Purification and properties of heterodisulfide reductase from Methanobacterium thermoautotrophicum (strain Marburg).
The reduction of the heterodisulfide of coenzyme M (H-S-CoM) and 7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80-fold to apparent homogeneity from Methanobacterium thermoautotrophicum. The native enzyme showed an apparent molecular mass of 550 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of three different subunits of apparent molecular masses 80 kDa, 36 kDa, and 21 kDa. The enzyme, which was brownish yellow, contained per mg protein 7 +/- 1 nmol FAD, 130 +/- 10 nmol non-heme iron and 130 +/- 10 nmol acid-labile sulfur, corresponding to 4 mol FAD and 72 mol FeS/mol native enzyme. The purified heterodisulfide reductase catalyzed the reduction of CoM-S-S-HTP (app. Km = 0.1 mM) with reduced benzylviologen at a specific rate of 30 mumol.min-1.mg protein-1 (kcat = 68 s-1) and the reduction of methylene blue with H-S-CoM (app. Km = 0.2 mM) plus H-S-HTP (app. Km less than 0.05 mM) at a specific rate of 15 mumol.min-1.mg-1. The enzyme was highly specific for CoM-S-S-HTP and H-S-CoM plus H-S-HTP. The physiological electron donor/acceptor remains to be identified.
Hedderich
R
R
Laboratorium für Mikrobiologie, Philipps-Universität Marburg, Federal Republic of Germany.
Berkessel
A
A
Thauer
R K
RK
D004220:Disulfides / Q000378:metabolism*; D019605:Euryarchaeota / Q000201:enzymology*; D015080:Mesna / Q000031:analogs & derivatives / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D010769:Phosphothreonine / Q000031:analogs & derivatives / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity
D004220:Disulfides; C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; C023349:methyl coenzyme M; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; D015080:Mesna