Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport

Hydrogenase was solubilized from the cytoplasmic membrane fraction of betaine-grown Sporomusa sphaeroides, and the enzyme was purified under oxic conditions. The oxygen-sensitive enzyme was partially reactivated under reducing conditions, resulting in a maximal activity of 19.8 mu mol H-2 oxidized min(-1) (mg protein)(-1) with benzyl viologen as electron acceptor and an apparent K-m value for H-2 of 341 mu M. The molecular mass of the native protein estimated by native PAGE and gel filtration was 122 and 130 kDa, respectively. SDS-PAGE revealed two polypeptides with molecular masses of 65 and 37 kDa, present in a 1:1 ratio. The native protein contained 15.6 +/- 1.7 mol Fe, 11.4 +/- 1.4 mol S2-, and 0.6 mol Ni per mol enzyme. The hydrogenase coupled with viologen dyes, but not with other various artificial electron carriers, FAD, FMN, or NAD(P)(+). The amino acid sequence of the N-termini of the subunits showed a high degree of similarity to eubacterial membrane-bound uptake hydrogenases. Washed membranes catalyzed a H-2-dependent cytochrome b reduction at a rate of 0.18 nmol min(-1) (mg protein)(-1).