The A-center of carbon monoxide dehydrogenase (CODH) resides in the enzyme's alpha-subunit and is responsible for the acetyl-CoA synthase activity. The center comprises a Ni site and an iron-sulfur cluster. We have isolated the alpha-subunit using both continuous and discontinuous electrophoresis methods. When incubated with CO, samples prepared using continuous gels attain the A(red)-CO state that exhibits an S = 1/2 EPR feature (g = 2.048, 2.046, 2.021) similar to the so-called NiFeC signal of native CODH. Both signals consistently quantify to <0.4 spin/alpha. In order to elucidate the structure of the A-cluster and to understand the cause of the substoichiometric spin intensities, we have studied the alpha-subunit with Mossbauer and EPR spectroscopy. As found for CODH, populations of isolated alpha are heterogeneous: they contain two major A-cluster forms designated nonlabile and Ni-labile. In native CODH, only the Ni-labile form develops the NiFeC signal and exhibits catalytic activity. Oxidized samples of alpha exhibit Mossbauer spectra (S = 0, Delta E-Q = 1.08 mm/s, delta = 0.45 mm/s) typical of [Fe4S4](2+) clusters. Upon reduction with dithionite, the Fe4S4 cluster of nonlabile A-clusters exhibits Mossbauer (average Delta E-Q = 1.0 mm/s, delta = 0.54 mm/s) and EPR properties similar to those of S = 3/2 [Fe4S4](+) cubanes; in contrast, Ni-labile clusters are not reducible by dithionite. Treatment with CO yielded a sample for which 40% of the Fe was associated with A(red)-CO, while 47% of the clusters (the nonlabile form) remained oxidized. Thus, the presence of nonlabile A-clusters is largely responsible for the low spin intensities of the NiFeC signal. Upon formation of A(red)-CO, the Fe4S4 portion of the A-cluster exhibits Fe-57 magnetic hyperfine interactions; the cluster sites divide into equivalent pairs with (isotropic) A(A) = A(B) = -34.2 MHz and A(C) = A(D) = +26.8 MHz. However, the values of Delta E-Q and delta show that the cluster has remained in the [Fe4S4](2+) state. We explain these observations with an electronic model that considers a Ni+-X-[Fe4S4](2+) assembly for which the Ni+ is exchange-coupled with one Fe site of the cube through a bridging ligand (X). The coupling was found to be substantial, namely \j\ approximate to 100 cm(-1) (H-NiFe = jS(Ni).S-Fe). The Mossbauer spectra provide no evidence that CO is bound ro the Fe4S4 cluster in the state A(red)-CO, as has been concluded from resonance Raman studies [Qiu, D.; Kumar, M.; Ragsdale, S. W.; Spiro, T. G. Science 1994, 264, 817-819]. We could not determine if the two metal centers are linked in the oxidized state (for either the Ni-labile or nonlabile form), but if they are, the Ni2+ must be low-spin (S-Ni = 0).