CO dehydrogenases catalyze the reversible oxidation of CO to CO2, at an active site (called the C-cluster) composed of an Fe4S4 cube with what appears to be a 5-coordinate Fe (called FCII), linked to a Ni (Hu, Z., Spangler, N. J., Anderson, M. E., Xia, J., Ludden, P. W., Lindahl, P. A., & Münck, E. (1996) J. Am. Chem. Soc. 118, 830-845). During catalysis, electrons are transferred from the C-cluster to an [Fe4S4]2+/1+ electron-transfer cluster called the B-cluster. An S = 1/2 form of the C-cluster (called Cred1) converts to another S = 1/2 form (called Cred2) upon reduction with CO, at a rate well within the turnover frequency of the enzyme (Kumar, M., Lu, W.-P., Liu, L., & Ragsdale, S. W. (1993) J. Am. Chem. Soc. 115, 11646-11647). This suggests that the conversion is part of the catalytic mechanism. Dithionite is reported in this paper to effect this conversion as well, but at a much slower rate (kso = 5.3 x 10(-2) M-1 s-1 for dithionite vs 4.4 x 10(6) M-1 s-1 for CO). By contrast, dithionite reduces the oxidized B-cluster much faster, possibly within the turnover frequency of the enzyme. Dithionite apparently effects the Cred1/Cred2 conversion directly, rather than through an intermediate. The conversion rate varies with dithionite concentration. The Cred1/Cred2 conversion occurs at least 10(2) times faster in the presence of CO2 than in its absence. CO2 alters the g values of the gav = 1.82 signal, indicating that CO2 binds to a C-cluster-sensitive site at mild potentials. CN- inhibits CO oxidation by binding to FCII (Hu et al., 1996), and CO, CO2 in the presence of dithionite, or CS2 in dithionite accelerate CN- dissociation from this site (Anderson, M. E., & Lindahl, P. A. (1994) Biochemistry 33, 8702-8711). The effect of CO, CO2, and CS2 on CN- dissociation suggested that these molecules bind at a site (called the modulator) other than that to which CN- binds. The effects of CO2, CS2, CO, and dithionite on the Cred1/Cred2 conversion rate followed a similar pattern, suggesting that this rate is also influenced by modulator binding. Some batches of enzyme cannot convert to the Cred2 form using dithionite, but pretreatment with CO or CO2/dithionite effectively "cures" such batches of this disability. The results presented suggest that the Ni of the C-cluster is the modulator and the substrate binding site for CO/CO2. The inhibitor CS2 in the presence of dithionite also accelerates the decline of Cred1, leading first to an EPR-silent state of the C-cluster, and eventually to a state yielding an EPR signal with gav = 1.66. CS2 binding thus shares some resemblance to CO2 binding. Approximately 90% of the absorbance changes at 420 nm that occur when oxidized CODHCt is reduced by dithionite occur within 2 min at 10 degrees C. This absorbance change occurs in concert with the gav = 1.94 signal development. The remaining 10% of the A420 changes occur over the course of approximately 50 min, apparently coincident with the Cred1/Cred2 conversion. One possibility is that the conversion involves reduction of an (unidentified) Fe-S cluster. A three-state model of catalysis is proposed in which Cred1 binds and oxidizes CO, Cred2 is two electrons more reduced than Cred1 and is the state that binds and reduces CO2, and Cint is a one-electron-reduced state that is proposed to exist because of constraints imposed by the nature of the CO/CO2 reaction and the properties of the clusters involved in catalysis.