Found 48 Abstracts CONTROL ID: 1672490TITLE: Influence of the PsbA1/PsbA2/PsbA3, Ca2+/Sr2+ and Cl−/Br−/I− exchanges on the Photosystem II function inThermosynechococcus elongatusAUTHORS/INSTITUTIONS: A. Boussac, iBiTec-S/SB2SM, UMR 8221, CNRS/CEA, Gif-sur-Yvette, FRANCE|F.Rappaport, IBPC, UMR 7141 , CNRS/Université Pierre et Marie Curie, Paris, FRANCE|M. Sugiura, Cell-Free Scienceand Technology Research Center, Ehime University, Matsuyama, JAPAN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Photosynthesis is the energy input in the biosphere and Photosystem II (PSII) is the enzyme thatinitiates the process by removing protons and electrons from water. The active site for water oxidation in PSII goesthrough five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5 cluster and TyrZ, aredox-active tyrosine residue. Chloride ions, although not structurally bound to the cluster, are also required for thefunction of the enzyme. The main cofactors involved in PSII oxygen evolution activity are borne by two proteins, D1(PsbA) and D2 (PsbD). The presence of several psbA genes is a common feature to cyanobacteria and there are 3genes (psbA1,2,3) encoding the D1 protein in the genome of the thermophilic cyanobacterium Thermosynechococcuselongatus. Among the 344 residues constituting each of the 3 possible PsbA variants there are 21 substitutionsbetween PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. These different genesare known to be differentially expressed depending on the environmental conditions, suggesting that the differences insequence translate into functional ones. In addition, the calcium ion of the Mn4CaO5 cluster can be biosyntheticallyexchanged for Sr2+ and the chloride ions can be exchanged for Br− and I−, thereby allowing to slightly affect thecatalytic properties of the oxygen evolution complex. The combination of the ionic exchange together with thepolypeptide sequence substitution provides a subtle and conservative way to tune the redox and catalytic properties ofthe enzyme and thereby gives new insights into the oxygen evolution mechanism. Results of such substitutions will bepresented and discussed.(No Image Selected)

CONTROL ID: 1685892TITLE: A New Redox Active Cofactor in Biology: Spectroscopic Studies Probe the Pyranopterin Cofactor and Its Rolein Molybdoenzyme CatalysisAUTHORS/INSTITUTIONS: M. Kirk, B. Stein, Department of Chemistry and Chemical Biology, The University of NewMexico, Albuquerque, New Mexico, UNITED STATES|R. Rothery, M. Solomonson, J. Weiner, Department ofBiochemistry, University of Alberta, Edmonton, Alberta, CANADA|S. Nieter-Burgmayer, Department of Chemistry,Bryn Mawr College, Bryn Mawr , Pennsylvania, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: The pyranopterin cofactor is an essential component of molybdoenzymes, which function to catalyzetwo-electron redox reactions coupled to formal oxygen atom transfer. These enzymes are essential to human life andtheir significance is underscored by the increasing realization of their roles in purine and amino acid catabolism, pro-drug activation and drug metabolism, oxidative stress due to the production of reactive oxygen species leading topostischemic reperfusion injury, and fatal diseases caused by pyranopterin molybdenum cofactor deficiency. Veryrecently, we provided evidence that pyranopterins from different molybdoenzymes possess conformational distortionsrelated to enzyme function (Pyranopterin Conformation Defines the Function of Molybdenum and Tungsten Enzymes,Proc. Natl. Acad. Sci. USA, 2012, 109, 4773–14778). This talk will highlight our most important studies as they relateto the redox state of the pyranopterin molybdenum cofactor (Moco) and its role in catalysis. Moco is comprised of amolybdenum ion and either one or two pyranopterin dithiolene ligands. The Mo ion (2 e- equivalents), the pterin (4 e-equivalents), and the dithiolene (2 e- equivalents) components of the cofactor ensure that the Mo-pyranopterin-dithiolene ensemble possesses one of the most complex electronic structures in biology. A number of important roleshave been suggested for the pyranopterin dithiolene in catalysis including acting as an electronic conduit that couplesthe molybdenum ion with other electron transfer centers, as well as a modulator of the Mo reduction potential.Specifically, we will present the latest results of our detailed spectroscopic (MCD, resonance Raman, electronicabsorption, EPR) and computational studies on enzymes and relevant small molecules, which provide new insight intopyranopterin molybdenum electronic structure contributions to reactivity. These results are important, since theysupport a remarkably complex and flexible electronic structure for this cofactor that suggests a critical contribution ofthe pyranopterin ditholene to enzyme function.

Top: Pyranopterin dithiolene distortions: red, tetrahydro (XO); green, dihydro (SO); blue, oxidized. Bottom: Thepyranopterin cofactor. A: reduced tetrahydro; B: oxidized dhihydro; C: protonated dihydro (zwitterion); D: protonateddihydro (thiol/thione).

CONTROL ID: 1701956TITLE: Visible Light-Driven O2 Reduction by Sensitizer-Laccase SystemsAUTHORS/INSTITUTIONS: T. Tron, Chemistry, Aix-Marseille Université, iSm2/BiosCiences UMR CNRS 7313,Marseille, FRANCE|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Laccases are very well known biocatalysts with great robustness, high oxidation power and substrateversatility (among other properties) [1]. They contain a unique set of copper ions made of one each of the three typesof biorelevant copper sites: type 1 (T1), type 2 (T2) and a binuclear type 3 (T3), and couple dioxygen reduction to theoxidation of substrates, either organic or metal ion [2]. They belong to the Blue Copper Binding Domain (BCBD) familyof proteins in which the archetypal members are the plant or bacterial electron transfer protein cupredoxins (CUP). Inthis family, function is modulated by the number of CUP domains, the number and type copper atoms and the fusionto non metalled domains. Taking natural plasticity within the BCBD family as a source of inspiration for theengineering of laccases [3, 4], we aim at shaping new catalysts based on a laccase platform functionalized with “plug-ins”.One of our targets is to develop a robust system where light absorption triggers electron transfer from a catalyticcentre to a renewable electron acceptor. We report here on the light driven four-electron reduction of a laccase thatultimately converts dioxygen into water using ruthenium(II) polypyridine or porphyrin type chromophores and asacrificial electron donor [5]. Prospects of creating renewable aerobic photo oxygenation catalysts will be discussed. [1] T. Tron, in Encyclopedia of Metalloproteins, Kretsinger, RH, Uversky, VN, Permyakov, EA. (Eds.) Springer, inpress.[2] E. Solomon, U. Sundaram, T. Machonkin, Chem. Rev., 1996, 96, 2563-2605.[3] V. Balland, C. Hureau, A. Cusano, Y. Liu, T. Tron, B, Limoges, Chem. Eu. J., 2008, 14, 7186-92.[4] V. Robert, Y. Mekmouche, P. Rousselot Pailley, T. Tron, Curr. Genomics, 2011, 12, 123-129.[5] a) A. Simaan, Y. Mekmouche, C. Herrero, P. Moreno, A. Aukauloo, J. Delaire, M. Réglier, T. Tron. Chem. Eu. J.,2011, 17, 11743-11746; b) T. Lazarides, I. V. Sazanovich, A. J. Simaan, M. C. Kafentzi, M. Delor, Y. Mekmouche, B.Faure, M. Réglier, J. A. Weinstein, A. G. Coutsolelos, T. Tron, J. Am. Chem. Soc. 2013, 135, 3095-3103.

CONTROL ID: 1702042TITLE: Reaction intermediates in bioengineered heme-nonheme diiron protein models of denitrifying nitric oxidereductasesAUTHORS/INSTITUTIONS: P. Moenne-Loccoz, H. Matsumura, T. Hayashi, Environmental and BiomolecularSystems, Oregon Health and Science University, Beaverton, Oregon, UNITED STATES|S. Chakraborty, Y. Lu,Chemistry, University of Illinois, Urbana, Illinois, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Denitrifying NO reductases (denNORs) protect symbiotic and pathogenic microorganisms againstmicromolar concentrations of NO and lead to resistance to the mammalian immune response. DenNORs aretransmembrane protein complexes evolutionarily related to terminal oxidases, but their active site consists of aheme/nonheme (heme/FeB) diiron center. We use resonance Raman (RR), FTIR, and EPR combined with stopped-flow and rapid-freeze-quench (RFQ) techniques to define the mechanism of NO reduction to N2O. Proposedmechanisms with different NO-binding and reduction steps will be described and briefly discussed.To test these mechanisms, we explore this reaction in two engineered myoglobins (FeBMbs) that mimic the structureof the reduced heme/FeB diiron site of denNORs. FeBMb1 possesses the 3His/1Glu required for the coordinationsphere of FeB, and FeBMb2 includes an additional non-coordinating glutamate peripheral to the diiron site. FTIRmeasurements show that the inclusion of this peripheral Glu in FeBMb2 results in >50% N2O production under single-turnover conditions. Stopped-flow and RFQ-RR monitoring of the reaction of FeBMb1 with NO delineate successivesteps leading to a 5-coordinate heme FeII(NO)/FeBII(NO) dead-end complex. Equivalent experiments with FeBMb2will be analyzed and discussed within the context of NOR activity gain, and the essential structural motifs in denNORs.

Resonance Raman spectra of rapid-freeze-quench samples of the reaction of the diferrous protein FeBMb2 withexcess nitric oxide at 6, 40 and 120 ms reveal the formation of a six-coordinate low-spin heme-nitrosyl species.

CONTROL ID: 1702622TITLE: Insights into the Nitrogenase Mechanism: ATP and Electron TransferAUTHORS/INSTITUTIONS: L.C. Seefeldt, K. Danyal, S. Duval, E. Antony, Chemistry and Biochemistry, Utah StateUniversity, Logan, Utah, UNITED STATES|B.M. Hoffman, Chemistry Department, Northwestern University, Evanston,Illinois, UNITED STATES|D.R. Dean, Biochemistry, Virginia Tech, Blacksburg, Virginia, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Nitrogenase catalyzes the biological nitrogen fixation reaction, the reduction of dinitrogen to ammonia.This reaction requires 8 electrons and 8 protons and the hydrolysis of a minimum of 16 ATP for each N2 reduced.New evidence on key steps in the process allows development of a full model of all key events in the nitrogenasecatalytic cycle. For example, new evidence will be presented showing that ATP hydrolysis follows electron transferand that a deficit spending model for electron transfer is utilized. In addition, a novel mechanism for activating the twoelectron transfer events will be presented. (No Image Selected)

CONTROL ID: 1705005TITLE: Heme Degradation without Releasing CO: Unique Reaction Mechanism of IsdG-type Heme DegradingEnzymesAUTHORS/INSTITUTIONS: T. Matsui, S. Nambu, Y. Ono, S. Takahashi, M. Ikeda-Saito, Institute of MultidisciplinaryResearch for Advanced Materials, Tohoku Univ., Sendai, Miyagi, JAPAN|C.W. Goulding, Departments of MolecularBiology and Biochemistry, and Pharmaceutical Sciences, University of California, Irvine, California, UNITEDSTATES|K. Tsumoto, Department of Medical Genome Sciences, School of Frontier Sciences, and the MedicalProteomics Laboratory, Institute of Medical Science, University of Tokyo, Tokyo, JAPAN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Heme degradation in bacteria is crucial to acquire iron from host heme molecules. An enzyme familytermed heme oxygenase (HO) is known to degrade heme into iron, CO and biliverdin (Fig.). Until recently, the HOfamily was thought to be the only heme degrading enzymes, however, a novel family of enzymes with heme degradingcapabilities has been identified in Staphylococcus aureus. IsdG and its paralogue, IsdI, are heme degrading enzymesof S. aureus having distinct structures compared to the HO-family enzymes. Especially, heme bound to the IsdG-typeenzyme exhibits severe distortion, best described as ruffled, in contrast to the flat heme in HO. This heme ruffling isexpected to modulate the O2 activation chemistry on the heme molecule, and in fact, a novel tetrapyrrole,staphylobilin, having an additional oxidation at a meso carbon is produced (Fig.). Nevertheless, the unique reactionmechanism of the IsdG-type enzymes remains unclear.In this study, we have examined heme degradation reactions by the IsdG-type enzymes, namely by MhuD fromMycobacterium tuberculosis. Spectroscopic and mutagenesis studies suggest that the 1:1 heme-MhuD complex hasan active site structure similar to those of IsdG and IsdI, including the heme ruffling. MhuD produces a noveltetrapyrrole product termed “mycobilin”, which retains the meso carbon at the ring cleavage site as an aldehyde group(Fig.). As expected from the mycobilin structure, the MhuD catalysis does not generate CO. This is the first report forenzymatic heme degradation without CO formation, and predicts a unique mechanism for MhuD. The drasticmechanistic change in MhuD is apparently induced by the heme ruffling even though S. aureus IsdG and IsdI wereassumed to generate CO. This assumption was, however, based only on removal of the meso carbon in staphylobilin,and our product analysis has revealed that the primary C1 product of the S. aureus enzymes is formaldehyde but notCO. We conclude that lack of CO formation is common to the IsdG-type enzymes and that heme ruffling is critical forthe drastic mechanistic change for these novel bacterial enzymes.

Figure. Heme degradation products of the HO- and IsdG-type enzymes

CONTROL ID: 1705825TITLE: Mediated Molybdoenzyme ElectrocatalysisAUTHORS/INSTITUTIONS: P.V. Bernhardt, Chemistry and Molecular Biosciences, University of Queensland,Brisbane, Queensland, AUSTRALIA|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Although in ideal cases direct electron transfer between an electrode and an enzyme can be achieved[1-3] this is often problematic with enzyme stability being compromised by (deliberate) adsorption on a workingelectrode (protein film voltammetry). The intrinsic catalytic properties of an enzyme may usually be preserved if itremains in solution. This demands the use of electron transfer mediators which shuttle electrons between theelectrode and the enzyme to sustain catalysis. These mediators can either be the enzyme’s natural electron transferpartner [4] or a small synthetic compound with a suitable redox potential [5,6]. The mononuclear Mo-enzymes, found in all forms of life, catalyse coupled two-electron, O-atom transfer reactions ofinorganic and organic substrates. They fall into three distinct families but each share an active site comprising one ortwo pterin-dithiolene ligands bound to Mo in addition to terminal oxo, hydroxo or aqua ligands exchanged with thesubstrate during catalysis. Here we shall illustrate how mediators (native and synthetic) can be used to enableelectrochemically driven Mo-enzyme catalysis where direct (protein film voltammetry) approaches have failed. References[1]K.-F. Aguey-Zinsou, P.V. Bernhardt, U. Kappler, A.G. McEwan, J. Am. Chem. Soc. 2003, 125, 530-5.[2]P.V. Bernhardt, P. V.; J.M. Santini, Biochemistry 2006, 45, 2804-9.[3]P.V. Bernhardt, Chem. Commun. 2011, 47, 1663-73.[4]N.L. Creevey, A.G. McEwan, P.V. Bernhardt, J. Biol. Inorg. Chem. 2008, 13,1231-8[5]K.-I. Chen, A.G. McEwan, P.V. Bernhardt, J. Biol. Inorg. Chem. 2011, 16, 227-34.[6] P. Kalimuthu, S. Leimkuehler, P.V. Bernhardt, Anal. Chem. 2012, 84, 10359-65

CONTROL ID: 1707115TITLE: The origin of high stability of the heme active site and catalysis of fatty acid hydroxylation by the thermostablecytochrome P450AUTHORS/INSTITUTIONS: S. Mazumdar, Chemical Sciences, Tata Institute of Fundamental Research, Mumbai,Maharashtra, INDIA|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: The proteins from thermostable organisms show higher stability compared to the mesophilicanalogues. The structure of the heme active site in the monooxygenase, CYP175A1 from T. thermophilus remainsintact even at 80 oC, making it a potential candidate for biocatalytic applications. Moreover, the natural substrate ofthis enzyme has not yet been identified. Our group has been involved in the study of the heme active site of thisorphan cytochrome P450 in order to understand the conformational properties of the heme cavity of the enzyme. Wehave shown that the high thermostability of CYP175A1 arises predominantly due to higher enthalpy of stabilization ofthe thermostable enzyme compared to the mesophilic analogue. The larger number of salt bridges around the metalcenter was shown to play important role for the higher enthalpy of stabilization of CYP175A1. CYP175A1 shows ~26%sequence homology with cytochrome P450BM3 that is known to catalyse hydroxylation of several long-chain fattyacids. Although CYP175A1 does not show any significant activity towards saturated fatty acids, it was found tocatalyze the oxygenation reaction of various mono-unsaturated fatty acids (chain length C16-C22). The productanalyses indicated that CYP175A1 catalyzed monooxygenation reaction exhibits regioselectivity, and the productdistribution depends on the configuration of the ethylenic double bond in the substrate. The cis- configuration wasshown to preferably form epoxide while the trans- configuration favors allylic hydroxylation of the fatty acid. The resultssupport molecular docking analyses, which suggest ‘U’-type conformation of the monoenoic fatty acids at the activesite of the enzyme. (No Image Selected)

CONTROL ID: 1707960TITLE: Ethane and Benzene Hydroxylation by Wild-type Cytochrome P450BM3 Assisted by Decoy Molecules AUTHORS/INSTITUTIONS: O. Shoji, T. Kunimatsu, Department of Chemistry, Graduate School of Science, NagoyaUniversity, Nagoya, JAPAN|N. Kawakami, Y. Watanabe, Research Center for Materials Science, Nagoya University,Nagoya, JAPAN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Cytochrome P450BM3 (P450BM3) isolated from Bacillus megaterium is one of the most promisingenzymes owing the highest monooxygenase activity among P450s reported thus far. Although P450BM3 exclusivelyhydroxylates long-alkyl-chain fatty acids, we have found that simple addition of perfluorocarboxylic acids (PFs) as inertdummy substrates (decoy molecules) turns P450BM3 into a small alkane hydroxylase.[1] PF-bound P450BM3oxidizes propane and butane to 2-propanol and 2-butanol, respectively. The coupling efficiency of small alkanehydroxylation, however, is very low compared with that of long-alkyl-chain fatty acid hydroxylation. In order to improvethe coupling efficiency and to realize the hydroxylation of their primary carbons, we performed small alkanehydroxylation under the high-pressure condition of 0.5 MPa small alkanes to increase the concentration of gaseoussubstrates in the reaction mixture. Propane hydroxylation under high-pressure conditions significantly improved thecoupling efficiency. It is noteworthy that a detectable amount of ethanol was observed in the ethane hydroxylationunder the pressure condition.[2] More recently, direct hydroxylation of benzene to phenol was also catalyzed by wild-type P450BM3 in the presence of “decoy molecules.”[3] The catalytic turnover rate reached 120/min/P450. Theselectivity toward phenol production was very high and no overoxidation products were detected, demonstrating theexclusive conversion of benzene to phenol by the “P450BM3-decoy molecule system.” References: [1] N. Kawakami, O. Shoji, Y. Watanabe, Angew. Chem. Int. Ed. 50, 5315-5318 (2011). [2] N. Kawakami,O. Shoji, Y. Watanabe, Chem. Sci., in press. [3] O. Shoji, T. Kunimatsu, N. Kawakami, Y. Watanabe, Angew. Chem.Int. Ed. Accepted.

Scheme. General reaction mechanism of P450BM3 (left) and the reaction mechanism of ethane and benzenehydroxylation catalyzed by P450BM3 with perfluorononanoic acid (PFC9) as a decoy molecule (right).

CONTROL ID: 1712214TITLE: Influence of the dynamic interplay between protein and solvent on the redox properties of blue copper proteins.AUTHORS/INSTITUTIONS: G. Battistuzzi, L. Paltrinieri, M. Borsari, Department of Chemical and GeologicalSciences, University of Modena and Reggio Emilia, Modena, ITALY|C.A. Bortolotti, M. Sola, Department of LifeSciences, University of Modena and Reggio Emilia, Modena, ITALY|C. Dennison, Institute for Cell and MolecularBiosciences, Medical School, Newcastle University, Newcastle upon Tyne, UNITED KINGDOM|S. Corni, CNR-NanoInstitute of Nanoscience, CNR, Modena, ITALY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Understanding the factors governing the electron transfer processes in proteins is crucial for a deeperunderstanding of redox processes in living organisms, as well as to improve the rational design of engineeredbiomolecules with tailored redox properties for nanobiotechnological applications. To understand the behavior of aprotein in vivo, time must be taken into account, extending the usual structure-function paradigm to include dynamics.Molecular dynamics (MD) simulations provide an unbeatable tool to investigate the effects of dynamics on thereactivity of a protein. We used direct electrochemistry [1, 2] and MD simulations [3] to investigate the redox reactivityof native azurin and four chimeric cupredoxins, in which the ligand-containing loop of azurin has been replaced eitherwith that of other members of the blue copper family (amicyanin and plastocyanin) or with synthetic sequencesfeaturing only Ala residues. It turns out that the dynamic interplay between protein and solvent is the key factordetermining the redox properties of these hallmark ET systems. In particular, the dynamics of the small, metal-bindingloop region controls the outer-sphere reorganization energy, not only by determining the exposure of the active site tosolvent but also through the modulation of the redox-dependent rearrangement of the whole protein scaffold and ofthe surrounding water molecules [3]. The molecular determinants to the reduction potential were also investigated,showing that the fluctuations of the solvent are tightly intertwined with those of the protein matrix and play a major rolein the modulation of redox entropy. Moreover, the dynamics between the protein scaffold and the surrounding solventproved to be crucial in determining the pKa of the protonation of the C-terminal copper binding His in the reducedproteins. [1] Battistuzzi G. et al. Biochim. Biophys. Acta 2009, 1794, 995–1000[2] Monari S. et al. J. Am. Chem. Soc. 2012, 134, 11848−11851[3] Paltrinieri L. et al. J. Phys. Chem. Lett. 2013, 4, 710−715(No Image Selected)

CONTROL ID: 1712402TITLE: Life in the Absence of Dioxygen: Reduction of Nitrite and Sulfite at Unique Heme Iron CentersAUTHORS/INSTITUTIONS: P.M. Kroneck, Biology, University of Konstanz, Konstanz, GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Many essential life processes on Earth, such as photosynthesis, respiration, or nitrogen fixation,depend on transition metals and their ability to catalyze multi-electron redox and hydrolytic transformations. The focusof this contribution will be on structural and functional aspects of two complex multi-site enzymes with unique hemeiron centers. These are involved in energy conserving processes of important branches within the biogeochemicalcycles of nitrogen and sulfur: (1) six-electron reduction of nitrite to ammonia catalyzed by pentaheme cytochrome cnitrite reductase (ccNiR), and (2) the six-electron reduction of sulfite to hydrogen sulfide by sulfite reductase (SIR).The structural and mechanistsic information has been obtained by X-ray crystallography and EPR spectroscopy (1,2). References1. K. Parey, G. Fritz, U. Ermler, and P.M.H. Kroneck, Metallomics, 2013, DOI: 10.1039/C2MT20225E.2. J. Simon and P.M.H. Kroneck, Adv, Microbial Physiol., 2013, 62, 45-117.

Active site heme of ccNir. Note the unusual Lys axial ligand.

CONTROL ID: 1712599TITLE: Electron Tunneling Rates In Complex I Are Optimized For Efficient Energy Conversion.AUTHORS/INSTITUTIONS: S. de Vries, M. Strampraad, Biotechnology, Delft University of Technology, Delft,NETHERLANDS|K. Dörner, T. Friedrich, Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg,GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Respiratory complex I converts the redox free energy of ubiquinone (Q) reduction by NADH into aprotonmotive force. FMN and a chain of seven iron-sulphur (FeS) centres located in the peripheral arm catalyse thereduction of Q. The membrane arm harbours four proton transfer pathways. Electron transfer and proton pumping arelinked via large conformational changes. Electron tunnelling rates are calculated as 25 ns to 5 µs, and ~ 100 µs for thelongest distance of 14.1 Å. Here the reduction kinetics of the FeS centres were determined by ultrafast freeze-quench experiments monitored byEPR and UV-vis spectroscopy. The multiphasic kinetics, three NADH turnovers, were quantitatively analysed. Electrontransfer from FMN to the most distal centre N2, t1/2 = 197 µs, is determined by the 14.1 Å tunnelling distance and theoverall driving force, ΔG0= -54 mV. The second electron crossing the 14.1 Å barrier reduces centre N4 with t1/2 =1200 µs. This six-fold lower rate reflects the change in ΔG0 to +38 mV when reducing N4 enabling uniquely the directdetermination of the reorganization energy, λ= 0.740 ± 0.004 eV, by the Marcus theory for electron transfer. By fine-tuning the midpoint potentials of the redox centres electron tunnelling rates are decreased to milliseconds, thetime scale for conformational changes required for proton pumping. Synchronization of electron tunnelling and protonpumping rates enables efficient energy conversion by complex I. Adjustment of electron and proton transfer rates byredox tuning is proposed as a general mechanism to enhance the catalytic efficiency in enzymes with long redoxchains such as present in hydrogenases, Mo/W-containing oxidoreductases, fumarate reductase and photosyntheticreaction centres. Electron and hydride transfer/tunnelling reactions are the only chemical reactions for which the rates are directlyrelated to the driving force. Long redox chains provide the structural basis and Marcus theory the theoretical basis fornature to exploit simple biophysical principles such as redox fine-tuning to evolve efficient biocatalysts and a highlyefficient energy converter, complex I.(No Image Selected)

CONTROL ID: 1712699TITLE: Pulsed EPR Spectroscopy of 33S-Labeled Molybdopterin in Sulfite OxidaseAUTHORS/INSTITUTIONS: J.H. Enemark, E.L. Klein, A.M. Raitsimring, A.C. Davis, A.V. Astashkin, Chemistry andBiochemistry, University of Arizona, Tucson, Arizona, UNITED STATES|E.L. Klein, T. Krämer, F. Neese, Max PlanckInstitute for Chemical Energy Conversion, Mülheim an der Ruhr, GERMANY|A.A. Belaidi, G. Schwarz, Institute ofBiochemistry, University of Cologne, Cologne, GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: All known molybdenum-containing enzymes, with the exception of nitrogenase, contain a pyranopterindithiolate (molybdopterin) moiety that coordinates to the metal through the two sulfur atoms of the ene-dithiolate(dithiolene) functionality to form the molybdenum cofactor. To date, molybdenum and tungsten enzymes are the onlyknown examples of dithiolene coordination in biology. During catalysis, molybdenum enzymes pass through theparamagnetic Mo(V) state, and high resolution pulsed electron paramagnetic resonance (EPR) measurements of thehyperfine and nuclear quadrupole interactions of nearby magnetic nuclei (I ≠ 0) provide important experimental dataabout the structures of these transient Mo(V) states. For sulfite oxidase (SO), pulsed EPR studies of the Mo(V) centerto determine the interactions of naturally abundant magnetic nuclei (1H, 14N, 31P) and isotopically enriched reagents(2H2O, H2

17O, 35Cl-, 37Cl-, [33SO3]2-, [S17O3]2-), in parallel with density functional theory (DFT) calculations, haveprovided important insights into the structure of the active site and the reaction mechanism of the enzyme. However,extension of pulsed EPR methods to dithiolene S atoms presents major challenges. Naturally abundant 32S has nonuclear spin (I = 0), and isotopic labeling of model Mo-ditholene compounds with 33S (I = 3/2) has not been feasible.However, the elucidation of the biosynthetic pathway of the molybdenum cofactor has opened up the possibility ofdirect incorporation of 33S-labeled sulfide into molybdopterin itself using controlled in vitro synthesis with purifiedproteins. Here we present the biosynthetic reactions for preparing 33S-labeled molybdenum cofactor in a catalyticallyactive SO variant. The 33S hyperfine and nuclear quadrupole parameters for the Mo(V) state of the construct aredetermined experimentally by pulsed EPR methods and are compared with results from DFT calculations. To ourknowledge, this is the first determination of the magnetic resonance parameters for a 33S atom in a dithiolene group.

<b>³³S-labeled molybdopterin</b>

CONTROL ID: 1713761TITLE: Stepwise molecular evolution of a synthetic heme b peroxidase based on the hemeless C-terminal domain of acatalase-peroxidaseAUTHORS/INSTITUTIONS: M. Zamocky, K.F. Pirker, P.G. Furtmüller, C. Obinger, Dept. Chemistry, DivisionBiochemistry, BOKU University, Vienna, AUSTRIA|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Bifunctional catalase-peroxidases are homodimeric heme b containing oxidoreductases presentamong prokaryotes and lower eukaryotes. Each subunit consists of a N-terminal heme containing domain connectedto a C-terminal domain without any prosthetic group [1]. We have separately cloned and expressed this C-terminaldomain from a fungal extracellular KatG2 [2] and designed de novo functional residues allowing heme binding andperoxidase catalysis. Performed stepwise design was based on phylogenetic [1] and structural studies [2]. Among theproduced mutants proteins with 5 and 8 substitutions at the distal and proximal heme sides were the most interestingand have been compared with the holoenzyme by a broad set of methods including UV-Vis, electronic circulardichroism, electronic paramagnetic resonance, differential scanning calorimetry and presteady-state and steady-statekinetics. It is demonstrated that heme is bound to the predominantly α-helical protein (Fig.) and enables one-electronoxidation reactions as well as increases the thermal stability of some variants. Biochemical/physical data will bepresented and discussed with respect to our knowledge on the bifunctional activity of KatG. The research was supported by the Austrian Funding Agency FWF with the project P23855-B11. References:[1] Zamocky, M., Gasselhuber, B., Furtmueller, P.G., Obinger, C. (2012) Molecular evolution of hydrogen peroxidedegrading enzymes. Arch. Biochem. Biophys. 525:131-144 [2] Zamocky, M., Garcia-Fernandez, M. Q., Gasselhuber, B., Furtmüller, P. G., Loewen, P. C., Fita, I., Obinger, C.,Carpena, X. (2012) High thermal and conformational stability of secretory eukaryotic catalase-peroxidases - Answersfrom first crystal structure and unfolding studies. J. Biol. Chem. 287:32254-32262

CONTROL ID: 1714752TITLE: Artificial Maturation of [FeFe] Hydrogenases, Biomimetic Chemistry and Biological Machinery in SynergyAUTHORS/INSTITUTIONS: G.O. Berggren, Dept. of Biochemistry & Biophysics, Stockholm University, Stockholm,SWEDEN|M. Fontecave, Collège de France, Paris, FRANCE|G.O. Berggren, T. Simmons, M. Atta, S. Gambarelli, J.Mouesca, V. Artero, M. Fontecave, CEA, Grenoble, FRANCE|C. Lambertz, J. Esselborn, T. Happe, Ruhr UniversitätBochum, Bochum, GERMANY|A. Adamska, E. Reijerse, W. Lubitz, Max-Planck Institut für ChemischeEnergiekonversion, Mülheim, GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Hydrogenases are the most efficient molecular catalysts known today with regards to hydrogenformation and combustion, and as a results these enzymes have been intensively studied as alternatives to noblemetals in (electro-)catalysts and fuel cells [1, 2]. In the case of [FeFe] hydrogenases (HydA) catalysis occurs at a,unique, diiron centre (the [2Fe] subunit), featuring a bridging dithiolate ligand as well as CO and CN- ligands [3].Through a complex, and as yet poorly understood, biosynthetic processes a precursor of the [2Fe] subunit isassembled onto the HydF protein. From there it is delivered to the apo-form of HydA, thereby generating the activeenzyme [4]. Synthetic chemistry has allowed the preparation of remarkably close mimics of the [2Fe] subunit, but sofar failed to reproduce its catalytic activities. Here I will describe how such mimics can be loaded onto HydF, and thentransferred to apo-HydA. Activation of HydA was achieved exclusively using the HydF hybrid protein containing the[2Fe] mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of enzyme [5, 6]. Toour knowledge this provides the first example of controlled metalloenzyme activation using the combination of aspecific protein scaffold and synthetic active site analogues. This simple methodology not only provides newmechanistic and structural insight into hydrogenase maturation but also provides a tool for producing recombinant[FeFe]-hydrogenases, with no requirement for co-expression of the maturation machinery. Moreover, we now have thecapacity to engineer the HydA active site through synthetic chemistry [7]. 1.C. Tard and C. J. Pickett, Chem.Rev., 2009, 109, 2245-2274.2.J. A. Cracknell et al, Chem. Rev., 2008, 108, 2439-2461.3.J. W. Peters et al, Science, 1998, 282, 1853-1858.4.D. W. Mulder et al, Nature, 2010, 465, 248-251.5.Y. Nicolet et al, J. Am. Chem. Soc., 2001, 123, 1596-1601.6.A. Silakov et al, Phys. Chem. Chem. Phys., 2009, 11, 6592-6599.7.G. Berggren et al, Nature, Accepted.

CONTROL ID: 1714816TITLE: Real-Time SpectroBioElectrochemistry on Mesoporous Metal Oxide ElectrodesAUTHORS/INSTITUTIONS: V. Balland, B. Limoges, Laboratoire d'Electrochimie Moléculaire, Université Paris Diderot-Paris 7, Paris , FRANCE|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Mesoporous transparent metal oxide electrodes offer new opportunities to develop time-resolvedspectroelectrochemical methods.[1] These new electrode materials exhibit a unique combination of high conductivity,high transparency in the visible range, and high surface area. Accordingly, they allow immobilization of a large amountof redox probe, up to a concentration easily detected by spectroscopy, with the possibility of direct electron transferand thus rapid redox conversion. It is thus possible to achieve real-time monitoring of the spectroscopic features of theimmobilized redox probe during a coupled electrochemical experiment such as cyclic voltammetry.We will illustrate the potential offered by mesoporous Indium Tin Oxide (ITO) electrodes prepared by glancing angledeposition for the adsorption and characterization of immobilized hemoproteins by various spectroscopictechniques.[2,3] We will then demonstrate how cross-correlations between spectroscopic and electrochemical dataobtained during real-time spectroelectrochemical experiments allow to unravel complex redox coupled chemicalreactions with the catalytic reduction of dioxygen by immobilized microperoxidase 11.[4] [1] P.A. Ash, K.A. Vincent; Chem. Commun., 2012, 48, 1400-1409[2] C. Renault, K.D. Harris, M.J. Brett, V. Balland, B. Limoges; Chem. Commun., 2011, 1863-5[3] D. Schaming, C. Renault, R. Tucker, S. Lau, J. Aubard, M.J. Brett, V. Balland, B. Limoges ; Langmuir, 2012, 28,14065-72[4] C. Renault, C. Andrieux, R. Tucker, M.J. Brett, V. Balland, B. Limoges ; J. Am. Chem. Soc., 2012, 134, 6834-45

CONTROL ID: 1714887TITLE: Structural Basis for the Functional Difference between Nitric Oxide Reductase and Oxygen ReductaseAUTHORS/INSTITUTIONS: T. Tosha, H. Sugimoto, Y. Shiro, RIKEN SPring-8 Center, Hyogo, JAPAN|N. Sato, KyotoUniversity, Kyoto, JAPAN|S. Ishii, Y. Shiro, University of Hyogo, Hyogo, JAPAN|T. Hino, Tottori University, Tottori,JAPAN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Bacterial nitric oxide reductase (NOR) is a membrane-integrated enzyme, which is involved inanaerobic nitrite respiration. On the basis of the phylogenetic analysis, NOR and aerobic oxygen reductase such ascytochrome c oxidase (COX) are suggested to belong to the same heme-copper oxidase superfamily. However, theserespiratory enzymes show distinctive function. COX shows proton pumping activity in a process coupled with catalyticO2 reduction (O2 + 4H+ + 4e- → 2H2O) at a heme/Cu binuclear center, whereas NOR has no proton pumping abilityand catalyzes nitrous oxide generation via reductive coupling of two NO molecules (2NO + 2H+ + 2e- → N2O + H2O)at a heme/non-heme Fe binuclear center. Recently, we solved the crystal structure of NOR in the resting oxidizedstate [1, 2], which provided the insights into the functional difference between NOR and COX. In this study, wecharacterized the structures of reduced and ligand-bound cytochrome c dependent NOR (cNOR) from Pseudomonasaeruginosa at 2.4-2.7 Å resolution, to further elucidate the structural elements required for the respective function inNOR and COX. Unlike COX, in which the redox-coupled conformational changes were reported, reduced cNORshowed no remarkable structural differences as compared with oxidized cNOR. The diatomic ligand, CO or CN,binding also induced no structural changes except for the elongation of the heme/non-heme Fe distance by ~0.5 Å.Such no substantial structural changes in response to the redox reaction and the ligand binding are consistent with thelack of the gating function for the proton pumping in cNOR. It is also interesting to note that the heme iron/non-hemeFe distance in the diatomic ligand-bound form (~4.4 Å) is shorter than the heme iron/Cu distance in COX (~5 Å).Confined space for the binuclear active center in cNOR could allow two NO molecules to be in short distance, therebyfacilitating the N-N coupling to produce a hyponitrite reaction intermediate during the catalytic reaction.[1] Hino et al. Science 330, 1666- (2010). [2] Matsumoto et al. Nat. Struct. Mol. Biol. 19, 238- (2012).(No Image Selected)

CONTROL ID: 1715151TITLE: STUDIES ON THE OXYGEN TOLERANT FORMATE DEYHDROGENASE FROM RHODOBACTERCAPSULATUS AUTHORS/INSTITUTIONS: S. Leimkuehler, Institute of Biochemistry and Biology, University of Potsdam, Potsdam,GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Formate dehydrogenase from Rhodobacter capsulatus (RcFDH) is a hexameric protein with a (αβγ)2structure which is localized in the cytoplasm. It belongs to the group of NAD+ dependent FDHs with coordination of amolybdenum containing cofactor (bis-MGD form) in its active site showing tolerance against oxygen. Several FeSclusters are coordinated in RcFDH (5x Fe4S4, 2x Fe2S2) bridging electron transfer from molybdenum to the flavinmononucleotide site. Beside the structural genes fdsGBA two additional genes fdsC and fdsD are encoded by the fdsoperon which are essential for the activity of FDH. Heterologously expressed RcFDH in E. coli shows high activitiesfor formate oxidation. The back reaction for CO2 reduction is possible with RcFDH, however, at significantly slowerrates. For the production of a NADH regeneration system we used the diaphorase subunits of the enzyme comprisingthe FMN and Fe4S4 containing beta subunit and the Fe2S2 containing gamma subunit. In summary, we characterizedthis oxygen tolerant enzyme to study the mechanism of maturation and substrate conversion. The goal is to producean oxygen tolerant CO2 reductase.(No Image Selected)

CONTROL ID: 1715533TITLE: NiFe hydrogenases: catalysis and inhibitionAUTHORS/INSTITUTIONS: C. Léger, S. Dementin, V. Fourmond, A. Abou Hamdan, C. Baffert, B. Guigliarelli, B.Burlat, P. Liebgott, P. Bertrand, CNRS / AMU, Marseille, FRANCE|O. Gutierrez-sanz, A.L. De Lacey, CSIC / ISP,Madrid, SPAIN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: The biological catalysts of hydrogen oxidation and production are large and structurally complexenzymes called hydrogenases. Their active site, which can oxidize thousands molecules of H2 per second, is a Fe2 orNiFe dinuclear inorganic cluster which is buried in the protein and connected to the solvent by a chain of redoxcofactors acting as a wire for transferring electrons, a tunnel that guides the diffusion of H2, and a series ofaminoacids for transferring protons. I will summarize some of our most recent results on the mechanism of NiFe hydrogenases, regarding the kinetics oflong range electron transfer (1,5), intramolecular diffusion along the substrate access channel and how this relates tooxygen resistance (7) and catalytic bias (1,4), the nature of the inactive species that are formed upon aerobic andanaerobic conditions (2,3), and the design of NiFe hydrogenase mutants that show increased resistance to O2 (3,6). http://bip.cnrs-mrs.fr/bip06/ https://twitter.com/BIP6_Marseille 1. Fourmond, et al, “Steady-state catalytic wave-shapes for 2-electron reversible electrocatalysts and enzymes” J. Am.Chem. Soc. 125 3926 (2013). 2. Abou Hamdan, et al, “O2-independent formation of the inactive states of NiFe hydrogenase” Nat. Chem. Biol. 9 15-17 (2013) 3. A Abou Hamdan, et al, “Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFehydrogenase mutants” PNAS USA 109 19916-19921 (2012) 4. A. Abou Hamdan, et al , “Understanding and tuning the catalytic bias of hydrogenase” J. Am. Chem. Soc. 1348368-8371 (2012) 5. S. Dementin, et al, “Rates of intra and intermolecular electron transfers in hydrogenase deduced from steady-stateactivity measurements” J. Am. Chem. Soc. 133 10211 (2011) 6. P-P Liebgott, et al, “Original design of an oxygen-tolerant [NiFe] hydrogenase : Major effect of a valine-to-cysteinemutation near the active site” J. Am. Chem. Soc. 133 986-997 (2011) 7. P.-P. Liebgott, et al, “Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase” Nat.Chem. Biol. 6(1) 63-70 (2010)

CONTROL ID: 1715967TITLE: Geometric and Electronic Structural Contributions to Fe/O2 ReactivityAUTHORS/INSTITUTIONS: E.I. Solomon, Chemistry, Stanford University, Stanford, California, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Non-heme iron enzymes either activate substrate using a ferric center or dioxygen using a ferrous site. The latter has been extremely difficult to study. Thus we have developed new spectroscopic methodologies thatprovide geometric and electronic structural insight into the ferrous center, its interaction with cosubstrates foractivation of dioxygen, and the nature of the Fe(III)-OOH and Fe(IV)=O intermediates generated in this reaction. (No Image Selected)

CONTROL ID: 1715979TITLE: New Families of Zinc Finger Proteins: Iron and Zinc Coordination and DNA and RNA RecognitionAUTHORS/INSTITUTIONS: S.L. Michel, J.L. Michalek, A. Besold, School of Pharmacy, Dept. of PharmaceuticalSciences, University of Maryland, Baltimore, Maryland, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Zinc finger proteins (ZFs) are the most common type of eukaryotic metal co-factored transcriptionfactor. ZFs have critically important roles in modulating transcription and translation and show a remarkable capacityfor highly selective nucleic acid recognition. We are studying two new families of ‘non-classical’ zinc finger proteins,Cys3His and Cys2His2Cys –type ZFs, for which mechanisms of metal-mediated RNA or DNA recognition are not wellunderstood. The two Cys3His ZFs that our lab has been studying, TTP and CPSF30, are involved in RNA regulationand contain 2 and 5 Cys3His domains, respectively. We have successfully isolated the ZF domains of TTP andCPSF30 and characterized their metal binding properties. Both proteins can function with either Zn(II) or Fe(II)coordinated, suggestive of a potential role for iron in function. Both proteins appear to specifically recognize adenineand uridine rich-RNA sequences in a metal dependent manner, and we will propose a mechanism for metal-mediated,selective RNA recognition by these ZFs. The two Cys2His2Cys –type ZFs that our lab has been studying, NZF-1 andMyT1, are involved in regulation of neuronal development. These two proteins are highly homologous; yet, theyrecognize completely different genes with different sequences. We have successfully isolated constructs of NZF-1 andMyT1. Using a combination of mutagenesis and UV-visible spectroscopy with Co(II) as a spectroscopic probe forZn(II), we have identified the ligands involved in metal coordination (Cys2HisCys) and have discovered that the non-coordinating His ligand is critical for DNA recognition. Moreover, we have discovered that mutagenesis of a singlenon-conserved residue present in MyT1 switches the DNA binding properties to those of NZF1. From these results,we will propose a novel DNA binding paradigm for the Cys2His2Cys ZF proteins. References:1.Besold, A.N., Oluyadi, A.A. Michel, S.L.J. Inorg. Chem. 2013 [Epub ahead of print]2.Michalek, J.L., Lee, S.J. Michel, S.L.J. J. Inorg. Biochem. 2012, 112, 32-383.Michalek, J.L., Besold, A.N., Michel, S.L.J. Dalton Trans. 2011, 40, 12619-32(No Image Selected)

CONTROL ID: 1716162TITLE: The histidine-heme posttranslational modification in Synechococcus hemoglobin alters its reactivity toward NO●

AUTHORS/INSTITUTIONS: J.T. Lecomte, M.R. Preimesberger, Biophysics, Johns Hopkins University, Baltimore,Maryland, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: GlbN, the hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, protects the cell fromthe buildup of reactive oxygen and nitrogen species (ROS/RNS), including NO● (1). Unlike other hemoglobins, GlbNcoordinates the ferric and ferrous heme iron with two histidines, a ligation scheme that facilitates electron transferprocesses. GlbN can also bind the ferrous heme covalently and irreversibly. Both non-crosslinked (GlbN) andcrosslinked (GlbN-A) versions of the holoprotein are found in vivo, but so far no clear role for the hemeposttranslational modification (PTM) has been established. NMR and optical spectroscopies were used to study NO● binding and its redox chemistry with GlbN and GlbN-A. Weobserved that GlbN and GlbN-A can form stable diamagnetic FeIII-NO● complexes that resist autoreduction to theparamagnetic FeII-NO● state over periods of days. NMR analysis shows that FeIII-NO● GlbN-A resembles moreclosely the isoelectronic FeII-CO protein than the reduced FeII-NO● form. GlbN and GlbN-A are both capable ofefficient nitrite reduction. The non crosslinked protein, however, loses heme immediately upon forming the FeII-NO●

complex. UV-visible data support that rapid heme dissociation is followed by slow heme rebinding. 15N NMR chaseexperiments indicate that unmodified GlbN is capable of reducing 15NO2

- via 15NO● to nitrosyl hydride (H15NO) inthe presence of excess dithionite. The diamagnetic HNO adduct of ferrous GlbN is stable, and NOESY data wereused to orient the ligand within the distal pocket. Upon reduction in the presence of NO2

-, H117A GlbN (which isincapable of PTM) exhibits similar behavior with respect to heme loss and HNO production. Formation of the heme-protein crosslink in the wild-type protein inhibits HNO formation completely and enables stable FeII-NO● binding.Altogether, it appears that the histidine-heme PTM modulates GlbN redox chemistry towards NO● and acts toextinguish undesirable reactions such as NO●-mediated heme dissociation and HNO production. (1) Scott et al. (2010) Biochemistry 49, 7000 Supported by NSF MCB 0843439

GlbN (PDB ID 4I0V)

CONTROL ID: 1716277TITLE: Artificial Metalloenzymes: Recent Advances and OpportunitiesAUTHORS/INSTITUTIONS: T. Ward, Chemistry, University of Basel, Basel, 0041, SWITZERLAND|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Artificial metalloenzymes result from incorporation of a catalytically competent organometallic moietywithin a host protein, Figure 1. We have been exploiting the potential of the biotin-avidin technology for the creation ofartificial metalloenzymes. Thanks to the remarkable affinity of biotin for either avidin or streptavidin, covalent linking ofa biotin anchor to a catalyst precursor ensures that, upon stoichiometric addition of (strept)avidin, the metal moiety isquantitatively incorporated within the host protein.Such artificial metalloenzymes are optimized either by chemical (variation of the biotin-spacer-ligand moiety) orgenetic (mutation of (strept)avidin) means. Such chemogenetic optimization schemes were applied to various organictransformations.Noteworthy features, reminiscent of homogeneous catalysis include: the straightforward access to both enantiomersof the product; broad substrate scope; organic solvent tolerance and reactions typical of homogeneous catalysis.Enzyme-like features include: genetic optimization; aqueous medium as the preferred solvent; Michaelis-Mentenbehaviour; single substrate derivatization. X-ray characterization of artificial metalloenzymes provides a fascinatinginsight into possible enantioselection mechanims involving a well defined second coordination sphere environment.Thus, such artificial metalloenzymes combine attractive features of both homogeneous and enzymatic kingdoms.After presenting an overview of recent results for the above reactions, the last part of the talk will outline our currentefforts on performing catalysis on crude cell extracts, thus paving the way towards in vivo catalysis and directedevolution of artificial metalloenzymes. Figure 1Artificial metalloenzymes result from incorporation of a catalytically competent artificial cofactor within a host protein.

CONTROL ID: 1716889TITLE: Nickel-dependent peptide bond hydrolysis in technology and toxicologyAUTHORS/INSTITUTIONS: W. Bal, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw,POLAND|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: The Ni(II)-dependent peptide bond hydrolysis occurs in peptides of a general sequence R-↓Ser/Thr-X-His-Z, with high reactivity for bulky X and Z. The reaction starts with the formation of the square-planar complexcontaining the 4-nitrogen Ni(II) coordination, anchored at the His residue. The reaction is controlled stereochemicallyand consists of the N-O acyl-shift, followed by hydrolysis of the intermediate oxygen ester [1-3].25% of human C2H2 zinc fingers contain a hydrolytic motif, confirmed experimentally for the 3rd zinc finger (ZF) ofSP1, a crucial transcription factor involved in the cell cycle control, cell differentiation and expression of functionalgene blocks. The hydrolysis proceeds without Zn(II) dissociation from the ZF, which makes it a potential mechanism ofepigenetic genotoxicity [4].The studied reaction is useful for protein purification methodology. Recombinant fusion proteins, can be purified withyields of 80-90%, using Ni(II)-dependent peptide bond hydrolysis at 45-50°C, pH 8.2 [5]. The method can also beapplied for oxidation-sensitive and membrane proteins. Acknowledgements. This work was supported in part by the project “Metal dependent peptide hydrolysis. Tools andmechanisms for biotechnology, toxicology and supramolecular chemistry.” carried out as part of the Foundation forPolish Science TEAM/2009-4/1 program, co-financed from European Regional Development Fund resources withinthe framework of Operational Program Innovative Economy. References[1] A. Krezel, E. Kopera, A. M. Protas, J. Poznanski, A. Wysloouch-Cieszynska, W. Bal, J. Am. Chem. Soc. 2010, 132,3355.[2] E. Kopera, A. Krezel, A. M. Protas, A. Belczyk, A. Bonna, A. Wyslouch-Cieszynska, J. Poznanski, W. Bal, Inorg.Chem. 2010, 49, 6636.[3] H. H. Ariani, A. Polkowska-Nowakowska, W. Bal, Inorg. Chem. 2013, 52, 2422.[4] E. Kurowska, J. Sasin-Kurowska, A. Bonna, M. Grynberg, J. Poznanski, L. Knizewski, K. Ginalski, W. Bal,Metallomics 2011, 3, 1227.[5] E. Kopera, A. Belczyk, W. Bal, PLoS One 2012, 7: e36350.(No Image Selected)

CONTROL ID: 1718343TITLE: The Mo- and Cu-Containing CO Dehydrogenase from Oligotropha carboxidovoransAUTHORS/INSTITUTIONS: R. Hille, Biochemistry, University of California, Riverside, Russ Hille, California, UNITEDSTATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Carbon monoxide dehydrogenase from the aerobic bacterium Oligotropha carboxidovorans catalyzesthe oxidation of CO to CO2, yielding two electrons and two H+ that enter the quinone pool of the organism. The activesite of theenzyme consists of a unique binuclear center ocnsisting of Mo(VI) and Cu(I) in the oxidized state, Mo(IV)and Cu(I) in the reduced. Work is presented describing the characteristics of an EPR-active Mo(V)/Cu(I) species seenin the course of enzyme reduction by CO that exhibits strong coupling to the copper of the active center (I = 3/2) and isfurther split when [13C]-CO is used to generate it, demonstrating that substrate (or product) is a component of thesignal-giving species. We also describe the properties of enzyme in which silver has been substituted for copper inthe active site that remains partially functional. Finally, the hydrogenase activity of the enzyme is described.

Teh active site of CO Dehydrogeanse from Oligotropha carboxidovorans

CONTROL ID: 1718735TITLE: Theoretical studies of molybdenum oxo-transfer enzymesAUTHORS/INSTITUTIONS: U. Ryde, J. Li, M. van Severen, Theoretical Chemistry, Lund University, Lund,SWEDEN|E. Nordlander, Chemical Physics, Lund University, Lund, SWEDEN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: We have studied a number of mononuclear Mo oxo-transfer enzymes with quantum mechanical (QM)methods. First, we have studied the enzyme DMSO reductase with DFT methods [J. Chem. Theory Comput., 2013, 9,1799]. The calculations show that the calculated energies are very sensitive to details in the calculations. Large basissets are needed and the results depend strongly on the DFT functional used for the calculation (because the oxidationstate of the Mo ion changes). By LCCSD(T) calibration calculations, we show that no single DFT functional giveaccurate results for all steps.Second, we have compared three different reaction mechanisms for two cluster models of sulfite oxidase([MoO2L(SCH3)]– , where L is either 1,2-dimethyldithiolene or a full molybdopterin ligand), as well as a functionalmodel of it, [MoO2(MNT)2]2– (MNT = maleonitrile dithiolate), both with either HSO3– or SO32– as the substrate. Theresults indicate that the simplest and energetically most favourable mechanism involves the attack of the substratesulfur atom on the equatorial oxo ligand. It involves a single Mo–sulfate intermediate and two transition states for theformation of the O–S bond and the cleavage of the Mo–O bond.Third, we have compared the active sites of the three families of Mo oxo-transfer enzymes, sulfite oxidase, DMSOreductase, and xanthine oxidase with the QM cluster approach to understand the intrinsic reactivity of the active sitesand to explain the differences in the design of the enzymes. The results indicate among other things that the sulfidoligand is crucial for the proton transfer involved in the xanthine oxidase reaction.Finally, we have run combined QM and molecular mechanical (QM/MM) calculations of the entire sulfite oxidase andDMSO reductase enzymes. The results indicate that conclusions from the QM cluster calculations are valid, but theactivation barriers are strongly reduced in sulfite oxidase, owing to the neutralisation of the Coulombic repulsionbetween the negatively charged active site and the negatively charged substrate.(No Image Selected)

CONTROL ID: 1718994TITLE: The Ferric Cytochrome P450cam Peroxide Shunt Reaction with Perbenzoic Acids: Probing the O-O BondCleavage Step to Form Compound I AUTHORS/INSTITUTIONS: J.H. Dawson, D.P. Collins, E. Johnson, E.D. Coulter, Z. Beharry, Chemistry &Biochemistry, Univ of South Carolina, Columbia, South Carolina, UNITED STATES|D.P. Ballou, Biological Chemistry,Univ of Michigan, Ann Arbor, Michigan, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: While monitoring the reaction of ferric cytochrome P450cam (Cyp101) with substituted (Cl, CH3,OCH3) perbenzoic acids using rapid scanning stopped flow (RSSF) spectroscopy, an intermediate appears en routeto the formation of the high-valent moiety known as Compound I [Fe(IV)=O/porphyrin radical cation] that is thought tobe the key catalytic species for oxygen atom transfer to substrate. It was suggested in previous studies (Spolitak, T.et al., J. Biol. Chem. 2005, 280, 20300-20309) that this is an acylperoxo-ferric heme adduct that subsequentlyundergoes O-O bond cleavage with release of the substituted benzoic acid to generate Compound I. Singular valuedecomposition analysis of the RSSF data for the formation of this intermediate shows that the energy of its Soretabsorption peak is sensitive to the electron donor properties of the aryl substituents. A linear Hammett correlation plotis seen for the energy of the Soret peak vs. the Hammett ρ constant. This correlation with the nature of thesubstituents requires that those substituents remain as part of the ligand bound to the heme iron, providing directevidence that this adduct is indeed a ferric acylperoxo derivative. Further support for this conclusion derives fromadditional linear Hammett correlation plots for both the rate of formation of the intermediate as well as for itsconversion to Compound I. Clearly, the electron donating/withdrawing properties of the substituents affect the donorproperties of the binding substrate, changing the observed rate of formation for the acylperoxo intermediate, as well asthe propensity and stability of the substituted benzoic acid to serve as the leaving group during O-O bond cleavageyielding Compound I. The ability to directly follow the O-O bond cleavage step in the formation of Compound I fromperacids provides a model for how this key step occurs in both P450 enzymes as well as in peroxidases.(No Image Selected)

CONTROL ID: 1719493TITLE: Studies on NColE7 - a potential platform for artificial metallonucleasesAUTHORS/INSTITUTIONS: B. Gyurcsik, A. Czene, E. Tóth, E. Németh, MTA-SZTE Bioinorganic Chemistry ResearchGroup / Department of Inorganic and Analytical Chemistry, University of Szeged, Szeged, HUNGARY|H. Otten, J.N.Poulsen, S. Larsen, Biophysical Chemistry Group / Department of Chemistry, University of Copenhagen,Copenhagen, DENMARK|H.M. Christensen, Department of Chemistry, Technical University of Denmark, Lyngby,DENMARK|K. Nagata, Department of Infection Biology / Graduate School of Comprehensive Human Sciences andFaculty of Medicine, University of Tsukuba, Tsukuba, JAPAN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Colicin E7 metallonuclease is a bacterial toxin of E. coli protecting the host cell from other relatedbacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. The cell killingactivity is attributed to the non-specific nuclease domain (NColE7), possessing the catalytic ββα-type metal ion-binding HNH motif at its C-terminus. Mutations affecting the positively charged amino acids at the N-terminus ofNColE7 (444-576) showed significantly reduced activity. The necessity of the N-terminal amino acids for the functionof the C-terminal catalytic center poses a possibility of an allosteric activation of the enzyme. The precise knowledgeof the intramolecular interactions of the residues affecting the catalytic activity could turn NColE7 into a potentialplatform for artificial nuclease design. Here we present the study on the N-terminal mutants of NColE7. Change incatalytic activity, metal ion and DNA binding, as well as, in the structure of distal amino acid sequences as aconsequence of mutations were monitored. Recombinant DNA technology, HPLC, gel mobility shift, massspectrometry, circular and linear dichroism and fluorescence spectroscopy were applied throughout the research. Oneof the mutant proteins was crystallized and the results from X-ray diffraction data lead to surprising conclusions aboutthe structural arrangement of the N-terminus of the mutant.Acknowledgement: Financial support of OTKA-NKTH CK80850, TAMOP-4.2.2/B-10/1-2010-0012, JSPS andDanscatt, as well as, the MAX-lab beamtime is greatly acknowledged.

The crystal structure of the nuclease domain of colicin E7 (PDB Id: 7CEI).

CONTROL ID: 1719830TITLE: Metallothioneins: a source of chemical and biological surprisesAUTHORS/INSTITUTIONS: M. Capdevila, Departament de Quimica, Universitat Autonoma de Barcelona, Cerdanyoladel Valles, Barcelona, SPAIN|S. Atrian, Departament de Genetica, Universitat de Barcelona, Barcelona, SPAIN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Metallothioneins (MTs) -a family of metalloproteins of low MW and high Cys content- have lavishlychallenged chemist and life scientists since their discovery in 1957 because their widespread occurrence in naturesuggests that they serve an important biological function, which has not been unambiguously defined yet.Initially, most works were devoted to the chemical and structural characterization of MTs and to the definition of theirmetal-binding properties. Currently, research points to the elucidation of their physiological function/s and the analysisof their molecular interactions with other cellular partners.At the beginning of the 90’s, there was a good body of data on two model MTs: the mammalian MT1, and the yeastCUP1. Since then, our groups have gathered data on the metal-binding abilities of a considerable number of MTsbelonging to evolutionary divergent organisms by always applying the same methodology for their recombinantsynthesis and the analysis of their metal binding abilities. The comprehensive analysis of all this data has revealedthat even small changes in the MT sequences led to surprising and unexpected differential properties. Also, these in-depth studies have revealed a clear gradation of their metal-binding features, which has afforded a robust newclassification: a stepwise gradation between what we call Zn- and Cu-thioneins. The intermediate positions areoccupied by a group of polyvalent MTs exhibiting a merging Zn-/Cu-thionein character, while the extreme positions arerespectively occupied by MTs highly specialized in either Zn- or Cu-binding.Taking into account that under physiological conditions, MTs are natively isolated as Zn(II)-, Cu(I)-, or mixed (Zn,Cu-)complexes, and that the bound metal ions drive the folding of the MT polypeptides, it is easy to conclude thatspecificity for Zn or Cu will determine the acquisition of a final 3D structure that very likely can be related with thefunctionality of the considered MT. This rationale supports our proposal of classification as a further step to unveil thedefinite function of these particular metalloproteins. (No Image Selected)

CONTROL ID: 1720516TITLE: Shining Light on the Metal Sites in Nitrogenase MoFe-protein by X-ray Anomalous DiffractionAUTHORS/INSTITUTIONS: L. Zhang, D.C. Rees, California Institute of Technology, Pasadena, California, UNITEDSTATES|D.C. Rees, Howard Hughes Medical Institute, Pasadena, California, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: In the process of biological nitrogen fixation, the nitrogenase MoFe-protein catalyzes the reductiondinitrogen to ammonia at the FeMo-cofactor, upon receiving the electrons from the donor via Fe-protein. Despitedecades of extensive biochemical, structural and spectral studies on the protein, many questions remain to beanswered regarding the mechanisms of electron transfer and substrate reduction at the FeMo-cofactor. We have usedmultiple-wavelength anomalous diffraction (MAD) to probe the identity and oxidization state of individual metals in theMoFe-protein. Our results demonstrate the presence of a mononuclear iron site, designated as Fe16, in addition to thefifteen iron sites present in the FeMo–cofactor and P–cluster. Fe16, which has been previously identified as eithercalcium or magnesium, is bound to a conserved set of protein and water oxygens forming a six–coordinate bindingsite between the two β–subunits of the MoFe–protein. The Fe K-edge absorption data of the Fe16 site indicates thatthe iron is in the ferrous oxidation state. The high sequence conservation of the residues coordinated to Fe16emphasizes the potential importance of the site in nitrogenase. The site-specific Fe K-edge absorption spectraobtained from the MAD data has been analyzed to assign oxidation states to irons in the metalloclusters. Theprogresses and challenges in this aspect of our work will be discussed. These studies will provide the structuralfoundation for a deeper understanding of the catalytic mechanism of nitrogenase. (No Image Selected)

CONTROL ID: 1720631TITLE: Advancing a Mechanism for Nitrogen Fixation by NitrogenaseAUTHORS/INSTITUTIONS: B. Hoffman, D. Lukoyanov, Chemistry, Northwestern, Evanston, Illinois, UNITEDSTATES|L. Seefeldt, Chemistry, Utah State University, Logan, Utah, UNITED STATES|D. Dean, Biochemistry,Virginia Tech University, Blacksburg, Virginia, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Biological nitrogen fixation — the reduction of N2 to two NH3 molecules — is catalyzed by theenzyme nitrogenase. An understanding of the nitrogenase catalytic mechanism requires, at minimum, identificationand characterization of each intermediate along the reaction pathway, plus an understanding of the kinetics/dynamicsthrough which these intermediates form. The kinetics are described by the Lowe-Thorneley (LT) model, whichprovides rate constants for transformations among intermediates, denoted En, as indexed by the number of electrons(and protons), n = 0-8, accumulated within the MoFe protein. The trapping and characterization of three of the eightelectron-reduction stages, including the key ‘Janus intermediate’, E4, the state at which N2 hydrogenation begins, andintermediate states that contain semi-reduced N2, have identified a form of the Alternating (A)’ pathway as most likelyoperative and unified the nitrogenase reaction pathway and LT kinetic scheme. One of the most puzzling aspects of nitrogenase function embodied in the LT scheme is an obligatory generationof H2 upon N2 binding, so that the limiting stoichiometry for ‘enzymological’ nitrogen fixation requires eightelectrons/protons to reduce each N2. As the delivery of each electron requires the hydrolysis of two MgATP, theformation of H2 thus apparently ‘wastes’ 25% of the total energy supplied by the hydrolysis of ATP. We recentlycompleted the unification of kinetics and pathway, thereby generating a first-draft mechanism for biological nitrogenfixation, by proposing that H2 release upon N2 binding involves reductive elimination of two hydrides, to yield N2bound to doubly-reduced FeMo-co, highly activated to achieve the most difficult step of nitrogen fixation, the initialhydrogenation of N2.This mechanism provides a framework to organize the vast body of data on which it builds, and as will be discussed,these provide constraints that have been successfully used to test the mechanism. As we will further show, themechanism also makes testable predictions. This talk describes these predictions and the experiments that inspired.

Janus intermediate

CONTROL ID: 1720870TITLE: A Universal Working Mechanism for the Ferroxidase Center of FerritinsAUTHORS/INSTITUTIONS: W.R. Hagen, K. Honarmand Ebrahimi, P.D. Verhaert, L. van der Weel, P. Hagedoorn,Biotechnology, Delft University of Technology, Delft, NETHERLANDS|E. Bill, MPI for Chemical Energy Conversion,Max Planck Institute, Muelheim an der Ruhr, GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Ferritins are ubiquitously distributed over the archaeal, bacterial, and eukaryal domains of life as a keymachine for cellular iron homeostasis. The 24-meric hollow spherical protein complex oxidizes Fe(II) and stores it as aferrihydrite-like nanoparticle in its interior for tuned reduction and release as required by cellular metallometabolism.According to communis opinio ferritins from different domains of life exhibit essential differences in their workingmechanism. It has long been thought that eukaryotic ferritins oxidize Fe(II) in their ferroxidase catalytic centers tostable μ-oxo bridged di-ferric units that would subsequently move spontaneously to the interior for core formation thusleaving behind ‘empty’ ferroxidase sites. In contrast, for several prokaryotic ferritins evidence suggested thatferroxidase centers were never completely free of iron. We have carried out comparative studies of a eukaryotic(human H-chain) ferritin and a prokaryotic (archaeal Pyrococcus furiosus) ferritin, from which we conclude that thesetwo – and by implication all – ferritins function under a universal principle according to which Fe(III) resides metastablyin the ferroxidase center until it is displaced towards core formation by incoming Fe(II). Our recent work on short-livedintermediates and the role of a conserved tyrosine residue in this process will be described. K Honarmand Ebrahimi, PL Hagedoorn, L van der Weel, PDEM Verhaert, WR Hagen (2012) J Biol Inorg Chem 17:975. A novel mechanism of iron-core formation by Pyrococcus furiosus archaeoferritin, a member of anuncharacterized branch of the ferritin-like superfamily.K Honarmand Ebrahimi, E Bill, PL Hagedoorn, WR Hagen (2012) Nature Chem Biol 8: 941. The catalytic center offerritin regulates iron storage via Fe(II)-Fe(III) displacement.K Honarmand Ebrahimi, P-L Hagedoorn, WR Hagen (2013) FEBS Lett 587: 220. Phosphate accelerates displacementof Fe(III) by Fe(II) in the ferroxidase center of Pyrococcus furiosus ferritin.K Honarmand Ebrahimi, P-L Hagedoorn, WR Hagen (2013) ChemBioChem, in press. A conserved tyrosine in ferritinis a molecular capacitor.

CONTROL ID: 1720979TITLE: Reactivity Patterns of Metal Oxo Enzymes and ReagentsAUTHORS/INSTITUTIONS: S. Shaik, The Hebrew University of Jerusalem, Jerusalem, ISRAEL|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: There are a few stories that concern bioinorganic chemical reactivity, and one of these will be chosenfor the talk close to the beginning of the conference:(a) The first story focuses on the reactivity of Cytochrome P450, its catalytic cycle, deactivation, etc.1(b) The second story describes the application of valence bond (VB) theory to bioinorganic chemistry.2(c) The third story concerns the oxy complexes of Myoglobin and Hemoglobin.3 It will be argued that VB theory andVB reading of complex multi reference wave functions is a productive future paradigm in the field.(d) The fourth story deals with exchange-enhanced reactivity (EER),4 which is the Hund’s rule analogue in chemicalreactivity. The utility of the concept will be outlined. [1] Shaik, S., Cohen, S., Wang, Y., Chen, H., Kumar, D., Thiel, W. Chem. Rev. 110 (2010) 949-1017.[2] Shaik, S., Lai, W.Z., Chen, H., Wang, Y. Acc. Chem. Res. 43 (2010) 1154-1165. (b) Lai, W.Z., Li, C.; Chen, H.;Shaik, S. Angew. Chem. Int. Ed. 51 (2012) 5556-5578. (c) Li, C.; Danovich, D.; Shaik, S. Chem. Sci. 3 (2012) 1903-1918. (d) Milko, P.; Schyman, P.; Dandamudi, D.; Chen, H.; Shaik, S. J. Chem. Theory Comput. 7 (2011) 327-339.[3] Chen, H., Ikeda-Saito, M., Shaik, S. J. Am. Chem. Soc., 130 (2008) 14778-14790.[4] Shaik, S., Chen, H., Janardanan, D. Nature Chem. 3 (2011) 19-27. (b) Chen, H., Lai, W.Z., Shaik, S., J. Phys.Chem. Lett. 1 (2010), 1533-1540. (c) Dandamudi, U.; Janardanan, D.; Li, C.; Shaik, S. Acc. Chem. Res. 46 (2013)471-482(No Image Selected)

CONTROL ID: 1721177TITLE: A radical alternative for methyl transfer reactions: Emergence of the B12-binding/radical SAM-domainenzymes.AUTHORS/INSTITUTIONS: O. Berteau, S. Pierre, A. Guillot, A. Benjdia, P. Langella, Institut Micalis (UMR 1319),INRA, Jouy-en-Josas, FRANCE|C. Sandström, Department of Chemistry, Swedish University of Agricultural Sciences,Uppsala, SWEDEN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Radical SAM (rSAM) enzymes are an emerging superfamily of enzymes involved in a wide range ofbiological processes and catalyzing chemically challenging reactions. This superfamily is characterized by thepresence of an atypical [4Fe-4S] cluster coordinated by three cysteinyl residues generally organized into the followingmotif: CXXXCXXC. It has been clearly established that this [4Fe-4S] cluster fulfills two major functions, (i) thecoordination of the S-adenosyl-L-methionine (SAM) cofactor and (ii) the injection of one electron resulting in the SAMhomolytic cleavage and the generation of the 5'-deoxyadenosyl (5'-dA) radical. We have undertaken the characterization of a novel rSAM enzyme, TsrM, predicted to possess an additional B12-binding domain and proposed to catalyze methyl transfer. This enzyme, which has the canonical rSAM motif, wasidentified in the biosynthetic pathway of the antibiotic Thiostrepton A. We performed its biochemical characterizationand confirmed its identity as a tryptophan methyltransferase. In an unexpected manner, we revealed that TsrM,contrary to all rSAM enzymes investigated so far, does not produce the canonical 5'-dA radical nor required anexternal electron donor for catalysis. We established that TsrM uses a novel mechanism, unique in enzymology, to transfer methyl groups to sp2-hybridized carbon atoms. This novel mechanism implies an unconventional use of the rSAM domain and the B12cofactor. At last, our current mechanistic hypothesis suggests that B12-binding/radical SAM enzymes produce amethyl radical during catalysis, which is an unprecedented reaction intermediate inside an enzyme active site. Reference:Pierre S, Guillot A, Benjdia A, Sandström C, Langella P & Berteau O*. (2012) Nat Chem Biol.:957-9

Proposed radical-based mechanism for the tryptophan methyltransferase (TsrM).

CONTROL ID: 1721369TITLE: New Tools for Copper(I) Biochemistry: Water Soluble High-Contrast Fluorescent Probes and Robust AffinityStandardsAUTHORS/INSTITUTIONS: C.J. Fahrni, M.T. Morgan, P. Bagchi, Chemistry and Biochemistry, Georgia Institute ofTechnology, Atlanta, Georgia, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: To cross cell membranes and reach specific cellular targets, fluorescent probes used in biologicalresearch must be sufficiently lipophilic. Dynamic light scattering studies on previously reported Cu(I)-responsivefluorescent probes revealed the formation of colloidal aggregates, presumably promoted by the high lipophilicity ofthese probes. Because the photophysical properties of fluorophores often change dramatically upon aggregation,lipophilic probes are prone to produce artifacts, especially within a mixed polarity environment as encountered inbiological applications. To address this problem, we designed and systematically optimized a series of new water-soluble Cu(I)-responsive fluorescent probes that respond with a robust, selective, and up to 180-fold emissionincrease upon saturation with Cu(I) in aqueous buffer. Furthermore, we developed a series of new water-solubleaffinity standards that can be utilized to deliver Cu(I) to biological ligands, to buffer aqueous Cu(I) concentrations inthe pico- to femtomolar range, or to reliably determine the Cu(I) stability constants of proteins and small moleculeligands through competition titrations. These new reagents offer a solid thermodynamic foundation for the reliabledetermination of Cu(I)-binding affinities of metalloproteins with kinetically accessible binding sites.(No Image Selected)

CONTROL ID: 1721502TITLE: New Insights into the Electronic Structure of the FeMo Cofactor of NitrogenaseAUTHORS/INSTITUTIONS: S. DeBeer, R. Bjornsson, F.A. Lima, T. Weyhermueller, F. Neese, Max Planck Institutefor Chemical Energy Conversion, Muelheim an der Ruhr, GERMANY|S. DeBeer, Cornell University, Ithaca, New York,UNITED STATES|O. Einsle, Albert-Ludwigs-Universität Freiburg, Freiburg, GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Nature utilizes the nitrogenase enzyme to effect the conversion of atmospheric dinitrogen to ammonia.Nitrogenase enzymes utilize a complex active site cluster consisting of 7 iron ions, 1 molybdenum ion and 9 sulfides,the so called FeMoco active site. Recently, valence-to-core spectroscopy X-ray emission spectroscopy, X-raycrystallography and ESEEM data have been utilized to reveal the presence of a central carbide in this cluster.[1,2]While a detailed description of the atomic composition of the FeMoco active site is now in hand, the electronicstructure of the active site remains elusive. Namely, the oxidation state distribution and charge of the cluster areunknown. At present, three main oxidation state assignments are discussed in the literature, all which assume adiamagnetic Mo(IV) in the active site of FeMoco. Recently, we have utilized high-energy resolution fluorescencedetected X-ray absorption spectroscopy (HERFD XAS) at the Mo K-edge in order to evaluate this assumption. HERFDXAS data allow for spectra at resolutions below the core hole lifetime broadening of the Mo 1s electron, allowing formore richly featured spectra, which can be quantitatively assessed. Mo HERFD XAS data on a series of Mo modelcomplexes and the FeMoco cofactor of nitrogenase will be presented. The experimental data are closely correlated todetailed theoretical calculations. On the basis of both experiment and theory the Mo oxidation state assignment inFeMoco is evaluated. The potential implications of for reactivity will be discussed. [1] Lancaster, K.M.; Roemelt, M.; Ettenhuber, P.; Hu, Y.; Ribbe, M.W.; Neese, F.; Bergmann, U.; DeBeer, S. Science334, 974 (2011).[2] Spatzal, T.; Aksoyoglu, M.; Zhang, L.M.; Andrade, S.L.A.; Schleicher, E.;Weber,S.; Rees, D.C.; Einsle, O. Science 334, 940 (2011). (No Image Selected)

CONTROL ID: 1721516TITLE: Crystal Structure of Dihydropyrimidinase from Tetraodon nigroviridis with Lysine Carboxylation: MetalRequirement for Post-translational Modification and FunctionAUTHORS/INSTITUTIONS: C. Chen, Y. Hsieh, Life Science Group, Scientific Research Division, NationalSynchrotron Radiation Research Center, Hsinchu, TAIWAN|Y. Yang, Department of Biological Science andTechnology, National Chiao Tung University, Hsinchu, TAIWAN|C. Chen, Institute of BIotechnology, National ChengKung University, Tainan, TAIWAN|S.I. Chan, Division of Chemistry and Chemical Engineering, California Institute ofTechnology, Pasadena, California, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Lysine carboxylation, a post-translational modification, facilitates metal coordination for specificenzymatic activities. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis(TnDhp) in various states: the apo enzyme as well as two forms of the holo enzyme with one and two metals at thecatalytic site. The essential active-site structural requirements have been identified with possible existence of fourmetal-mediated stages of lysine carboxylation. Only one metal is sufficient for lysine carboxylation. However, the post-translational lysine carboxylation facilitates additional metal coordination for the regulation of specific enzymaticactivities through controlling the conformations of two dynamic loops, Ala69–Arg74 and Met158–Met165, located inthe tunnel for the substrate entrance. The substrate/product tunnel is in the “open form” in the apo-TnDhp, in the“intermediate state” in the mono-metal TnDhp, and in the “close form” in the di-metal TnDhp structure, respectively.Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactionswith the Ala69–Arg74 dynamic loop. These structural results provide an illustration of how a protein exploits uniquelysines and the metal distribution to accomplish lysine carboxylation as well as subsequent enzymatic functions.(No Image Selected)

CONTROL ID: 1721854TITLE: Construction of a Methionine Synthase Model by Apomyoglobin–Cobalt Corrin ComplexAUTHORS/INSTITUTIONS: T. Hayashi, Y. Morita, K. Oohora, Applied Chemistry, Osaka University, Suita, JAPAN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Myoglobin, a dioxygen storage hemoprotein, binds one heme molecule as a prosthetic group in theprotein matrix via Fe–His coordination and non-covalent interaction. Apomyoglobin prepared by removal of heme willprovide a useful cavity (heme pocket) as a metal complex binding site. Our group has focused on preparation andcharacterization of reconstituted myoglobins with an artificial prosthetic group to generate a new biocatalyst.Methionine synthase having cobalamin, a vitamin B12 derivative, as a cofactor catalyzes the synthesis of methioninefrom homocysteine via Co–CH3 bond formation. The catalytic reaction contains two key intermediates, Co(I) fourcoordinate species with highly nucleophilicity and an organocobalt complex including a Co–C bond. Although thecobalamin-depenent enzyme responsible for the methyl transfer is quite interesting in the field of bioinorganic andorganometallic chemistry, no real and simple model has been presented. Thus, we have recently replaced thecobalamin and cobalamin binding domain with cobalt tetradehydrocorrin and apomyoglobin, respectively, todemonstrate a new model of methionine synthase. First, we prepared Co(II) tetradehydrocorrin with two propionateside chains and then inserted it into the heme pocket of apomyoglobin. The obtained reconstituted myoglobin wasfully characterized by spectroscopic methods and X-ray crystal structure analysis. The Co(II) complex can be easilyreduced to Co(I) species in the heme pocket upon the addition of dithionite. The crystal structural analysis of Co(I)species reveals the tetra-coordinate low spin state. Furthermore, addition of methyl iodide to a solution of Co(I)species gave the photoactive methylated cobalt complex. In addition, the methyl group was transferred from thecobalt atom to the imidazole of His 64 in the heme pocket. These results indicate that the cobalt complex in the hemepocket forms the Co–C bond and then transfers the methyl group, which is seen in methionine synthase. Here, wereport on the design, synthesis, characterization and reactivity of myoglobin reconstituted with tetradehydrocorrincobalt complex.

CONTROL ID: 1721863TITLE: Isoprenoid biosynthesis in pathogenic bacteria: Substrate and Inhibitor interaction of the 4Fe-4S center of theLytB protein investigated by nuclear inelastic scattering AUTHORS/INSTITUTIONS: V. Schünemann, I. Faus, S. Rackwitz, J.A. Wolny, Department of Physics, University ofKaiserslautern, Kaiserslautern, GERMANY|M. Rohmer, M. Seemann, Institut de Chimie UMR CNRS UDS 7177,Université de Strasbourg, Strasbourg, FRANCE|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Nuclear Inelastic Scattering (NIS; also called Nuclear Resonance Vibrational spectroscopy; NRVS) hasbeen shown to be a useful tool in detecting selectively iron-ligand vibrations in proteins. Currently we are applyingthese biophysical tools to the 4Fe-4S enzyme LytB, also called IspH, which is involved in the last steps of isoprenoidsynthesis via the methylerythritol phosphate pathway (MEP) pathway. This pathway does not exist in humans, andtherefore LytB is a promising target for the development of new specific antibacterial and antiparasitic drugs. We havepreviously shown by conventional Mössbauer spectroscopy that substrate as well as potential inhibitors bind directlyto the 4Fe-4S cluster of LytB. Now we present results concerning the experimental detection and theoreticalcalculation of functional relevant iron ligand modes in LytB via combined quantum chemical and molecular mechanics(QM/MM) calculations by assuming model structures of the active site. Acknowledgements. The support of the German Federal Ministry of Education andResearch under 05K10UKA is gratefully acknowledged.

Top: Experimental pDOS of HMBPP bound LytB taken at T=20 K. Bottom: pDOS simulations obtained via combinedquantum chemical and molecular mechanics (QM/MM) calculations by using the X-ray structure data (3KE8.pdb) ofHMBPP bound LytB.

CONTROL ID: 1722073TITLE: The Mechanism of Solar Water Oxidation: A High Resolution Molecular and Electronic Structure of theOxygen-Evolving Complex of Photosystem IIAUTHORS/INSTITUTIONS: K.V. Lakshmi, S. Milikisiyants, C. Coates, R. Chatterjee, Chemistry and ChemicalBiology, Rensselaer Polytechnic Institute, Troy, New York, UNITED STATES|F. Koua, J. Shen, School of NaturalScience and Technology, Okayama University, Okayama, JAPAN|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: The recent 1.9 Angstrom resolution X-ray structure of Photosystem II (PSII) [Umena et al. Nature,2011,473, 55] provides a detailed molecular geometry of the oxygen-evolving complex (OEC) in the dark stable S1state. Despite the remarkable breakthrough in the resolution achieved by the new X-ray crystal structure of the OEC inthe S1 state, its structure in the higher oxidation states remain largely unknown. Also, there is limited knowledge of theelectronic structure of the tetra-nuclear manganese calcium-oxo (Mn4Ca-oxo) cluster in the OEC of PSII. Weakmagnetic interactions between the paramagnetic Mn4Ca-oxo cluster and surrounding magnetic nuclei are verysensitive to both molecular geometry and electronic structure of the OEC. Two-dimensional hyperfine sublevelcorrelation (HYSCORE) spectroscopy is a powerful technique to resolve hyperfine interactions in multinuclearparamagnetic centers, such as, the OEC of PSII. In this study, we describe HYSCORE spectroscopy measurementsof the hyperfine couplings in the S2 state of the OEC. In order to relate the experimental parameters to the electronicand geometric structure of the OEC, we also examine the spectroscopic properties of water-splitting model systemsby HYSCORE spectroscopy. We report, for the first time, the high-resolution molecular and electronic structure of theOEC in the S2 state of PSII.

Two-dimensional proton HYSCORE spectrum of the OEC of photosystem II.

CONTROL ID: 1726545TITLE: Biogenesis of Fe-S proteins in Escherichia coli under fluctuating environmental conditionsAUTHORS/INSTITUTIONS: F. Barras, Université de Aix-Marseille, Marseille, FRANCE|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Iron-sulphur (Fe-S) clusters are ubiquitous cofactors present in a myriad of proteins controllingprocesses and pathways (DNA replication and repair, gene expression, photosynthesis, respiration). Fe-S clustersassembly and delivery into apo-proteins are catalysed by multi-protein systems conserved throughout prokaryotes andeukaryotes1. Because so many cellular processes are dependent upon Fe-S proteins, alteration of the Fe-S clusters,or of the systems that make them, has profound impact on cellular physiology. Remarkably, Escherichia colipossesses two Fe-S biogenesis systems, ISC and SUF, which are thought to function under non-stress and stressconditions, respectively. In addition, “non-ISC, non-SUF” components, such as NfuA, ErpA, Mrp or CyaY mightintervene at different steps in the process. After a brief overview of the overall Fe-S biogenesis process, I will discussthe maturation processes of selected Fe-S proteins. I will focus in particular on Fe-S proteins that act as stress-sensing transcriptional regulators2 and on Fe-S proteins that participate to traffic across membranes. A special focuswill be put on stress conditions provoked by the presence of poisonous chemicals and/or antibiotics3,4. This shouldprovide us with a model of the molecular strategies E. coli uses to make and deliver Fe-S clusters to approximately160 proteins species under fluctuating conditions. 1Py B. and F. Barras (2010) Building Fe/S proteins : Bacterial strategies.Nature Rev. Microbiol. 8:436-446.2Vinella, D. et al. (2013) In vivo (Fe-S) cluster acquisition by IscR and NsrR, two stress regulators in Escherichia coli.Mol. Microbiol. 87:493-508.3Roche B. et al. (2013) Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity. BBA.Bioenergetics. 1827:455-4694 Barras, F., and M. Fontecave (2011) Cobalt stress in Escherichia coli and Salmonella enterica : molecular bases fortoxicity and resistance. Metallomics 3:1130-1134.(No Image Selected)

CONTROL ID: 1726644TITLE: PerR: a bacterial resistance regulator or what else?AUTHORS/INSTITUTIONS: J. Latour, iRTSV, CEA, Grenoble, FRANCE|J. Latour, Université de Grenoble, Grenoble,FRANCE|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: PerR belongs to the Fur family of bacterial metalloregulators,[1] but it is unique within this family sinceits purported role is to detect hydrogen peroxide whereas all other members are metal sensors (Mur: Mn, Fur: Fe, Nur: Ni and Zur: Zn). As all members of the family PerR is a homodimer, the dimer structure being locked in its case bythe binding of a zinc ion in a tetracysteinate site,[2] and requires a regulatory metal (Fe, Mn) to bind its cognate DNAsequence (the so-called "PerR box") (Figure). Its sensing function has been shown to operate through peroxideoxidation of an histidine ligand catalyzed by the iron.[3,4] All structural and physical data highlight the strong similarity between PerR and Fur[5] whereas Fur is not sensitive tohydrogen peroxide. The cause of this difference will be identified through a combination of in vivo and in vitroexperiments using mass spectrometric and spectroscopic studies and rationalized with the help of DFT calculations.Furthermore the mechanism of PerR (an)aerobic interaction with hydrogen peroxide will be addressed.[1] C.M. Moore, J.D. Helmann Curr. Op. Microbiol. 2005, 8, 188[2] D.A.K. Traoré et al. Mol. Mic. 2006, 61, 1211.[3] J.W. Lee, J.D. Helmann Nature 2006, 440, 363[4] D.A.K. Traoré et al. Nature Chem. Biol. 2009, 5, 53[5] L. Jacquamet et al. Mol. Mic. 2009, 73, 20.

Structure of the regulatory site of PerR-Zn-Mn

CONTROL ID: 1727679TITLE: Biosynthesis of the H-Cluster of the [FeFe]-HydrogenaseAUTHORS/INSTITUTIONS: J.B. Broderick, J.W. Peters, E. Shepard, B.R. Duffus, A. Byer, S. Ghose, Chemistry &Biochemistry, Montana State University, Bozeman, Montana, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: The hydrogenase H-cluster is an unusual iron-sulfur cluster assembly that exists as a [4Fe-4S] clusterbridged to a 2Fe cluster containing multiple inorganic (CO and CN-) ligands as well as an exogenous bridgingdithiolate ligand; the proper assembly of this H-cluster is necessary for hydrogenase activity, and is carried out by thehydrogenase-specific accessory proteins HydE, HydF, and HydG. HydE and HydG are radical SAM enzymes thatsynthesize the ligands of the 2Fe subcluster, while HydF is a GTPase that appears to serve as a scaffold for assemblyof the 2Fe subcluster prior to its transfer to the hydrogenase containing a pre-formed [4Fe-4S] cluster in the active sitecavity. HydG has been shown to synthesize the CO and CN- ligands using tyrosine as a substrate. Both the N-terminal radical SAM [4Fe-4S] cluster and a second site-differentiated [4Fe-4S] cluster are required for catalysis, withthe former required for tyrosine cleavage to p-cresol and the latter required for the ultimate generation of the diatomicligands. The function of HydE remains more elusive, although evidence supports a role in which this enzyme utilizesan amino acid (probably cysteine) as a substrate for synthesis of the dithiolate bridging ligand. HydF can bind both[2Fe-2S] and [4Fe-4S] clusters, and it has been suggested that a [2Fe-2S] cluster bound to HydF is modified by theactions of the radical SAM enzymes HydE and HydG to generate the 2Fe precursor of the H-cluster. The latestresults from our lab regarding the functions and mechanisms of these three Hyd accessory proteins will be presentedin this talk.(No Image Selected)

CONTROL ID: 1729522TITLE: The Chemistry of Oxygen Sensing in Humans and other AnimalsAUTHORS/INSTITUTIONS: C.J. Schofield, chemistry, University of Oxford, Oxford, UNITED KINGDOM|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: The lecture will describe studies aimed at identifying the role of oxygenases in the regulation of proteinbiosynthesis. Attempts to correlate the biochemical properties of oxygenases with their physiological roles, inparticular with respect to oxygen sensing will be described. In addition to work on enzymes involved in transcriptionalregulation, including via the oxygen-dependent modification of ribosomes will be described.(No Image Selected)

CONTROL ID: 1734791TITLE: [NiFe] and [FeFe] hydrogenases: Active site structures and catalytic mechanismsAUTHORS/INSTITUTIONS: W. Lubitz, Max Planck Institute for Chemical Energy Conversion, Muelheim an der Ruhr,GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Hydrogenases catalyze the reversible heterolytic splitting of molecular hydrogen at a binuclear (NiFe,FeFe) metal center. A detailed understanding of the enzymatic mechanisms requires knowledge of the intermediatesof the catalytic cycle. These can be trapped and spectroscopically characterized using magnetic resonance andvibrational techniques, the obtained parameters are verified by DFT calculations. For the anaerobically isolated [NiFe]hydrogenase of Desulfovibrio vulgaris Miyazaki F (DvMF) a crystallographic structure with a resolution ≤ 0.9Å ispresented(1) in which for the first time the hydrogens could be located. In the reduced Ni-R state the products of theheterolytic H2 splitting are seen, i.e. the hydride in the bridge between Ni and Fe and a proton at a terminal cysteineligand. Furthermore the CO and CN ligands of the Fe could be clearly assigned (see figure). The [NiFeSe]hydrogenase from DvMF could be isolated and first spectroscopic and electrochemical results are presented.(2) Theoxygen sensitivity of these hydrogenases is discussed and it is demonstrated that the oxygen tolerance is significantlyincrease by embedding the enzyme in a tailor-made polymer matrix. For the [FeFe] hydrogenase a new intermediatecould be spectroscopically identified completing the catalytic cycle.(3) It is further demonstrated how inorganic modelcomplexes mimicking the [2Fe] subcluster of the active site in [FeFe] hydrogenases can be incorporated into theapoenzyme.(4) The vast prospects of this finding for hydrogenase research in general and possible applications willbe discussed. 1. H. Ogata, K. Nishikawa, W. Lubitz (2013), submitted.2. J. Riethausen, O. Rüdiger, W. Gärtner, W. Lubitz, H.S. Shafaat Chem. Bio. Chem. (2013), in press, doi:10.1002/cbic.201300120.3. A. Adamska, Silakov, O. Rüdiger, C. Lambertz, T. Happe, E.J. Reijerse, W. Lubitz Angew. Chem. Int. Ed. (2012)51: 11458.4. G. Berggren, A. Adamska, T. Simmons, C. Lambertz, J. Esselborn, M. Atta, S. Gambarelli, J.M. Mouesca, E.Reijerse, W. Lubitz, T. Happe, V. Artero, M. Fontecave Nature (2013), in press, doi: 10.1038/nature12239.

X-ray crystallographic structure of the active site (Ni-R state) in the [NiFe] hydrogenase of DvMF at 0.9Å resolution

CONTROL ID: 1747606TITLE: Nitrogenase: A case Study of Metalloprotein Assembly and MechanismAUTHORS/INSTITUTIONS: M.W. Ribbe, Dpt of Chemistry, University of California, Irvine, California, UNITEDSTATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Nitrogenase is highly complex in structure and uniquely versatile in function. It is capable of catalyzingtwo reactions that parallel two important industrial processes: the reduction of nitrogen (N2) to ammonia (NH3), whichparallels the Haber-Bosch process in ammonia production; and the reduction of carbon monoxide (CO) tohydrocarbons (C1-C4 alkanes and alkenes), which parallels the Fischer-Tropsch process in carbon fuel production.Despite decades of dedicated research, the biosynthetic and catalytic mechanisms of nitrogenase have remainedelusive, largely due to the complexity of the metal centers within this enzyme. Taking a combined genetic,biochemical, spectroscopic and structural approach, we have made progress in piecing together the biosyntheticpathway of nitrogenase, as well as in identifying homologous systems for mechanistic studies of this enzyme. Thesedevelopments not only afford a better understanding of how nitrogenase is assembled into a functional unit, but alsoestablish a useful paradigm for biosynthetic and functional studies of other metalloenzyme systems.(No Image Selected)

CONTROL ID: 1747620TITLE: New Cofactors, Hot Intermediates, and Unexpected Reactivities: Exploring the Mechanistic Diversity of Non-Heme-Iron EnzymesAUTHORS/INSTITUTIONS: C. Krebs, M.J. Bollinger Jr, Penn state University, University Park, Pennsylvania, UNITEDSTATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Enzymes that activate dioxygen at a mononuclear or dinuclear non-heme-iron cofactor catalyze a widevariety of chemically difficult and biologically important oxidation reactions.1-3 Our joint group is particularly interestedin non-heme-iron enzymes that cleave aliphatic C-H bonds. We study their reaction mechanisms by a combination ofbiochemical, kinetic, and spectroscopic methods.4 This approach led to the identification of new cofactors,5,6 trappingand characterization of many reactive intermediates,7,8 and discovery of unexpected reactivities. The mechanisticdiversity of these enzymes will be presented. Specifically, the role of 57Fe-Mössbauer spectroscopy in these studieswill be highlighted. (1)Hausinger, R. P. Crit. Rev. Biochem. Mol. Biol. 2004, 39, 21-68.(2)Costas, M.; Mehn, M. P.; Jensen, M. P.; Que, L., Jr. Chem. Rev. 2004, 104, 939-986.(3)Krebs, C.; Bollinger, J. M., Jr.; Booker, S. J. Current Opinion in Chemical Biology 2011, 15, 291-303.(4)Bollinger, J. M., Jr.; Krebs, C. J. Inorg. Biochem. 2006, 100, 586-605.(5)Jiang, W.; Hoffart, L. M.; Krebs, C.; Bollinger, J. M., Jr. Biochemistry 2007, 46, 8709-8716.(6)Bollinger, J. M., Jr.; Diao, Y.; Matthews, M. L.; Xing, G.; Krebs, C. Dalton Trans. 2009, 6, 905-914.(7)Krebs, C.; Galonić Fujimori, D.; Walsh, C. T.; Bollinger, J. M., Jr. Acc. Chem. Res. 2007, 40, 484-492.(8)van der Donk, W. A.; Krebs, C.; Bollinger, J. M. Current Opinion in Structural Biology 2010, 20, 673-683. (No Image Selected)

CONTROL ID: 1748398TITLE: Alternative ground states in the CuA center and its possible role in electron transferAUTHORS/INSTITUTIONS: A.J. Vila, Institute for Molecular and Cellular Biology , University of Rosario, Rosario,ARGENTINA|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: CuA is a binuclear copper site which acts as the electron entry port in terminal heme-copper oxidasesand in N2O reductases. The high efficiency of this metal site in long range intra- and intermolecular electron transferhas been attributed to its unusual coordination features, which ensure a low reorganization energy and an electronicstructure poised to meet the physiological requirements. The electronic structure of the oxidized CuA center can bedescribed by a σu* ground state wavefunction, i.e., involving an anti-bonding σ interaction between the two copperions, which is associated with a short Cu-Cu distance (2.5 Å). An alternative ground state of πu symmetry with higherenergy is partially populated at room temperature. The πu state has been associated to much longer Cu-Cu distances(2.9-3.0 Å), and its role in electron transfer has been considered as marginal.Minor perturbations are able to increase the population of the πu state without disrupting the mixed valence features,as shown by NMR experiments in a series of mutants. Moreover, the πu state stabilized by the protein matrix differsfrom the one found in model complexes in the sense that is compatible with a short Cu-Cu distance, thus being able tomaintain a low reorganization energy and ET efficiency.(No Image Selected)

Found 10 Abstracts CONTROL ID: 1677920TITLE: Biosynthesis of Nicotinamide Adenine Dinucleotide: an iron-sulfur cluster as catalyst and therapeutic targetAUTHORS/INSTITUTIONS: S. Ollagnier de Choudens, CNRS, Grenoble, FRANCE|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Nicotinamide adenine dinucleotide (NAD) is an essential cofactor involved in a myriad of biologicaloxidation-reduction reactions. Its biosynthesis involves the formation of quinolinic acid (QA), the biosynthesis of whichis different in prokaryotes and eukaryotes1. In eukaryotic organisms, QA is produced by the degradation of L-tryptophan, whereas in most prokaryotes, QA is generated by the condensation reaction between dihydroxyacetonephosphate (DHAP) and iminoaspartate (IA). This reaction requires the concerted actions of two proteins, L-aspartateoxidase, which first converts L-aspartate into IA, and quinolinate synthase (NadA), a Fe4S4 enzyme, whichcondenses IA with DHAP to form QA2,3. Besides these de novo syntheses of NAD, a salvage pathway exists in someorganisms that enable NAD to be recycled. However, some pathogens such as Mycobacterium leprae andHelicobacter pylori were reported to lack this salvage pathway. The presence in prokaryotes and eukaryotes of distinctpathways for QA biosynthesis, along with the absence of the salvage pathway in some pathogenic microorganisms,makes NadA a target for the development of antibacterial agents.One of our objectives is to shed light on the mechanism catalysed by NadA at a molecular level, which so far hasbeen a stumbling block in the elucidation of the de novo QA biosynthesis pathway in bacteria. Using substrate andpostulated intermediate analogs of the mechanism we demonstrated for the first time, the involvement of the Fe4S4cluster in catalysis through a differentiated iron site. In addition, one analog proved to be a selective inhibitor of QAbiosynthesis pathway in vivo on E. coli raising the possibility of this molecule to act as a new antibacterial agent onNadA from M. leprae and H. pylori (1)Begley, T. P. et al. Vitamins and hormones 2001, 61, 103.(2)Cicchillo, R. M. et al. Journal of the American Chemical Society 2005, 127, 7310.(3)Ollagnier-de Choudens, S. et al. FEBS letters 2005, 579, 3737. (No Image Selected)

CONTROL ID: 1711116TITLE: INSIGHTS INTO THE CATALYTICCYCLE OF Pseudomonas nautica NITROUS OXIDE REDUCTASEIsabel Moura1,Sofia R. Pauleta1, Simone Dell'Acqua1,2, Rute F. Nunes1, Susana Ramos1, Oliver Einsle3, José J.G.Moura1AUTHORS/INSTITUTIONS: I. Moura, C. Carreira, S. Pauleta, R.F. Nunes, J.J. Moura, S. Ramos, ChemistryDepartment, Requimte/CQFB, Caparica, PORTUGAL|S. Dell?acqua, Dipartimento di Chimica, Università di Pavia,Università di Pavia, Pavia, ITALY|O. Einsle, BIOSS Centre for Biological Signalling Studies, Institute of OrganicChemistry and Biochemistry , Friburg, GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Denitrification is an important biological pathway with environmental implications. Nitrate accumulationand release of nitrous oxide in the atmosphere due to use of excess fertilizers are examples of two environmentalproblems, where denitrification plays a central role. The reduction of nitrate to nitrogen gas uses four different types ofmetalloenzymes in four simple steps: nitrate is reduced to nitrite, then to nitric oxide, followed by the reduction tonitrous oxide and by a final reduction to di-nitrogen: (2 NO3- → 2 NO2- → 2 NO → N2O → N2).The 3D structures of all these enzymes are known.We present a concise updated review of the bioinorganic aspects of denitrification with emphasis on spectroscopicfeatures, structural and mechanistic aspects of the relevant enzymes involved, with emphasis on those involved in thelast step of this complex pathway. The metal diversity detected in this pathway is also acknowledged. Nitratereductase is a molybdenum containing enzyme, Nitric oxide reductase, a membrane enzyme contains a non-hemeiron coupled to a b-type heme and Nitrous oxide reductase contains a new tetranuclear copper center (CuZ). AcknowledgementContributions from BIOIN/BIOPROT and project PTD/QUI-BIQ/116481/2010 FCT-MCTES for financial support. References[1] S. Dell’Acqua, et al., Encyclop. of Metalloproteins, 2012, in press.[2] P. Gonzalez, et al., Coordination Chemistry Reviews, 2012, in press.[3] S. Dell’Acqua, et al., 2011, J.Biol.Inorg.Chem. 16, 1241-1254.[4] C. Timóteo, et al., 2011, Biochem. 50, 4251-4262.[5] P. Tavares et al. , 2006, J. Inorg. Biochem. 100, 2087-100. (No Image Selected)

CONTROL ID: 1715532TITLE: The great diversity of cytochrome P450 reactions : recently discovered reactions and molecular bases of thisdiversity(Invited Keynote lecture) AUTHORS/INSTITUTIONS: D. Mansuy, UMR 8601, Université Paris Descartes, Paris, FRANCE|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Diversity and adaptability are two main key-words that well characterize the cytochromes P450superfamily. These ubiquitous hemeproteins exhibit an extreme diversity of biological functions, as they are involvedin the biosynthesis and/or catabolism of a huge number of important endogenous molecules such as steroidhormones, prostaglandins and vitamin D in man, alkaloids in plants or terpenes in microorganisms, as well as in themetabolism of xenobiotics. The extreme substrate diversity of the cytochromes P450 involved in xenobioticsmetabolism is a key element in the adaptation of living organisms to their always changing chemical environment.Cytochromes P450 also exhibit a surprising diversity of reactions that they are able to catalyze. A series of newreactions quite different from the usual oxidations that they most often catalyze have been discovered recently. Thisincludes for instance hydrolysis, hydration, and decarboxylation reactions, and 2,2-cycloadditions. Some of thesereactions need the consumption of NADPH whereas others do not need the presence of this cofactor. Our studies todetermine the mechanisms of several of these new reactions will be presented, and the molecular bases of the greatdiversity of cytochrome P450 reactions will be discussed.(No Image Selected)

CONTROL ID: 1715803TITLE: How bacteria enjoy bioinorganic chemistryAUTHORS/INSTITUTIONS: H. Kozlowski, M. Rowinska-Zyrek, D. Witkowska, S. Potocki, Faculty of Chemistry,University of Wroclaw, Wroclaw, POLAND|D. Valensin, Department of Biotechnology, Chemistry and Pharmacy,University of Siena, Siena, ITALY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Recently, a series of findings were described on the homeostasis of nickel in Helicobacter pylori,bacterium that colonizes the gastric mucosa in humans, and is the causative agent of acute and chronic gastritis andpeptic ulcer disease. The homeostasis of Ni2+ is crucial for the survival of H. pylori in the extremely acidicenvironment of the stomach, and, unluckily, this bacterium is highly specialised in handling nickel ions - delivered tourease and hydrogenase by a set of accessory proteins, and most of the bacterium's metal metabolism is centredupon their expression and maturation [1].Cys and His residues are tempting binding sites for Ni2+, which coordinates to the sulfur of Cys and amide nitrogenatoms, or to His imidazoles and amides. Knowledge about the mechanism of such coordination can be the cluetowards its suppression. Bi3+, one of the treatments for stomach ulcer disease, can serve as an example; it has a verystrong affinity towards Cys thiol groups, and can also coordinate an additional His imidazole. The affinity of bismuthtowards Cys side groups is much stronger than the affinity of nickel towards the same sites, therefore Bi3+ is able todisplace nickel from its binding site, causing the inhibition of nickel chaperones [2]. Zn2+, the metal which often playsa structural or regulatory role in those nickel chaperones [3], is also able to bind to both thiolates and imidazoles.Not only poly-His and poly-Cys, but also poly-Gln sites have a surprising impact on the stability of metal ion binding[4]. Poly-aminoancid regions are typically unstructured, and the binding of a cation to such regions might result in astructural rearrangement of a distinct site of the same protein. References:[1] Witkowska D., Rowinska-Zyrek M., Valensin G., Kozlowski H. Coord. Chem. Rev., 2012, 256, 133-148.[2] Rowinska-Zyrek M., Witkowska D., Valensin D., Kamysz W., Kozlowski H. Dalton Trans., 2010, 39, 5814-5826.[3] Rowinska-Zyrek M., Potocki S., Wtkowska D., Valensin D., Kozlowski H., Dalton Trans, 2013, 6010-6019.[4] Chiera N.M., Rowinska-Zyrek M., Wieczorek R., Guerrini R., Witkowska D., Remelli M., Kozlowski H., Metallomics,2013, 5, 214-221.(No Image Selected)

CONTROL ID: 1721110TITLE: New insights on the reactivity of the molybdenum cofactor in bacterial nitrate reductasesAUTHORS/INSTITUTIONS: B. Guigliarelli, J. Jacques, P. Ceccaldi, B. Burlat, F. Biaso, S. Grimaldi, C. Léger, V.Fourmond, BIP-UMR7281, Aix-Marseille University, CNRS, Marseille, FRANCE|A. Magalon, LCB-UMR7283, Aix-Marseille University, CNRS, Marseille, FRANCE|D. Pignol, P. Arnoux, M. Sabaty, LBC-UMR7265, Aix-MarseilleUniversity, CEA, CNRS, St Paul Lez Durance, FRANCE|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Molybdenum enzymes constitute a wide enzyme family found in nearly all organisms, in all kingdomsof life. In prokaryotes, the vast majority of these enzymes contain a mononuclear Mo-cofactor in which the Mo ion iscoordinated by two pyranopterin guanosine dinucleotide (PGD) moieties and by an additional protein ligand which canbe Ser, Cys (or Se-cys) or Asp. These enzymes are very diverse in terms of structure and subunit composition andare able to use a broad diversity of substrates, being involved in the major biogeochemical cycle of nitrogen, sulfur,carbon and metalloids (1). They catalyze redox reactions which can be considered for most of them as oxygen atomtransfer between substrate and solvent, sulfur atom transfer and hydrogen atom transfer, but non-redox reactionswere also identified. Thus, in spite of the similarity of their Mo-bisPGD cofactor, these enzymes catalyze a widediversity of reactions but the molecular factors which trigger their reactivity remains largely debated (2). To addressthis question, the periplasmic nitrate reductase from Rhodobacter sphaeroides and the respiratory nitrate reductasefrom Escherichia coli, two enzymes catalyzing the same reaction but with different proteic ligands of the Mo cofactorwere used as model systems. By combining site-directed mutagenesis, EPR spectroscopy, electrochemistry and DFTcalculations, new insights on the activation process of the enzyme and on the role of the various spectroscopicallydetected Mo species in catalysis were brought. The potential role of pyranopterin in these processes is clearlyemphasized and these results give prominent information for the future design of artificial molybdo-enzymes withcontrolled reactivity. 1- S. Grimaldi, B. Schoepp-Cothenet, P. Ceccaldi, B. Guigliarelli, A. Magalon, Biochim. Biophys. Acta (2013) in press.2- S. Metz, W. Thiel, Coord. Chem. Rev. 255 (2011) 1085-1103.

CONTROL ID: 1721815TITLE: Theoretical Spectroscopy of Open Shell Transition Metals in Enzymes and Model ComplexesAUTHORS/INSTITUTIONS: F. Neese, Max-Planck-Institut fuer Chemische Energiekonversion, Muelheim an derRuhr, GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Open Shell transition metal ions play fundamental roles in the active sites of enzymes, in catalysis, inmaterials science and molecular magnetism to only name a few important research fields. The reactivities andspectroscopic properties of these open-shell transition metal ions are complex and represent a substantial challengefor theoretical chemistry. In the past years we have been involved in developing and applying theoretical methodsranging from density functional theory to multireference wavefunction approaches to problems in transition metalchemistry (e.g.1-6). The talk will present selected examples from this work.(No Image Selected)

CONTROL ID: 1721998TITLE: Particulate methane monooxygenaseAUTHORS/INSTITUTIONS: A. Rosenzweig, Departments of Molecular Biosciences and of Chemistry, NorthwesternUniversity, Evanston, Illinois, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Methane gas is underutilized as a feedstock for production of liquid fuels due to low conversionefficiencies and high capital costs. Increasing natural gas reserves combined with an ongoing price spread betweennatural gas and gasoline have led to renewed interest in bioconversion of methane. In nature, methanotrophic bacteriaactivate methane with high selectivity under mild conditions using methane monooxygenases (MMOs). However, theuse of MMOs in bioconversion processes is hindered by low efficiency, low carbon yields, and suboptimal kinetics.Improving these properties requires detailed understanding of the MMO enzyme systems, which has not beenachieved for the primary MMO in nature, particulate MMO (pMMO). pMMO comprises three subunits, pmoA, pmoB,and pmoC, arranged in a trimeric complex. Despite extensive research and the availability of multiple crystalstructures, the nature of the pMMO active site remains controversial and the chemical mechanism has not beenelucidated. Mounting evidence indicates that methane is oxidized at a dinuclear copper center in the pmoB subunit,but several key questions remain unresolved. In addition, neither the roles of additional metal centers observed bycrystallography nor the function of the transmembrane domains has been established. Progress toward answeringthese fundamental questions, including the development of new experimental systems, will be reported.(No Image Selected)

CONTROL ID: 1728479TITLE: Metalloenzymes and Catalytic Concepts in the Conversions of Nitrogen CompoundsAUTHORS/INSTITUTIONS: O. Einsle, Dpt of Biochemistrty, Albert-Ludwigs-Universität Freiburg, Freiburg,GERMANY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: The biogeochemical nitrogen cycle is a metabolic network that unites a broad variety of metal-containing enzymes able to catalyze the chemically challenging conversions of small, inert substrate molecules.Nature uses the entire range of metals and metals sites, realizing different catalytic strategies and concepts. As arecurring concept, bioinorganic active sites combine the inorganic metal site and its protein ligand to yield emergentproperties that exceed the individual capacities of their components. Such principles will be highlighted in several examples. Nitrous oxide reductase is a copper enzyme that activates andreduces N2O in bacterial denitrification [1,3]. It uses a [4Cu:2S] center, CuZ, to activate its substrate that then isreduced through a second site, CuA (Fig. 1A). Nitrogenase, the sole enzyme able to break the N2 triple bond, has theunique FeMo cofactor as a catalytic site [2], while multiheme oxidoreductases couple heme centers to create largemulti-electron transfer arrays to drive complex redox chemistry.[1]Wüst, A., Schneider, L., Pomowski, A., Zumft, W. G., Kroneck, P. M. H. & Einsle, O. (2012) Nature’s way ofhandling a greenhouse gas: The copper-sulfur cluster of purple nitrous oxide reductase. Biol. Chem., 393, 1067-1077.[2]Spatzal, T., Aksoyoglu, M., Zhang, L., Andrade, S.L.A., Schleicher, E., Weber, S., Rees, D.C. & Einsle, O. (2011)Evidence for Interstitial Carbon in Nitrogenase FeMo Cofactor. Science, 334, 940.[3]Pomowski, A., Zumft, W.G., Kroneck, P.M.H. & Einsle, O. (2011) N2O binding at a [4Cu:2S] copper-sulphur clusterin nitrous oxide reductase. Nature, 477, 234-237.[4]Einsle, O. (2011) Structure and function of formate-dependent cytochrome c nitrite reductase. Meth. Enzymol., 496,399-422

Figure 1: Bio-inorganic active sites. (A) The [4Cu:2S] CuZ cluster is the binding site for N2O in nitrous oxidereductase.

CONTROL ID: 1729233TITLE: Non-haem Iron Enzymes in the Biosyntheses of Bioactive Natural Products AUTHORS/INSTITUTIONS: M.J. Bollinger, W. Chang, biochemistry, Penn State University, University Park,Pennsylvania, UNITED STATES|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Of the many crucial roles of iron in biology, one of its most important is the removal of hydrogen atoms(H.s) and installation of new functional groups upon unactivated carbon centers.1-3 In the classic case, a Fe(IV)-oxo(ferryl) complex forms from Fe(II), O2 and two electrons from a reducing co-substrate, it removes H. from an aliphaticcarbon (R-H), and the resultant Fe(III)-OH/R. complex decays by transfer of HO. to the R., yielding the hydroxylatedproduct (R-OH). The bewildering array of chemically distinct reaction types mediated by enzymes in the class arisesfrom the remarkable variations on this canonical mechanism, in which (1) the ferryl complex forms without a reducingco-substrate, as the two redox-balancing electrons are provided by another source; (2) the ferryl abstracts H. from aheteroatom other than carbon to initiate a different type of oxidative transformation; and/or (3) the X-Fe(III)-OH/R.state resulting from H. abstraction by the ferryl decays by (i) radical-group transfer of a different ligand (X.) to form R-X, (ii) removal of a second H. to form a double bond, or (iii) return of H. to R. to effect stereoinversion. The centralissues driving our ongoing study of these enzymes are the sequence of chemical events leading to the divergentoutcomes and how the structures of the enzymes tune the common cofactor to direct these outcomes. I will presentrecent progress toward an understanding of structure-function relationships for this most versatile enzyme class,focusing on members that catalyze reactions in the biosyntheses of antibiotic drugs and other bioactive naturalproducts.(1) Krebs, C.; Galonić Fujimori, D.; Walsh, C. T.; Bollinger, J. M., Jr. Acc. Chem. Res. 2007, 40, 484-492.(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) Costas, M.; Mehn, M. P.; Jensen, M. P.; Que, L., Jr. Chem. Rev. 2004, 104, 939-986.(No Image Selected)

CONTROL ID: 1748373TITLE: A MOLECULAR APPROACH TO SYSTEMS BIOLOGY OF METAL TRANSPORT: FROM STRUCTURES TOPATHWAYSAUTHORS/INSTITUTIONS: L. Banci, CERM, Sesto Fiorentino, ITALY|CURRENT CATEGORY: Metalloproteins and MetalloenzymesABSTRACT BODY: Abstract Body: Cellular processes commonly require the concerted action of a number of proteins and biomolecules,each of which should have the suitable conformation(s), be located in the proper cellular compartment and able tointeract with each other in the correct mode. The characterization of processes therefore requires a comprehensiveknowledge of all the players, of their properties and of their interactions, both at system level (e.g. a cell) and atmolecular level. On this respect NMR, with its multiple approaches and applications, is quite powerful in describingfunctional biological processes in atomic details and in a cellular context. NMR indeed not only characterizes thestructure and dynamics of biomolecules but, even more importantly, can describe functional events. Along a functionalprocess, most interactions are transient in nature, effectively studied by NMR, which can also characterize processesin living cells with atomic resolution. Among these transient events are the metal transfer processes, in which metaltransfer, from metal transporters to the final recipient proteins, occurs through a series of protein-protein interactions1,2. These transfer processes are determined by metal affinity gradients within the protein transfer chain, with kineticfactors contributing to the selectivity of the processes3. They often also involve proteins whose folding and maturationare tightly linked to redox reactions4. The power of this integrated structural/functional approach to describe cellularpathways at atomic resolution will be presented for a few pathways responsible for copper trafficking in the cell and forthe folding of the involved proteins, with a particular focus on mitochondria as well as for iron-sulfur biogenesis. Newmajor advancements in the application of in cell NMR will be also discussed5.1 Banci L, Bertini I et al. Cell Mol Life Sci: 67, 2563-89, 2010. 2 Banci L, Bertini I et al. Nat Prod Rep 27: 695-710,2010 3 Banci L, et al. Nature 465: 645-48, 2010 4 Banci, L., et al. Proc.Natl.Acad.Sci.USA, 107, 20190-95, 2010 5Banci, L., et al. Nat.Chem.Biol. 9, 297-299, 2013.(No Image Selected)