The two available crystallographic structures of cobalamin dependent enzymes, the 27 kDa fragment of the methylcobalamin-dependent enzyme, methionine synthase, from Escherichia coli [Drennan, C. L. et al. Science 1994, 266, 1669] and the 5‘-deoxyadenosylcobalamin-dependent enzyme methylmalonyl-coenzyme A mutase from Propionibacterium shermanii [Mancia, F. et al. Structure 1996, 4, 339], show striking similarities despite the differences in reaction mechanism. In particular, the 5,6-dimethylbenzimidazole group is detached and replaced by a histidine group of the enzyme. Here we present an analysis of Extended X-ray Absorption Fine Structure (EXAFS) spectroscopic data for both 5‘-deoxyadenosylcobalamin and aquocobalamin bound to methylmalonyl-coenzyme A mutase in the absence of substrate. The analysis is conducted with a suite of programs 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 EXAFS code FEFF 6.01. The X-ray edge data indicate an increase in effective nuclear charge of the metal ion of the enzyme bound 5‘-deoxyadeonsylcobalamin 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 coordination of the nitrogenous axial ligand engineered by the enzyme does 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.