VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

This reduction leads to a significant rearrangement of three distinct loops,resulting in an accessible catalytic site [3]. The dissimilatory metal-reducingbacterium Geobacter sulfurreducens possesses two genes with sequencehomology to bacterial Ccps, whose expression increases dramatically underoxidative stress [4]. The proteins were isolated and crystallized afterheterologous expression in Escherichia coli. Additional biochemicalcharacterization confirmed peroxidase activity. For a better understanding ofthe reaction mechanism we created several variants of these two proteinswhich on one hand mimic critical regions of the Nitrosomonas europaeaenzyme and one the other hand differ with respect to the ligands of the twoheme groups. The crystal structures of these variants provide new insightsinto the mechanism of bacterial Ccps [5,6].[1] Fülöp, V. et al (1995): Crystal structure of the di-haem cytochrome cperoxidase from Pseudomonas aeruginosa. Structure 3, 1225-1233.[2] Shimizu, H. et al (2001): Crystal structure of Nitrosomonas europaeacytochrome c peroxidase and the structural basis for ligand switching inbacterial di-heme peroxidases. Biochemistry 40, 13483-13490.[3] Pettigrew, G. W. et al (2006): Structure and mechanism in the bacterialdihaem cytochrome c peroxidases. J. Inorg. Biochem. 100, 551-567.[4] DiDonato, L. N. et al (2006): Role of rel GSU in stress response and Fe(III)reduction in Geobacter sulfurreducens. J. Bacteriol. 188, 8469-8478.[5] Hoffmann, M. et al (2009): CcpA from Geobacter sulfurreducens is abasic di-heme cytochrome c peroxidase. J. Mol. Biol. 393, 951-965.[6] Seidel, J et al. (unpublished data)OTP019Characterization and crystallization of YhjA, a predictedcytochrome c peroxidase from Escherichia coliA. Wuest* 1 , J. Seidel 1 , M. Hoffmann 2 , O. Einsle 11 Institute for Biochemistry and Molecular Biology, Albert-Ludwigs-University, Freiburg, Germany2 University of Technology, Braunschweig, GermanyThe yhjA gene of Escherichia coli encodes a putative cytochrome cperoxidase (CCP), a protein containing 3 heme groups, with a molecularweight of 53 kDa. The heme groups are covalently attached to the proteinchain via two thioether bonds. Cysteine residues occur in the amino acidsequence as a CxxCH heme binding motif. It is known from previous workthat the expression of the yhjA gene is regulated by the oxygen-sensitivetranscription factor FNR and the regulator OxyR [1]. Thus YhjA probablyserves to protect the cell against reactive oxygen species (ROS) and acts as acytochrome c peroxidase, by reducing hydrogen peroxide to water [2]. Theamino acid sequence shows a high similarity to known diheme CCPs, suchas MacA and CcpA from Geobacter sulfurreducens [3]. Aggregatibacteractinomycetemcomitans contains a homologus triheme cytochrome c thatcatalyzes the peroxidation reaction in the respiratory chain and uses quinolas the physiological electron donor, but this activity could not be detectedfor YhjA [4].For the isolation of the gene product it was necessary to express YhjAtogether with the cytochrome c maturation system (ccm) of Escherichia coli,which is encoded by the plasmid pEC86. 5 This system is physiologicallyactive only under anaerobic conditions, but was placed under the control of aconstitutive tet promoter, allowing for cytochrome c expression underaerobic conditions. YhjA shows a low peroxidase activity with ABTS 2-[2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] as an electron donor.The protein could only be crystallized in its reduced state under anaerobicconditions, which points towards conformational changes between theoxidation states, that may be required to activate the enzyme.[1] Partridge, J. D. (2007): The Escherichia coli yhjAgene, encoding a predicted cytochrome cperoxidase, is regulated by FNRand OxyR. Microbiology. 153. 1499 - 1507.[2] Pettigrew, G. W. et al (2006): Structure andmechanism of bacterial dihaem cytochrome cperoxidases. J Inorg Biochem. 100. 551 - 567.[3] Hoffmann, M. et al (2009): CcpA from Geobacter sulfurreducens Is a Basic Di-Heme Cytochromec Peroxidase. J. Mol. Biol. 393, 951-965[4] Takashima, E. et al (2009): Recombinant expressionand redoxproperties of triheme c membraneboundquinol peroxidase. FEMS Microbiol Lett 302. 52-57[5] Schulz, H. (1998): Prototype of a hemechaperone essential for cytochrome c maturation. Science.281. 1197 – 1200.OTP020Incorporation of the prosthetic heme group intocytoplasmatic and membrane proteinsS. Huhn*, M. Jahn, D. JahnInstitute of Microbiology, University of Technology, Braunschweig,GermanyModified tetrapyrroles are complex macrocycles and the most abundantpigments found in nature. They play a central role in electron transferdependentenergy generating processes such as photosynthesis andrespiration. They further function as prosthetic groups for a variety ofenzymes, including catalases, peroxidases, cytochromes of the P450 classand in sensor molecules. Heme is a hydrophobic molecule and associatesnon-specifically with lipids and proteins in aqueous solution where itpromotes peroxidations. Due to its hydrophobicity und toxiticity, heme hasto be transported to its target proteins by different mechanisms, e.g.transport by transmembrane proteins, heme binding proteins and hemechaperones.The aim is to identify heme-binding and/or heme-transporting proteins /invivo/ using the /P. aeruginosa/ Bacterial Adenylate Cyclase Two-Hybridsystem. The interaction between the probable candidates for heme-bindingand/or heme-transport and their target proteins are then further analysed by/in vitro/ translation and further more assays.A /Lactococcus lactis/ Δ/hemW/ mutant showed accumulation of free hemeand failed to respire upon hemin supplementation. Further it was possible tocomplement a /E. coli/ Δ/yggW/ mutant with /Lactococcus lactis/ hemW/. Toverify that /E. coli/ /yggW/ is a heme-transporting protein, the /E. coli/Δ/yggW/ mutant was physiologically characterized. The incorporation of atransporter provides the opportunity to monitor the ability of respirationupon addition of heme and its derivatives.OTP021Characterization of the electronic properties of thenitrogenase Fe protein [1-3]D. Sippel*, T. Spatzal, E.-M. Roth, S. Andrade, O. EinsleInstitute for Biochemistry and Molecular Biology, Albert-Ludwigs-University, Freiburg, GermanyBiological nitrogen fixation is carried out by the enzyme complexnitrogenase. It consists of two metalloproteins, the MoFe protein and the Feprotein. Whereas the MoFe protein is involved in substrate reduction ofnitrogen to ammonia, the Fe protein is the physiological electron donor forthe MoFe protein. The MoFe protein contains the active site, amolybdenum-iron metal cluster. The Fe protein is a homodimer with amolecular mass of 64 kDa and contains one [4Fe-4S]-cluster. Mechanisticunderstanding of the reduction of the MoFe protein by the Fe proteindepends on the elucidation of the distinct oxidation states of the Fe atoms ofthe [4Fe-4S]-cluster. Due to the oxygen sensitivity of the protein, ananaerobic purification strategy of the wild-type enzyme from Azotobactervinelandii was established that yields high amounts of protein of high purity.Crystallization of the protein provides the basis for performing X-raydiffraction, single crystal electron paramagnetic resonance (EPR)spectroscopy and a combination of X-ray diffraction and X-ray absorptionspectroscopy (XAS) [4] to gain a more detailed insight into the electronicstructure of the [4Fe-4S]-cluster. Refining the structure at high resolution incombination with single crystal EPR spectroscopy offers the possibility tocorrelate the cluster orientation with its g-tensor. XAS offers the opportunityof assigning different oxidation states of the Fe atoms in the [4Fe-4S]-cluster. Combining these techniques may provide new insights into the Feprotein being the unique electron donor for the MoFe protein.[1] Georgiadis, M. M. et al (1992): Crystallographic Structure of the Nitrogenase Iron Protein fromAzotobacter vinelandii. Science, 257, 1653-1659.[2] Tezcan, F. A. et al (2005): Nitrogenase Complexes: Multiple Docking Sites for a NucleotideSwitch Protein. Science, 309, 1377-1380.[3] Strop, P. et al (2001): Chrystal structre of the all-ferrous [4Fe-4S] 0 form of the nitrogenase ironprotein from Azotobacter vinelandii. Biochemistry, 40, 651-656.[4] Einsle, O. et al (2007): Assignment of Individual Metal Redox States in a Metalloprotein byCrystallographic Refinement at Multiple X-ray Wavelengths. J. Am. Chem. Soc., 129, 2210-2211.spektrum | Tagungsband 2011