Two isolated α subunit mutants (A110C and A222L) of the α2β2 acetyl coenzyme A synthase (ACS)/carbon monoxide dehydrogenase (CODH) from Moorella thermoacetica were designed to block the CO-migrating tunnel in the α subunit, allowing comparison with equivalent mutants in ACS/CODH. After Ni activation, both mutants exhibited electron paramagnetic resonance spectra indicating that the A-cluster was properly assembled. ACS activities were similar to those of the wild-type recombinant Ni-activated α subunit, suggesting that CO diffuses directly to the A-cluster from solvent rather than through the tunnel as is observed for the “majority” activity of ACS/CODH. Thus, CO appears to migrate to the A-cluster through two pathways, one involving and one not involving the tunnel. The kinetics and extent of reduction of the Fe4S4 cubane in the apo-α subunit and the Ni-activated α subunit upon exposure to titanium(III) citrate were examined using the stopped-flow method. The extent of reduction was independent of Ni, whereas the kinetics of reduction was Ni-dependent. Apo-α subunit reduction was monophasic while Ni-activated α subunit reduction was biphasic, with the more rapid phase coincident with that of apo-α subunit reduction. Thus, binding of Ni to the A-cluster slows the reduction kinetics of the [Fe4S4]2+ cubane. An upper limit of two electrons per α subunit are transferred from titanium(III) citrate to the Ni subcomponent of the A-cluster during reductive activation. These electrons are accepted quickly relative to the reduction of the [Fe4S4]2+ cubane. This reduction is probably a prerequisite for methyl group transfer. CO appears to bind to reduced nonfunctional subunits, thereby inhibiting reduction (or promoting reoxidation) of the cubane subcomponent of the A-cluster.