The number of sequenced genomes of sulfate reducing organisms (SRO) has increased significantly in the recent years, providing an opportunity for a broader perspective into their energy metabolism. In this work we carried out a comparative survey of energy metabolism genes found in 25 available genomes of SRO. This analysis revealed a higher diversity of possible energy conserving pathways than classically considered to be present in these organisms, and permitted the identification of new proteins not known to be present in this group. The Deltaproteobacteria (and Thermodesulfovibrio yellowstonii) are characterized by a large number of cytochromes c and cytochrome c-associated membrane redox complexes, indicating that periplasmic electron transfer pathways are important in these bacteria. The Archaea and Clostridia groups contain practically no cytochromes c or associated membrane complexes. However, despite the absence of a periplasmic space, a few extracytoplasmic membrane redox proteins were detected in the Gram-positive bacteria. Several ion-translocating complexes were detected in SRO including H+-pyrophosphatases, complex I homologs, Rnf, and Ech/Coo hydrogenases. Furthermore, we found evidence that cytoplasmic electron bifurcating mechanisms, recently described for other anaerobes, are also likely to play an important role in energy metabolism of SRO. A number of cytoplasmic [NiFe] and [FeFe] hydrogenases, formate dehydrogenases, and heterodisulfide reductase-related proteins are likely candidates to be involved in energy coupling through electron bifurcation, from diverse electron donors such as H2, formate, pyruvate, NAD(P)H, β-oxidation, and others. In conclusion, this analysis indicates that energy metabolism of SRO is far more versatile than previously considered, and that both chemiosmotic and flavin-based electron bifurcating mechanisms provide alternative strategies for energy conservation.

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Hydrogenases are highly active enzymes for hydrogen production and oxidation. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H production. In contrast to [NiFe] hydrogenases, they display reduced H inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for production of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.

The ACS journal of surfaces and colloids 27: 6449-6457 (2011) 2 ABSTRACTThe interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble than with membrane enzymes due to the higher structural complexity and instability of the latter proteins. In this work we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H 2 -oxidation reversibly and is associated to the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, only allowing mediated electron transfer between enzyme and electrode. On the other hand, a one-step immobilization procedure favours formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted in a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.

[NiFeSe] hydrogenases are a sub-group of the large family of [NiFe] hydrogenases in which a selenocysteine ligand is coordinating the Ni at the active site. As observed for other selenoproteins, the [NiFeSe] hydrogenases display much higher catalytic activities than their Cys-containing homologues. Here we review the biochemical, catalytic, spectroscopic and structural properties of known [NiFeSe] hydrogenases, namely from the Hys, Fru and Vhu families. A survey of new [NiFeSe] hydrogenases present in the databases showed that all enzymes belong to either group 1 periplasmic uptake hydrogenases (Hys) or to group 3 cytoplasmic hydrogenases (Fru and Vhu), and are present in either sulfate-reducing or methanogenic microorganisms. In both kinds of organisms the [NiFeSe] hydrogenases are preferred over their Cyscontaining homologues if selenium is available. Since no structural information is available for the Vhu and Fru enzymes, we have modelled the large subunit of these enzymes and analyzed the area surrounding the active site. Three [NiFeSe] hydrogenases of the Hys and Vhu types were identified in which the selenocysteine residue is found in a different location in the sequence, which should result in a surprising coordination to the Ni as a bridging, rather than terminal, ligand. The high activity and fast reactivation, together with a degree of oxygen tolerance for the H 2 -production activity, make the Hys hydrogenases attractive catalysts for technological applications. ____________ [a]Protein Modeling