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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.

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.)

The 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. 2014, 4, 336-342] 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.

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 H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 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 H2 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 O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.

By analysing the results of experiments carried out with two FeFe hydrogenases and several "channel mutants" of a NiFe hydrogenase, we demonstrate that whether or not hydrogen evolution is significantly inhibited by H2 is not a consequence of active site chemistry, but rather relates to H2 transport within the enzyme.

[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.

Using direct electrochemistry to learn about the mechanism of electrocatalysts and redox enzymes requires that kinetic models be developed. Here we thoroughly discuss the interpretation of electrochemical signals obtained with adsorbed enzymes and molecular catalysts that can reversibly convert their substrate and product. We derive analytical relations between electrochemical observables (overpotentials for catalysis in each direction, positions, and magnitudes of the features of the catalytic wave) and the characteristics of the catalytic cycle (redox properties of the catalytic intermediates, kinetics of intramolecular and interfacial electron transfer, etc.). We discuss whether or not the position of the wave is determined by the redox potential of a redox relay when intramolecular electron transfer is slow. We demonstrate that there is no simple relation between the reduction potential of the active site and the catalytic bias of the enzyme, defined as the ratio of the oxidative and reductive limiting currents; this explains the recent experimental observation that the catalytic bias of NiFe hydrogenase depends on steps of the catalytic cycle that occur far from the active site [Abou Hamdan et al., J. Am. Chem. Soc. 2012, 134, 8368]. On the experimental side, we examine which models can best describe original data obtained with various NiFe and FeFe hydrogenases, and we illustrate how the presence of an intramolecular electron transfer chain affects the voltammetry by comparing the data obtained with the FeFe hydrogenases from Chlamydomonas reinhardtii and Clostridium acetobutylicum, only one of which has a chain of redox relays. The considerations herein will help the interpretation of electrochemical data previously obtained with various other bidirectional oxidoreductases, and, possibly, synthetic inorganic catalysts.

Direct electron transfer between enzymes and electrodes is now commonly achieved, but obtaining protein films that are very stable may be challenging. This is particularly crucial in the case of hydrogenases, the enzymes that catalyze the biological conversion between dihydrogen and protons, because the instability of the hydrogenase films may prevent the use of these enzymes as electrocatalysts of H(2) oxidation and production in biofuel cells and photoelectrochemical cells. Here we show that two different FeFe hydrogenases (from Chamydomonas reinhardtii and Clostridium acetobutylicum) can be covalently attached to functionalized pyrolytic graphite electrodes using peptidic coupling. In both cases, a surface patch of lysine residues makes it possible to favor an orientation that is efficient for fast, direct electron transfer. High hydrogen-oxidation current densities are maintained for up to one week, the only limitation being the intrinsic stability of the enzyme. We also show that covalent attachment has no effect on the catalytic properties of the enzyme, which means that this strategy can also used be for electrochemical studies of the catalytic mechanism.

We investigated di-hydrogen transport between the solvent and the active site of FeFe hydrogenases. Substrate channels supposedly exist and serve various functions in certain redox enzymes which use or produce O2, H2, NO, CO, or N2, but the preferred paths have not always been unambiguously identified, and whether a continuous, permanent channel is an absolute requirement for transporting diatomic molecules is unknown. Here, we review the literature on gas channels in proteins and enzymes and we report on the use of site-directed mutagenesis and various kinetic methods, which proved useful for characterizing substrate access to the active site of NiFe hydrogenase to test the putative "static" H2 channel of FeFe hydrogenases. We designed 8 mutations in attempts to interfere with intramolecular diffusion by remodeling this putative route in Clostridium acetobutylicum FeFe hydrogenase, and we observed that none of them has a strong effect on any of the enzyme's kinetic properties. We suggest that H2 may diffuse either via transient cavities, or along a conserved water-filled channel. Nitrogenase sets a precedent for the involvement of a hydrophilic channel to conduct hydrophobic molecules.