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 thermoaceti cum functions as a methyl carrier in the Wood-Ljungdahl pathway of ace tyl-CoA synthesis. The small subunit (33 kDa) contains cobalt in a cor rinoid cofactor, and the large subunit (55 kDa) contains a [4Fe-4S] cl uster. The cobalt center is methylated by methyltetrahydrofolate (CH3- H(4)folate) to form a methylcobalt intermediate and, subsequently, is demethylated by carbon monoxide dehydrogenase/acetyl-CoA synthase (COD H/ ACS). The work described here demonstrates that the [4Fe-4S] cluste r is required to facilitate the reactivation of oxidatively inactivate d 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 po tential of the cluster in the C20A variant was similar to 500 mV more positive than that of the [4Fe-4S] cluster in the native enzyme. Accor dingly, it was found that the Co center in the C20A mutant protein cou ld 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 fo rce needed to reduce Co(II) to Co(I), since the Co(II/I) midpoint pote ntial is -504 mV. The C20A valiant also was unable to catalyze the ste ady-state synthesis of acetyl-CoA when CH3-H(4)folate or methyl iodide were provided as methyl donors and CO and CODH/ACS as reductants. Add ition of chemical reductants rescued the catalytically crippled varian t form in both of these reactions. On the other hand, in single-turnov er reactions, the methyl-Co state of the altered protein was fully act ive in methylating H(4)folate and in synthesizing acetyl-CoA in the pr esence of CO and CoA, The combined results strongly indicate that the FeS cluster of the CFeSP is necessary for reductive activation of Co(I I) to Co(I) by physiological reductants but is not required for cataly sis, e.g., demethylation of CH3-H(4)folate 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 ferredox in (Fd)) to the FeS cluster which then directs electrons to the cobalt center for catalysis. These results also support earlier hypotheses t hat the methylation and demethylation reactions involving the CFeSP ar e S(N)2-type nucleophilic displacement reactions and do not involve ra dical chemistry.