The carbon monoxide dehydrogenase complex was purified from acetate-grown Methanosarcina thermophila. This complex made up greater than 10% of the cellular protein and the native enzyme formed aggregates with a Mr of approximately 1,000,000. The enzyme contained five subunits of different molecular weight suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The electron paramagnetic resonance spectrum of CO-reduced enzyme at 113K contained g values of 2.073, 2.049, and 2.028. Isotopic substitution with ⁶¹Ni, ⁵⁷Fe, or ¹³Co resulted in broadening of the spectrum consistent with a Ni-Fe-C spin-coupled complex. Acetyl-CoA caused a perturbation of the signal that was not caused by acetyl-phosphate or mercaptoethanol indicating acetyl-CoA is a physiological substrate. Cell extracts from acetate-grown M. thermophila contained CO-oxidizing:H₂-evolving activity 16-fold greater than extracts of methanol-grown cells. CO-oxidizing:H₂-evolving activity was reconstituted upon combination of: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase and b-type cytochrome. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The molecular weight of the isolated protein was 16,400, and the apparent minimum molecular weight was 4,900. The ferredoxin contained 2.8 ± 0.56 Fe atoms and 1.98 ± 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with a A₃₉₅/A₂₉₅ ratio range of 0.80 to 0.88. The N-terminal amino acid sequence revealed a 4-cysteine cluster, similar to other Fe:S centers that coordinate a Fe:S center. A CH₃-B₁₂:HS-CoM methyltransferase activity was characterized in extracts of acetate- and methanol-grown cells. The activity from extracts of acetate-grown M. thermophila was stable at 70°C for 30 minutes. The activity in cell extracts of acetate- and methanol-grown cells was fractionated with ammonium sulfate treatment and FPLC phenyl superose chromatography. Two peaks of methyltransferase activity were observed in each cell extract sample following phenyl superose fractionation. / Ph. D.