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

Abstract

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.

Evidence for Intersubunit Communication during Acetyl-CoA Cleavage by the Multienzyme CO Dehydrogenase/Acetyl-CoA Synthase Complex from Methanosarcina thermophila

“…9, the next step is attack by Ni(I) on the methyl group of methylcob(III)amide (bound to the corrinoid iron-sulfur component of the complex) to form methyl-Ni(III) (Reaction 5). Recent results strongly indicate that this methyl transfer is a nucleophilic displacement, not a radical, reaction (46,48). We propose that it is the next step at which the CODH subunit is required.…”

ATP-induced electron transfer by redox-selective partner recognition

“…The physiological role of the [4Fe4S] cluster of CoFeSP is enigmatic, as it does not directly contribute to the chemistry of methyl transfer neither from methyltransferase to CoFeSP nor from CoFeSP to acetyl-CoA synthase. It was suggested that the [4Fe4S] cluster was essential for the communication between CoFeSP and physiological electron donors providing a conduit for electron flow from, for example, CO-reduced carbon monoxide dehydrogenase or a ferredoxin to the cobalamin 13,14 . In the RACo:CoFeSP complex, the [4Fe4S] cluster domain of CoFeSP interacts extensively with the RACo dimer ( Supplementary Fig.…”

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

“…The absence of protein ligands is the probable reason why the redox potential of the Co 2þ ∕Co 1þ couple of CoFeSP is about 50-100 mV more positive than in other corrinoid containing methyltransferases (25,33), rendering it reducible by electrons generated from the oxidation of CO by carbon monoxide dehydrogenases (26). The necessity for an ATP-dependent reductive activator has therefore not been anticipated.…”

“…The occasional oxidation of Co(I) to Co(II) inactivates CoFeSP, which has to be reactivated by a one-electron reduction (23,24). The low midpoint potential needed to reduce Co 2þ to Co 1þ (< − 504 mV at pH 7.4) (25) can be achieved in vitro using either chemical reducing agents such as Na-dithionite (DT), Ti 3þ citrate, via photoreduction with deazariboflavin as a catalyst or enzymatically with electrons generated by the oxidation of CO to CO 2 by carbon monoxide dehydrogenase (22,26). An ATP-dependent reactivation of CoFeSP has not been reported so far.…”