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1. Rivers2Restore : How restored rivers strengthen our resilience to climate change

Author: Baffert, C., Casey, Z., Baffert, C., and Casey, Z.
Abstract: Europe’s rivers are in crisis. They have been deeply altered from their natural form and function with dams, embankments, dredged river beds and drained floodplains. Their mismanagement means they are prone to devastating floods and damaging dry spells, worsened by climate change. Restoring the natural shape, habitats, flow and functioning of the river system, including its floodplains, by removing river barriers and creating more space for nature in and along the river, offers a multitude of benefits and is urgently needed to improve our resilience to climate change impacts and bring back biodiversity.
Published: 2024

2. Water for nature, water for life : adapting to Europe's water scarcity challenge

Author: Interwies, E., Schwörer, S., Görlitz, S., Baffert, C., Rousselot, J., Seiz, R., Interwies, E., Schwörer, S., Görlitz, S., Baffert, C., Rousselot, J., and Seiz, R.
Abstract: From crop failures to shrinking lakes, nature and people in Europe are increasingly suffering from a lack of water. This report presents four case studies from across Europe which reveal different water mismanagement issues: illegal, excessive and/or uncontrolled water abstraction for agriculture (Spain; The Netherlands); illegal filling and operation of water reservoirs for agriculture (France); and illegal construction and irregular operation of hydropower plants without considering flows of water necessary for nature and people (Bulgaria). These case studies are only a snapshot of the more profound and widespread management issues across the continent.
Published: 2023

3. Photochemistry and photoinhibition of the H-cluster of FeFe hydrogenases

Author: Sensi, M, Baffert, C, Fourmond, V, De Gioia, L, Bertini, L, Leger, C, Sensi M., Baffert C., Fourmond V., De Gioia L., Bertini L., Leger C., Sensi, M, Baffert, C, Fourmond, V, De Gioia, L, Bertini, L, Leger, C, Sensi M., Baffert C., Fourmond V., De Gioia L., Bertini L., and Leger C.
Abstract: Hydrogenases are enzymes that catalyze the oxidation and production of molecular hydrogen. For about fifteen years, there have been many reports about the successful connection of these enzymes to photosensitizers with the aim of designing H2 photoproduction systems, but relatively little attention has been paid to whether and why illumination may affect the catalytic properties of the enzyme. In all hydrogenases, hydrogen activation occurs at an inorganic active site that includes at least one Fe-carbonyl motif, which may make it sensitive to irradiation. Here we review the evidence that hydrogenases are indeed photosensitive. We focus mainly on the so-called FeFe hydrogenases; their active site, called the H-cluster, consists of a [4Fe4S] cluster that is bound by a cysteine sulfur to a diiron site. The iron atoms of the binuclear cluster are coordinated by carbonyl and cyanide ligands and an azadithiolate group. We describe the effects of UV-visible light irradiation on the enzyme under cryogenic or turnover conditions and the photoreactivity of model complexes that mimic the diiron site. We emphasize the dependence of the photochemical processes on wavelength, and warn about FeFe hydrogenase photoinhibition, which should probably be considered when attempts are made to use FeFe hydrogenases for the artificial photosynthesis of solar fuels. We also underline the relevance of studies of synthetic mimics of the H-cluster for understanding at atomistic level the photochemical processes observed in the enzyme.
Published: 2021

4. Author Correction: The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster (Nature Chemistry, (2014), 6, 4, (336-342), 10.1038/nchem.1892)

Author: Fourmond V., Fourmond, V, Greco, C, Sybirna, K, Baffert, C, Wang, P, Ezanno, P, Montefiori, M, Bruschi, M, Meynial-Salles, I, Soucaille, P, Blumberger, J, Bottin, H, De Gioia, L, Leger, C, Fourmond V., Greco C., Sybirna K., Baffert C., Wang P. -H., Ezanno P., Montefiori M., Bruschi M., Meynial-Salles I., Soucaille P., Blumberger J., Bottin H., De Gioia L., Leger C., Fourmond V., Fourmond, V, Greco, C, Sybirna, K, Baffert, C, Wang, P, Ezanno, P, Montefiori, M, Bruschi, M, Meynial-Salles, I, Soucaille, P, Blumberger, J, Bottin, H, De Gioia, L, Leger, C, Fourmond V., Greco C., Sybirna K., Baffert C., Wang P. -H., Ezanno P., Montefiori M., Bruschi M., Meynial-Salles I., Soucaille P., Blumberger J., Bottin H., De Gioia L., and Leger C.
Abstract: In the version of this Article originally published, in several instances throughout the text, the Chlamydomonas reinhardtii amino acid residue phenylalanine was incorrectly labelled F234; it should have been F290.
Published: 2019

5. Experimental and theoretical study of the reaction of FeFe hydrogenases with dioxygen

Author: Orain, C, Sensi, M, Baffert, C, Fourmond, V, Léger, C, Orain, C, Sensi, M, Baffert, C, Fourmond, V, and Léger, C
Subjects: CHIM/02 - CHIMICA FISICA, Hydrogenase, Dihydrogen, Enzyme kinetic, Chemistry (all), Electrochemistry, Chemical biology, Theoretical chemistry, Bioinorganic chemistry
Abstract: Hydrogenases are enzymes that catalyze hydrogen oxidation and production. The so-called "FeFe hydrogenases", the active site of which is a [Fe6(CN)2(CO)3] cluster, are particularly efficient. Their inhibition by O2 prevents them from being used for H2 production. Combining electrochemical experiments, site-directed mutagenesis, molecular dynamics calculations and quantum chemistry calculations allowed to elucidate all steps of the reaction with O2 and to predict the rate of inhibition. These results will pave the way for engineering enzymes that resist O2.
Published: 2018

6. Experimental and theoretical study of the reaction of FeFe hydrogenases with dioxygen

Author: Orain, C., Sensi, M., Baffert, C., Fourmond, V., and Leger, C.
Subjects: Hydrogenase, Dihydrogen, Enzyme kinetics, Electrochemistry, Bioinorganic chemistry, Chemical biology, Theoretical chemistry
Published: 2018

7. Electrochemical Investigations of Hydrogenases and Other Enzymes That Produce and Use Solar Fuels

Author: Del Barrio, M, Sensi, M, Orain, C, Baffert, C, Dementin, S, Fourmond, V, Léger, C, Del Barrio, Melisa, Sensi, Matteo, Orain, Christophe, Baffert, Carole, Dementin, Sébastien, Fourmond, Vincent, Léger, Christophe, Del Barrio, M, Sensi, M, Orain, C, Baffert, C, Dementin, S, Fourmond, V, Léger, C, Del Barrio, Melisa, Sensi, Matteo, Orain, Christophe, Baffert, Carole, Dementin, Sébastien, Fourmond, Vincent, and Léger, Christophe
Abstract: Conspectus Many enzymes that produce or transform small molecules such as O2, H2, and CO2 embed inorganic cofactors based on transition metals. Their active site, where the chemical reaction occurs, is buried in and protected by the protein matrix, and connected to the solvent in several ways: chains of redox cofactors mediate long-range electron transfer; static or dynamic tunnels guide the substrate, product and inhibitors; amino acids and water molecules transfer protons. The catalytic mechanism of these enzymes is therefore delocalized over the protein and involves many different steps, some of which determine the response of the enzyme under conditions of stress (extreme redox conditions, presence of inhibitors, light), the catalytic rates in the two directions of the reaction and their ratio (the "catalytic bias"). Understanding all the steps in the catalytic cycle, including those that occur on sites of the protein that are remote from the active site, requires a combination of biochemical, structural, spectroscopic, theoretical, and kinetic methods. Here we argue that kinetics should be used to the fullest extent, by extracting quantitative information from the comparison of data and kinetic models and by exploring the combination of experimental kinetics and theoretical chemistry. In studies of these catalytic mechanisms, direct electrochemistry, the technique which we use and contribute to develop, has become unescapable. It simply consists in monitoring the changes in activity of an enzyme that is wired to an electrode by recording an electric current. We have described kinetic models that can be used to make sense of these data and to learn about various aspects of the mechanism that are difficult to probe using more conventional methods: long-range electron transfer, diffusion along gas channels, redox-driven (in)activations, active site chemistry and photoreactivity under conditions of turnover. In this Account, we highlight a few results that illustra
Published: 2018

8. Photoinhibition of FeFe hydrogenase

Author: Sensi, M, Baffert, C, Fradale, L, Gauquelin, C, Soucaille, P, Meynial Salles, I, Bottin, H, De Gioia, L, Bruschi, M, Fourmond, V, Léger, C, Bertini, L, Sensi, M, Baffert, C, Fradale, L, Gauquelin, C, Soucaille, P, Meynial Salles, I, Bottin, H, De Gioia, L, Bruschi, M, Fourmond, V, Léger, C, and Bertini, L
Abstract: In the enzyme FeFe hydrogenase, hydrogen oxidation and production occur at the H-cluster, a Fe6S6 active site that bears intrinsic carbonyl and cyanide ligands. This enzyme has been coupled to photosensitizers to design H2 photoproduction systems, and yet, according to earlier reports, the enzyme from Desulfovibrio desulfuricans is "easily destroyed" in "normal laboratory light". Here we report direct electrochemistry measurements of the effect of light on the activity of the enzymes from Chlamydomonas reinhardtii and Clostridium acetobutylicum, together with TDDFT and DFT calculations of the reactivity of the excited states of the H-cluster. We conclude that visible light does not inhibit these enzymes, but absorption of UV-B (280-315 nm) irreversibly damages the H-cluster by triggering the release of an intrinsic CO ligand; the resulting unsaturated species rearranges and protonates to form a stable, inactive dead-end. Answering the question of which particular hydrogenase can resist which particular wavelengths is important regarding solar H2 production, and our results show that some but not all FeFe hydrogenases can actually be combined with photosensitizers that utilise the solar spectrum, provided a UV screen is used. We suggest that further investigations of the compatibility of hydrogenases or hydrogenase mimics with light-harvesting systems should also consider the possibility of irreversible photoinhibition
Published: 2017

9. New perspectives in hydrogenase direct electrochemistry

Author: Sensi, M, del Barrio, M, Baffert, C, Fourmond, V, Léger, C, SENSI, MATTEO, Léger, C., Sensi, M, del Barrio, M, Baffert, C, Fourmond, V, Léger, C, SENSI, MATTEO, and Léger, C.
Abstract: Electrochemical studies of hydrogenases, the biological catalysts of H2 oxidation and production, have proven wrong the old saying that enzymes do not easily transfer electrons to electrodes in the absence of mediators. Many distinct hydrogenases have actually been directly connected to electrodes or particles, for studying their catalytic mechanism or for designing solar-fuels catalysts. In this review, we list the electrodes that have proved successful for direct electron transfer to hydrogenases, and we discuss recent results which illustrate new directions in this research field: the study of the biosynthesis of FeFe hydrogenase, the electrochemical characterization of non-standard NiFe or FeFe hydrogenases, the general discussion of what makes a catalyst better in one particular direction of the reaction, and the elucidation of the molecular mechanisms of hydrogenase catalysis by combining electrochemistry and theoretical chemistry, spectroscopy or photochemistry. The electrochemical methods described herein will probably prove useful for studying or using other redox enzymes.
Published: 2017

10. Mechanism of O2 diffusion and reduction in FeFe hydrogenases

Author: Kubas, A, Orain, C, Sancho, D, Saujet, L, Sensi, M, Gauquelin, C, Meynial-Salles, I, Soucaille, P, Bottin, H, Baffert, C, Fourmond, V, Best, R, Blumberger, J, Léger, C, Kubas, A, Orain, C, Sancho, D, Saujet, L, Sensi, M, Gauquelin, C, Meynial-Salles, I, Soucaille, P, Bottin, H, Baffert, C, Fourmond, V, Best, R, Blumberger, J, and Léger, C
Abstract: FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage
Published: 2017

11. Covalent attachment of FeFe hydrogenases to graphite electrode and inhibition studies

Author: Baffert, C., Fourmond, V., Leger, C., Meynial-Salles, I., Soucaille, P., Sybirna, K., Bottin, H., claudio greco, Gioia, L., Baffert, C, Fourmond, V, Leger, C, Meynial Salles, I, Soucaille, P, Sybirna, K, Bottin, H, Greco, C, DE GIOIA, L, Université de la Méditerranée - Aix-Marseille 2, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Università degli Studi di Milano = University of Milan (UNIMI), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Università degli Studi di Milano [Milano] (UNIMI)
Subjects: [SDV]Life Sciences [q-bio], [SDV.IDA]Life Sciences [q-bio]/Food engineering, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, hydrogenase, ComputingMilieux_MISCELLANEOUS
Abstract: National audience
Published: 2014

12. Covalent attachment of FeFe hydrogenase to graphite electrode and inhibition studies

Author: Baffert, C, Sybirna, K, Greco, C, Meynial Salles, I, Fourmond, V, DE GIOIA, L, Bottin, H, Soucaille, P, Léger, C, Baffert C., Sybirna K., Meynial Salles I., Fourmond V., Bottin H., Soucaille P., Léger C., GRECO, CLAUDIO, DE GIOIA, LUCA, Baffert, C, Sybirna, K, Greco, C, Meynial Salles, I, Fourmond, V, DE GIOIA, L, Bottin, H, Soucaille, P, Léger, C, Baffert C., Sybirna K., Meynial Salles I., Fourmond V., Bottin H., Soucaille P., Léger C., GRECO, CLAUDIO, and DE GIOIA, LUCA
Published: 2013

13. Covalent attachment of FeFe hydrogenase to graphite electrode and inhibition studies

Author: Baffert C., Sybirna K., Meynial Salles I., Fourmond V., Bottin H., Soucaille P., Léger C., GRECO, CLAUDIO, DE GIOIA, LUCA, Baffert, C, Sybirna, K, Greco, C, Meynial Salles, I, Fourmond, V, DE GIOIA, L, Bottin, H, Soucaille, P, and Léger, C
Subjects: idrogeno, idrogenasi
Published: 2013

14. Reactivity of the Excited States of the H-Cluster of FeFe Hydrogenases

Author: Sensi, M, Baffert, C, Greco, C, Caserta, G, Gauquelin, C, Saujet, L, Fontecave, M, Roy, S, Artero, V, Soucaille, P, Meynial Salles, I, Bottin, H, DE GIOIA, L, Fourmond, V, Léger, C, Bertini, L, SENSI, MATTEO, GRECO, CLAUDIO, DE GIOIA, LUCA, BERTINI, LUCA, Sensi, M, Baffert, C, Greco, C, Caserta, G, Gauquelin, C, Saujet, L, Fontecave, M, Roy, S, Artero, V, Soucaille, P, Meynial Salles, I, Bottin, H, DE GIOIA, L, Fourmond, V, Léger, C, Bertini, L, SENSI, MATTEO, GRECO, CLAUDIO, DE GIOIA, LUCA, and BERTINI, LUCA
Abstract: FeFe hydrogenases catalyze H2 oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe2(CO)3(CN)2(dithiomethylamine)] subcluster covalently attached to a Fe4S4 subcluster. This active site is photosensitive: visible light has been shown to induce the release of exogenous CO (a reversible inhibitor of the enzyme), shuffle the intrinsic CO ligands, and even destroy the H-cluster. These reactions must be understood because they may negatively impact the use of hydrogenase for the photoproduction of H2. Here, we explore in great detail the reactivity of the excited states of the H-cluster under catalytic conditions by examining, both experimentally and using TDDFT calculations, the simplest photochemical reaction: the binding and release of exogenous CO. A simple dyad model can be used to predict which excitations are active. This strategy could be used for probing other aspects of the photoreactivity of the H-cluster.
Published: 2016

15. CO disrupts the reduced H-cluster of FeFe Hydrogenase. A combined DFT and PFV study

Author: Baffert, C., Bertini, Lorenzo, Lautier, T., Greco, C., Sybirna, K., Ezanno, Pierre, Etienne, Emilien, Soucaille, P., Bertrand, Patrick, Bottin, H., Meynial-Salles, I., De Gioia, Luca, Léger, Christophe, Bioénergétique et Ingénierie des Protéines (BIP ), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Azzopardi, Laure, and Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [CHIM.INOR] Chemical Sciences/Inorganic chemistry, [CHIM.INOR]Chemical Sciences/Inorganic chemistry, ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2011

16. Comparing the efficiency of the laccase-NHA and laccase-HBT systems in eucalyptus pulp bleaching

Author: Valls, C., Colom, J.F., Baffert, C., Gimbert, I., Roncero, M.B., Sigoillot, J.C., and Azzopardi, Laure
Subjects: [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS
Published: 2010

17. The oxidative inactivation of FeFe hydrogenase reveals the plasticity of the H-cluster

Author: Fourmond, V, Baffert, C, Ezanno, P, Leger, C, Greco, C, Bruschi, M, DE GIOIA, L, Sybirna, K, Bottin, H, Meynial Salles, I, Soucaille, P, Wang, P, Montefiori, M, Blumberger, J, Blumberger, J., GRECO, CLAUDIO, DE GIOIA, LUCA, Fourmond, V, Baffert, C, Ezanno, P, Leger, C, Greco, C, Bruschi, M, DE GIOIA, L, Sybirna, K, Bottin, H, Meynial Salles, I, Soucaille, P, Wang, P, Montefiori, M, Blumberger, J, Blumberger, J., GRECO, CLAUDIO, and DE GIOIA, LUCA
Published: 2014

18. Covalent attachment of FeFe hydrogenases to graphite electrode and inhibition studies

Author: Baffert, C, Fourmond, V, Leger, C, Meynial Salles, I, Soucaille, P, Sybirna, K, Bottin, H, Greco, C, DE GIOIA, L, GRECO, CLAUDIO, DE GIOIA, LUCA, Baffert, C, Fourmond, V, Leger, C, Meynial Salles, I, Soucaille, P, Sybirna, K, Bottin, H, Greco, C, DE GIOIA, L, GRECO, CLAUDIO, and DE GIOIA, LUCA
Published: 2014

19. Combining experimental and theoretical methods to learn about the reactivity of gas-processing metalloenzymes

Author: Greco, C, Fourmond, V, Baffert, C, Wang, P, Dementin, S, Bertrand, P, Bruschi, M, Blumberger, J, DE GIOIA, L, Léger, C, GRECO, CLAUDIO, BRUSCHI, MAURIZIO, DE GIOIA, LUCA, Léger, C., Greco, C, Fourmond, V, Baffert, C, Wang, P, Dementin, S, Bertrand, P, Bruschi, M, Blumberger, J, DE GIOIA, L, Léger, C, GRECO, CLAUDIO, BRUSCHI, MAURIZIO, DE GIOIA, LUCA, and Léger, C.
Abstract: After enzymes were first discovered in the late XIX century, and for the first seventy years of enzymology, kinetic experiments were the only source of information about enzyme mechanisms. Over the following fifty years, these studies were taken over by approaches that give information at the molecular level, such as crystallography, spectroscopy and theoretical chemistry (as emphasized by the Nobel Prize in Chemistry awarded last year to M. Karplus, M. Levitt and A. Warshel). In this review, we thoroughly discuss the interplay between the information obtained from theoretical and experimental methods, by focussing on enzymes that process small molecules such as H2 or CO2 (hydrogenases, CO-dehydrogenase and carbonic anhydrase), and that are therefore relevant in the context of energy and environment. We argue that combining theoretical chemistry (DFT, MD, QM/MM) and detailed investigations that make use of modern kinetic methods, such as protein film voltammetry, is an innovative way of learning about individual steps and/or complex reactions that are part of the catalytic cycles. We illustrate this with recent results from our labs and others, including studies of gas transport along substrate channels, long range proton transfer, and mechanisms of catalysis, inhibition or inactivation
Published: 2014

20. The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster

Author: Fourmond, V, Greco, C, Sybirna, K, Baffert, C, Wang, P, Ezanno, P, Montefiori, M, Bruschi, M, Meynial Salles, I, Soucaille, P, Blumberger, J, Bottin, H, DE GIOIA, L, Léger, C, GRECO, CLAUDIO, Wang P, BRUSCHI, MAURIZIO, DE GIOIA, LUCA, Léger, C., Fourmond, V, Greco, C, Sybirna, K, Baffert, C, Wang, P, Ezanno, P, Montefiori, M, Bruschi, M, Meynial Salles, I, Soucaille, P, Blumberger, J, Bottin, H, DE GIOIA, L, Léger, C, GRECO, CLAUDIO, Wang P, BRUSCHI, MAURIZIO, DE GIOIA, LUCA, and Léger, C.
Abstract: Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H 2. In FeFe hydrogenases, H 2 oxidation occurs at the H-cluster, and catalysis involves H 2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H 2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O 2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H 2 oxidation. © 2014 Macmillan Publishers Limited.
Published: 2014

21. 'Étude par électrochimie directe de la sensibilité à l'O2 de l'hydrogénase à [FeFe] de Clostridium acetobutylicum '

Author: Baffert, C. and Azzopardi, Laure
Published: 2008

22. Does the environment around the H-cluster allow coordination of the pendant amine to the catalytic iron center in [FeFe] hydrogenases? Answers from theory

Author: Miyake, T, Bruschi, M, Cosentino, U, Baffert, C, Fourmond, V, Léger, C, Moro, G, DE GIOIA, L, Greco, C, BRUSCHI, MAURIZIO, COSENTINO, UGO RENATO, MORO, GIORGIO, DE GIOIA, LUCA, GRECO, CLAUDIO, Miyake, T, Bruschi, M, Cosentino, U, Baffert, C, Fourmond, V, Léger, C, Moro, G, DE GIOIA, L, Greco, C, BRUSCHI, MAURIZIO, COSENTINO, UGO RENATO, MORO, GIORGIO, DE GIOIA, LUCA, and GRECO, CLAUDIO
Abstract: [FeFe] hydrogenases are H2-evolving enzymes that feature a diiron cluster in their active site (the [2Fe]H cluster). One of the iron atoms has a vacant coordination site that directly interacts with H 2, thus favoring its splitting in cooperation with the secondary amine group of a neighboring, flexible azadithiolate ligand. The vacant site is also the primary target of the inhibitor O2. The [2Fe]H cluster can span various redox states. The active-ready form (Hox) attains the FeIIFeI state. States more oxidized than Hox were shown to be inactive and/or resistant to O2. In this work, we used density functional theory to evaluate whether azadithiolate-to-iron coordination is involved in oxidative inhibition and protection against O2, a hypothesis supported by recent results on biomimetic compounds. Our study shows that Fe-N(azadithiolate) bond formation is favored for an FeIIFeII active-site model which disregards explicit treatment of the surrounding protein matrix, in line with the case of the corresponding FeIIFeII synthetic system. However, the study of density functional theory models with explicit inclusion of the amino acid environment around the [2Fe]H cluster indicates that the protein matrix prevents the formation of such a bond. Our results suggest that mechanisms other than the binding of the azadithiolate nitrogen protect the active site from oxygen in the so-called H oxinact state. © 2013 SBIC.
Published: 2013

23. CO Disrupts the Reduced H-Cluster of FeFe Hydrogenase. A Combined DFT and Protein Film Voltammetry Study

Author: Baffert, C, Bertini, L, Lautier, T, Greco, C, Sybirna, K, Ezanno, P, Etienne, E, Philippe Soucaille, P, Bertrand, P, Bottin, H, Meynial Salles, I, DE GIOIA, L, Leger, C, BERTINI, LUCA, GRECO, CLAUDIO, DE GIOIA, LUCA, Leger C., Baffert, C, Bertini, L, Lautier, T, Greco, C, Sybirna, K, Ezanno, P, Etienne, E, Philippe Soucaille, P, Bertrand, P, Bottin, H, Meynial Salles, I, DE GIOIA, L, Leger, C, BERTINI, LUCA, GRECO, CLAUDIO, DE GIOIA, LUCA, and Leger C.
Abstract: Carbon monoxide is often described as a competitive inhibitor of FeFe hydrogenases, and it is used for probing H2 binding to synthetic or in silico models of the active site H-cluster. Yet it does not always behave as a simple inhibitor. Using an original approach which combines accurate electrochemical measurements and theoretical calculations, we elucidate the mechanism by which, under certain conditions, CO binding can cause permanent damage to the H-cluster. Like in the case of oxygen inhibition, the reaction with CO engages the entire H-cluster, rather than only the Fe2 subsite.
Published: 2011

24. Two New Terpyridine Dimanganese Complexes:u A Manganese(III,III) Complex with a Single Unsupported Oxo Bridge and a Manganese(III,IV) Complex with a Dioxo Bridge. Synthesis, Structure, and Redox Properties.

Author: Baffert, C., Collomb, M.-N., Deronzier, A., Pecaut, J., Limburg, J., Crabtree, R.H., and Brudvig, G.W.
Subjects: *MATHEMATICAL transformations, *MANGANESE, *PHYSICAL & theoretical chemistry, *MATHEMATICAL complexes
Abstract: Two new terpyridine dimanganese oxo complexes [Mn2[supIII,IV](7-O)2(terpy)2(CF3CO2)2][sup+] (3) and [Mn2[supIII,III](7-O)(terpy)2(CF3CO2)4] (4) (terpy = 2,2`:6,2`u`-terpyridine) have been synthesized and their X-ray structures determined. In contrast to the corresponding mixed-valent aqua complex [Mn2[supIII,IV](7-O)2(terpy)2(H2O)2][sup3+] (1), the two Mn atoms in 3 are not crystallographically equivalent. The neutral binuclear monooxo manganese(III,III) complex 4 exhibits two crystallographic forms having cis and trans configurations. In the cis complex, the two CF3CO2[sip-] ligands on each manganese adopt a cis geometry to each other; one CF3CO2[sup-] is trans to the oxygen of the oxo bridge while the second is cis. In the trans complex, the two coordinated CF3CO2[sup-] have a trans geometry to each other and are cis to the oxo bridge. The electrochemical behavior of 3 in organic medium (CH3CN) shows that this complex could be oxidized into its corresponding stable manganese(IV,IV) species while its reduced form manganese(III,III) is very unstable and leads by a disproportionation process to Mn(II) and Mn(IV) complexes. Complex 4 is only stable in the solid state, and it disproportionates spontaneously in CH3CN solution into the mixed-valent complex 3 and the mononuclear complex [Mn[supII](terpy)2][sup2+] (2), thereby preventing the observation of its electrochemical behavior.
Published: 2002
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25. Electrochemical properties of [MnIII(terpy)(N3)3] (terpy=2,2prime:6prime,2Prime-terpyridine) in CH3CN

Author: Baffert, C., Chen, H., Crabtree, R. H., Brudvig, G. W., and Collomb, M. N.
Published: 2001
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26. The oxidative inactivation of FeFe hydrogenase reveals the plasticity of the H-cluster

Author: Fourmond, V., Baffert, C., Ezanno, P., Leger, C., claudio greco, Bruschi, M., Gioia, L., Sybirna, K., Bottin, H., Meynial-Salles, I., Soucaille, P., Wang, P., Montefiori, M., Blumberger, J., Fourmond, V, Baffert, C, Ezanno, P, Leger, C, Greco, C, Bruschi, M, DE GIOIA, L, Sybirna, K, Bottin, H, Meynial Salles, I, Soucaille, P, Wang, P, Montefiori, M, and Blumberger, J
Subjects: hydrogenase

27. NiFe hydrogenases: catalysis and inhibition

Author: Leger, C., Dementin, S., Fourmond, V., Abou Hamdan, A., Baffert, C., Guigliarelli, B., Burlat, B., Pierre-Pol LIEBGOTT, Bertrand, P., Gutierrez-Sanz, O., and Lacey, A. L.

28. Photochemistry and photoinhibition of the H-cluster of FeFe hydrogenases

Author: Matteo Sensi, Vincent Fourmond, Carole Baffert, Christophe Léger, Luca De Gioia, Luca Bertini, University of Modena and Reggio Emilia, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Sensi, M, Baffert, C, Fourmond, V, De Gioia, L, Bertini, L, Leger, C, and Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB)
Subjects: Hydrogenase, Photoinhibition, Cyanide, Energy Engineering and Power Technology, chemistry.chemical_element, catalysi, 010402 general chemistry, Photochemistry, 01 natural sciences, Catalysis, Artificial photosynthesis, chemistry.chemical_compound, hydrogenase, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], biology, 010405 organic chemistry, Renewable Energy, Sustainability and the Environment, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Active site, Sulfur, 0104 chemical sciences, Fuel Technology, chemistry, biology.protein, sense organs, Cysteine
Abstract: Hydrogenases are enzymes that catalyze the oxidation and production of molecular hydrogen. For about fifteen years, there have been many reports about the successful connection of these enzymes to photosensitizers with the aim of designing H2 photoproduction systems, but relatively little attention has been paid to whether and why illumination may affect the catalytic properties of the enzyme. In all hydrogenases, hydrogen activation occurs at an inorganic active site that includes at least one Fe-carbonyl motif, which may make it sensitive to irradiation. Here we review the evidence that hydrogenases are indeed photosensitive. We focus mainly on the so-called FeFe hydrogenases; their active site, called the H-cluster, consists of a [4Fe4S] cluster that is bound by a cysteine sulfur to a diiron site. The iron atoms of the binuclear cluster are coordinated by carbonyl and cyanide ligands and an azadithiolate group. We describe the effects of UV-visible light irradiation on the enzyme under cryogenic or turnover conditions and the photoreactivity of model complexes that mimic the diiron site. We emphasize the dependence of the photochemical processes on wavelength, and warn about FeFe hydrogenase photoinhibition, which should probably be considered when attempts are made to use FeFe hydrogenases for the artificial photosynthesis of solar fuels. We also underline the relevance of studies of synthetic mimics of the H-cluster for understanding at atomistic level the photochemical processes observed in the enzyme.
Published: 2021
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29. New perspectives in hydrogenase direct electrochemistry

Author: Vincent Fourmond, Carole Baffert, Christophe Léger, Melisa del Barrio, Matteo Sensi, Sensi, M, del Barrio, M, Baffert, C, Fourmond, V, Léger, C, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)
Subjects: Hydrogenase, Chemistry, Hydrogenases, Electrochemistry, Kinetics, PFV, Electrode, Nanotechnology, 02 engineering and technology, 010402 general chemistry, 021001 nanoscience & nanotechnology, Electrochemistry, 7. Clean energy, 01 natural sciences, 0104 chemical sciences, Analytical Chemistry, Catalysis, Electron transfer, Redox enzymes, Molecular mechanism, Theoretical chemistry, [CHIM.OTHE]Chemical Sciences/Other, 0210 nano-technology
Abstract: International audience; Electrochemical studies of hydrogenases, the biological catalysts of H 2 oxidation and production, have proven wrong the old saying that enzymes do not easily transfer electrons to electrodes in the absence of mediators. Many distinct hydrogenases have actually been directly connected to electrodes or particles, for studying their catalytic mechanism or for designing solar fuels catalysts. In this review, we list the electrodes that have proved successful for direct electron transfer to hydrogenases, and we discuss recent results which illustrate new directions in this research field: the study of the biosynthesis of FeFe hydrogenase, the electrochemical characterization of non-standard NiFe-or FeFe hydrogenases, the general discussion of what makes a catalyst better in one particular direction of the reaction, and the elucidation of the molecular mechanism of hydrogenase catalysis by combining electrochemistry and theoretical chemistry, spectroscopy or photochemistry. The electrochem-ical methods described herein will probably prove useful for studying or using other redox enzymes.
Published: 2017
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30. Mechanism of O2 diffusion and reduction in FeFe hydrogenases

Author: Philippe Soucaille, Matteo Sensi, Christophe Léger, Isabelle Meynial-Salles, Charles Gauquelin, Carole Baffert, Jochen Blumberger, Laure Saujet, Robert B. Best, David De Sancho, Adam Kubas, Christophe Orain, Hervé Bottin, Vincent Fourmond, Kubas, A, Orain, C, Sancho, D, Saujet, L, Sensi, M, Gauquelin, C, Meynial-Salles, I, Soucaille, P, Bottin, H, Baffert, C, Fourmond, V, Best, R, Blumberger, J, Léger, C, Department of Physics and Astronomy [UCL London], University College of London [London] ( UCL ), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Department of Chemistry [Cambridge, UK], University of Cambridge [UK] ( CAM ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie et de Technologies de Saclay ( IBITECS ), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), and Institut de Microbiologie de la Méditerranée ( IMM )
Subjects: Hydrogenase, General Chemical Engineering, Mutant, Nanotechnology, Molecular Dynamics Simulation, Molecular Dynamics, 010402 general chemistry, Electrochemistry, [ CHIM ] Chemical Sciences, 01 natural sciences, Catalysis, Diffusion, Molecular dynamics, Metalloproteins, Site-Directed, Clostridium, CHIM/03 - CHIMICA GENERALE E INORGANICA, chemistry.chemical_classification, biology, 010405 organic chemistry, Chemistry, Mutagenesis, Active site, Electrochemical Techniques, General Chemistry, 0104 chemical sciences, Oxygen, CHIM/02 - CHIMICA FISICA, Enzyme mecanisms, Enzyme, Hydrogen, Mutagenesis, Site-Directed, Oxidation-Reduction, Quantum Theory, Density functional theory, Electrocatalysis, Enzyme mechanisms, Metalloproteins, Molecular dynamics, Density functional theory, Biophysics, biology.protein, Electrocatalysis, Cysteine
Abstract: International audience; FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.
Published: 2016
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31. Electrochemical Investigations of Hydrogenases and Other Enzymes That Produce and Use Solar Fuels

Author: Carole Baffert, Vincent Fourmond, Christophe Léger, Christophe Orain, Melisa del Barrio, Matteo Sensi, Sébastien Dementin, Bioénergétique et Ingénierie des Protéines (BIP ), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), ANR-14-CE05-0010,HEROS,Hydrogénases résistantes à l'Oxygène(2014), ANR-17-CE11-0027,MeCO2Bio,Études mécanistiques de la réduction du CO2: exploration de la biodiversité des CO déshydrogénases(2017), ANR-12-BS08-0014,ECCHYMOSE,Etudes d'hydrogénases à Fer par électrochimie: mécanisme et optimisation pour la photoproduction d'hydrogène(2012), Del Barrio, M, Sensi, M, Orain, C, Baffert, C, Dementin, S, Fourmond, V, and Léger, C
Subjects: [SDV]Life Sciences [q-bio], Molecular Dynamics Simulation, 010402 general chemistry, 01 natural sciences, Chemical reaction, Redox, Catalysis, Diffusion, Electron transfer, Hydrogenase, Humans, [CHIM]Chemical Sciences, Electrodes, Density Functional Theory, chemistry.chemical_classification, biology, 010405 organic chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Sulfite Oxidase, Active site, Substrate (chemistry), General Medicine, General Chemistry, Electrochemical Techniques, Combinatorial chemistry, Biocatalysis, Sunlight, 0104 chemical sciences, CHIM/02 - CHIMICA FISICA, Enzyme, chemistry, Catalytic cycle, biology.protein, Electrochemistry, Hydrogenases, Solar Fuels, Hydrogen, Enzyme Kinetics
Abstract: Conspectus Many enzymes that produce or transform small molecules such as O2, H2, and CO2 embed inorganic cofactors based on transition metals. Their active site, where the chemical reaction occurs, is buried in and protected by the protein matrix, and connected to the solvent in several ways: chains of redox cofactors mediate long-range electron transfer; static or dynamic tunnels guide the substrate, product and inhibitors; amino acids and water molecules transfer protons. The catalytic mechanism of these enzymes is therefore delocalized over the protein and involves many different steps, some of which determine the response of the enzyme under conditions of stress (extreme redox conditions, presence of inhibitors, light), the catalytic rates in the two directions of the reaction and their ratio (the "catalytic bias"). Understanding all the steps in the catalytic cycle, including those that occur on sites of the protein that are remote from the active site, requires a combination of biochemical, structural, spectroscopic, theoretical, and kinetic methods. Here we argue that kinetics should be used to the fullest extent, by extracting quantitative information from the comparison of data and kinetic models and by exploring the combination of experimental kinetics and theoretical chemistry. In studies of these catalytic mechanisms, direct electrochemistry, the technique which we use and contribute to develop, has become unescapable. It simply consists in monitoring the changes in activity of an enzyme that is wired to an electrode by recording an electric current. We have described kinetic models that can be used to make sense of these data and to learn about various aspects of the mechanism that are difficult to probe using more conventional methods: long-range electron transfer, diffusion along gas channels, redox-driven (in)activations, active site chemistry and photoreactivity under conditions of turnover. In this Account, we highlight a few results that illustrate our approach. We describe how electrochemistry can be used to monitor substrate and inhibitor diffusion along the gas channels of hydrogenases and we discuss how the kinetics of intramolecular diffusion relates to global properties such as resistance to oxygen and catalytic bias. The kinetics and/or thermodynamics of intramolecular electron transfer may also affect the catalytic bias, the catalytic potentials on either side of the equilibrium potential, and the overpotentials for catalysis (defined as the difference between the catalytic potentials and the open circuit potential). This is understood by modeling the shape of the steady-state catalytic response of the enzyme. Other determinants of the catalytic rate, such as domain motions, have been probed by examining the transient catalytic response recorded at fast scan rates. Last, we show that combining electrochemical investigations and MD, DFT, and TD-DFT calculations is an original way of probing the reactivity of the H-cluster of hydrogenase, in particular its reactions with CO, O2, and light. This approach contrasts with the usual strategy which aims at stabilizing species that are presumed to be catalytic intermediates, and determining their structure using spectroscopic or structural methods.
Published: 2018
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32. Does the environment around the H-cluster allow coordination of the pendant amine to the catalytic iron center in [FeFe] hydrogenases? Answers from theory

Author: Carole Baffert, Ugo Cosentino, Christophe Léger, Toshiko Miyake, Vincent Fourmond, Giorgio Moro, Maurizio Bruschi, Luca De Gioia, Claudio Greco, Begegnungszentren der IGAFA, Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Miyake, T, Bruschi, M, Cosentino, U, Baffert, C, Fourmond, V, Léger, C, Moro, G, DE GIOIA, L, and Greco, C
Subjects: Iron-Sulfur Proteins, Stereochemistry, Iron–sulfur cluster, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, Redox, Catalysis, Inorganic Chemistry, chemistry.chemical_compound, Hydrogenase, Cluster (physics), Amines, Hydrogen, hydrogenase, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, 010405 organic chemistry, Ligand, Chemistry, MESH: Hydrogen, MESH: Amines, Active site, MESH: Iron-Sulfur Proteins, 0104 chemical sciences, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, MESH: Hydrogenase, Biocatalysis, biology.protein, Quantum Theory, Density functional theory, MESH: Biocatalysis, MESH: Quantum Theory, Hydrogen
Abstract: International audience; [FeFe] hydrogenases are H2-evolving enzymes that feature a diiron cluster in their active site (the [2Fe]H cluster). One of the iron atoms has a vacant coordination site that directly interacts with H2, thus favoring its splitting in cooperation with the secondary amine group of a neighboring, flexible azadithiolate ligand. The vacant site is also the primary target of the inhibitor O2. The [2Fe]H cluster can span various redox states. The active-ready form (Hox) attains the Fe(II)Fe(I) state. States more oxidized than Hox were shown to be inactive and/or resistant to O2. In this work, we used density functional theory to evaluate whether azadithiolate-to-iron coordination is involved in oxidative inhibition and protection against O2, a hypothesis supported by recent results on biomimetic compounds. Our study shows that Fe-N(azadithiolate) bond formation is favored for an Fe(II)Fe(II) active-site model which disregards explicit treatment of the surrounding protein matrix, in line with the case of the corresponding Fe(II)Fe(II) synthetic system. However, the study of density functional theory models with explicit inclusion of the amino acid environment around the [2Fe]H cluster indicates that the protein matrix prevents the formation of such a bond. Our results suggest that mechanisms other than the binding of the azadithiolate nitrogen protect the active site from oxygen in the so-called H ox (inact) state.
Published: 2013
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33. Reactivity of the Excited States of the H-Cluster of FeFe Hydrogenases

Author: Carole Baffert, Claudio Greco, Luca Bertini, Souvik Roy, Charles Gauquelin, Isabelle Meynial-Salles, Matteo Sensi, Vincent Artero, Hervé Bottin, Luca De Gioia, Marc Fontecave, Laure Saujet, Christophe Léger, Philippe Soucaille, Vincent Fourmond, Giorgio Caserta, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Department of Earth and Environmental Sciences [Milano], Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Laboratoire de Chimie des Processus Biologiques (LCPB), Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF)-Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Institut de Biologie et de Technologies de Saclay (IBITECS), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Department of Earth and Environmental Sciences, Università degli Studi di Milano-Bicocca [Milano], Laboratoire de Chimie des Processus Biologiques ( LCPB ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Collège de France ( CdF ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie et de Technologies de Saclay ( IBITECS ), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Laboratoire de Chimie et Biologie des Métaux ( LCBM - UMR 5249 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Collège de France - Chaire Chimie des processus biologiques, Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), CNRS, Aix Marseille Universite, INSA, CEA, ANR-12-BS08-0014,ECCHYMOSE,Etudes d'hydrogénases à Fer par électrochimie: mécanisme et optimisation pour la photoproduction d'hydrogène(2012), ANR-14-CE05-0010,HEROS,Hydrogénases résistantes à l'Oxygène(2014), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), ANR-11-IDEX-0001,Amidex,INITIATIVE D'EXCELLENCE AIX MARSEILLE UNIVERSITE(2011), Chaire Chimie des processus biologiques, Université Pierre et Marie Curie - Paris 6 (UPMC)-Collège de France (CdF)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Sensi, M, Baffert, C, Greco, C, Caserta, G, Gauquelin, C, Saujet, L, Fontecave, M, Roy, S, Artero, V, Soucaille, P, Meynial Salles, I, Bottin, H, DE GIOIA, L, Fourmond, V, Léger, C, and Bertini, L
Subjects: Hydrogenase, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, [ CHIM ] Chemical Sciences, Catalysis, [ CHIM.CATA ] Chemical Sciences/Catalysis, Colloid and Surface Chemistry, Cluster (physics), [CHIM]Chemical Sciences, Reactivity (chemistry), Hydrogen, hydrogenase, biology, 010405 organic chemistry, Chemistry, Active site, General Chemistry, Time-dependent density functional theory, [CHIM.CATA]Chemical Sciences/Catalysis, 0104 chemical sciences, [ PHYS.PHYS.PHYS-CHEM-PH ] Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], Covalent bond, Excited state, biology.protein
Abstract: International audience; FeFe hydrogenases catalyze H-2 oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe-2(CO)(3)(CN)(2)(dithiomethylamine)] subcluster covalently attached to a Fe4S4 subcluster. This active site is photosensitive: visible light has been shown to induce the release of exogenous CO (a reversible inhibitor of the enzyme), shuffle the intrinsic CO ligands, and even destroy the H-cluster. These reactions must be understood because they may negatively impact the Use of hydrogenase for the photoproduction of H-2. Here, we explore in great detail the reactivity of the excited states of the H-duster under catalytic conditions by examining, both experimentally and using TDDFT calculations, the simplest photochemical reaction: the binding and release of exogenous CO. A simple dyad model can be used to predict which excitations are active. This could be used for probing other, aspects of the photoreactivity of the H-cluster.
Published: 2016
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34. Combining experimental and theoretical methods to learn about the reactivity of gas-processing metalloenzymes

Author: Claudio Greco, Vincent Fourmond, Po-hung Wang, Sébastien Dementin, Carole Baffert, Maurizio Bruschi, Patrick Bertrand, Luca De Gioia, Christophe Léger, Jochen Blumberger, Department of Earth and Environmental Sciences, Università degli Studi di Milano-Bicocca [Milano], Institut de Microbiologie de la Méditerranée ( IMM ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Institute of Oceanography [Taipei], National Taiwan University [Taiwan] ( NTU ), Dipartimento di Biotecnologie e Bioscienze, Department of Physics and Astronomy [UCL London], University College of London [London] ( UCL ), Department of Earth and Environmental Sciences [Milano], Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Institut de Microbiologie de la Méditerranée (IMM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), National Taiwan University [Taiwan] (NTU), University College of London [London] (UCL), Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Greco, C, Fourmond, V, Baffert, C, Wang, P, Dementin, S, Bertrand, P, Bruschi, M, Blumberger, J, DE GIOIA, L, and Léger, C
Subjects: enzymes, Context (language use), [CHIM.INOR]Chemical Sciences/Inorganic chemistry, DFT, theoretical chemistry, bioinorganic chemistry, Molecular level, catalytic power of enzymes, computational chemistry, spectroscopy, Computational chemistry, Theoretical chemistry, Environmental Chemistry, Reactivity (chemistry), [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Renewable Energy, Sustainability and the Environment, Chemistry, [ CHIM.INOR ] Chemical Sciences/Inorganic chemistry, Pollution, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, QMMM, Nuclear Energy and Engineering, electrochemistry, Protein film voltammetry, Theoretical methods, [ CHIM.THEO ] Chemical Sciences/Theoretical and/or physical chemistry, Physical chemistry, Experimental methods
Abstract: International audience; After enzymes were first discovered in the late XIX century, and for the first seventy years of enzymology, kinetic experiments were the only source of information about enzyme mechanisms. Over the following fifty years, these studies were taken over by approaches that give information at the molecular level, such as crystallography, spectroscopy and theoretical chemistry (as emphasized by the Nobel Prize in Chemistry awarded last year to M. Karplus, M. Levitt and A. Warshel). In this review, we thoroughly discuss the interplay between the information obtained from theoretical and experimental methods, by focussing on enzymes that process small molecules such as H 2 or CO 2 (hydrogenases, CO-dehydrogenase and carbonic anhydrase), and that are therefore relevant in the context of energy and environment. We argue that combining theoretical chemistry (DFT, MD, QM/MM) and detailed investigations that make use of modern kinetic methods, such as protein film voltammetry, is an innovative way of learning about individual steps and/or complex reactions that are part of the catalytic cycles. We illustrate this with recent results from our labs and others, including studies of gas transport along substrate channels, long range proton transfer, and mechanisms of catalysis, inhibition or inactivation. Broader context Some reactions which are very important in the context of energy and environment, such as the conversion between CO and CO2 , or H+ and H2 , are catalyzed in living organisms by large and complex enzymes that use inorganic active sites to transform substrates, chains of redox centers to transfer electrons, ionizable amino acids to transfer protons, and networks of hydrophobic cavities to guide the diffusion of substrates and products within the protein. This highly sophisticated biological plumbing and wiring makes turnover frequencies of thousands of substrate molecules per second possible. Understanding the molecular details of catalysis is still a challenge. We explain in this review how a great deal of information can be obtained using an interdisciplinary approach that combines state-of-the art kinetics and computational chemistry. This differs from—and complements—the more traditional strategies that consist in trying to see the catalytic intermediates using methods that rely on the interaction between light and matter, such as X-ray diffraction and spectroscopic techniques.
Published: 2014
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35. The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster

Author: Isabelle Meynial-Salles, Jochen Blumberger, Vincent Fourmond, Carole Baffert, Marco Montefiori, Luca De Gioia, Pierre Ezanno, Claudio Greco, Philippe Soucaille, Kateryna Sybirna, Po-hung Wang, Hervé Bottin, Christophe Léger, Maurizio Bruschi, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Department of Earth and Environmental Sciences [Milano], Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Department of Physics and Astronomy [UCL London], University College of London [London] (UCL), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Department of Biotechnologies and Biosciences, Centre National de la Recherche Scientifique, Aix-Marseille Universite, Agence Nationale de la Recherche [ANR-12-BS08-0014, ANR-2010-BIOE-004], Ministero dell'Istruzione, dell'Universita e della Ricerca [Prin 2010M2JARJ], Ministry of Education, Republic of China (Taiwan), Engineering and Physical Sciences Research Council [EP/J015571/1, EP/F067496], Royal Society, Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), University of Milano-Bicocca, Fourmond, V, Greco, C, Sybirna, K, Baffert, C, Wang, P, Ezanno, P, Montefiori, M, Bruschi, M, Meynial Salles, I, Soucaille, P, Blumberger, J, Bottin, H, DE GIOIA, L, Léger, C, Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Department of Earth and Environmental Sciences ( DEES ), Service de Bioénergétique, Biologie Stucturale, et Mécanismes ( SB2SM ), Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie et de Technologies de Saclay ( IBITECS ), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), University College of London [London] ( UCL ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), and Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS )
Subjects: Iron-Sulfur Proteins, Hydrogenase, Coordination sphere, Protein Conformation, General Chemical Engineering, Phenylalanine, Oxidative phosphorylation, Hydrogenase mimic, Photochemistry, Electrocatalyst, [ CHIM ] Chemical Sciences, Catalysis, Oxidizing agent, [CHIM]Chemical Sciences, chemistry.chemical_classification, General Chemistry, Combinatorial chemistry, Kinetics, Enzyme, chemistry, Mutation, Enzyme mechanisms, Tyrosine, hydrogenases, hydrogen, density functional theory, molecular dynamics, Electrocatalysis, Oxidation-Reduction, Hydrogen
Abstract: Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H 2. In FeFe hydrogenases, H 2 oxidation occurs at the H-cluster, and catalysis involves H 2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H 2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O 2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H 2 oxidation. © 2014 Macmillan Publishers Limited.
Published: 2014
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36. CO Disrupts the Reduced H-Cluster of FeFe Hydrogenase. A Combined DFT and Protein Film Voltammetry Study

Author: Emilien Etienne, Claudio Greco, Philippe Soucaille, Kateryna Sybirna, Christophe Léger, Luca Bertini, Thomas Lautier, Patrick Bertrand, Hervé Bottin, Luca De Gioia, Pierre Ezanno, Isabelle Meynial-Salles, Carole Baffert, Centre National de la Recherche Scientifique (CNRS), Università degli Studi di Milano [Milano] (UNIMI), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR, Pole de competitivite Capenergies, European Commission [SolarH2 212508], Università degli Studi di Milano = University of Milan (UNIMI), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Baffert, C, Bertini, L, Lautier, T, Greco, C, Sybirna, K, Ezanno, P, Etienne, E, Philippe Soucaille, P, Bertrand, P, Bottin, H, Meynial Salles, I, DE GIOIA, L, and Leger, C
Subjects: Hydrogenase, Stereochemistry, In silico, [SDV]Life Sciences [q-bio], Chlamydomonas reinhardtii, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, CHLAMYDOMONAS-REINHARDTII, CARBON-MONOXIDE BINDING, chemistry.chemical_compound, Colloid and Surface Chemistry, ONLY HYDROGENASE, Catalytic Domain, [SDV.IDA]Life Sciences [q-bio]/Food engineering, Electrochemistry, Cluster (physics), Protein Film Voltammetry, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Density Functional theory, CO binding, CHIM/03 - CHIMICA GENERALE E INORGANICA, Carbon Monoxide, ANALOGS, biology, 010405 organic chemistry, ACTIVE-SITE, Active site, General Chemistry, biology.organism_classification, 0104 chemical sciences, CHIM/02 - CHIMICA FISICA, chemistry, Iron-hydrogenasi, Protein film voltammetry, biology.protein, Quantum Theory, Carbon monoxide binding, CLOSTRIDIUM-PASTEURIANUM, Oxidation-Reduction, ENZYMES, Carbon monoxide
Abstract: International audience; Carbon monoxide is often described as a competitive inhibitor of FeFe hydrogenases, and it is used for probing H-2 binding to synthetic or in silico models of the active site H-cluster. Yet it does not always behave as a simple inhibitor. Using an original approach which combines accurate electrochemical measurements and theoretical calculations, we elucidate the mechanism by which, under certain conditions, CO binding can cause permanent damage to the H-cluster. Like in the case of oxygen inhibition, the reaction with CO engages the entire H-cluster, rather than only the Fe-2 subsite.
Published: 2011
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37. Corrigendum to "Transport limited adsorption experiments give a new lower estimate of the turnover frequency of Escherichia coli hydrogenase 1" BBA Advances 3 (2023) 100090.

Author: Aldinio-Colbachini A, Fasano A, Guendon C, Jacq-Bailly A, Wozniak J, Baffert C, Kpebe A, Léger C, Brugna M, and Fourmond V
Published: 2024
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38. Kinetic Modeling of the Reversible or Irreversible Electrochemical Responses of FeFe-Hydrogenases.

Author: Fasano A, Baffert C, Schumann C, Berggren G, Birrell JA, Fourmond V, and Léger C
Subjects: Oxidation-Reduction, Electron Transport, Spectrum Analysis, Hydrogen chemistry, Hydrogenase chemistry, Iron-Sulfur Proteins chemistry, Chlamydomonas reinhardtii
Abstract: The enzyme FeFe-hydrogenase catalyzes H 2 evolution and oxidation at an active site that consists of a [4Fe-4S] cluster bridged to a [Fe 2 (CO) 3 (CN) 2 (azadithiolate)] subsite. Previous investigations of its mechanism were mostly conducted on a few "prototypical" FeFe-hydrogenases, such as that from Chlamydomonas reinhardtii (Cr HydA1), but atypical hydrogenases have recently been characterized in an effort to explore the diversity of this class of enzymes. We aim at understanding why prototypical hydrogenases are active in either direction of the reaction in response to a small deviation from equilibrium, whereas the homologous enzyme from Thermoanaerobacter mathranii (Tam HydS) shows activity only under conditions of very high driving force, a behavior that was referred to as "irreversible catalysis". We follow up on previous spectroscopic studies and recent developments in the kinetic modeling of bidirectional reactions to investigate and compare the catalytic cycles of Cr HydA1 and Tam HydS under conditions of direct electron transfer with an electrode. We compare the hypothetical catalytic cycles described in the literature, and we show that the observed changes in catalytic activity as a function of potential, pH, and H 2 concentration can be explained with the assumption that the same catalytic mechanism applies. This helps us identify which variations in properties of the catalytic intermediates give rise to the distinct "reversible" or "irreversible" catalytic behaviors.
Published: 2024
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39. A Chimeric NiFe Hydrogenase Heterodimer to Assess the Role of the Electron Transfer Chain in Tuning the Enzyme's Catalytic Bias and Oxygen Tolerance.

Author: Fasano A, Guendon C, Jacq-Bailly A, Kpebe A, Wozniak J, Baffert C, Barrio MD, Fourmond V, Brugna M, and Léger C
Subjects: Catalysis, Oxygen, Electrons, Escherichia coli genetics
Abstract: The observation that some homologous enzymes have the same active site but very different catalytic properties demonstrates the importance of long-range effects in enzyme catalysis, but these effects are often difficult to rationalize. The NiFe hydrogenases 1 and 2 (Hyd 1 and Hyd 2) from E. coli both consist of a large catalytic subunit that embeds the same dinuclear active site and a small electron-transfer subunit with a chain of three FeS clusters. Hyd 1 is mostly active in H 2 oxidation and resistant to inhibitors, whereas Hyd 2 also catalyzes H 2 production and is strongly inhibited by O 2 and CO. Based on structural and site-directed mutagenesis data, it is currently believed that the catalytic bias and tolerance to O 2 of Hyd 1 are defined by the distal and proximal FeS clusters, respectively. To test these hypotheses, we produced and characterized a hybrid enzyme made of the catalytic subunit of Hyd 1 and the electron transfer subunit of Hyd 2. We conclude that catalytic bias and sensitivity to CO are set by the catalytic subunit rather than by the electron transfer chain. We confirm the importance of the proximal cluster in making the enzyme Hyd 1 resist long-term exposure to O 2 , but we show that other structural determinants, in both subunits, contribute to O 2 tolerance. A similar strategy based on the design of chimeric heterodimers could be used in the future to elucidate various structure-function relationships in hydrogenases and other multimeric metalloenzymes and to engineer useful hydrogenases that combine the desirable properties of distinct, homologous enzymes.
Published: 2023
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40. Transport limited adsorption experiments give a new lower estimate of the turnover frequency of Escherichia coli hydrogenase 1.

Author: Aldinio-Colbachini A, Fasano A, Guendon C, Jacq-Bailly A, Wozniak J, Baffert C, Kpebe A, Léger C, Brugna M, and Fourmond V
Abstract: Protein Film Electrochemistry is a technique in which a redox enzyme is directly wired to an electrode, which substitutes for the natural redox partner. In this technique, the electrical current flowing through the electrode is proportional to the catalytic activity of the enzyme. However, in most cases, the amount of enzyme molecules contributing to the current is unknown and the absolute turnover frequency cannot be determined. Here, we observe the formation of electrocatalytically active films of E. coli hydrogenase 1 by rotating an electrode in a sub-nanomolar solution of enzyme. This process is slow, and we show that it is mass-transport limited. Measuring the rate of the immobilization allows the determination of an estimation of the turnover rate of the enzyme, which appears to be much greater than that deduced from solution assays under the same conditions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)
Published: 2023
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41. Correction to "Steady-State Catalytic Wave-Shapes for 2-Electron Reversible Electrocatalysts and Enzymes".

Author: Fourmond V, Baffert C, Sybirna K, Lautier T, Abou Hamdan A, Dementin S, Soucaille P, Meynial-Salles I, Bottin H, and Léger C
Published: 2023
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42. An essential role of the reversible electron-bifurcating hydrogenase Hnd for ethanol oxidation in Solidesulfovibrio fructosivorans .

Author: Kpebe A, Guendon C, Payne N, Ros J, Khelil Berbar M, Lebrun R, Baffert C, Shintu L, and Brugna M
Abstract: The tetrameric cytoplasmic FeFe hydrogenase Hnd from Solidesulfovibrio fructosivorans (formely Desulfovibrio fructosovorans ) catalyses H 2 oxidation and couples the exergonic reduction of NAD + to the endergonic reduction of a ferredoxin by using a flavin-based electron-bifurcating mechanism. Regarding its implication in the bacterial physiology, we previously showed that Hnd, which is non-essential when bacteria grow fermentatively on pyruvate, is involved in ethanol metabolism. Under these conditions, it consumes H 2 to produce reducing equivalents for ethanol production as a fermentative product. In this study, the approach implemented was to compare the two S. fructosivorans WT and the hndD deletion mutant strains when grown on ethanol as the sole carbon and energy source. Based on the determination of bacterial growth, metabolite consumption and production, gene expression followed by RT-q-PCR, and Hnd protein level followed by mass spectrometry, our results confirm the role of Hnd hydrogenase in the ethanol metabolism and furthermore uncover for the first time an essential function for a Desulfovibrio hydrogenase. Hnd is unequivocally required for S. fructosivorans growth on ethanol, and we propose that it produces H 2 from NADH and reduced ferredoxin generated by an alcohol dehydrogenase and an aldehyde ferredoxin oxidoreductase catalyzing the conversion of ethanol into acetate. The produced H 2 could then be recycled and used for sulfate reduction. Hnd is thus a reversible hydrogenase that operates in H 2 -consumption by an electron-bifurcating mechanism during pyruvate fermentation and in H 2 -production by an electron-confurcating mechanism when the bacterium uses ethanol as electron donor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kpebe, Guendon, Payne, Ros, Khelil Berbar, Lebrun, Baffert, Shintu and Brugna.)
Published: 2023
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43. NMR-based metabolomic analysis of the physiological role of the electron-bifurcating FeFe-hydrogenase Hnd in Solidesulfovibrio fructosivorans under pyruvate fermentation.

Author: Payne N, Kpebe A, Guendon C, Baffert C, Maillot M, Haurogné T, Tranchida F, Brugna M, and Shintu L
Subjects: Electrons, Fermentation, Hydrogen metabolism, Oxidation-Reduction, Pyruvic Acid, Desulfovibrionaceae chemistry, Desulfovibrionaceae metabolism, Hydrogenase genetics, Hydrogenase chemistry, Hydrogenase metabolism
Abstract: Solidesulfovibrio fructosivorans (formely Desulfovibrio fructosovorans), an anaerobic sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of hydrogen gas (H 2 ) into protons and electrons. One of these, named Hnd, was demonstrated to be an electron-bifurcating hydrogenase Hnd (Kpebe et al., 2018). It couples the exergonic reduction of NAD + to the endergonic reduction of a ferredoxin with electrons derived from H 2 and whose function has been recently shown to be involved in ethanol production under pyruvate fermentation (Payne 2022). To understand further the physiological role of Hnd in S. fructosivorans, we compared the mutant deleted of part of the hnd gene with the wild-type strain grown on pyruvate without sulfate using NMR-based metabolomics. Our results confirm that Hnd is profoundly involved in ethanol metabolism, but also indirectly intervenes in global carbon metabolism and additional metabolic processes such as the biosynthesis of branched-chain amino acids. We also highlight the metabolic reprogramming induced by the deletion of hndD that leads to the upregulation of several NADP-dependent pathways., Competing Interests: Declaration of interest The authors have no conflict of interest to declare., (Copyright © 2022 Elsevier GmbH. All rights reserved.)
Published: 2023
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44. The electron-bifurcating FeFe-hydrogenase Hnd is involved in ethanol metabolism in Desulfovibrio fructosovorans grown on pyruvate.

Author: Payne N, Kpebe A, Guendon C, Baffert C, Ros J, Lebrun R, Denis Y, Shintu L, and Brugna M
Subjects: Desulfovibrio, Electrons, Ethanol, Ferredoxins metabolism, Hydrogen metabolism, NAD metabolism, Oxidation-Reduction, Pyruvic Acid, Hydrogenase genetics, Hydrogenase metabolism
Abstract: Desulfovibrio fructosovorans, a sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of H 2 into protons and electrons. Among them, Hnd is an electron-bifurcating hydrogenase, coupling the exergonic reduction of NAD + to the endergonic reduction of a ferredoxin with electrons derived from H 2 . It was previously hypothesized that its biological function involves the production of NADPH necessary for biosynthetic purposes. However, it was subsequently demonstrated that Hnd is instead a NAD + -reducing enzyme, thus its specific function has yet to be established. To understand the physiological role of Hnd in D. fructosovorans, we compared the hnd deletion mutant with the wild-type strain grown on pyruvate. Growth, metabolite production and consumption, and gene expression were compared under three different growth conditions. Our results indicate that hnd is strongly regulated at the transcriptional level and that its deletion has a drastic effect on the expression of genes for two enzymes, an aldehyde ferredoxin oxidoreductase and an alcohol dehydrogenase. We demonstrated here that Hnd is involved in ethanol metabolism when bacteria grow fermentatively and proposed that Hnd might oxidize part of the H 2 produced during fermentation generating both NADH and reduced ferredoxin for ethanol production via its electron bifurcation mechanism., (© 2022 John Wiley & Sons Ltd.)
Published: 2022
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45. The dyad of the Y-junction- and a flavin module unites diverse redox enzymes.

Author: Zuchan K, Baymann F, Baffert C, Brugna M, and Nitschke W
Subjects: Bacterial Proteins chemistry, Bacterial Proteins genetics, Electron Transport, Flavins chemistry, Formate Dehydrogenases chemistry, Formate Dehydrogenases genetics, Hydrogenase chemistry, Hydrogenase genetics, Phylogeny, Bacterial Proteins metabolism, Electrons, Flavins metabolism, Formate Dehydrogenases metabolism, Hydrogenase metabolism
Abstract: The concomitant presence of two distinctive polypeptide modules, which we have chosen to denominate as the "Y-junction" and the "flavin" module, is observed in 3D structures of enzymes as functionally diverse as complex I, NAD(P)-dependent [NiFe]-hydrogenases and NAD(P)-dependent formate dehydrogenases. Amino acid sequence conservation furthermore suggests that both modules are also part of NAD(P)-dependent [FeFe]-hydrogenases for which no 3D structure model is available yet. The flavin module harbours the site of interaction with the substrate NAD(P) which exchanges two electrons with a strictly conserved flavin moiety. The Y-junction module typically contains four iron-sulphur centres arranged to form a Y-shaped electron transfer conduit and mediates electron transfer between the flavin module and the catalytic units of the respective enzymes. The Y-junction module represents an electron transfer hub with three potential electron entry/exit sites. The pattern of specific redox centres present both in the Y-junction and the flavin module is correlated to present knowledge of these enzymes' functional properties. We have searched publicly accessible genomes for gene clusters containing both the Y-junction and the flavin module to assemble a comprehensive picture of the diversity of enzymes harbouring this dyad of modules and to reconstruct their phylogenetic relationships. These analyses indicate the presence of the dyad already in the last universal common ancestor and the emergence of complex I's EFG-module out of a subgroup of NAD(P)- dependent formate dehydrogenases., (Copyright © 2021 Elsevier B.V. All rights reserved.)
Published: 2021
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46. Electrochemical Characterization of a Complex FeFe Hydrogenase, the Electron-Bifurcating Hnd From Desulfovibrio fructosovorans .

Author: Jacq-Bailly A, Benvenuti M, Payne N, Kpebe A, Felbek C, Fourmond V, Léger C, Brugna M, and Baffert C
Abstract: Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans , is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H 2 to both the exergonic reduction of NAD + and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases. In this study, we describe the purification of the enzyme under O 2 -free atmosphere and its biochemical and electrochemical characterization. Despite its complexity due to its multimeric composition, Hnd can catalytically and directly exchange electrons with an electrode. We characterized the catalytic and inhibition properties of this electron-bifurcating hydrogenase using protein film electrochemistry of Hnd by purifying Hnd aerobically or anaerobically, then comparing the electrochemical properties of the enzyme purified under the two conditions via protein film electrochemistry. Hydrogenases are usually inactivated under oxidizing conditions in the absence of dioxygen and can then be reactivated, to some extent, under reducing conditions. We demonstrate that the kinetics of this high potential inactivation/reactivation for Hnd show original properties: it depends on the enzyme purification conditions and varies with time, suggesting the coexistence and the interconversion of two forms of the enzyme. We also show that Hnd catalytic properties (Km for H 2 , diffusion and reaction at the active site of CO and O 2 ) are comparable to those of standard hydrogenases (those which cannot catalyze electron bifurcation). These results suggest that the presence of the additional subunits, needed for electron bifurcation, changes neither the catalytic behavior at the active site, nor the gas diffusion kinetics but induces unusual rates of high potential inactivation/reactivation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jacq-Bailly, Benvenuti, Payne, Kpebe, Felbek, Fourmond, Léger, Brugna and Baffert.)
Published: 2021
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47. Hybrid cluster proteins in a photosynthetic microalga.

Author: van Lis R, Brugière S, Baffert C, Couté Y, Nitschke W, and Atteia A
Subjects: Algal Proteins genetics, Algal Proteins metabolism, Binding Sites, Chlamydomonas reinhardtii classification, Chlamydomonas reinhardtii genetics, Chlamydomonas reinhardtii metabolism, Cloning, Molecular, Desulfovibrio chemistry, Escherichia coli genetics, Escherichia coli metabolism, Evolution, Molecular, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Iron-Sulfur Proteins genetics, Iron-Sulfur Proteins metabolism, Microalgae genetics, Microalgae metabolism, Models, Molecular, Nitrates metabolism, Photosynthesis physiology, Phylogeny, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Structural Homology, Protein, Algal Proteins chemistry, Chlamydomonas reinhardtii chemistry, Iron-Sulfur Proteins chemistry, Microalgae chemistry, Multigene Family
Abstract: Hybrid cluster proteins (HCPs) are metalloproteins characterized by the presence of an iron-sulfur-oxygen cluster. These proteins occur in all three domains of life. In eukaryotes, HCPs have so far been found only in a few anaerobic parasites and photosynthetic microalgae. With respect to all species harboring an HCP, the green microalga Chlamydomonas reinhardtii stands out by the presence of four HCP genes. The study of the gene and protein structures as well as the phylogenetic analyses strongly support a model in which the HCP family in the alga has emerged from a single gene of alpha proteobacterial origin and then expanded by several rounds of duplications. The spectra and redox properties of HCP1 and HCP3, produced heterologously in Escherichia coli, were analyzed by electron paramagnetic resonance spectroscopy on redox-titrated samples. Both proteins contain a [4Fe-4S]-cluster as well as a [4Fe-2O-2S]-hybrid cluster with paramagnetic properties related to those of HCPs from Desulfovibrio species. Immunoblotting experiments combined with mass spectrometry-based proteomics showed that both nitrate and darkness contribute to the strong upregulation of the HCP levels in C. reinhardtii growing under oxic conditions. The link to the nitrate metabolism is discussed in the light of recent data on the potential role of HCP in S-nitrosylation in bacteria., (© 2019 Federation of European Biochemical Societies.)
Published: 2020
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48. Author Correction: The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster.

Author: Fourmond V, Greco C, Sybirna K, Baffert C, Wang PH, Ezanno P, Montefiori M, Bruschi M, Meynial-Salles I, Soucaille P, Blumberger J, Bottin H, De Gioia L, and Léger C
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Published: 2019
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49. Hydrogenases and H 2 metabolism in sulfate-reducing bacteria of the Desulfovibrio genus.

Author: Baffert C, Kpebe A, Avilan L, and Brugna M
Subjects: Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Biocatalysis, Desulfovibrio enzymology, Desulfovibrio genetics, Electrons, Gene Expression Regulation, Bacterial, Genetic Variation, Hydrogenase chemistry, Hydrogenase genetics, Hydrogenase metabolism, Models, Biological, Bacterial Proteins physiology, Desulfovibrio metabolism, Hydrogen metabolism, Hydrogenase physiology
Abstract: Hydrogen metabolism plays a central role in sulfate-reducing bacteria of the Desulfovibrio genus and is based on hydrogenases that catalyze the reversible conversion of protons into dihydrogen. These metabolically versatile microorganisms possess a complex hydrogenase system composed of several enzymes of both [FeFe]- and [NiFe]-type that can vary considerably from one Desulfovibrio species to another. This review covers the molecular and physiological aspects of hydrogenases and H 2 metabolism in Desulfovibrio but focuses particularly on our model bacterium Desulfovibrio fructosovorans. The search of hydrogenase genes in more than 30 sequenced genomes provides an overview of the distribution of these enzymes in Desulfovibrio. Our discussion will consider the significance of the involvement of electron-bifurcation in H 2 metabolism., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
Published: 2019
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50. A new mechanistic model for an O 2 -protected electron-bifurcating hydrogenase, Hnd from Desulfovibrio fructosovorans.

Author: Kpebe A, Benvenuti M, Guendon C, Rebai A, Fernandez V, Le Laz S, Etienne E, Guigliarelli B, García-Molina G, de Lacey AL, Baffert C, and Brugna M
Subjects: Amino Acid Sequence, Biocatalysis, Desulfovibrio genetics, Ferredoxins genetics, Ferredoxins metabolism, Hydrogenase chemistry, Hydrogenase genetics, NAD metabolism, Spectrophotometry, Ultraviolet, Spectroscopy, Fourier Transform Infrared, Desulfovibrio enzymology, Electrons, Hydrogenase metabolism, Models, Biological, Oxygen metabolism
Abstract: The genome of the sulfate-reducing and anaerobic bacterium Desulfovibrio fructosovorans encodes different hydrogenases. Among them is Hnd, a tetrameric cytoplasmic [FeFe] hydrogenase that has previously been described as an NADP-specific enzyme (Malki et al., 1995). In this study, we purified and characterized a recombinant Strep-tagged form of Hnd and demonstrated that it is an electron-bifurcating enzyme. Flavin-based electron-bifurcation is a mechanism that couples an exergonic redox reaction to an endergonic one allowing energy conservation in anaerobic microorganisms. One of the three ferredoxins of the bacterium, that was named FdxB, was also purified and characterized. It contains a low-potential (E m  = -450 mV) [4Fe4S] cluster. We found that Hnd was not able to reduce NADP + , and that it catalyzes the simultaneous reduction of FdxB and NAD + . Moreover, Hnd is the first electron-bifurcating hydrogenase that retains activity when purified aerobically due to formation of an inactive state of its catalytic site protecting against O 2 damage (H inact ). Hnd is highly active with the artificial redox partner (methyl viologen) and can perform the electron-bifurcation reaction to oxidize H 2 with a specific activity of 10 μmol of NADH/min/mg of enzyme. Surprisingly, the ratio between NADH and reduced FdxB varies over the reaction with a decreasing amount of FdxB reduced per NADH produced, indicating a more complex mechanism than previously described. We proposed a new mechanistic model in which the ferredoxin is recycled at the hydrogenase catalytic subunit., (Copyright © 2018 Elsevier B.V. All rights reserved.)
Published: 2018
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