The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g x 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(II1). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and x 2.0 (component I) and at 2.33, 2.24, and x 2.0 (component 11). In the reduced state, after exposure to an Hz atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers : center I (2.03, 1.89 and 1.86) detectable below 10 K, and center I1 (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 89421. During the course of a redox titration at pH 7.6 using Hz gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fastrelaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity. This indicates that the redox state of the isolated enzyme is important for the full expression of enzymatic activity. The catalytic properties were also followed by the proton-deuterium exchange reaction. The isolated hydrogenases produced Hz/HD ratios higher than those observed for nonselenium-containing hydrogenases.The enzyme responsible for the biological activation of H2, termed hydrogenase [l, 21, has a central role in many relevant anaerobic processes where molecular hydrogen is oxidized or evolved. Also, molecular hydrogen, via the hydrogenase system, is a link between different bacterial consortia w...

Three types of hydrogenases have been isolated from the sulfate‐reducing bacteria of the genus Desulfobibrio. They differ in their subunit and metal compositions, physico‐chemical characteristics, amino acid sequences, immunological ractivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as ‘iron only’ hydrogenases and nickel‐containing hydrogenases. The iron‐sulfur‐containg hydrogenase ([Fe] hydrogenase) contains two ferredoxin‐type (4Fe‐4S) clusters and an atypical iron‐sulfur center belived to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton‐deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2−. It is not present in all species of Desulfovibrio The nickel‐(iron‐sulfur)‐containing hydrogenases ([NiFe] hydrogenase) posses two (4Fe‐4S) centers and one (3Fe‐xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2−. The genes encoding the large and small subunits of a periplasmic and a membrane‐bound species of the [NiFe] hydrogenase have been cloned in Eschierichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal‐binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel‐iron‐sulfur)‐selenium‐containing hydrogenases ([NiFe‐Se] hydrohenases) which contain nickel and selenium in equimoleular amounts plus (4Fe‐4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfrovibio (D) baculatus (DSM 1743) (for abbrviations see appendix) have been cloned

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