Electrochemical measurements of the kinetics of inhibition of two FeFe hydrogenases by O2 demonstrate that the reaction is partly reversible

International audienceThe mechanism of reaction of FeFe hydrogenases with oxygen has been debated. It is complex, apparently very dependent on the details of the protein structure, and difficult to study using conventional kinetic techniques. Here we build on our recent work on the anaerobic inactivation of the enzyme [Fourmond et al, Nat. Chem. 4 336 (2014)] to propose and apply a new method for studying this reaction. Using electrochemical measurements of the turnover rate of hydrogenase, we could resolve the first steps of the inhibition reaction and accurately determine their rates. We show that the two most studied FeFe hydrogenases, from Chlamydomonas reinhardtii and Clostridium acetobutylicum, react with O2 according to the same mechanism, despite the fact that the former is much more O2 sensitive than the latter. Unlike often assumed, both enzymes are reversibly inhibited by a short exposure to O2. This will have to be considered to elucidate the mechanism of inhibition, before any prediction can be made regarding which mutations will improve oxygen resistance. We hope that the approach described herein will prove useful in this respect

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

International audienceAfter 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

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

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Synthèse, propriétés rédox et photorédox de complexes polypyridiniques de manganèse et de ruthénium, modèles des sites actifs du photosystème II et de catalases à manganèse

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Cobaloximes as Functional Models for Hydrogenases (Part 2): Proton Electroreduction Catalyzed by Difluoroboryl-bis(dimethylglyoximato)cobalt(II) Complexes in Organic Media

International audienc

Hydrogenases and H2 metabolism in sulfate-reducing bacteria of the Desulfovibrio genus

International audienceHydrogen 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 H2 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 H2 metabolism

L’électrochimie, un outil pour étudier les mécanismes enzymatiques

International audienceLe fonctionnement des enzymes qui catalysent des réactions redox fait intervenir des étapes très diverses (diffusion du substrat à l’intérieur de l’enzyme, réactions chimiques au site actif, transferts à longue distance d’électrons et de protons) et qui impliquent des sites de la protéine distants les uns des autres.Cet article illustre, en prenant pour exemple les enzymes qui catalysent l’oxydation réversible du dihydrogène, comment l’électrochimie peut maintenant être utilisée en combinaison avec d’autres approches comme la chimie théorique et la mutagenèse dirigée, pour étudier des aspects variés du mécanisme moléculaire des enzymes redox

L’électrochimie, un outil pour étudier les mécanismes enzymatiques

International audienceLe fonctionnement des enzymes qui catalysent des réactions redox fait intervenir des étapes très diverses (diffusion du substrat à l’intérieur de l’enzyme, réactions chimiques au site actif, transferts à longue distance d’électrons et de protons) et qui impliquent des sites de la protéine distants les uns des autres.Cet article illustre, en prenant pour exemple les enzymes qui catalysent l’oxydation réversible du dihydrogène, comment l’électrochimie peut maintenant être utilisée en combinaison avec d’autres approches comme la chimie théorique et la mutagenèse dirigée, pour étudier des aspects variés du mécanisme moléculaire des enzymes redox