Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum.
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., & Münck, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Harder
S R
SR
Department of Chemistry, University of Wisconsin, Milwaukee 53201.
Lu
W P
WP
Feinberg
B A
BA
Ragsdale
S W
SW
D000105:Acetyl Coenzyme A / Q000096:biosynthesis*; D003013:Clostridium / Q000201:enzymology* / Q000254:growth & development; D003035:Cobalt; D045728:Corrinoids; D004563:Electrochemistry; D007506:Iron-Sulfur Proteins / Q000502:physiology; D008667:Metalloproteins / Q000502:physiology; D008745:Methylation; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet; D014805:Vitamin B 12 / Q000502:physiology*
D045728:Corrinoids; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D003035:Cobalt; D000105:Acetyl Coenzyme A; D014805:Vitamin B 12