Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum

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Abstract
An 88-kDa corrinoid/iron-sulfur protein (C/Fe-SP) is the methyl carrier protein in the acetyl-CoA pathway of Clostridium thermoaceticum. In previous studies, it was found that this C/Fe-SP contains (5-methoxybenzimidazolyl)cobamide and a [4Fe-4S]2+/1+ center, both of which undergo redox cycling during catalysis, and that the benzimidazole base is uncoordinated to the cobalt (base off) in all three redox states, 3+, 2+, and 1+ [Ragsdale, S. W., Lindahl, P. A., and Munck, E. (1987) J. Biol. Chem. 262, 14289-14297]. In this paper, we have determined the midpoint reduction potentials for the metal centers in this C/Fe-SP by electron paramagnetic resonance and UV-visible spectroelectrochemical methods. The midpoint reduction potentials for the Co3+/2+ and the Co2+/1 couples of the corrinoid were found to be 300-350 and -504 mV (.+-. 3 mV) in Tris-HCl at pH 7.6, respectively. We also removed the (5-methoxybenzimidazolyl)cobamide cofactor from the C/Fe-SP and determined that its Co3+/2+ reduction potential is 207 mV at pH 7.6. The midpoint potential for the [4Fe-4S]2+/1+ couple in the C/Fe-SP was determined to be -523 mV (.+-. 5 mV). Removal of this cluster totally inactivates the protein; however, there is little effect of cluster removal on the midpoint potential of the Co2+/1+ couple. In addition, removal of the cobamide has an insignificant effect on the midpoint reduction potential of the [4Fe-4S] cluster. A 27-kDa corrinoid protein (CP) also was studied since it contains (5-methoxybenzimidazolyl)cobamide in the base-on form. The reduction potentials for the Co3+/2+ and Co2+/1+ couples of the 27-kDa CP were found to be 215 and -630 mV, respectively, at pH 7.6. Our work provides evidence that the c/Fe-SP stabilizes the active Co1+ state of the C/Fe-SP relative to that of the 27-kDa CP and free corrinoids by control of the state of ligation of the benzimidazole base to Co2+. The implications of this increased stability in the context of the methyl-transfer reaction catalyzed by this enzyme are discussed. Our results are consistent with a mechanism of methyl transfer to CO dehydrogenase in which a nucleophile on CODH performs a heterolytic displacement of the methyl group of the methylCo3+ corrinoid, resulting in Co1+ and methylated CODH.