molecule. Solution of the X-ray crystallographic structure of HSA facilitated 

the location of the two major drug binding sites, site I and site II proposed originally by Sudlow et al., in 

subdomains IIA and IIIA of the protein, respectively [1, 2]. 

The lanthanide-based pharmaceuticals to date are either non-specific agents or have suffered from toxicity or 

efficacy problems. Recent developments in the fields of coordination chemistry and biotechnology have taken 

great strides towards tissue targeted diagnostic and therapeutic agents. The interactions of lanthanide complexes 

with blood constituents, particularly with serum albumin indicates the importance of the molecular shape of the 

complexes. 

Studies of gadolinium(III) complexes with serum albumin [3] suggest that the rate of water exchange in the 

complex may be slowed upon binding to HSA. This would presumably be the result of secondary interactions 

between protein and the chelate that hinder access of the bulk water to coordinated water site in the complex. 

Human Serum Albumin Molecular structure of Eu(III)–EDTMP–carbonate complex 

In the present work a mechanism of the interaction of Eu(III)–EDTMP complex (where EDTMP is 

ethylenediaminetetra(methylenephosphonate) ligand) with HSA has been considered. The identification of 

binding sites and the nature of forces involved in the interaction were studied using fluorescence and CD 

spectroscopic techniques. The decrease of relative fluorescence intensity of the Eu(III)–EDTMP–bound HSA 

suggests that perturbation around the Trp 214 residue takes place. This was confirmed by the destabilization of 

the warfarin binding site located in subdomain IIA. CD spectroscopic results showed a discernible reduction in 

the affinity of albumin for bilirubin upon Eu(III)–EDTMP binding. These results may indicate that one of the 

binding sites of the complex is subdomain IIA. 

Recently it was shown that at physiological pH a partial hydrolysis of the Eu(III)–EDTMP complex occurs [4] as 

well as the equilibrium between species of [Eu(EDTMP)(H2O)2] 5– and [Eu(EDTMP)(H2O)(OH)] 6– exists [5]. It 

was also revealed that the replacement of water molecules and/or hydroxy groups by a carbonate anion in the 

Eu(III)–EDTMP complex at pH 7.5 results in the formation of thermodynamically stable and kinetically inert 

[Eu(EDTMP)(CO3)] 7– species of which crystal structure has been determined [5]. Probably formation of 

hydrogen bond between HSA and inner-sphere water molecules of Eu(III) ion in the energetically unfavorable 

[Eu(EDTMP)(H2O)2] 5– species is a reason of a weak hydrogen interaction between this species and protein. The 

lack of fluorescence intensity changes in the phosphate buffered solution between the spectra of HSA and 

Eu(III)–EDTMP–HSA may indicate the replacement of inner-sphere water molecules onto phosphate anion. 

The presented investigations may be helpful in understanding of the uptake mechanism of the 153 Sm(III)– 

EDTMP complex by metastatic bones and may provide some indications for future ligand design for therapeutic 

complexes with lanthanide radionuclides. 

References: 

[1] D.C. Carter, J.X. Ho, Adv. Protein Chem., 45, 152 (1994). 

[2] X.M. He, D.C Carter, Nature, 358, 209 (1992). 

[3] S. Aime, M. Botta, M. Fasano, S.G. Crich, E. Terreno, J. Biol. Inorg. Chem, 1, 312 (1996). 

[4] G.C. de Witt, P.M. May, J. Webb, G. Hefter, BioMetals, 9, 351 (1996). 

[5] A. Mondry, R. Janicki, Dalton Trans., 4702 (2006). 

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315


P197. Coordination Ability of Niacin Towards Nickel(II) Ions 

J. Urbańska and H. Podsiadły 


Niacin is a water-soluble type of vitamin B including nicotinic acid and nicotinamide. Nicotinic acid is converted 

in vivo to nicotinamide and although the two compounds are identical in their vitamin functions, their 

pharmacologic and toxic effects are different. 

_____________________________________________________________________ 

316 

N 

O 

OH 

Nicotinamide is an reactive moiety of two coenzymes with similar structures: nicotinamide adenine dinucleotide 

(NAD) and nicotinamide adenine dinucleotide phosphate (NADP) which are necessary for enzymatic catalysis of 

several vitally important redox processes. 

Metal complexes of biologically important ligands are sometimes more effective than the free ligands. 

In spite of the great biological importance of nicotinic acid and nicotinamide their interaction with metal ions is 

rarely studied polarographically. This is probable due to the electrochemical activity of these ligands which 

reduction waves overlap with the reduction wave of some metal ions or their complexes [1, 2, 3]. 

In this communication we present the polarographic, potentiometric and spectroscopic (UV-VIS) results on the 

coordination ability of these ligands towards Ni(II) ions. 

Ni(II) ions in non-complexing electrolyte undergo reduction process at mercury electrode irreversible at about - 

1.0 V. Addition of very small amounts of nicotinamide or nicotinate ions causes the appearence of the new wave 

(prewave), at less negative potentials (- 0.7 V). This prewave increase with concentration of ligands in the 

solution, whereas Ni(II) wave decreases and finally disappears. The appearance of this prewave implies the 

formation of at least one Ni(II)-niacin complex which is reduced at a lower potential than that required for the 

reduction of aquaion. Detailed analysis polarographic curves on the concentration of reagents (ligands and Ni(II) 

ions), pH values and mercury drop time allow us to establish the stoichiometry of the complexes reduced at the 

electrode and those existing in the solution as well as their stability constants. Such a peculiar behaviour of 

nickel(II) ions at the dropping mercury electrode was already observed in the presence of the other N and N, O 

donor ligands [4, 5, 6, 7]. 

Potentiometric and spectroscopic studies display that nicotinic acid and nicotinamide are relatively week ligands 

towards nickel(II) ions. The complexation occurs in the pH range 3 - 6; at higher pH the formation of poorly 

soluble species (probably Ni(OH)2) has been observed. The stability constants of complexes determined from 

potentiometric data are in good accordance with that obtained polarographically. 

UV-VIS spectroscopy confirms monodentate coordination mode of both ligands to nickel(II) ions, by the 

nitrogen of pyridine rings. 

References: 

[1] L. Campanella, P. Cignini and G. De Angelis, Rev.Roumaine Chim., 18, 1269 (1973). 

[2] R. Rodriguez-Amaro, R. Perez, V. Lopez and J.J. Ruiz, J. Electroanal. Chem., 278, 307 (1990). 

[2] E. Mathieu, R. Meunier-Prest and E. Laviron, Electrochim. Acta, 42, 331 (1997). 

[4] D.R. Crow and M.E. Rose, Electrochimica Acta, 24, 41 (1979). 

[5] P.K. Aggarwal and D.R. Crow, Electrochimica Acta, 25, 411 (1980). 

[6] J. Urbańska, H. Kozłowski, A. Delannoy and J. Henion, Anal. Chim. Acta, 207, 85 (1988). 

[7] J. Urbańska and H. Kozłowski, J.Coord.Chem., 42, 197 (1992). 

N 

O 

NH 2


P198. Enantiomers of a Cr(III) Polypyridyl Complex: 

Photophysics and DNA Intercalation Studies 

S. Vasudevan a , S. Quinn a , N. Fletcher b , M. Wojdyla a , J. Kelly a 

a 

School of Chemistry, Trinity College, College Green, 2, Dublin, Ireland 

e-mail: vasudevs@tcd.ie 

c 

School of Chemistry and Chemical Engineering, Queens University, University Road, BT7 1NN, Belfast, 

United Kingdom 

Transition metal heteroleptic complexes of dipyridophenazine (DPPZ) have acquired wide acclaim as DNAbinding 

agents due to their strong binding, light-switching and photocleaving effects. The most studied of these 

are the Ruthenium complexes [1]. However, Kane-Maguire revealed the chromium analogues to be particularly 

interesting due to their long emission lifetimes, strong oxidising power and higher intercaltion with DNA [2]. In 

this work, the photophysics of Cr(III) polypyridyl complex, [Cr(phen)2dppz] 3+ has been explored using nanosecond 

transient absorption and fluorescence lifetime measurements. The single crystal X-ray diffraction studies 

of the complex revealed two enantiomers, which were successfully separated by means of size-exclusion 

chromatography. The interactions of these enantiomers with DNA was investigated by emission and optical 

spectroscopy, including thermal denaturation experiments and circular dichroism studies. 

References: 

[1] J. G. Vos, J. M. Kelly; Dalton Trans., 2006, 4869 - 4883. 

[2] N. A. P. Kane-Maguire, J. F. Wheeler; Coord. Chem. Rev., 2001, 145 - 162. 

_____________________________________________________________________ 

317


P199. A Novel Respiratory Complex in Desulfovibrio vulgaris 

Hildenborough 

S. Venceslau, I. Cardoso Pereira 

a Microbial Biochemistry, Instituto de Tecnologia Química e Tecnológica, U, Av. da República, EAN, 2780-157, 

Oeiras, Portugal 

e-mail: sofiav@itqb.unl.pt 

Sulfate-reducing organisms are anaerobic prokaryotes found ubiquitously in nature. They use a respiratory 

mechanism with sulfate as the terminal electron acceptor, but the intervenients in the respiratory chain have not 

been fully elucidated. Here, we describe a novel multihemic cytochrome complex isolated from the membranes 

of Desulfovibrio vulgaris Hildenborough, composed by four subunits (72, 48, 31 and 24 kDa). The 24 kDa 

protein is a periplasmic penta or hexaheme c membrane anchored subunit, the 31 kDa protein is an FeS protein 

that may also contain one heme c, and the 48 kDa subunit is an integral membrane protein. Although the 

periplasmic 72 kDa subunit is annotated as a molybdopterin oxidoreductase subunit, no Mo was detected. This 

complex is the first example described of the so-called MFIc complexes, proposed to be oxidoreductases of the 

respiratory electron transfer chains. These complexes are related to the MFIcc class, which have already been 

reported as an alternative complex III, when this one is absent: in the anoxigenic phototrophic bacterium 

Chloroflexus aurentiacus, and in the aerobic non-phototrophic bacterium Rhodothermus marinus[1, 2]. The D. 

vulgaris MFIc complex is present in large amounts suggesting an important role in the energy metabolism. This 

is supported by expression studies, where the complex behaves similarly to other proteins directly involved in 

the sulphate respiratory chain[3]. However, its electron donor and acceptor are still not known. 

References: 

[1] Yanyushin, M.F., et al., New class of bacterial membrane oxidoreductases. Biochemistry, 2005. 44(30): 

p. 10037-45. 

[2] Pereira, M.M., et al., The alternative complex III from Rhodothermus marinus - a prototype of a new family 

of quinol:electron acceptor oxidoreductases. FEBS Lett, 2007. 581(25): p. 4831-5. 

[3] Pereira, P.M., et al., Transcriptional response of Desulfovibrio vulgaris Hildenborough to oxidative stress 

mimicking environmental conditions. Arch Microbiol, 2008. 189(5): p. 451-61. 

_____________________________________________________________________ 

318


P200. Improving Platinum Chemotherapy Through Controlled and 

Targeted Drug Delivery 

N. Wheate 

Strathclyde Institute of Pharmacy and Biomedical S,University of Strathclyde,27 Taylor Street,G40NR, 

Glasgow,United Kingdom 

e-mail: nial.wheate@strath.ac.uk 

Cisplatin, carboplatin and oxaliplatin are the only three platinum based drugs with wide approval for the 

treatment of human cancers [1]. Whilst several new drugs are in various stages of clinical trials including 

satraplatin, picoplatin and multinuclear drugs [1], the biggest advances in the coming decade will come from the 

development of controlled and targeted drug delivery systems. Over the last 5 years our work has focussed on 

macromolecules which can encapsulate mono- and multinuclear platinum complexes and platinum based DNA 

intercalators [2-3]. This includes cyclodextrins, calixarenes, cucurbiturils and PAMAM dendrimers. Many of 

these macromolecules are able to prevent drug degradation by thiol peptides and proteins and can be used to tune 

the cytotoxicity and toxicity of the drugs. In vivo animal trials also demonstrated that one drug delivery vehicle, 

cucurbituril was able to nearly double the maximum tolerated dose of a multinuclear platinum drug. The first 

phase of our research is nearly complete and we are now examining two-fold drug encapsulation and suitable 

targeting groups. In the end, the goal of our research is the development of a targeted platinum drug delivery 

system that can specifically recognise and bind cancer cells, thus eliminating many of the side-effect of platinum 

drugs from that arise from non-specific cellular attack, and improve drug efficacy. 

References: 

[1]L. Kelland. The resurgence of platinum-based cancer chemotherapy, Nature Reviews Cancer, 2007, 7, 573- 

584. 

[2] N. J. Wheate, D. P. Buck, A. I. Day, J. G. Collins, Cucurbit[n]uril binding of platinum anticancer complexes, 

Dalton Transactions, 2006, 451-458. 

[3] S. Kemp, N. J. Wheate, S. Wang, J. G. Collins, S. F. Ralph, A. I. Day, V. J. Higgins, J. R. Aldrich-Wright, 

Encapsulation of platinum(II)-based DNA intercalators within cucurbit[6,7,8]urils, Dalton Transactions, 2007, 

12, 969-979. 

_____________________________________________________________________ 

319


P201. Electrocatalytic Oxygen Reduction by Cytochrome C Oxidase 

F.G.M. Wiertz a , O.M. Richter b , B. Ludwig b , H.A. Heering a 

a 

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, Netherlands 

e-mail: h.a.heering@chem.leidenuniv.nl 

b 

Institute of Biochemistry, J.W. Goethe Universität, Max-von Laue-Str. 9, D-60438, Frankfurt am Main, 

Germany 

Cytochrome c oxidase (CcO) is the final electron acceptor in the respiratory chain. It oxidizes four ferrous 

cytochome c while reducing oxygen to water without releasing intermediates. Paracoccus denitrificans CcO is a 

four-subunit membrane enzyme, containing two heme groups (a, a3) and two copper centres (CuA, CuB). The 

mixed-valence binuclear CuA centre is the primary electron acceptor from cytochrome c. Heme a3 and 

mononuclear CuB form the site for oxygen reduction. Spectroscopic methods revealed that oxygen binding to the 

four-electron-reduced enzyme is followed by rapid reductive O=O bond splitting. The reduced enzyme is 

regenerated by uptake of four protons from the cytoplasm, oxidation of four cytochrome c, and release of two 

water molecules. In addition, four protons are translocated across the membrane, converting redox free energy 

into a transmembrane electrochemical potential. [1, 2] 

Due to the relatively slow F → OH transition, it is difficult to obtain information on subsequent electron-uptake 

events and coupled proton pumping with pre-steady state kinetics. In contrast, these events may be observed by 

protein film voltammetry. We have immobilized active P. denitrificans CcO on gold electrodes. Voltammetry in 

presence of oxygen yields a characteristic sigmoidal but non-nernstian catalytic wave. Analysis of the wave 

shape under various conditions is in progress, with the aim to derive novel mechanistic information on the 

reductive phase of the catalytic cycle. 

References: 

[1] F.G.M. Wiertz, O.M. Richter, B. Ludwig, S. de Vries, J. Biol. Chem. 282, 31580 (2007). 

[2] I. Belevich, M.I. Verkhovsky, Antioxidants & Redox Signaling 10, 1 (2008). 

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320


P202. The Crystal Structure of Tryptophan Hydroxylase with Bound 

Amino Acid Substrate 

M.S. Windahl a , C.R. Petersen b , P. Harris b , H.E.M. Christensen b 

a 

Department of Natural Science, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, 

Denmark 

e-mail:mskn@life.ku.dk 

b 

Department of Chemistry, Technical University of Denmark, Kemitorvet 207, 2800, Kgs. Lyngby, Denmark 

Tryptophan hydroxylase (TPH) is part of the small enzyme family of tetrahydrobiopterin (BH4) dependent 

aromatic amino acid hydroxylases [1]. The TPH catalysed formation of 5-hydroxytryptophan is the first and ratelimiting 

step in the biosynthesis of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). Although 

serotonin has many physiological functions, it is mainly known as a neurotransmitter. Abnormalities in the 

serotonergic neurons are implicated in a wide range of neuropsychiatric disorders such as depression, obsessivecompulsive 

disorder and schizophrenia [2]. Two isoforms of TPH exist: isoform 1 (TPH1) is primarily found in 

the mast cells, pineal gland and enterochromaffin cells, while isoform 2 (TPH2) appears mostly in the 

serotonergic neurons of the brain [3]. TPH is a homotetrameric three domain enzyme; its three domains are an 

N-terminal regulatory domain, a catalytic domain and a small C-terminal tetramerisation domain. 

We have previously reported the expression, purification and crystallisation of the catalytic domain (∆1- 

100/∆415-445) of chicken tryptophan hydroxylase isoform 1 [4]. We here present the 1.9 Å resolution crystal 

structure in complex with the tryptophan substrate and an iron bound imidazole. The iron coordination can be 

described as a distorted trigonal bipyramidal coordination with His273, His278 and Glu318 (partially bidentate) 

and one imidazole as ligands. The tryptophan binding pocket is distinct from the BH4 binding pocket and the 

tryptophan stacks against Pro269 with a distance of 3.9 Å between the iron and the tryptophan C5 that is 

hydroxylated. The binding of tryptophan and imidazole have caused structural changes in the catalytic domain 

compared to the structure of the human TPH1 with bound dihydrobiopterin [5]. The structure of chicken TPH1 is 

more compact and the loops of residues Leu124-Asp139 and Ile367-Thr369 closes around the active site. The 

same structural changes are seen in the catalytic domain of phenylalanine hydroxylase (PAH) upon binding of 

substrate analogues norleucine and thienylalanine to the PAH•BH4 complex [6]. In fact the chicken TPH1•Trp 

structure resembles the PAH•BH4•thienylalanine structure (r.m.s.d. of Cα atoms 0.94 Å) more than the human 

TPH1 structure (r.m.s.d. 1.47 Å). 

References: 

[1] Fitzpatrick, P. F. (1999) Annu. Rev. Biochem. 68, 355-381. 

[2] Lucki, I. (1998) Biol. Psychiatry 44, 151-162. 

[3] Walther, D. J., and Bader, M. (2003) Biochem. Pharmacol. 66, 1673-1680. 

[4] Nielsen, M. S., Petersen, C. R., Munch, A., Vendelboe, T. V., Boesen, J., Harris, P., and Christensen, H. E. 

M. (2008) Prot. Expr. Purif. 57, 116-126. 

[5] Wang, L., Erlandsen, H., Haavik, J., Knappskog, P. M., and Stevens, R. C. (2002) Biochemistry 41, 12569- 

12574. 

[6] Andersen, O. A., Stokka, A. J., Flatmark, T., and Hough, E. (2003) J. Mol. Biol. 333, 747-757. 

_____________________________________________________________________ 

321


P203. Complexation of Pb(II) Ions with Humic Acids and Naturally 

Occurring Antioxidants; EPR and Relativistic DFT Study 

M. Witwicki a , A. Jaszewski b , J. Jezierska b , A. Ożarowski c , A. Jezierski b , M. Jerzykiewicz b 

a 

Department of Chemistry, Wroclaw University, F. Joliot-Curie 14, Wroclaw 50-283, Poland 

e-mail:mck@eto.wchuwr.pl 

b 

Department of Chemistry, Wroclaw University, F. Joliot-Curie 14, Wroclaw 50-283, Poland, 

c 

National High Magnetic Field Laboratory, Florida State University, 1800 E. Paul Dirac Drive, 

Tallahassee, FL 32310, USA, 

Complexation of Pb(II) with functional groups of humic acids [1, 2] and with their models, polihydroxybenzoic 

acids which are present in natural products as strong antioxidants, results in the formation of new radicals whose 

g values (observed in EPR spectra) are unusually low in comparison with those of parent semiquinone radicals 

[2, 3]. 

To show the reasons of this effect we underwent the EPR studies of Pb(II) complexes with the model radical 

derived from of 3, 4-dihydroxybenzoic acid. The 3, 4-dihydroxybenzoic acid is representative for other 

polihydroxybenzoic acids substituted with at least two vicinal phenolic OH and one carboxylic groups which 

form the radicals able to shift characteristically g parameters after Pb(II) complexation. 

The role of the OH and COOH substituents of the model radical in Pb(II) coordination, leading to the 

characteristic shift of g parameter, was studied by us using the relativistic DFT calculations of the expected 

complexes geometries, unpaired electron delocalizations and electronic structures with ADF program. The 

calculations were verified by the best agreement between the predicted and experimental g parameters. 

Acknowledgement: The work was supported by Grant No. 6 PO4G 06730. 

References: 

[1] M. Jerzykiewicz, Geoderma 122 (2004) 305 

[2] E. Giannakopoulos, K.C. Christoforidis, A. Tsipis, M. Jerzykiewicz, Y. Deligiannakis, Journal of Physical 

Chemistry Part A 109 (2005) 2223. 

[3] F. Czechowski, I. Golonka, A. Jezierski, Spectrochimica Acta Part A 60 (2004) 1387 

_____________________________________________________________________ 

322


P204. NMR Study of Heme Binding to HmuY Protein 

from Porphyromonas gingivalis 

J. Wojaczyński, a H. Połata, b T. Olczak, b and L. Latos-Grażyński a 

a 

Department of Chemistry, University of Wrocław, 14 F. Joliot-Curie St., 50 383 Wrocław, Poland 

e-mail: jw@wchuwr.pl 

b 

Faculty of Biotechnology, University of Wrocław, Tamka 2, 50 138 Wrocław, Poland 

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression 

of chronic periodontitis, requires iron and heme for growth. One of the mechanisms of heme uptake in this 

bacterium comprises the outer-membrane heme transporter HmuR and a putative heme-binding lipoprotein 

HmuY. 

The aim of this study was to characterize the nature of heme binding to HmuY using 1 H NMR spectroscopy. The 

protein was expressed, purified and detailed magnetic resonance investigations were performed. We found that 

the heme complexed to HmuY is in a low-spin Fe(III) hexa-coordinate environment. The nature of coordinating 

ligands and the possibility of additional heme binding by HmuY was thoroughly explored. 

heme methyl signals 

30 20 10 0 -10 

δ [ppm] 

D 2 O, 323 K 

Using site-directed mutagenesis, several single and double HmuY mutants were constructed with the methionine, 

histidine, cysteine, and tyrosine residues replaced by an alanine residue. The ability of the mutated proteins to 

bind heme was reflected by their 1 H NMR spectra which gave a closer insight into the heme-protein interactions. 

_____________________________________________________________________ 

323


P205. New Binucleating Ligands for Modelling the Dizinc 

Metallo-β-Lactamase Active Site 

S. Wöckel a , B. Burger a , M. Jarenmark c , S. Dechert a , E. Nordlander c , F. Meyer a 

a Institute for Inorganic Chemistry, University of Göttingen, Tammanstr. 4, D-37077, Göttingen, Germany 

e-mail: Simone.Woeckel@chemie.uni-goettingen.de 

c Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-22100, Lund, Sweden 

β-lactamases are enzymes that mediate hydrolytic ring cleavage of β-lactams. They are thus responsible for the 

increasing resistance towards widely used β-lactam antibiotics [1]. One group of these enzymes, so called 

metallo-β-lactamases, contain one or two zinc atoms in their active site, which are ligated by amino acid residues 

[2, 3]. The Lewis acidic character of zinc allows water to be deprotonated at physiological pH, generating 

a nucleophilic hydroxide that is able to attack the β-lactam ring of the antibiotic substrate. Other roles of the 

metal ions and details of the catalytic process are still controversial. In view of the importance of this class of 

enzymes, further insight in the mechanism of β-lactam hydrolysis at dizinc sites is highly desirable. 

We have developed a general class of ligands that can hold two metal ions in close proximity suitable for 

cooperative reactivity. These tunable ligands are based on a central pyrazole bridge, substituted with chelating 

side arms in 3- and 5- position of the heterocycle. Initial studies had provided some first insight into the binding 

and cleavage of penicillin G at dizinc complexes of those ligands [4]. In order to more closely emulate 

characteristic of the enzyme active site, we have now prepared several new ligand scaffolds that bear biomimetic 

N- (imidazole or benzimidazole) or O- (carboxylate) side arm donor functions. Their synthesis and coordination 

chemistry relevant to the metallo-β-lactamases will be presented. 

References: 

[1] I. Massova, S. Mobashery, Acc. Chem. Res. 1997, 30, 162-168. 

[2] A. Badarau, M. I. Page, Biochemistry 2006, 45, 10654-10666. 

[3] M. W. Crowder, J. Spencer, A. J. Vila, Acc. Chem. Res. 2006, 39, 721-728. 

[4] B. Bauer-Siebenlist, S. Dechert, F. Meyer, Chem. Eur. J. 2005, 11, 5343-5352. 

_____________________________________________________________________ 

324


P206. Chiral Interactions in Metal Complexes Containing Amino Acids 

and its Derivatives 

T. Yajima a , S. Ito a , J. Morita a , M. Yumoto a , Y. Shimazaki b , O. Yamauchi a , T. Shiraiwa a 

a Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680, Japan. 

b College of Science, Ibakaki Univeristy, Mito, Ibaraki 310-8512, Japan 

e-mail: t.yajima@ipcku.kansai-u.ac.jp 

A number of bio-active compounds have chirality, and their stereoisomers exhibit various activities in biological 

systems and play key roles in sustaining life. Especially, optically active amino acids have been used in foods, 

medicines, pesticides, cosmetics, and even as chiral reagents for asymmetric syntheses. Enantiomeric 

compounds have been obtained by refining natural products, asymmetric sysntheses, optical resolution from 

racemates, and so on. Optical resolution of racemic amino acids (AAs) is achieved by various procedures to give 

their enantiomers. However, the procedures are limited to resolution of certain AAs, and there is a strong 

demand for the method of general applicability. Transition metal ions can bind two or more ligands, so that a 

metal complex containing a chiral AA may coordinate the second ligand (AA’) enantioselectively for steric or 

other reasons, enabling separation of the racemic mixture of this ligand by differences in solubility or reactivity. 

We first studied selective incorporation of an enantiomer of a racemic AA’ into Cu(II) complexes with an active 

AA, Cu(AA)(AA’). 

The ternary Cu(II) complex containing L-isoleucine (ile) and D-alanine (ala), [Cu(L-ile)(D-ala)], is less soluble 

than [Cu(L-ile)(L-ala)], because of the difference in configuration: [Cu(L-ile)(D-ala)] has a cis-configuration, 

whereas [Cu(L-ile)(L-ala)] has a trans-configuration.[1] Cis-trans isomerism may have an influence on the 

properties of ternary Cu(II) complexes; for complexes containing D/L-aminobutanoic acid (abu) instead of D/Lala, 

[Cu(L-ile)(L-abu)] is over 10 times more soluble than [Cu(L-ile)(D-abu)]. Such a wide difference in solubility 

may arise from the stability difference between the diastereomers of the ternary complexes, whose stability 

constants, logβ, were found to be 15.692(9) for [Cu(L-ile)(D-abu)] and 15.166(25) for [Cu(L-ile)(L-abu)]. 

On the other hand, two ternary complexes containing L-alloisoleucine (aile) and D/L-ala, [Cu(L-aile)(D/L-ala)], 

exhibit similar solubilities and the same cis-configuration. 

O 

H2 O N 

Cu 

N O 

H2 O 

L-ala O O 

D-ala 

OH2 Cu 

N OH 

H 2 

2 

In order to attain efficient separation of racemic AAs, we studied 

potentiometrically and spectroscopically the M-L-AA ternary systems, where M 

refers to Zn 2+ , Cu 2+ , and Ni 2+ and L to tris(2-pyridylmethyl)amine (TPA) and 

(S)-N, N-bis(2-pyridylmethyl)-1-(2-pyridyl)ethylamine ((S)-MeTPA).[2] 

Potentiometric titrations of the M-TPA-AA systems revealed that stable ternary 

complexes, [M(TPA)(L-AA)], are formed for M = Ni 2+ , while ternary 

complexes with M = Zn 2+ or Cu 2+ N 

N 

N 

are not formed or less stable. The stability 

constants of [Ni(TPA)(AA)] for AA = chiral amino acids are slightly smaller 

R = H 

CH3 

TPA 

(S)-MeTPA 

than the value of [Ni(TPA)(gly)], suggesting that the side chain of AA may give rise to steric hindrance with the 

pyridine ring of TPA and that it may be possible to resolve efficiently a number of amino acids by a chiral TPAtype 

ligand. The circular dichroism spectra of Cu-(S)-MeTPA-L-Phe and Cu-(S)-MeTPA-D-Phe systems 

exhibited a large negative and a positive extremum at ~600 nm, respectively, as compared with Cu-TPA-D/L- 

Phe, which may indicate that AA has a higher affinity for Cu-(S)-MeTPA than Cu-TPA. 

Acknowledgement: We would like to thank Professor Akira Odani, Kanazawa University, for kind advice on 

potentiometric titrations. 

References: 

[1] T. Shiraiwa, H. Fukuoka, M. Yoshida, H. Kurokawa, Bull. Chem. Soc. Jpn., 57, 1675 (1984). 

[2] J.W. Canary, Y. Wang, R. Roy, Jr., Inorg. Synth., 32, 70 (1998). 

O 

Cu 

N 

H2 N 

R 

[Cu(L-ile)(L-ala)] [Cu(L-ile)(H2O) 2] 

[Cu(L-ile)(D-ala)] 

_____________________________________________________________________ 

325 

O 

N 

H 2 

O O


P207. Photochemical Reduction of Nitrite Catalyzed by Ruthenium 

Complex or Zinc Porphyrin-linked Nitrite Reductases and the Model Cu 

Complexes 

K. Yamaguchi a , N. Isoda a , A. Tani a , T. Okada a , S. Suzuki a , N. Nakamura b , H. Ohno b , 

N. Ikeda a 

a 

Department of Chemistry, Grad. Sch. of Science, Osaka University, 1-1 Machikaneyama, 560-0043, Toyonaka, 

Osaka, Japan 

e-mail: kazu@ch.wani.osaka-u.ac.jp 

b 

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, 

184-8588, Tokyo, Japan 

Copper-containing nitrite reductase (CuNIR), which is a key enzyme in biological denitrification, catalyzes the 

reduction of nitrite to nitrogen monoxide. In this paper, we investigated that and the photochemical reduction of 

nitrite to nitrogen monoxide by Ru(bpy)3 or Zn prophirin complex modified CuNIRs and the CuNIR model 

complexes [Cu(Me2bpa)] 2+ linked to Ru(bpy)3 analogue or Zn porphirin in the presence of sacrificial electron 

donor reagents under acidic condition at room temperature. Transient absorption spectra of Ru-Cu complex were 

observed by nanosecond laser flash photolysis (λex = 532 nm, fwhm 4 ns) at 298 K. The spectral change at 503 

nm due to Ru(III) moiety was observed, it is due to the intramolecular electron transfer from the excited Ru(II) 

to Cu(II) moieties in Ru-Cu complex. The same profiles were observed in the Zn prophirin linked Cu complex 

and the Ru complex modified CuNIR. Photo chemical NO production from the dinuclear complexes or the 

complex modified CuNIR solutions including nitrite and sacrificial electron donor reagent was observed under 

irradiation of visible light and the continuous flow of Ar at room temperature. This is the first example of the 

photochemical reduction of nitrite to nitrogen monoxide catalyzed by dinuclear complex and complex modified 

enzyme. 

References: 

[1]K. Yamaguchi, T. Okada, and S. Suzuki, Inorganic Chemistry Commun., 9, 989-991 (2006). 

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326


P208. Reinvestigation of Dioxygen Activation by α-Ketoglutarate 

Dependent Dioxygenase 

S. Ye a , C. Riplinger a , C. Krebs b , M. Bollinger b , and F. Neese a 

a Institute of Physical and Theoretical Chemistry, Bonn University, Germany 

b Department of Biochemistry, Penn State University, USA 

The TauD/α-ketoglutarate (α-KG)-dependent dioxygenase is a member of the superfamily of α-ketoglutaratedependent 

dioxygenase, a large and diverse class of mononuclear non-heme iron enzymes that require hs-Fe(II), 

α-KG and dioxygen for catalysis. The reaction mechanism for dioxygen activation in this enzyme system has 

been studied by hybrid density functional theory (DFT) at septet, quintet and triplet potential energy surfaces. 

The calculations showed that the dioxygen activation proceeds at the septet potential surface through only one 

transition state for a concerted O-O and C-C bond cleavage, which is consistent with the experimental findings 

that oxidative decarboxylation of the α-KG is the rate-limiting step. Detailed analysis of the electron pathways 

for the four-electron reduction process of dioxygen provided new insights into the catalytic mechanism: Why is 

only one transition state needed at the septet surface? Why can the similar process not take place at the quintet 

and triplet potential surfaces? What kind of roles do the hs-Fe(II) and α-KG play for the dioxygen activation? 

_____________________________________________________________________ 

327


P209. Cytotoxicity, Cellular Uptake and DNA Binding Mode of a New 

Dinuclear Platinum(II) Complex 

L. Zerzankova a , J. Kasparkova a , N.P. Farrell b , V. Brabec a 

a 

Institute of Biophysics ASCR, v.v.i., Kralovopolska 135, CZ-612 65, Brno, Czech Republic 

e-mail: zerzankova@ibp.cz 

b 

Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Virginia-232 84, 

Richmond, United States 

One concept of designing new platinum drugs is based on the observation that carrier amine ligands of cisplatin 

can modulate its anticancer properties. This concept has resulted in a new structural analog of cisplatin - 

oxaliplatin that is currently used in the clinic. Another series of antitumor platinum compounds is based on 

polynuclear geometry. From this large family, the sub-class [{PtCl(NH3)2}2µ-(H2N-Y-NH2)] n+ , where the 

bridging linker contains a diamine, polyamine or a third coordination sphere, was chosen for clinical 

development. We examined the biological properties of novel dinuclear Pt II complex BBR3610-DACH (figure 

1). In this compound, the structural features of two classes of the platinum compounds with proven antitumor 

activity are combined, namely DACH carrier ligands and polynuclear platinum geometry with a polyamine 

linker. DNA binding mode of this new complex was analyzed by biophysical and biochemical methods. These 

modifications have been compared with the cytotoxicity and mutagenicity of this new complex in several tumor 

cell lines, cellular uptake and inhibition of DNA synthesis. The results show that the new complex coordinates 

DNA in a unique way, different from that of mononuclear analog as well as from that of dinuclear spermine 

complex. These results are also consistent with the observation that the new dinuclear complex shows different 

biological properties in human tumor cell lines. 

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328


P210. Interaction of Cap43 Protein Fragment with Ni(II) and Cu(II) Ions 

M.A. Zoroddu a , M. Peana a , and S. Medici a , T. Kowalik-Jankowska b , H. Kozłowski b 

a 

Department of Chemistry, University of Sassari, Via Vienna 2, 07100, Sassari, Italy 

e-mail: zoroddu@uniss.it 

b 

Faculty of Chemistry, University of Wrocław, 14 Joliot-Curie, 50-383 Wrocław, Poland 

One of the research topics in our group is protein Cap 43, which seems directly related to the cellular response 

after nickel exposure, [1, 2] as well as to a number of cancers.[1, 3] The studies we carried out for several years 

started from a small peptide model of Cap 43 and were successively expanded to include a two- 

(TRSRSHTSEG-TRSRSHTSEG) and a three-repeated (TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG) 

monohistidinic decapeptide fragment in its C-terminus.[4-6] Such 20- and 30-amino acid sequences were tested 

for Ni(II)- and Cu(II)- coordination at different pH values, by both potentiometry and spectroscopic techniques 

(NMR, EPR, UV-Vis, CD). The two metals showed a slightly different behaviour towards coordination, and the 

interaction of Cu(II) ions with the two peptides started at pH values lower than those for Ni(II). What appeared 

clear was that in both cases each 10-amino acid fragment TRSRSHTSEG was able to coordinate a single metal 

ion to form a square planar 4N {4N} chromophore. The coordination mode involved an imidazole nitrogen from 

the His residue, and the amidic nitrogens from His, Ser, and Arg. At high pH values, further deprotonations were 

observed for the Cu(II) species. 

References: 

[1] D. Zhou, K. Salnikow, M. Costa; Cancer Res., 58, 2182 (1998) 

[2] K. Salnikow, D. Zhou, T. Kluz, C. Wang, M. Costa, in: Metal and Genetics, (Sarkar Bed), New York, 131 

(1999) 

[3] K. Salnikow, T. Kluz, M. Costa, Toxicol. Appl. Pharmacol., 160, 127 (1999) 

[4] M.A. Zoroddu, T. Kowalik-Jankowska, H. Kozlowski, K. Salnikow, M. Costa, J. Inorg. Biochem., 84, 47 

(2001) 

[5] M.A. Zoroddu, M. Peana, T. Kowalik-Jankowska, H. Kozlowski, M. Costa, J. Chem. Soc. Dalton Trans., 458 

(2002) 

[6] M.A. Zoroddu, M. Peana, T. Kowalik-Jankowska, H. Kozlowski, M. Costa, J. Inorg Biochem., 98, 931 

(2004) 

_____________________________________________________________________ 

329


P211. Polymorphic Forms of Zn(II) Compound with Biological Active 

Diethyl (Pyridin-3-Ylmethyl)Phosphonate 

(3-Pmpe) Ligand: Zn(3-Pmpe)Cl2 

B. Żurowska a , K. Ślepokura a , T. Lis a , J. Ochocki b 

a Faculty of Chemistry, University of Wroclaw, 50-383 Wroclaw, Poland 

b Department of Bioinorganic Chemistry, Faculty of Pharmacy, Medical University, 90-151 Lodz, Poland 

e-mail: zurowska@wchuwr.pl 

Zinc is one of the most important trace elements playing a versatile role in biological systems due to its structural 

and catalytic roles in enzymes. On the other hand, cis-Platinum(II) complexes of N-heterocyclic phosphonate 

ligands exhibit biological activity [1, 2]. Therefore we have now studied spectroscopy and the crystal structures 

of different crystalline forms of the compound of the empirical formula Zn(3-pmpe)Cl2. 

X-ray analyses show that in the reaction of ZnCl2 with didentate ligand diethyl (pyridin-3-ylmethyl) phosphonate 

(3-pmpe) in methanol three crystalline polymorphs are formed: [Zn(3-pmpe)Cl2]2 (1) and [Zn(3-pmpe)Cl2]n (2 

and 3). In these crystals 3-pmpe acts as a didentate N, O-bridging ligand and Zn(II) are in a slightly distorted 

tetrahedral ZnNOCl2 environment. Zn(II) ions in 1 are doubly bridged by the 3-pmpe ligands, resulting in the 

formation of dinuclear species. In polymeric compounds 2 and 3 Zn(II) ions are singly bridged by the 3-pmpe, 

resulting in the formation of one-dimensional chains. 

1 2 

Crystals of compound 1 are triclinic, space group P1̅, Z = 2, with cell parameters: a = 8.228(2), 

b = 8.982(2), c = 10.329(3) Å, α = 78.97(3), β = 89.92(3), γ = 81.04(3)º, V = 739.8(3) Å 3 . 

Crystals of compound 2 are monoclinic P21, Z = 4, with cell parameters: a = 7.742(2), 

b = 28.246(6), c = 7.864(2)Å, β = 117.60(3)º, V = 739.8(3) Å 3 . 

The third polymorphous form (polymeric 3) is triclinic, P1̅, Z = 2 with cell parameters: a = 8.850(3) Å, 

b = 9.060(4) Å, c = 10.305(4) Å, α = 113.50(4)°, β = 98.91(3)°, γ = 95.27(3)°, V = 1524.0(6)Å 3 . 

References: 

[1] K. Aranowska, J. Graczyk, L. Chęcińska, W. Pakulska, J. Ochocki, Pharmazie 61 (2006) 457. 

[2] B. Kostka, J. Sikora, K. Aranowska, J. Para, J. Ochocki, Acta Toxicologica 13 (2005) 113. 

_____________________________________________________________________ 

330 

3

Author Index 


_____________________________________________________________________ 

331


Aartsma...............................................117, 123, 308 

Abad Andrade .................................................... 120 

Abbas ................................................................. 248 

Abdelhamid...........................................30, 214, 260 

Adolph ............................................................... 195 

Ahmedova.......................................................... 121 

AlAgha................................................................. 74 

Alarcón-Payer .................................................... 122 

Alberto ............................................................... 232 

Aldrich-Wright................................................... 222 

Alessio ................................................................. 23 

Alfonso-Prieto...................................................... 80 

Alham................................................................. 178 

Almeida.............................................................. 295 

Alpoim ............................................................... 150 

Amara................................................................... 62 

Andberg ............................................................. 192 

Anđelković......................................................... 247 

Anderlund .......................................................... 130 

Andersson .............................84, 112, 197, 265, 313 

Andolfi ............................................................... 170 

Andrade.............................................................. 295 

Andreoni ............................................................ 123 

Andreozzi........................................................... 166 

Annalora............................................................... 88 

Antonyuk.............................................................. 89 

Anwar................................................................. 248 

Arakawa ............................................................. 261 

Archer ................................................................ 264 

Arellano ............................................................. 180 

Armstrong .............................28, 183, 269, 282, 284 

Arnoux ............................................................... 114 

Asada ................................................................. 245 

Atrian ............................................36, 133, 266, 268 

Atta....................................................................... 62 

Bacco ................................................................... 77 

Bachurin............................................................... 47 

Badea ......................................................... 124, 263 

Baffert ................................................................ 125 

Bal................................................................ 76, 271 

Balenci ............................................................... 126 

Ballmann.............................................................. 51 

Baranowska........................................................ 157 

Barda.................................................................... 25 

Barnett................................................................ 251 

Barone................................................................ 127 

Barra..................................................................... 84 

Barragán............................................................. 128 

Barszczewski...................................................... 306 

Bartosz-Bechowski .................................... 215, 279 

Barys .................................................................. 164 

Basallote............................................................. 108 

Basinski.............................................................. 233 

Basran ................................................................ 280 

Battistoni.............................................................. 64 

Battistuzzi .......................................................... 118 

Becker ........................................................ 129, 223 

Bell..................................................................... 313 

Bellandi.............................................................. 314 

_____________________________________________________________________ 

332 

Berends ................................................................ 96 

Bergan.................................................................. 84 

Berggren............................................................. 130 

Bermejo.............................................................. 169 

Berry .................................................................... 90 

Bertoncini............................................................. 78 

Bertrand ......................................113, 114, 125, 229 

Besson................................................................ 301 

Bhachu ................................................................. 20 

Biega .......................................................... 131, 132 

Biernat................................................................ 152 

Bill ....................................................................... 51 

Binolfi .................................................................. 78 

Bjerrum ...........................................56, 99, 143, 196 

Blanford ............................................................. 269 

Blasco................................................................. 108 

Blindauer...................................................... 67, 230 

Blondin................................................................. 94 

Boersma ............................................................. 286 

Bofill .................................................................. 133 

Bogusz ............................................................... 208 

Böhme................................................................ 207 

Boiry .................................................................. 114 

Bollinger ............................................................ 327 

Bonechi .............................................................. 126 

Bonifacio............................................................ 252 

Bonna ................................................................. 201 

Borel..................................................................... 94 

Borsari................................................................ 118 

Botelho............................................................... 134 

Bothe.................................................................... 51 

Boucher.............................................................. 265 

Bourles ............................................................... 135 

Boussac .............................................................. 107 

Boyko................................................................... 50 

Brabec ...........................57, 193, 219, 239, 258, 328 

Brandi-Blanco ............................................ 122, 136 

Brasuń .........................................137, 138, 139, 243 

Bratsos ................................................................. 23 

Bregier-Jarzebowska.......................................... 205 

Bren.................................................................... 112 

Breukink............................................................. 308 

Brillouet ............................................................. 160 

Brindell ........................................................ 69, 140 

Brondino .............................................249, 254, 283 

Brouwer ............................................................. 240 

Brown................................................................. 260 

Bruijnincx .......................................................... 191 

Brynda................................................................ 267 

Bryszewska ........................................................ 141 

Bubacco ............................................................. 308 

Bubnov................................................................. 86 

Budzisz............................................................... 142 

Buglyó.................................................................. 60 

Bukh................................................................... 143 

Burdjiev ............................................................. 121 

Burger ........................................................ 144, 324 

Burkholz............................................................. 248 

Burlat ..........................................113, 114, 125, 229

Bursakov .................................................... 145, 180 

Butler ................................................................... 74 

Cabral................................................................. 145 

Calvete ....................................................... 145, 180 

Camara ............................................................... 141 

Cancines............................................................. 314 

Cannistraro......................................................... 170 

Canters ........................................117, 123, 307, 308 

Capdevila ......................................36, 133, 266, 268 

Cardo.................................................................. 104 

Cardoso Pereira.......................................... 264, 318 

Carepo.........................................146, 187, 283, 295 

Carlsson ............................................................... 75 

Carpena ................................................................ 80 

Carvalho............................................................. 148 

Casella........................................................ 154, 165 

Castillo Gonzalez ............................................... 108 

Castiñeiras...........122, 136, 158, 179, 185, 272, 294 

Caux-Thang.......................................................... 94 

Cebrat..........................................137, 147, 243, 279 

Cerveñansky......................................................... 78 

Chakraborty........................................................ 305 

Chauhan ............................................................. 280 

Cheesman........................................................... 280 

Chifiriuc Balotescu .................................... 124, 263 

Cho..................................................................... 228 

Chojnacki ........................................................... 225 

Chong................................................................... 31 

Choquesillo-Lazarte ............122, 136, 158, 185, 294 

Christensen H.E.M............................................. 321 

Christensen N.J. ................................................. 305 

Christianen ......................................................... 117 

Chumakov.......................................................... 203 

Ciattini ............................................................... 121 

Ciesiołka ...................................................... 70, 164 

Cieślak-Golonka ................................................ 278 

Ciudad................................................................ 128 

Ciunik................................................................. 164 

Ciurli .................................................................... 21 

Coelho.................................................148, 184, 254 

Coggins ................................................................ 27 

Collins................................................................ 248 

Comba.......................................................... 46, 149 

Contreras............................................................ 314 

Costa .................................................................. 150 

Courbon ............................................................. 160 

Cournac.............................................................. 113 

Crichton ............................................................... 26 

Crisponi.............................................................. 272 

Crook ................................................................. 151 

Csapó ................................................................... 60 

Cun....................................................................... 63 

Cydzik................................................................ 152 

Czeluśniak.......................................................... 153 

D’Alfonso G....................................................... 159 

D’Alfonso L. ...................................................... 159 

D’Amelio ................................................... 126, 224 

Dallavalle ........................................................... 102 

Dallinger ............................................................ 268 

Dawson ................................................................ 27 


De Gioia............................................................... 29 

De Martino ......................................................... 307 

de Oliveira Silva ................................................ 155 

de Val................................................................... 26 

de Vries .............................................................. 253 

DeBeer George............................................. 73, 200 

Dechert..........................................51, 144, 172, 324 

Declercq ............................................................... 26 

Deeth.................................................................. 168 

Degerman........................................................... 256 

DeGrado............................................................. 166 

Dell’Acqua......................................................... 154 

Delord ................................................................ 160 

Dementin.............................................113, 114, 229 

Demuez .............................................................. 125 

den Dulk............................................................. 240 

Dennison .............................................................. 54 

Derat..................................................................... 80 

Dertz................................................................... 200 

Di Nardo..................................................... 156, 170 

Di Venere........................................................... 156 

Dijkstra................................................................. 81 

Dimitrakopoulou .................................................. 41 

Djinović-Carugo .................................................. 54 

Djoko ................................................................... 31 

Doering .............................................................. 248 

Dohmae.............................................................. 261 

Dolla................................................................... 283 

Dołęga................................................................ 157 

Domínguez-Martín..................................... 158, 294 

Donghi ............................................................... 159 

Donnadieu.......................................................... 182 

Dooley................................................................ 260 

Dorbes................................................................ 160 

Dorlet ................................................................... 93 

Dou..................................................................... 285 

Dowling ............................................................. 161 

Drew................................................................... 162 

Duarte....................................................94, 146, 238 

Düpre ..................................................163, 207, 270 

Duran ................................................................... 78 

Durand ............................................................... 283 

Dziuba........................................................ 126, 164 

Efimov ............................................................... 280 

Egg ..................................................................... 268 

Egmond................................................................ 81 

El Ghazouani........................................................ 94 

Engelen .............................................................. 165 

Engelkamp ......................................................... 117 

Ensign ................................................................ 112 

Erat..................................................................... 218 

Eriksson ............................................................. 130 

Faiella................................................................. 166 

Faller ............................................................ 93, 167 

Fantuzzi.............................................................. 170 

Farkas................................................................... 60 

Farrell......................................................... 105, 328 

Farrer.................................................................. 168 

Feese .................................................................. 101 

Feringa ............................................................... 286 

_____________________________________________________________________ 

333


Fernandes ........................................................... 171 

Fernández............................................................. 78 

Fernandez-Garcia............................................... 169 

Ferrari................................................................. 165 

Ferrer............................................................ 94, 108 

Ferrero................................................................ 170 

Figueiredo .......................................................... 146 

Filimonova ........................................................... 47 

Filipov................................................................ 203 

Fita ....................................................................... 80 

Fletcher .............................................................. 317 

Florek ................................................................. 306 

Fontal ................................................................. 314 

Fontecave ................................................37, 62, 288 

Fontecilla-Camps ..................................62, 113, 229 

Fontes Costa Lima ............................................. 171 

Formaggio .......................................................... 285 

Fourmond........................................................... 114 

Fregona .............................................................. 285 

Freisinger ..............................................35, 178, 230 

Freitas................................................................. 171 

Friedrich............................................................. 116 

Frison ................................................................. 109 

Fritsky ...........................................50, 237, 289, 304 

Fritz.................................................................... 134 

Fuchs............................................................ 51, 172 

Fuglewicz........................................................... 137 

Fujii............................................................ 173, 199 

Fujita .................................................................. 260 

Fujiwara ............................................................. 245 

Fuks............................................................ 174, 175 

Funabiki ..................................................... 115, 217 

Funahashi ..............................98, 199, 244, 262, 297 

Gabriel ............................................................... 292 

Gabriela Almeida ............................................... 301 

Gaggelli E. ....................................44, 126, 176, 226 

Gaggelli N.......................................................... 126 

Gahan ................................................................... 22 

Gajda.............................................................. 39, 64 

Gajewska............................................................ 177 

Galstyan ............................................................. 178 

Gałęzowska .................................................. 58, 304 

Gambino............................................................. 127 

Garcia................................................................. 128 

García-España .................................................... 108 

García-Santos............................................. 179, 185 

Garcia-Tojal ....................................................... 182 

Garino ................................................................ 291 

Garner .................................................................. 20 

Gasowska ........................................................... 233 

Gavel.......................................................... 145, 180 

Ge......................................................................... 63 

George................................................................ 283 

Gharib ................................................................ 181 

Ghiladi ............................................................... 101 

Ghosh ................................................................. 251 

Gianferrara ........................................................... 23 

Gilardi ........................................................ 156, 170 

Gil-Garcia .......................................................... 182 

Ginanneschi........................................................ 139 

_____________________________________________________________________ 

334 

Girbal ................................................................. 125 

Glazer................................................................... 88 

Glueck.................................................................. 97 

Gładysz .............................................................. 138 

Gniazdowska...................................................... 174 

Gobetto............................................................... 291 

Goldet................................................................. 183 

Golenia................................................................. 50 

Gomes ................................................................ 134 

González .............................108, 148, 184, 249, 254 

González-Noya .................................................. 169 

González-Pérez ...122, 136, 158, 179, 185, 272, 294 

Goodin ................................................................. 88 

Görbitz ............................................................... 197 

Gómez Quiroga.................................................. 293 

Gómez-Herrero .................................................. 163 

Górniak .............................................................. 188 

Górny ................................................................. 235 

Grabowski.......................................................... 278 

Graff................................................................... 146 

Gralka........................................................... 77, 186 

Gräslund............................................................... 16 

Gray ..................................................................... 88 

Grazina............................................................... 187 

Griesinger............................................................. 78 

Grimling..................................................... 188, 189 

Gros...................................................................... 81 

Grossmann ........................................................... 89 

Grzonka.............................................................. 131 

Gudat.................................................................. 157 

Guedes da Silva.......................................... 177, 302 

Güell..................................................................... 45 

Guerrini................................................................ 77 

Guigliarelli ..................................113, 114, 125, 229 

Guilloreau .......................................................... 167 

Gumienna-Kontecka ...............................50, 58, 289 

Guo....................................................................... 24 

Guzow................................................................ 215 

Habib.................................................................. 190 

Habtemariam...................................................... 191 

Hadler................................................................... 22 

Hagen ................................................................. 253 

Hakulinen........................................................... 192 

Halamikova ........................................................ 193 

Hamada .............................................................. 217 

Hamelin.............................................................. 288 

Hannam.............................................................. 141 

Hannon..................................................10, 104, 293 

Hanson ............................................................... 162 

Harbitz ............................................................... 112 

Harmer ............................................................... 284 

Harris ................................................................. 321 

Hartwig .............................................................. 271 

Hashimoto .......................................................... 194 

Hasnain ................................................................ 89 

Hatzakis ............................................................. 117 

Haukka ..................................................75, 204, 256 

Hedman.............................................................. 200 

Heering....................................................... 253, 320 

Heinz.................................................................. 195

Hemmingsen ...............................195, 196, 290, 305 

Heringova........................................................... 193 

Herman............................................................... 157 

Hersleth...................................................... 197, 265 

Hewener ............................................................. 203 

Hewison ............................................................. 198 

Heydari............................................................... 211 

Higa.................................................................... 199 

Hildebrandt ................................................ 116, 221 

Hine...................................................................... 20 

Hirota ................................................................. 244 

Hitomi ........................................................ 115, 217 

Hocking.............................................................. 200 

Hockner.............................................................. 268 

Hodgson ............................................................. 200 

Hoffmann ........................................................... 312 

Holm-Jørgensen ................................................... 56 

Hori .................................................................... 261 

Hough................................................................... 89 

Hsiao .................................................................. 197 

Hu....................................................................... 110 

Huang................................................................. 130 

Hughes ............................................................... 161 

Hureau.................................................................. 93 

Husted ................................................................ 259 

Hutyra ................................................................ 235 

Ikeda................................................................... 326 

Imagawa............................................................. 245 

Inomata ........................................................ 98, 199 

Ishida.................................................................. 107 

Isoda................................................................... 326 

Ito....................................................................... 325 

Itoh..................................................................... 209 

Ivancich................................................................ 82 

Jacob Claus ........................................................ 248 

Jacquamet............................................................. 94 

Jahangir ............................................................. 181 

Jameson................................................................ 83 

Jancsó............................................................. 39, 64 

Janicka-Kłos....................................................... 201 

Janicki ........................................................ 202, 315 

Jankowska.................................................. 131, 309 

Janoschka ........................................................... 203 

Jańczyk................................................................. 69 

Jański ................................................................. 225 

Jarenmark......................................75, 144, 204, 324 

Jarjayes............................................................... 287 

Jaroszewicz ........................................................ 225 

Jastrzab............................................................... 205 

Jaszewski............................................................ 322 

Jensen K......................................................... 56, 99 

Jensen M ............................................................ 206 

Jensen M. ....................................................... 56, 99 

Jerzykiewicz....................................................... 322 

Jezierska......................................208, 242, 267, 322 

Jezierski ............................................................. 322 

Jeżowska-Bojczuk...............126, 164, 216, 224, 250 

Jingwei ................................................................. 47 

Jitsukawa............................................................ 262 

João Romão........................................................ 148 


Johannsen................................................... 207, 270 

Jozwiak .............................................................. 142 

Jyunichi.............................................................. 173 

Kafarski...................................................... 242, 274 

Kajita.....................................................98, 199, 244 

Kajiya................................................................. 255 

Kallio ................................................................. 192 

Kamecka ............................................................ 208 

Kamysz ...................................................... 186, 226 

Kano........................................................... 209, 217 

Kapczyńska ........................................................ 213 

Karotki ............................................................... 100 

Kasparkova ...................................57, 193, 219, 328 

Kasuno ............................................................... 245 

Katayama ........................................................... 261 

Kaur ................................................................... 112 

Kawakami .......................................................... 173 

Kayal.................................................................. 210 

Kelly................................................................... 317 

Keppler................................................................. 32 

Kessissoglou ................................................ 41, 277 

Ketomäki.............................................................. 39 

Khorasani-Motlagh .................................... 211, 257 

Khutia................................................................. 212 

Kierzenkowska................................................... 215 

Kijewska ............................................................ 213 

Kikkeri ................................................................. 25 

Kimura ............................................................... 214 

Kitagawa ............................................................ 260 

Kitagishi............................................................. 209 

Kladova.............................................................. 145 

Kleffmann ............................................................ 83 

Klein Gebbink...................................................... 81 

Klijn ................................................................... 286 

Kluczyk.......................................152, 215, 216, 279 

Knipp ................................................................... 90 

Knobloch............................................................ 250 

Knör ..................................................................... 68 

Koch................................................................... 134 

Kochel................................................................ 306 

Kodera........................................................ 115, 217 

Kohzuma...............................................30, 214, 260 

Koivula............................................................... 192 

Kolozsi ................................................................. 64 

Komeda.............................................................. 105 

Konopińska ........................................................ 132 

Kooijman............................................................ 240 

Kopera.......................................................... 76, 271 

Korbas................................................................ 283 

Korth .................................................................. 218 

Köse ................................................................... 220 

Kostrhunova....................................................... 219 

Kothari ............................................................... 175 

Kowalik-Jankowska ....................131, 132, 242, 329 

Kozłowski ..44, 50, 58, 77, 186, 201, 226, 237, 250, 

289, 309, 310, 329 

Kozminski.......................................................... 174 

Krämer ................................................................. 12 

Kranich............................................................... 221 

Krause-Heuer ..................................................... 222 

_____________________________________________________________________ 

335


Krebs.................................................................. 327 

Krężel................................................................... 76 

Kropidłowska............................................. 129, 223 

Kroutil.................................................................. 97 

Król .................................................................... 306 

Kruus.................................................................. 192 

Kubiak................................................................ 241 

Kucharczyk ........................................................ 224 

Kuczer................................................................ 132 

Kuduk-Jaworska ........................................ 225, 241 

Kulon ................................................................. 226 

Kumar Patel ....................................................... 185 

Kurz ..................................................................... 96 

Kurzak................................................................ 208 

Kuznetsova................................................. 117, 123 

Lachowicz.......................................................... 272 

Lai ...................................................................... 107 

Lamberto .............................................................. 78 

Lara ...................................................................... 97 

Lascoux................................................................ 94 

Latorre................................................................ 108 

Latos-Grażyński................................................. 323 

Latour........................................................... 94, 135 

Lau ..................................................................... 227 

Lazar .......................................................... 124, 263 

Lee ..................................................................... 299 

Lee H.I. .............................................................. 228 

Lee J.E. .............................................................. 228 

Léger ...........................................113, 114, 125, 229 

Leino .................................................................... 39 

Leitgeb ................................................................. 92 

Lendzian............................................................. 116 

Lenz ................................................................... 116 

Leroux.................................................113, 125, 229 

Leszczyszyn ................................................. 67, 230 

Licandro ............................................................. 159 

Lim....................................................................... 26 

Lippert...................................................38, 178, 212 

Lis .............................................................. 241, 330 

Lisowski............................................................. 152 

Lista ................................................................... 231 

Liu...................................................................... 232 

Loewen................................................................. 80 

Lombard............................................................. 265 

Lombardi.................................................... 166, 307 

Lomozik ..................................................... 205, 233 

Lönnberg.............................................................. 39 

Lopes.................................................................. 283 

Lorenz ........................................................ 142, 234 

Luchinat ................................................................. 9 

Luchter-Wasylewska.......................................... 235 

Ludwig ....................................................... 116, 320 

Luis ...................................................................... 45 

Lutz ...................................................................... 81 

Ly....................................................................... 221 

Łabuz ................................................................... 69 

Łakomska..................................................... 66, 236 

Macedo................................................................. 54 

Maciąg ............................................................... 237 

Maciejewska ...................................................... 278 

_____________________________________________________________________ 

336 

Macyk .................................................................. 69 

Maddelein .......................................................... 167 

Maglio................................................................ 166 

Magnussen ......................................................... 196 

Maia ................................................................... 238 

Maiorana ............................................................ 159 

Makowski........................................................... 138 

Malina ...................................................57, 239, 258 

Maneiro.............................................................. 169 

Máñez................................................................. 108 

Mann .......................................................... 151, 198 

Mannila ................................................................ 39 

Mansuy................................................................. 15 

Marchiò.............................................................. 102 

Marcinkowska.................................................... 309 

Maret.................................................................... 13 

Marinescu................................................... 124, 263 

Marini................................................................. 219 

Marques-Gallego................................................ 240 

Mastalarz A........................................................ 241 

Mastalarz H........................................................ 241 

Masuda............................................................... 262 

Masuda H......................................98, 199, 244, 297 

Masuda M. ......................................................... 255 

Matczak-Jon....................................................... 242 

Matera-Witkiewicz..................................... 139, 243 

Mathevon ............................................................. 62 

Matias................................................................. 264 

Matsumoto ......................................................... 244 

Matsushita.......................................................... 245 

Mattsson............................................................... 91 

Mayer ................................................................. 142 

McKee.................................................................. 42 

Mecklenburg ...................................................... 248 

Medici ................................................................ 329 

Megger ............................................................... 246 

Mei..................................................................... 156 

Meler.................................................................. 189 

Melman ................................................................ 25 

Meloni................................................................ 167 

Merkx................................................................. 103 

Messori............................................................... 139 

Meunier................................................................ 43 

Meyer ....................................51, 144, 172, 237, 324 

Meynial-Sall....................................................... 125 

Mikkelsen........................................................... 290 

Milacic ............................................................... 285 

Milaeva ................................................................ 47 

Milenković ......................................................... 247 

Millo................................................................... 116 

Milosavljević...................................................... 247 

Mino................................................................... 261 

Miodragović Dj.................................................. 247 

Miodragović Z ................................................... 247 

Mitewa ............................................................... 121 

Mitić............................................................. 22, 247 

Młynarz.............................................................. 224 

Mochizuki .......................................................... 115 

Mockel ............................................................... 167 

Mohammed ........................................................ 248

Mokhir ................................................................. 71 

Molteni....................................................... 126, 310 

Monari................................................................ 118 

Mondry....................................................... 202, 315 

Montaña ............................................................. 128 

Monteiro............................................................. 266 

Monzani ..................................................... 154, 165 

Moreno....................................................... 128, 150 

Morita................................................................. 325 

Mota........................................................... 249, 283 

Motterlini ................................................... 151, 198 

Moura I.48, 145, 146, 148, 154, 184, 187, 249, 252, 

254, 283, 295 

Moura J.49, 145, 146, 148, 154, 184, 187, 238, 249, 

254, 283, 295, 301 

Mucha ................................................................ 250 

Mukherjee .......................................................... 251 

Mukhopadhyay .......................................... 184, 252 

Müller............................72, 163, 207, 246, 270, 296 

Munoz Olivas..................................................... 141 

Murata................................................................ 255 

Murgida.............................................................. 221 

Mutti..................................................................... 97 

Nadolny.............................................................. 226 

Naghi Torabi ...................................................... 257 

Nagy..................................................................... 60 

Nahid Hasan....................................................... 253 

Najmudin.............................................148, 184, 254 

Nakamura............................................217, 255, 326 

Nakayama .......................................................... 261 

Nam...................................................................... 11 

Nastri.................................................................. 166 

Natile.......................................................... 193, 258 

Navarro Ranninguer........................................... 293 

Neese............................................................ 79, 327 

Negom Kouodom............................................... 285 

Nerdal................................................................. 206 

Neves ................................................................. 175 

Niclós-Gutiérrez..122, 136, 158, 179, 185, 272, 294 

Nicolet.................................................................. 62 

Nidetzky............................................................... 92 

Niittymäki ............................................................ 39 

Nilsson ......................................................... 84, 256 

Nishikawa ............................................................ 98 

Noe..................................................................... 128 

Noguchi.............................................................. 194 

Nolan.................................................................... 74 

Nolte................................................................... 117 

Nordlander ............................75, 144, 204, 256, 324 

Noroozifar.................................................. 211, 257 

Novakova ........................................................... 258 

Nunez ................................................................... 74 

Nurchi ................................................................ 272 

Nymark Hegelund .............................................. 259 

O’Halloran ............................................................. 8 

Obara............................................................ 30, 260 

Obrist ................................................................. 110 

Ochocki.............................................................. 330 

Odaka ......................................................... 194, 261 

Odani.......................................................... 105, 205 


Ohenessian ......................................................... 109 

Ohno........................................................... 255, 326 

Okada ......................................................... 260, 326 

Okumura ............................................................ 262 

Olar ............................................................ 124, 263 

Olbert-Majkut .................................................... 224 

Olczak ........................................................ 275, 323 

Oleksińska.......................................................... 303 

Olędzki............................................................... 271 

Oliveira .............................................................. 264 

Ollagnier de Choudens....................................... 288 

Olsbu.................................................................. 265 

Olsen .................................................................. 305 

Ołdziej................................................................ 139 

Orihuela ............................................................. 266 

Orkey ................................................................. 222 

Ortolani .............................................................. 156 

Osborne................................................................ 27 

Ozawa ...................................98, 199, 244, 262, 297 

Ożarowski .................................................. 267, 322 

Pagani................................................................. 268 

Paksi..................................................................... 64 

Palacios .............................................................. 268 

Paluma ............................................................... 271 

Panigati .............................................................. 159 

Papadopoulos ..................................................... 174 

Papagiannopoulou.............................................. 174 

Pappalardo............................................................ 60 

Parkin ................................................................. 269 

Pasikowski ......................................................... 152 

Pauleta........................................................ 146, 154 

Paulsen ............................................................... 203 

Paulus................................................................. 270 

Pavone................................................................ 307 

Peana.................................................................. 329 

Pechlaner.............................................................. 54 

Pecoraro ....................................................... 29, 305 

Pelecanou ........................................................... 174 

Pellecchia ........................................................... 307 

Penkova.............................................................. 237 

Pereira A. ........................................................... 283 

Pereira A.S. ................................................ 146, 154 

Perez Del Valle .................................................. 287 

Peroza................................................................. 230 

Petersen.............................................................. 321 

Philouze ............................................................. 287 

Piątek ................................................................. 271 

Picot ................................................................... 109 

Pignol ................................................................. 114 

Pirmettis ............................................................. 174 

Pivetta ................................................................ 272 

Pizarro................................................................ 291 

Plińska................................................................ 137 

Pluta ........................................................... 188, 189 

Podsiadły.................................................... 273, 316 

Podstawka .......................................................... 274 

Poijärvi-Virta ....................................................... 39 

Poirot.................................................................. 160 

Polikarpov.......................................................... 180 

Polonius ............................................................. 246 

_____________________________________________________________________ 

337


Połata ......................................................... 275, 323 

Pombeiro.................................................... 177, 302 

Porto................................................................... 171 

Posewitz ............................................................... 62 

Powell .................................................................. 40 

Poznański ............................................................. 76 

Prahl................................................................... 201 

Pratesi................................................................. 139 

Predko ................................................................ 216 

Prencipe ............................................................. 159 

Prieto.......................................................... 128, 150 

Prinson ............................................................... 276 

Proniewicz.......................................................... 274 

Pruchnik ............................................................. 302 

Psomas ............................................................... 277 

Puchalska ........................................................... 306 

Puszyńska-Tuszkanow....................................... 278 

Que..................................................................... 281 

Quinn ................................................................. 317 

Quintanar.............................................................. 78 

Radecka-Paryzek.................................................. 85 

Rafice ................................................................. 280 

Ranieri................................................................ 118 

Rappaport........................................................... 107 

Raptopoulou............................................... 174, 277 

Ratajska.............................................................. 279 

Ravanat ................................................................ 94 

Raven ................................................................. 280 

Ray..................................................................... 281 

Raymond ............................................................ 200 

Reedijk ......................................................... 18, 240 

Regan ................................................................. 161 

Regiec ................................................................ 241 

Rehder................................................................ 256 

Reisner ............................................................... 282 

Remelli..................................................77, 102, 272 

Reyes.................................................................. 314 

Richter................................................................ 320 

Riplinger ............................................................ 327 

Rivas .......................................................... 249, 283 

Rodríguez-Doutón.............................................. 169 

Roelfes ....................................................... 106, 286 

Roessler.............................................................. 284 

Røhr ............................................................. 84, 313 

Roig.................................................................... 180 

Romão C.C................................................. 184, 254 

Romão M.J..........................................184, 252, 254 

Romero............................................................... 314 

Ronconi.............................................................. 285 

Rosati ................................................................. 286 

Roshani.............................................................. 181 

Rotthaus ............................................................. 287 

Rousset................................................113, 229, 288 

Rouvinen............................................................ 192 

Rovira................................................................... 80 

Rowan ................................................................ 117 

Rowińska-Żyrek................................................. 289 

Roy..................................................................... 210 

Rubach ................................................................. 62 

Rubka ................................................................. 202 

_____________________________________________________________________ 

338 

Rud-Petersen ...................................................... 290 

Ruggirello .......................................................... 127 

Rutherford.......................................................... 107 

Rutten................................................................... 81 

Ryde ......................................................51, 172, 197 

Sabaty................................................................. 114 

Sadeghi....................................................... 156, 170 

Sadlej-Sosnowska .............................................. 174 

Sadler ............................33, 168, 191, 219, 251, 291 

Saggu ................................................................. 116 

Salassa................................................................ 291 

Salifoglou........................................................... 292 

Sanchez Cano..................................................... 293 

Sánchez de Medina-Revilla........................ 158, 294 

Sancho Oltra....................................................... 106 

Sandvik ................................................................ 84 

Sanna.................................................................... 60 

Santos................................................................. 155 

Sanz.................................................................... 180 

Sarkar ................................................................. 299 

Sato ...................................................................... 54 

Sauer .................................................................. 290 

Sawle.......................................................... 151, 198 

Sawoska ............................................................. 140 

Scapens .............................................................. 151 

Schenk.................................................................. 22 

Schiller ............................................................... 259 

Schjoerring......................................................... 259 

Schmid ................................................................. 54 

Schmutz ............................................................. 232 

Schneider.............................................................. 29 

Schulzke................................................53, 120, 276 

Schünemann....................................................... 203 

Schwerdtle.......................................................... 271 

Scozzafava ........................................................... 55 

Sénèque.............................................................. 135 

Serra................................................................... 295 

Seubert ............................................................... 296 

Shaaban.............................................................. 248 

Shaik .................................................................... 80 

Shanzer................................................................. 25 

Shen ................................................................... 178 

Shevtsova ............................................................. 47 

Shibayama.......................................................... 297 

Shimazaki................................................... 298, 325 

Shimomaki ......................................................... 115 

Shin .................................................................... 299 

Shiraiwa ............................................................. 325 

Shnyrov.............................................................. 180 

Shpakovsky.......................................................... 47 

Shteinman ............................................................ 75 

Sigel H. .............................................................. 300 

Sigel R.K.O...........................17, 207, 218, 250, 270 

Silveira ............................................................... 301 

Silvente-Poirot ................................................... 160 

Silvestri .............................................................. 127 

Singh .................................................................. 222 

Siwek ................................................................. 118 

Skała................................................................... 126 

Sladić ................................................................... 65

Smirnova............................................................ 271 

Smith S................................................................. 22 

Smith W. .............................................................. 89 

Smoleński........................................................... 302 

Sochacka ............................................................ 147 

Solà .............................................................. 45, 118 

Solomon ..................................................... 200, 313 

Sono ..................................................................... 27 

Sonois................................................................. 167 

Soriano ............................................................... 108 

Søtofte.................................................................. 99 

Soucaille............................................................. 125 

Sousa.................................................................. 187 

Souza.................................................................... 78 

Sowińska J ......................................................... 303 

Sowińska M. ...................................................... 304 

Sóvágó ................................................................. 95 

Spek ................................................................... 240 

Spingler.............................................................. 232 

Stachura ..................................................... 196, 305 

Starosta............................................................... 306 

Staszewska ................................................. 216, 224 

Stefanowicz.................147, 213, 215, 216, 224, 279 

Stewart ................................................................. 20 

Stochel ......................................................... 69, 140 

Stout ..................................................................... 88 

Straganz ............................................................... 92 

Strand ................................................................... 84 

Strange ................................................................. 89 

Strianese..................................................... 307, 308 

Styring................................................................ 130 

Suárez................................................................. 314 

Sugiura ............................................................... 107 

Sumithran............................................................. 27 

Sun ....................................................................... 63 

Suyama............................................................... 311 

Suzuki ........................................................ 194, 326 

Swart .................................................................... 45 

Szaciłowski .......................................................... 69 

Szczepanik ..........................................126, 164, 224 

Szewczuk ....152, 213, 215, 216, 224, 279, 289, 309 

Szilágyi ................................................................ 39 

Szłyk .................................................................... 66 

Szyrwiel ............................................................. 309 

Ślepokura ........................................................... 330 

Świątek-Kozłowska ....137, 138, 188, 201, 243, 304 

Tabares....................................................... 123, 308 

Tabata................................................................. 190 

Tai ...................................................................... 168 

Takahashi ........................................................... 244 

Talmard.............................................................. 167 

Tanaka................................................................ 260 

Tangoulis.............................................................. 41 

Tani .................................................................... 326 

Tanigawa............................................................ 115 

Taniguchi ........................................................... 194 

Taraszkiewicz .................................................... 310 

Tarushi ............................................................... 277 

Taura .................................................................. 311 

Teat .................................................................... 240 


Tegoni ................................................................ 102 

Teissie ................................................................ 167 

Tepper ................................................................ 123 

Teramaoto .......................................................... 115 

Terenzi ............................................................... 127 

Thapper .............................................................. 130 

Thérisod ............................................................. 288 

Therrien................................................................ 91 

Thomas............................................................... 287 

Thulstrup ...............................................99, 196, 312 

Tomter.......................................................... 84, 313 

Torres................................................................. 314 

Traoré................................................................... 94 

Trimoteo............................................................. 252 

Trincão ........................................148, 184, 252, 254 

Trynda-Lemiesz ................................................. 315 

Tsoukalas ........................................................... 174 

Tsuzuki................................................................. 14 

Tuczek.................................................................. 96 

Turano.................................................................. 61 

Turco Liveri ....................................................... 127 

Turel..................................................................... 59 

Turowska-Tyrk .................................................. 223 

Urbańska ............................................................ 316 

Valensin D. .............................44, 77, 176, 186, 226 

Valensin G. ...................44, 126, 176, 186, 226, 310 

van Eldik ............................................................ 140 

van Koten............................................................. 81 

Varzatskii ............................................................. 86 

Vašák ..............................................34, 90, 100, 167 

Vasile ......................................................... 124, 263 

Vasudevan.......................................................... 317 

Vazquez-Fernandez............................................ 169 

Venceslau................................................... 264, 318 

Verdejo............................................................... 108 

Verma................................................................. 251 

Vibenholt............................................................ 196 

Vidossich.............................................................. 80 

Vignes ................................................................ 167 

Virta ..................................................................... 39 

Volbeda...................................................... 113, 229 

Voloshin............................................................... 86 

Vonrhein ............................................................ 264 

Walker.......................................................... 90, 203 

Walkowiak ......................................................... 202 

Wang.................................................................... 39 

Wasada-Tsutsui............................................ 98, 244 

Wedd............................................................ 31, 146 

Weik..................................................................... 54 

Weiner................................................................ 269 

Welte.................................................................. 163 

Wheate ............................................................... 319 

Wiczk ................................................................. 215 

Wieczorek .......................................................... 111 

Wieczorek B......................................................... 81 

Wiertz................................................................. 320 

Wilbanks .............................................................. 83 

Willaims............................................................. 251 

Williams............................................................. 105 

Wilson.......................................................... 88, 251 

_____________________________________________________________________ 

339


Windahl.............................................................. 321 

Wisitruangsakul ................................................. 116 

Witkiewicz-Kucharczyk..................................... 271 

Witwicki............................................................. 322 

Wöckel ....................................................... 144, 324 

Wojaczyński....................................................... 323 

Wojciechowski........................................... 267, 303 

Wojdyla.............................................................. 317 

Wolny................................................................. 203 

Woods ................................................................ 168 

Woźna ................................................................ 208 

Wrzesiński.......................................................... 164 

Wysłouch-Cieszyńska.................................. 76, 271 

Xiao...................................................................... 31 

Xu....................................................................... 200 

Yajima................................................................ 325 

Yamaguchi ......................................................... 326 

Yamauchi ........................................................... 325 

Yang..................................................................... 90 

Yano................................................................... 297 

Yashiro............................................................... 173 

Ye ....................................................................... 327 

_____________________________________________________________________ 

340 

Yohda......................................................... 194, 261 

Yong..................................................................... 89 

Yoshii................................................................... 98 

Yumoto .............................................................. 325 

Zajaczkowski ..................................................... 149 

Zamora ............................................................... 163 

Zamorano ........................................................... 180 

Zampella .............................................................. 29 

Zauner ........................................................ 117, 308 

Zebger ................................................................ 116 

Zefirov ................................................................. 47 

Zeng ..................................................................... 63 

Zerzankova......................................................... 328 

Zhadan ............................................................... 180 

Zhang ................................................................... 90 

Zhukova ............................................................. 271 

Zimmermann........................................................ 31 

Zoppellaro.......................................................... 112 

Zoroddu.............................................................. 329 

Zümreoğlu-Karan............................................... 220 

Zweckstetter......................................................... 78 

Żurowska............................................................ 330

ISBN: 978-83-60043-10-3 - eurobic9

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