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.