The biological synthesis of acetyl-coenzyme A (acetyl-CoA), catalyzed by acetyl-CoA synthase (ACS), is of biological significance and chemical interest acting as a source of c energy and carbon. The catalyst contains an unusual hexa-metal cluster with two nickel ions and a [Fe4S4] cluster. DFT calculations have been performed to investigate the ACS reaction mechanism starting from three different oxidation states (+2, +1, and 0) of Ni-p, the nickel proximal to [Fe4S4]. The results indicate that the ACS reaction proceeds first through a methyl radical transfer from cobalamin (CIA) to Ni randomly accompanying with the CO binding. After that, C-C bond formation occurs between the Ni-p bound methyl and CO, forming Ni-p-acetyl. The substrate CoA-S- then binds to Ni-p, allowing C-S bond formation between the Ni-p bound acetyl and CoA-S-. Methyl transfer is rate-limiting with a barrier of similar to 14 kcal/mol, which does not depend on the presence or absence of CO. Both the Ni(p)(2+ )and Ni(p)(1+)states are chemically capable of catalyzing the ACS reaction independent of the state (+2 or +1) of the [Fe4S4] cluster. The [Fe4S4] cluster is not found to affect the steps of methyl transfer and C-C bond formation but may be involved in the C-S bond formation depending on the detailed mechanism chosen. An ACS active site containing a Ni-p (0) state could not be obtained. Optimizations always led to a Ni-p(1+) state coupled with [Fe4S4](1+). The calculations show a comparable activity for Ni-p(1+)/[Fe4S4](1+), Ni-p(1+)/[Fe4S4](2+), and Ni-p(2+)/[Fe4S4](2+). The results here give significant insights into the chemistry of the important ACS reaction.