Characterization of a CO: heterodisulfide oxidoreductase system from acetate-grown Methanosarcina thermophila.
During the methanogenic fermentation of acetate by Methanosarcina thermophila, the CO dehydrogenase complex cleaves acetyl coenzyme A and oxidizes the carbonyl group (or CO) to CO2, followed by electron transfer to coenzyme M (CoM)-S-S-coenzyme B (CoB) and reduction of this heterodisulfide to HS-CoM and HS-CoB (A. P. Clements, R. H. White, and J. G. Ferry, Arch. Microbiol. 159:296-300, 1993). The majority of heterodisulfide reductase activity was present in the soluble protein fraction after French pressure cell lysis. A CO:CoM-S-S-CoB oxidoreductase system from acetate-grown cells was reconstituted with purified CO dehydrogenase enzyme complex, ferredoxin, membranes, and partially purified heterodisulfide reductase. Coenzyme F420 (F420) was not required, and CO:F420 oxidoreductase activity was not detected in cell extracts. The membranes contained cytochrome b that was reduced with CO and oxidized with CoM-S-S-CoB. The results suggest that a novel CoM-S-S-CoB reducing system operates during acetate conversion to CH4 and CO2. In this system, ferredoxin transfers electrons from the CO dehydrogenase complex to membrane-bound electron carriers, including cytochrome b, that are required for electron transfer to the heterodisulfide reductase. The cytochrome b was purified from solubilized membrane proteins in a complex with six other polypeptides. The cytochrome was not reduced when the complex was incubated with H2 or CO, and H2 uptake hydrogenase activity was not detected; however, the addition of CO dehydrogenase enzyme complex and ferredoxin enabled the CO-dependent reduction of cytochrome b.
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061-0305.
C W
CW
Peer
M H
MH
Painter
M E
ME
Rasche
J G
JG
Ferry
D000085:Acetates / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism*; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism; D004579:Electron Transport; D005288:Ferredoxins / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D015080:Mesna / Q000378:metabolism; D017020:Methanosarcina / Q000201:enzymology* / Q000254:growth & development; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010769:Phosphothreonine / Q000031:analogs & derivatives / Q000378:metabolism; D013053:Spectrophotometry
D000085:Acetates; D003573:Cytochrome b Group; D005288:Ferredoxins; D009097:Multienzyme Complexes; C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; D002248:Carbon Monoxide; D010088:Oxidoreductases; C087171:heterodisulfide reductase; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D015080:Mesna