Abstract
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 μmol H2 oxidized min–1 (mg protein)–1 with benzyl viologen as electron acceptor and an apparent K m value for H2 of 341 μM. 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 H2-dependent cytochrome b reduction at a rate of 0.18 nmol min–1 (mg protein)–1.
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Received: 7 September 1995 / Accepted: 4 December 1995
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Dobrindt, U., Blaut, M. Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport. Arch Microbiol 165, 141–147 (1996). https://doi.org/10.1007/s002030050309
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DOI: https://doi.org/10.1007/s002030050309