The genome of Desulfovibrio vulgaris Hildenborough (DvH) encodes for six hydrogenases (Hases), making it an interesting organism to study the role of these proteins in sulphate respiration. In this work we address the role of the [NiFeSe] Hase, found to be the major Hase associated with the cytoplasmic membrane. The purified enzyme displays interesting catalytic properties, such as a very high H(2) production activity, which is dependent on the presence of phospholipids or detergent, and resistance to oxygen inactivation since it is isolated aerobically in a Ni(II) oxidation state. Evidence was obtained that the [NiFeSe] Hase is post-translationally modified to include a hydrophobic group bound to the N-terminal, which is responsible for its membrane association. Cleavage of this group originates a soluble, less active form of the enzyme. Sequence analysis shows that [NiFeSe] Hases from Desulfovibrionacae form a separate family from the [NiFe] enzymes of these organisms, and are more closely related to [NiFe] Hases from more distant bacterial species that have a medial [4Fe4S](2+/1+) cluster, but not a selenocysteine. The interaction of the [NiFeSe] Hase with periplasmic cytochromes was investigated and is similar to the [NiFe](1) Hase, with the Type I cytochrome c (3) as the preferred electron acceptor. A model of the DvH [NiFeSe] Hase was generated based on the structure of the Desulfomicrobium baculatum enzyme. The structures of the two [NiFeSe] Hases are compared with the structures of [NiFe] Hases, to evaluate the consensual structural differences between the two families. Several conserved residues close to the redox centres were identified, which may be relevant to the higher activity displayed by [NiFeSe] Hases.

Sulphate-reducing bacteria are important players in the global sulphur and carbon cycles, with considerable economical and ecological impact. However, the process of sulphate respiration is still incompletely understood. Several mechanisms of energy conservation have been proposed, but it is unclear how the different strategies contribute to the overall process. In order to obtain a deeper insight into the energy metabolism of sulphate-reducers whole-genome microarrays were used to compare

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Desulfovibrio vulgarisHydrogen plays a central role in the energy metabolism of sulfate-reducing bacteria (17). H 2 is one of the major energy sources for these organisms in their natural habitats, but it may also be a product of their fermentative metabolism. Furthermore, a chemiosmotic mechanism involving production and oxidation of H 2 on opposite sides of the membrane has been proposed to explain energy transduction during sulfate respiration with lactate (21). In agreement with the important metabolic role of H 2 , hydrogenases (Hases) are particularly abundant proteins in sulfate-reducing bacteria, and many species have several different Hases (39 (25,36). This raises the question of what distinguishes these three Hases in physiological terms, and one possibility could be differences in expression conditions. Interestingly, the genes coding for the [NiFe]

Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum-and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC 3 protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC 3 specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage.Formate is a key metabolite in anaerobic habitats, arising as a metabolic product of bacterial fermentations and functioning as a growth substrate for many microorganisms (for example, methanogens and sulfate-reducing bacteria [SRB]). Formate is also an intermediate in the energy metabolism of several prokaryotes and a crucial compound in many syntrophic associations, whereby organisms live close to the thermodynamic limit (30,45). Recent reports indicate that formate plays an even more important role in anaerobic microbial metabolism than previously considered (14,24,27). The key enzyme in formate metabolism is formate dehydrogenase (FDH) (50), a member of the dimethyl sulfoxide (DMSO) reductase family. It catalyzes the reversible two-electron oxidation of formate or reduction of CO 2 and plays a role in energy metabolism and carbon fixation. In anaerobic microorganisms, FDH includes a molybdenum or tungsten bis-(pyranopterin guanidine dinucleotide) cofactor and iron-sulfur clusters (20, 41) and shows great variability in quaternary structure, physiological redox partner, and cellular location (7,23,38,50).FDH was the first enzyme shown to naturally incorporate tungsten, at a time when this element was considered to be mostly an antagonist to molybdenum (2, 52). Since then, several tungstoenzymes have been isolated and characterized, mainly but not exclusively from archaeal organisms, including FDHs, formylmethanofuran dehydrogenases (FMDH), aldehyde oxidoreductases (AOR) (not belonging to the xanthine oxidase family), and acetylene hydratase (3,4,20,25,31,41). FDHs and FMDHs can naturally incorporate either tungsten or molybdenum. Since these two elements have very similar chemical and catalytic properties,...