Physiological Substrates and Products of Carbon Monoxide Dehydrogenase from Methanosarcina barkeri

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Current evidence indicates that, during methanogenesis from acetate, carbon monoxide dehydrogenase from Methanosarcina barkeri mediates cleavage of acetylCoA to methyl and carbonyl intermediates, then oxidizes the carbonyl moiety to provide electrons for the reduction of methylCoM to methane. However, isolated carbon monoxide dehydrogenase is known to catalyze only the methylviologen linked oxidation of carbon monoxide to carbon dioxide. This research will establish the physiological substrates and products of carbon monoxide dehydrogenase, and directly test the proposed role of this enzyme in methanogenesis. An assay has been developed to measure carbon dioxide evolution from acetate dependent upon the addition of a methyl accepting coenzyme, such as coenzyme M, to extracts in which methylreductase is inhibited. Using this assay methylated intermediates of the pathway will be identified by their ability to replace CoM as well as by radiotracer studies. The methyl intermediate proximal to the cleavage of acetyl-CoA will then be used to monitor purification of proteins capable of cleaving acetyl-CoA to that methyl intermediate and carbon dioxide.%%% Formation of methane and carbon dioxide from acetate is an essential reaction in the bio-degradation of organic matter in many anaerobic environments. Acetate is a key intermediate in the breakdown of organic polymers generated both by the growth of organisms and by human industrial and agricultural activities. Acetic acid is the direct precursor of the majority of biologically generated methane in a reaction mediated by microorganisms. Some microorganisms can synthesize methane in a reaction requiring that the carbon- carbon bond be broken. The enzyme which carries out this biologically important step is the subject of this investigation.***