Spectroscopic States of the CO Oxidation/CO2 Reduction Active Site of Carbon Monoxide Dehydrogenase and Mechanistic Implications

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• 作者：Mark E. Anderson and Paul A. Lindahl
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：June 25, 1996
• 年：1996
• 卷：35
• 期：25
• 页码：8371 - 8380
• 全文大小：458K
• 年卷期：v.35,no.25(June 25, 1996)
• ISSN：1520-4995

文摘

CO dehydrogenases catalyze the reversible oxidation of CO toCO₂, at an active site (calledthe C-cluster) composed of an Fe₄S₄ cube withwhat appears to be a 5-coordinate Fe (called FCII), linkedto 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 theC-clusterto an [Fe₄S₄]^2+/1+electron-transfer cluster called the B-cluster. An S= ¹/₂ form of the C-cluster(calledC_red1) converts to another S =¹/₂ form (called C_red2) uponreduction with CO, at a rate well within theturnover frequency of the enzyme (Kumar, M., Lu, W.-P., Liu, L., &Ragsdale, S. W. (1993) J. Am.Chem. Soc. 115, 11646-11647). This suggests thatthe conversion is part of the catalytic mechanism.Dithionite is reported in this paper to effect this conversion aswell, but at a much slower rate (k_so =5.3× 10^-2 M^-1s^-1 for dithionite vs 4.4 ×10⁶ M^-1s^-1 for CO). By contrast, dithionitereduces the oxidizedB-cluster much faster, possibly within the turnover frequency of theenzyme. Dithionite apparently effectsthe C_red1/C_red2 conversion directly, ratherthan through an intermediate. The conversion rate varieswithdithionite concentration. The C_red1/C_red2conversion occurs at least 10² times faster in the presenceofCO₂ than in its absence. CO₂ alters theg values of the g_av = 1.82 signal,indicating that CO₂ binds to aC-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, orCS₂ in dithionite accelerate CN^- dissociationfromthis site (Anderson, M. E., & Lindahl, P. A. (1994)Biochemistry 33, 8702-8711). The effect ofCO,CO₂, and CS₂ on CN^- dissociationsuggested that these molecules bind at a site (called themodulator)other than that to which CN^- binds. The effects ofCO₂, CS₂, CO, and dithionite on theC_red1/C_red2conversion rate followed a similar pattern, suggesting that this rateis also influenced by modulator binding.Some batches of enzyme cannot convert to the C_red2form using dithionite, but pretreatment with CO orCO₂/dithionite effectively "cures" such batches of thisdisability. The results presented suggest that theNi of the C-cluster is the modulator and the substrate binding site forCO/CO₂. The inhibitor CS₂ inthepresence of dithionite also accelerates the decline ofC_red1, leading first to an EPR-silent state oftheC-cluster, and eventually to a state yielding an EPR signal withg_av = 1.66. CS₂ binding thussharessome resemblance to CO₂ binding. Approximately 90% ofthe absorbance changes at 420 nm that occurwhen oxidized CODH_Ct is reduced by dithionite occur within2 min at 10 C. This absorbance changeoccurs in concert with the g_av = 1.94 signaldevelopment. The remaining 10% of the A₄₂₀changes occurover the course of ~50 min, apparently coincident with theC_red1/C_red2 conversion. One possibility isthatthe conversion involves reduction of an (unidentified) Fe-S cluster.A three-state model of catalysis isproposed in which C_red1 binds and oxidizes CO,C_red2 is two electrons more reduced than C_red1and is thestate that binds and reduces CO₂, and C_int is aone-electron-reduced state that is proposed to existbecauseof constraints imposed by the nature of the CO/CO₂ reactionand the properties of the clusters involvedin catalysis.