The Role of an Iron-Sulfur Cluster in an Enzymatic Methylation Reaction

Abstract

This paper focuses on how a methyl group is transferred from a methyl-cobalt(III) species on one protein (the corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase). This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO and CO 2 fixation. The results described here strongly indicate that transfer of methyl group to carbon monoxide dehydrogenase/acetyl-CoA synthase occurs by an S N 2 pathway. They also provide convincing evidence that oxidative inactivation of Co(I) competes with methylation. Under the conditions of our anaerobic assay, Co(I) escapes from the catalytic cycle one in every 100 turnover cycles. Reductive activation of the CFeSP is required to regenerate Co(I) and recruit the protein back into the catalytic cycle. Our results strongly indicate that the [4Fe-4S] cluster of the CFeSP is required for reductive activation. They support the hypothesis that the [4Fe-4S] cluster of the CFeSP does not participate directly in the methyl transfer step but provides a conduit for electron flow from physiological reductants to the cobalt center.

Spectroscopic Studies of the Corrinoid/Iron−Sulfur Protein from Moorella thermoacetica

“…To achieve and maintain the Co 1+ oxidation state, human ATR forces the Co 2+ corrinoid substrate into a four-coordinate conformation, which helps to lower the thermodynamic barrier for cobalt-ion reduction (E° = −490 mV for the Co 2+/1+ couple of Co 2+ Cbi + ) by the in vivo flavodoxin reductant (E° = −285 mV). 74 CFeSP instead employs a low-potential [Fe 4 S 4 ] cluster (E° = −523 mV) as an internal reducing system that is bound to the enzyme through four cysteine residues, 14,15 as further indicated by our resonance Raman (rR) data ( Figure 5). This [Fe 4 S 4 ] 2+/1+ potential is intermediate between that of the C cluster of CODH (−530 mV) 79 and the Co 2+/1+ couple of the bound Factor III m (−504 mV).…”

“…13 Therefore, it has been postulated that reductive activation begins with the CO-dependent reduction of the CODH C cluster that then transfers an electron to the [Fe 4 S 4 ] 2+ cluster of the CFeSP, which reduces the bound Factor III m to the Co 1+ state. 14,15,80 Reduction activation is enhanced by replacement of the lower axial MBI ligand of Factor III m by a weaker water ligand when the cofactor binds to the CFeSP, generating a fivecoordinate Co 2+ species that resembles Co 2+ Cbi + (see above). This ligand switch has a profound influence in the redox properties of the Co center, increasing the Co 2+/1+ reduction potential by ~120 mV relative to that of the MBI-bound form of the cofactor found in solution (~−620 mV).…”

“…However, the Co 2+/1+ reduction potential of the CFeSP-bound corrinoid is very negative, E° = -504 mV, 13 leading to oxidation of the Co 1+ intermediate to the catalytically inactive Co 2+ state about 1 out of every 100 turnovers. 14 Intriguingly, metal analysis as well as Mössbauer and EPR spectroscopic studies revealed that the 48 kDa subunit of CFeSP contains a [Fe 4 S 4 ] cluster that functions to reduce the Co 2+ form of the corrinoid back to the active Co 1+ state. 12,14,15 The [Fe 4 S 4 ] 2+/1+ reduction potential of this cluster is E° = -523 mV, which is nearly isopotential with the Co 2+/1+ couple of the corrinoid.…”

“…14 Intriguingly, metal analysis as well as Mössbauer and EPR spectroscopic studies revealed that the 48 kDa subunit of CFeSP contains a [Fe 4 S 4 ] cluster that functions to reduce the Co 2+ form of the corrinoid back to the active Co 1+ state. 12,14,15 The [Fe 4 S 4 ] 2+/1+ reduction potential of this cluster is E° = -523 mV, which is nearly isopotential with the Co 2+/1+ couple of the corrinoid. Removal of Factor III m from the CFeSP by treatment with urea has no noticeable effect on the Mössbauer and EPR properties of the [Fe 4 S 4 ] cluster and raises the [Fe 4 S 4 ] 2+/1+ reduction potential by a mere ~6 mV.…”

Review: Biological methylation of less‐studied elements

“…8,139,140 Acetylcoenzyme A was involved in reaction(s) leading to formation of both a methylnickel linkage in the cofactor and also an iron±carbon monoxide linkage at a nearby site. 140,141 EPR studies suggested that the nickel in methylcoenzyme M reductase existed as nickel(I). 142±145 Like cobalt in II, the nickel±methyl bond must be stabilized by a special chelating environment in order to form without immediate decomposition.…”