A key step in the catalytic cycle of coenzyme Bit-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. Ln 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. Sec. 1997, 119, 12192-12200). Here, we report the results of an EXAFS study on the related coenzyme B-12-dependent enzymes glutamate mutase from Clostridium cochlearium and 2-methyleneglutarate mutase from Clostridium barkeri. Both apoenzymes were overproduced from E. coil and reconstituted with methylcobalamin (MeCbl) to yield inactive enzymes, whose stability toward (substrate-induced) cobalt-carbon bond homolysis should be higher than for the enzymatically active forms obtained by reconstitution with 5'-desoxyadenosylcobalamin. X-ray absorption data were collected around the cobalt K-absorption edge at 20 K on freeze-dried and frozen protein preparations. in addition to the two recombinant enzymes, we also collected XAS data on recombinant glutamate mutase, reconstituted with MeCbl in the presence of the inhibitor (2S,4S)-4-fluoroglutamate. As a reference compound for the interpretation of the EXAFS spectra, absorption data were also collected from crystalline MeCbl, whose crystal structure is known (Rossi et al. J. Am. Chem. Sec. 1985, 107, 1729-1738). The XANES parts of the XAS spectra for the four samples look very similar to one another and deviate significantly from the corresponding spectra of aquocob(III)alamin and for cob(II)alamin. Moreover, all four methyl-B-12 spectra show a pronounced preedge peak, indicating the presence of a covalently attached sixth carbon ligand to the cobalt center. The EXAFS region of the protein spectra were simulated by deriving the amplitude reduction factor, the energy shift, and the Debye-Waller factors for each scattering path from the spectrum of the model compound. All geometrical parameters were assumed to be equal between model compound and enzyme-bound B-12 cofactor, with the exception of the axial cobalt-nitrogen distance, which was varied between 1.7 and 2.9 ft. The resulting optimization profiles show the deepest minimums between 2.1 and 2.2 Angstrom, close to the value observed for methylcobalamin. This is also true fur the spectrum of GLM in the presence of inhibitor. In all simulations (including the one for the model compound), a second minimum appeared around 2.8 Angstrom. In conclusion, our EXAFS evidence suggests a "normal" axial Co-N bond in the two coenzyme B-12-dependent enzymes glutamate mutase and 2-methyleneglutarate mutase. Although a very long (2.6-2.8 Angstrom) Co-N bond would also be compatible with the observed spectra, it is considered unlikely.