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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Role of the [4Fe-4S] cluster in reductive activation of the cobalt center of the corrinoid iron-sulfur protein from Clostridium thermoaceticum during acetate biosynthesis.

The corrinoid iron-sulfur protein (CFeSP) from Clostridium thermoaceticum functions as a methyl carrier in the Wood-Ljungdahl pathway of acetyl-CoA synthesis. The small subunit (33 kDa) contains cobalt in a corrinoid cofactor, and the large subunit (55 kDa) contains a [4Fe-4S] cluster. The cobalt center is methylated by methyltetrahydrofolate (CH3-H4folate) to form a methylcobalt intermediate and, subsequently, is demethylated by carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). The work described here demonstrates that the [4Fe-4S] cluster is required to facilitate the reactivation of oxidatively inactivated Cob(II)amide to the active Co(I) state. Site-directed mutagenesis of the large subunit gene was used to change residue 20 from cysteine to alanine, which resulted in formation of a cluster with EPR and redox properties consistent with those of [3Fe-4S] clusters. The midpoint potential of the cluster in the C20A variant was approximately 500 mV more positive than that of the [4Fe-4S] cluster in the native enzyme. Accordingly, it was found that the Co center in the C20A mutant protein could be reduced artificially but was severely crippled in its ability to be reduced by physiological electron donors. This is probably because the reduced cluster of the C20A protein cannot provide the driving force needed to reduce Co(II) to Co(I), since the Co(II/I) midpoint potential is -504 mV. The C20A variant also was unable to catalyze the steady-state synthesis of acetyl-CoA when CH3-H4folate or methyl iodide were provided as methyl donors and CO and CODH/ACS as reductants. Addition of chemical reductants rescued the catalytically crippled variant form in both of these reactions. On the other hand, in single-turnover reactions, the methyl-Co state of the altered protein was fully active in methylating H4folate and in synthesizing acetyl-CoA in the presence of CO and CoA. The combined results strongly indicate that the FeS cluster of the CFeSP is necessary for reductive activation of Co(II) to Co(I) by physiological reductants but is not required for catalysis, e.g., demethylation of CH3-H4folate or methylation of CODH/ACS. We propose that, during reductive activation, electrons flow from the reduced electron-transfer protein (e.g., CODH/ACS or reduced ferredoxin ( Fd)) to the FeS cluster which then directs electrons to the cobalt center for catalysis. These results also support earlier hypotheses that the methylation and demethylation reactions involving the CFeSP are SN2-type nucleophilic displacement reactions and do not involve radical chemistry.[1]


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