STRUCTURAL AND ELECTRONIC FACTORS IN HETEROLYTIC CLEAVAGE - FORMATIONOF THE CO(I) INTERMEDIATE IN THE CORRINOID IRON-SULFUR PROTEIN FROM CLOSTRIDIUM-THERMOACETICUM/

We have completed the first direct structural characterization of an e nzyme-bound four-coordinate Co(I) intermediate, in this case for the c orrinoid/iron-sulfur protein (C/Fe-SP) from Clostridium thermoaceticum . Extended X-ray absorption fine structure and X-ray edge spectroscopy of the active Co(I) state of the C/Fe-SP indicates a four-coordinate (distorted) square-planar structure where the best fit gives average C o-N(equatorial) distances of 1.87 +/- 0.01 Angstrom, corresponding to 4.2 +/- 0.3 ligands. The X-ray edge spectrum of Co(I) C/Fe-SP contains a moderate intensity 1s-4p + ''shake-down'' (SD) transition and no 1s -3d peak (where SD transitions are indicative of square-planar geometr ies). X-ray edge results for the methyl-Co(III) form, reported earlier [Wirt, M. D., Kumar, M., Ragsdale, S. W., & Chance, M. R. (1993) J. A m. Chem. Soc. 115, 2146-2150], are consistent with a base-off methylco bamide structure. The absence of a ligated 5-methoxybenzimidazole base in the methyl-Co(III) state is important since the base-off form is p redicted to predispose the Co-C bond toward heterolytic cleavage to fo rm the four-coordinate Co(I) species concurrent with methyl transfer. Additionally, we have examined first-derivative X-ray edge spectra of Co(I) C/Fe-SP, relative to edge spectra of a cobalt foil, as an indica tor of effective nuclear charge on cobalt. The Co(I) C/Fe-SP edge posi tion at 7720.5 +/- 0.3 eV is less than, but very close to, the value s een for the corresponding free Co(I) cobalamin. The observed reduction in effective nuclear charge for protein-bound cobamides in Co(I), Co( II), and Co(III) oxidation states may promote heterolytic Co-C bond cl eavage by increasing the electrophilic nature of the donated methyl gr oup and increase the nucleophilicity of enzyme bound Co1+ to facilitat e remethylation of the cofactor.