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

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