EXTENDED X-RAY-ABSORPTION FINE-STRUCTURE ANALYSIS OF COENZYME B-12 BOUND TO METHYLMALONYL-COENZYME-A MUTASE USING GLOBAL MAPPING TECHNIQUES

The two available crystallographic structures of cobalamin dependent e nzymes, the 27 kDa fragment of the methylcobalamin-dependent enzyme, m ethionine synthase, from Escherichia coli [Drennan, C. L. et al. Scien ce 1994, 266, 1669] and the 5'-deoxyadenosylcobalamin-dependent enzyme methylmalonyl-coenzyme A mutase from Propionibacterium shermanii Manc ia, F. et al. Structure 1996, 4, 339], show striking similarities desp ite the differences in reaction mechanism. In particular, the 5,6-dime thylbenzimidazole group is detached and replaced by a histidine group of the enzyme. Here we present an analysis of Extended X-ray Absorptio n Fine Structure (EXAFS) spectroscopic data for both 5'-deoxyadenosylc obalamin and aquocobalamin bound to methylmalonyl-coenzyme A mutase in the absence of substrate. The analysis is conducted with a suite of p rograms called AUTOFIT 1.0 [Chance, et al. Biochemistry 1996, 35, 9014 ], which allows an evenhanded comparison of the goodness-of-fit of the EXAFS data to a varied grid of simulations based on the ab initio EXA FS code FEFF 6.01. The X-ray edge data indicate an increase in effecti ve nuclear charge of the metal ion of the enzyme bound 5'-deoxyadeonsy lcobalamin compared to the corresponding free cobalamin, and the EXAFS results show small decreases in equatorial and no significant change in the Co-C bond length (despite the potential elongation of the Co-N( His) bond) upon cofactor binding to the enzyme. Thus, the change in co ordination of the nitrogenous axial ligand engineered by the enzyme do es not significantly contribute to a trans effect in the ground state. Weakening of the Co-C bond must be initiated by substrate binding. In addition, the global mapping technique resolves discrepancies between previous EXAFS results and crystallographic data on aquocobalamin.