Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex found in methanogens and certain other Archaea that carries out the overall synthesis and cleavage of the acetyl C-C and C-S bonds of acetyl-CoA. The reaction is involved both in the autotrophic fixation of carbon and in the process of methanogenesis from acetate, and takes place at a unique active site metal center known as the A cluster, located on the beta subunit of the ACDS complex and composed of a binuclear Ni-Ni site bridged by a cysteine thiolate to an Fe4S4 center. In this work, a high rate of acetyl-CoA synthesis was achieved with the recombinant ACDS beta subunit by use of methylcobinamide as an appropriate mimic of the physiological base-off corrinoid substrate. The redox dependence of acetyl-CoA synthesis exhibited one-electron Nernst behavior, and the effects of pH on the observed midpoint potential indicated that reductive activation of the A cluster also involves protonation. Initial burst kinetic studies indicated the formation of stoichiometric amounts of an A cluster-acetyl adduct, further supported by direct chromatographic isolation of an active enzyme-acetyl species. Titration experiments indicated that two electrons are required for activation of the enzyme in the process of forming the enzyme-acetyl intermediate. The results also established that the A cluster-acetyl species undergoes reductive elimination of the acetyl group with the simultaneous release of two, low potential electron equivalents. Thus, the one-electron Nernst behavior can be interpreted as the sum of two separate, low potential, one-electron steps. The results tend to exclude reaction mechanisms involving either one- or three-electron reduced forms of the A cluster as immediate precursors to the acetyl species. A scheme involving a [Fe4S4]1+-Ni1+ species is favored over a [Fe4S4]2+-Ni0 form. The role of proton uptake in the possible formation of a Ni2+-hydride intermediate is also discussed. Trapping of electrons during the formation of the A cluster-acetyl species from substrates CO and methylcobinamide was found to be highly favorable, thus presenting a means for extensive activation of the enzyme under otherwise nonpermissive physiological redox potentials.