A soluble hydrogenase from the halophilic sulfate reducing bacterium Desulfovibrio salexigens, strain British Guiana (NCIB 8403) has been purified to apparent homogeneity with a final specific activity of 760 μmoles H 2 evolved/min/mg (an overall 180-fold purification with 20 % recovery yield). The enzyme is composed of two non-identical subunits of molecular masses 62 and 36 kDa, respectively, and contains approximately 1 Ni, 12–15 Fe and 1 Se atoms/mole. The hydrogenase shows a visible absorption spectrum typical of an iron-sulfur containing protein (A 400 /A 280 = 0.275) and a molar absorbance of 54 mM −1 cm −1 at 400 nm. In the native state (as isolated, under aerobic conditions), the enzyme is almost EPR silent at 100 K and below. However, upon reduction under H 2 atmosphere a rhombic EPR signal develops at g-values 2.22, 2.16 and around 2.0, which is optimally detected at 40 K. This EPR signal is reminiscent of the nickel signal C (g-values 2.19, 2.16 and 2.02) observed in intermediate redox states of the well characterized D. gigas nickel containing hydrogenase and assigned to nickel by 61 Ni isotopic substitution (J.J.G. Moura, M. Teixeira, I. Moura, A.V. Xavier and J. LeGall (1984), J. Mol. Cat., 23, 305–314). Upon longer incubation with H 2 the “2.22” EPR signal decreases. During the course of a redox titration under H 2 , this EPR signal attains a maximal intensity around −380 mV. At redox states where this “2.22” signal develops (or at lower redox potentials), low temperature studies (below 10 K) reveals the presence of other EPR species with g-values at 2.23, 2.21, 2.14 with broad components at higher fields. This new signal (fast relaxing) exhibits a different microwave power dependence from that of the “2.22” signal, which readily saturates with microwave power (slow relaxing). Also at low temperature (8 K) typical reduced iron-sulfur EPR signals are concomitantly observed with g med ∼ 1.94. The catalytic properties of the enzyme were also followed by substrate isotopic exchange D 2 /H + and H 2 production measurements. The general properties of the D. salexigens hydrogenase are compared with those of [NiFe] hydrogenases isolated from other sulfate reducers from the genus Desulfovibrio.
- centres fer-soufre
- Desulfovibrio sp.
- electron paramagnetic resonance
- espèce Desulfovibrio
- iron-sulfur clusters