EXAFS Data Indicate a “Normal” Axial Cobalt−Nitrogen Bond of the Organo-B12 Cofactor in the Two Coenzyme B12-Dependent Enzymes Glutamate Mutase and 2-Methyleneglutarate Mutase

Abstract

A key step in the catalytic cycle of coenzyme B12-dependent enzymes is the homolysis of the cofactor's organometallic bond, leading to the formation of a 5‘-deoxyadenosyl radical. For the adenosylcobalamin-dependent enzyme methylmalonyl CoA mutase (MCM), it has been suggested that this step is mediated by a protein-induced lengthening of the cofactor's axial cobalt−nitrogen bond, in trans position to the scissile organometallic bond. In fact, such a lengthening was first observed in the crystal structure of MCM (Mancia et al. Structure 1996, 4, 339−350) and was later confirmed by an analysis of EXAFS data on the same protein in frozen solution (Scheuring et al. J. Am. Chem. Soc. 1997, 119, 12192−12200). Here, we report the results of an EXAFS study on the related coenzyme B12-dependent enzymes glutamate mutase from Clostridium cochlearium and 2-methyleneglutarate mutase from Clostridium barkeri. Both apoenzymes were overproduced from E. coli and reconstituted with methylcobalamin (MeCbl) to yield inactive...