SPECTROSCOPIC STATES OF THE CO OXIDATION CO2 REDUCTION ACTIVE-SITE OFCARBON-MONOXIDE DEHYDROGENASE AND MECHANISTIC IMPLICATIONS

CO dehydrogenases catalyze the reversible oxidation of CO to CO2, at a n active site (called the C-cluster) composed of an Fe4S4 cube with wh at 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 ca talysis, electrons are transferred from the C-cluster to an [Fe4S4](2/1+) electron-transfer cluster called the B-cluster. An S = 1/2 form o f the C-cluster (called C-red1) converts to another S = 1/2 form (call ed 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 th e conversion is part of the catalytic mechanism. Dithionite is reporte d in this paper to effect this conversion as well, but at a much slowe r 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-c luster much faster, possibly within the turnover frequency of the enzy me. 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 al ters the g values of the g(av) = 1.82 signal, indicating that CO2 bind s to a C-cluster-sensitive site at mild potentials. CN- inhibits CO ox idation by binding to FCII (Hu et al., 1996), and CO, CO2 in the prese nce of dithionite, or CS2 in dithionite accelerate CN- dissociation fr om this site (Anderson, M. E., Br Lindahl, P. A. (1994) Biochemistry 3 3, 8702-8711). The effect of CO, CO2, and CS2 on CN- dissociation sugg ested that these molecules bind at a site (called the modulator) other than that to which CN- binds. The effects of CO2, CS2, CO, and dithio nite on the C-red1/C-red2 conversion rate followed a similar pattern, suggesting that this rate is also influenced by modulator binding. Som e batches of enzyme cannot convert to the C-red2 form using dithionite , but pretreatment with CO or CO2/dithionite effectively ''cures'' suc h batches of this disability. The results presented suggest that the N i of the C-cluster is the modulator and the substrate binding site for CO/CO2. The inhibitor CS2 in the presence of dithionite also accelera tes 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. App roximately 90% of the absorbance changes at 420 nm that occur when oxi dized 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 sig nal development. The remaining 10% of the A(420) changes occur over th e course of similar to 50 min, apparently coincident with the C-red1/C -red2 conversion. One possibility is that the conversion involves redu ction of an (unidentified) Fe-S cluster.A three-stare model of catalys is is proposed in which C-red1 binds and oxidizes CO, C-red2 is two el ectrons more reduced than C-red1 and is the state that binds and reduc es 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 react ion and the properties of the clusters involved in catalysis.