CO DEHYDROGENASE AND ACETOCLASTIC METHANOGENESIS IN METHANOSARCINA BARKERI

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In experiments with Methanosarcina barkeri it was determined that an intact and energized cell membrane is required for carbon-carbon bond cleavage of acetate as measured by isotopic exchange of carbon dioxide. Although extensive isotopic exchange was not detected in soluble preparations, methanogenesis and methyl-SCoM formation from acetate were readily observed in the presence of ATP and hydrogen. The rate of in vitro methanogenesis was dependent upon both ATP and acetate concentrations. The lag before onset of methanogenesis was not due to a slow production of methyl-SCoM, but rather to activation of step(s) needed for reduction of methyl-SCoM to HSCoM and methane. Both processes of methanogenesis and methyl-SCoM formation from acetate were stimulated by coenzyme A which indicates that acetyl-CoA is the activated form of acetate required for cleavage in methanogenesis. A convenient method was developed for analysis of the enzyme responsible for formation of methyl-SCoM from a methylated corrinoid compound and HSCoM. Cells of M. barkeri grown on acetate exhibited essentially the same level of this enzyme as cells grown on methanol. This suggests a common methyl group transfer step in both pathways of acetate and methanol metabolism. The methyltransferase enzyme was purified to near homogeneity and was found to be monomeric with a molecular weight of 37,000. It showed a high affinity for HSCoM (Km = 0.086 mM), but weakly bound methylcobalamin (Km = 13.5 mM). Metal ion chelators inhibited the enzyme but reactivation was brought about by removal of the chelator or addition of divalent metal ions. Supplementation of extracts producing methane and methyl-SCoM from acetate with purified carbon monoxide dehydrogenase (CODH) and the methyltransferase, respectively, failed to stimulate these processes. This suggests that the levels of CODH and methyltransferase were saturating under the conditions tested and that another unidentified step(s) was predominantly rate limiting.