title: Mössbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl coenzyme A by Clostridium thermoaceticum.
abstract: We have purified to homogeneity the 88-kDa corrinoid protein from Clostridium thermoaceticum which acts as a methyl carrier in the synthesis of acetyl-CoA. As shown here, this protein contains a [4Fe-4S]1+/2+ cluster in addition to a corrinoid. The corrinoid is 5-methoxybenzimidazolylcobamide, with an OH- group probably present as the upper axial ligand. Co+ is present in the reduced form, Co2+ in the as-isolated form, and Co3+ in the methylated form of the protein. The as-isolated corrinoid/Fe-S protein exhibits a Co2+ EPR signal lacking nitrogen superhyperfine splittings, indicating that the benzimidazole base is uncoordinated ("base-off") in the Co2+ state. Optical studies suggest that the Co3+-CH3 corrinoid is also base-off. In the as-isolated and methylated forms, the iron-sulfur cluster is diamagnetic, with quadrupole splittings and isomer shifts characteristic of [4Fe-4S]2+ clusters. The protein can be reduced by CO and CO dehydrogenase in the absence of ferredoxin. The EPR spectra of the reduced cluster exhibit two components: one with principal g-values at 2.07, 1.93, and 1.82 and the other at 2.02, 1.94, and 1.86. The Mössbauer data show that these signals result from [4Fe-4S]1+ clusters. Chemical analysis shows that the iron:cobalt atomic ratio is close to 4:1, suggesting that a single [4Fe-4S]1+ cluster occurs in two distinct S = 1/2 spin states in the reduced state. Treatment with 1-2.5 M urea converts the two cluster forms into a single one, with EPR and Mössbauer spectra of typical [4Fe-4S]1+ clusters. A 27-kDa corrinoid protein (Ljungdahl, L.G., LeGall, J., and Lee, J.P. (1973) Biochemistry 12, 1802-1808) also was purified and found to be inactive in the synthesis of acetyl-CoA, contrary to the suggestion of Ljungdahl et al. (1973).
authors.affiliation: Department of Chemistry, University of Wisconsin-Milwaukee 53201.
authors.forename: S W
authors.initials: SW
authors.lastname: Ragsdale
authors.forename: P A
authors.initials: PA
authors.lastname: Lindahl
authors.forename: E
authors.initials: E
authors.lastname: Münck
mesh_terms: D000105:Acetyl Coenzyme A / Q000096:biosynthesis*; D003013:Clostridium / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008667:Metalloproteins / Q000302:isolation & purification*; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D013053:Spectrophotometry; D013057:Spectrum Analysis
chemical_list: D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D000105:Acetyl Coenzyme A; D008780:Methyltransferases