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., & Munck, E. (1996) J. Ant. Chem. Sec. 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 C-red1) converts to another S = 1/2 form (called C-red2) upon reduction with CO, at a rate well within the turnover frequency of the enzyme (Kumar, M., Lu, W.-P., Liu, L., st Ragsdale, S. W. (1993) J. Am. Chem. Sec. 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 (k(50) = 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 C-red1/C-red2 conversion occurs at least 10(2) times faster in the presence of CO2 than in its absence. CO2 alters the g values of the g(av) = 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., Br 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 C-red1/C-red2 conversion rate followed a similar pattern, suggesting that this rate is also influenced by modulator binding. Some batches of enzyme cannot convert to the C-red2 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 C-red1, leading first to an EPR-silent state of the C-cluster, and eventually to a state yielding an EPR signal with g(av) = 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 g(av) = 1.94 signal development. The remaining 10% of the A(420) changes occur over the course of similar to 50 min, apparently coincident with the C-red1/C-red2 conversion. One possibility is that the conversion involves reduction of an (unidentified) Fe-S cluster. A three-stare model of catalysis is proposed in which C-red1 binds and oxidizes CO, C-red2 is two electrons more reduced than C-red1 and is the state that binds and reduces CO2, and C-int 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.
