title: Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport.
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 &mu;mol H2 oxidized min-1 (mg protein)-1 with benzyl viologen as electron acceptor and an apparent Km value for H2 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 H2-dependent cytochrome b reduction at a rate of 0.18 nmol min-1 (mg protein)-1.
authors.affiliation: Institut für Mikrobiologie der Universität Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Dobrindt
authors.forename: M
authors.initials: M
authors.lastname: Blaut
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D003580:Cytochromes / Q000378:metabolism; D004579:Electron Transport; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid
chemical_list: D003580:Cytochromes; D006859:Hydrogen; D006864:Hydrogenase