Purification of a two-subunit cytochrome-b-containing heterodisulfide reductase from methanol-grown Methanosarcina barkeri.
Heterodisulfide reductase catalyzes the terminal step in the energy-conserving electron-transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol-grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q-Sepharose and Superdex-200 yielded a high-molecular-mass fraction (> 700 kDa) which was dissociated by dodecyl beta-D-maltoside. After chromatography on Q-Sepharose, an active heterodisulfide reductase preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa. For each 69 kDa, the enzyme contained 0.6 mol cytochrome b, 0.2 mol FAD, 20 mol non-heme iron and 20 mol acid-labile sulfur. The 23-kDa subunit possessed heme-derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N-7-mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating that the cytochrome b served as electron donor for heterodisulfide reduction.
Heiden
S
S
Max-Planck-Institut für Terrestrische Mikrobiologie, Philipps-Universität, Marburg, Germany.
Hedderich
R
R
Setzke
E
E
Thauer
R K
RK
D000595:Amino Acid Sequence; D003573:Cytochrome b Group / Q000032:analysis; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007501:Iron / Q000032:analysis; D017021:Methanosarcina barkeri / Q000201:enzymology* / Q000254:growth & development; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D003573:Cytochrome b Group; D005182:Flavin-Adenine Dinucleotide; D007501:Iron; D010088:Oxidoreductases; C087171:heterodisulfide reductase