The results indicate that oxidation of CO to CO 2 supplies electrons for reduction of CO 2 to a methyl group by steps and enzymes of the pathway for CO 2 reduction determined for other methane-producing species. However, proteomic and quantitative RT-PCR results suggest that reduction of the methyl group to methane involves novel methyltransferases and a coenzyme F 420H2:heterodisulfide oxidoreductase system that generates a proton gradient for ATP synthesis not previously described for pathways reducing CO 2 to methane. Biochemical assays support a role for the oxidoreductase, and transcriptional mapping identified an unusual operon structure encoding the oxidoreductase. The proteomic results further indicate that acetate is synthesized from the methyl group and CO by a reversal of initial steps in the pathway for conversion of acetate to methane that yields ATP by substrate level phosphorylation. The results indicate that M. acetivorans utilizes a pathway distinct from all known CO 2 reduction pathways for methane formation that reflects an adaptation to the marine environment. Finally, the pathway supports the basis for a recently proposed primitive CO-dependent energy-conservation cycle that drove and directed the early evolution of life on Earth.anaerobic ͉ Archaea ͉ carbon monoxide C arbon monoxide (CO), an atmospheric pollutant that binds tightly to hemoglobin, is held below toxic levels in part by both aerobic and anaerobic microbes (1). The microbial metabolism of CO is an important component of the global carbon cycle (1, 2), and CO is believed to have been present in the atmosphere of early Earth that fueled the evolution of primitive metabolisms (3-7). Investigations of aerobic species from the Bacteria domain have contributed important insights into microbial CO oxidation (8, 9), as have investigations of anaerobes from the Bacteria domain that conserve energy by coupling CO oxidation to H 2 evolution (10-12). Further understanding has been derived from studies of CO-using anaerobes from the Bacteria domain that conserve energy by oxidizing CO and reducing CO 2 to acetate (13,14) or reducing sulfate to sulfide (15). Far less is known for pathways of the few CO-using species in the Archaea domain that have been described. Methanothermobacter thermautotrophicus, Methanosarcina barkeri, and Methanosarcina acetivorans obtain energy for growth by converting CO to methane (16)(17)(18)(19)(20). Although methane formation from CO first was reported in 1947 (21), a comprehensive understanding of the overall pathway for any species has not been reported. It is postulated that M. barkeri oxidizes CO to H 2 , and the H 2 is reoxidized to provide electrons for reducing CO 2 to methane (16). It is postulated further that H 2 production is essential for ATP synthesis during growth on CO (16,22,23). M. acetivorans was isolated from marine sediments where giant kelp is decomposed to methane (24). The flotation bladders of kelp contain CO that is a presumed substrate for M. acetivorans in nature. M. acetivorans produ...