Redox properties and activity studies on a nickel-containing hydrogenase isolated from a halophilic sulfate reducer Desulfovibrio salexigens

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.