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Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H2 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 O2-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 H2, diffusion and reaction at the active site of CO and O2) 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.

The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni-Fe-S cluster employed to catalyze the reduction of CO2 to CO as part of the Wood-Ljungdahl carbon fixation pathway. Using X-ray crystallography, we have observed unprecedented conformational dynamics in the C-cluster of the CODH from Desulfovibrio vulgaris, providing the first view of an oxidized state of the cluster. Combined with supporting spectroscopic data, our structures reveal that this novel, oxidized cluster arrangement plays a role in avoiding irreversible oxidative degradation at the C-cluster. Furthermore, mutagenesis of a conserved cysteine residue that binds the C-cluster in the oxidized state but not in the reduced state suggests that the oxidized conformation could be important for proper cluster assembly, in particular Ni incorporation. Together, these results lay a foundation for future investigations of C-cluster activation and assembly, and contribute to an emerging paradigm of metallocluster plasticity., eLife digest Life relies on countless chemical reactions, almost all of which need to be sped up by enzymes. About half of all enzymes carry metal ions that expand the range of the reactions that they can catalyze. In some enzymes these metal ions assemble with sulfur ions to form so-called metalloclusters. These structures can carry out many different types of reactions, including converting simple forms of elements like nitrogen and carbon into other forms that can be used to make more complicated biological molecules. One enzyme that contains metalloclusters is carbon monoxide dehydrogenase. Known as CODH for short, this enzyme uses a metallocluster called the “C-cluster” to interconvert two gases: the pollutant carbon monoxide and the greenhouse gas carbon dioxide. CODH enzymes are found inside certain bacteria, but they are also of interest for humans, who wish to use them to remove the harmful gases from the environment. But this is not as simple as it may at first seem: CODH enzymes usually become inactive when exposed to air because the metalloclusters fall apart in the presence of oxygen. One CODH enzyme from a widespread bacterium called Desulfovibrio vulgaris, however, is an attractive target for industrial use because it can tolerate oxygen better. Yet, it is still unclear why this enzyme does not get inactivated the way other CODHs do. Wittenborn et al. have now characterized the CODH enzyme from D. vulgaris in more depth via a technique called X-ray crystallography, which can reveal the location of individual atoms within a molecule. By a happy accident, the structures revealed that the C-cluster can adopt a dramatically different arrangement of metal and sulfur ions after being exposed to oxygen. This rearrangement is fully reversible; when oxygen is removed, the metal and sulfur ions move back to their normal positions. This ability to flip between different arrangements appears to protect the metallocluster from losing its metal ions when exposed to oxygen. By providing structural snapshots of how CODH responds to oxygen these results provide a more complete understanding of an enzyme that plays a key role in the global carbon cycle. This understanding could help scientists to develop bioremediation tools to remove carbon monoxide and carbon dioxide from the atmosphere and to engineer bacteria to capture carbon to make biofuels.

Hydrogenases catalyze the conversion between 2H+ + 2e− and H2. Most of these enzymes are inhibited by O2, which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O2 tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. A few bacteria, however, contain hydrogenases that activate H2 even in the presence of O2. Intriguingly, kinetic and spectroscopic studies lead to assuming that different mechanisms might be responsible for the resistance, depending on the enzyme type. The various hypotheses that emerged from these studies are still a matter of debate. In order to better understand the molecular bases of resistance to O2 inhibition, we explored different methods to improve the O2-tolerance of the O2-sensitive [Ni–Fe] hydrogenase from Desulfovibrio fructosovorans. A whole bunch of mutants has been studied and fully characterized, which revealed that actually, different mechanisms can lead to O2 tolerance. These mechanisms are described in this review and compared to the current hypothesis of O2 tolerance.

Acid mine drainage (AMD) waters are highly acidic (pH < 4), contain high concentrations of sulfate and dissolved metals, and are very toxic to many living organisms. The development of technologies to treat sulfate contaminated wastewaters by using sulfate-reducing bacteria (SRB) has produced a cost-effective route to treat AMD. Notwithstanding, the SRB sensitivity to acid limits their use in AMD remediation. In the current study, acidophilic strains of SRB were isolated from an AMD followed by their molecular characterization. One SRB-culture was able to grow at pH 4.5 in Postgate C modified medium containing ethanol as carbon source, indicating that such bacterium has the potential for the bioremediation of acidic waters. Following, the strains were characterized by molecular biology techniques. The characterization was done by PCR amplification, cloning and sequencing of the genes coding for parts of the alpha and beta subunits of dissimilatory sulfite reductase (dsrAB) and hydrogenase (hyd), which encode key enzymes of the SRB energy metabolism. Phylogenetic analysis suggested a line of SRB descent from the delta-Proteobacteria among the strains identified as Desulfovibrio fructosovorans.

We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O2 is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O2 inactivation and O2 tolerance in NiFe hydrogenases., This work was funded by the CNRS, the Agence Nationale de la Recherche, the Aix-Marseille Université, the City of Marseilles and the Spanish Ministerio de Ciencia e Innovación (MICINN) (project CTQ2009-12649) and was supported by the Pôle de Compétitivité Capénergies. A.A.H. thanks the CNRS and the Région Provence-Alpes Côte d'Azur for financial support. O.G.-S. thanks the MICINN for a Formación de Personal Investigador (FPI) grant.

Reproducing the naturally occurring O2-tolerant hydrogenases is a potential strategy to make the oxygen sensitive enzymes, produced by organisms of biotechnological interest, more resistant. The search for resistance “hotspots” that could be transposed into sensitive hydrogenases is underway. Here, we replaced two residues (Y77 and V78) of the oxygen sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans with Gly and with Cys, respectively, to copy the active site pocket of the resistant membrane-bound [NiFe] enzyme from Ralstonia eutropha and we examined how this affected oxygen sensitivity. The results are discussed in the light of a short review of the recent results dealing with the reactivity of hydrogenases towards oxygen.

A strain devoid of the three hydrogenases characterized for Desulfovibrio fructosovorans was constructed using marker exchange mutagenesis. As expected, the H 2 -dependent methyl viologen reduction activity of the strain was null, but physiological studies showed no striking differences between the mutated and wild-type strains. The H + -D 2 exchange activity measured in the mutated strain indicates the presence of a fourth hydrogenase in D. fructosovorans .

A new halotolerant Desulfovibrio, strain CVLT (T = type strain), was isolated from a solar saltern in California. The curved, gram-negative, nonsporeforming cells (0.3 × 1.0–1.3 μm) occurred singly, in pairs, or in chains, were motile by a single polar flagellum and tolerated up to 12.5% NaCl. Strain CVLT had a generation time of 60 min when grown in lactate-yeast extract medium under optimal conditions (37°C, pH 7.6, 2.5% NaCl). It used lactate, pyruvate, cysteine, or H2/CO2 + acetate as electron donors, and sulfate, sulfite, thiosulfate, or fumarate as electron acceptors. Elemental sulfur, nitrate, or oxygen were not used. Sulfite and thiosulfate were disproportionated to sulfate and sulfide. The G+C content of the DNA was 62 mol%. Phylogenetic analysis revealed that Desulfovibrio fructosovorans was the nearest relative. Strain CVLT is clearly different from other Desulfovibrio species, and is designated Desulfovibrio senezii sp. nov. (DSM 8436).