Crystal structures of several proteins with a B 12 cofactor show abnormally long axial bonds between the cofactor's Co atom and its`lower' ligand, which is typically a protein-derived imidazole from a histidine residue. X-ray absorption spectroscopy (XAS) experiments were carried out with the following cofactor derivatives to examine the question of whether the bond elongation might be due to an X-ray-induced reduction of the cofactor's cobalt centre: aquocobalamin, cyanocobalamin, methylcobalamin, 5H -desoxyadenosylcobalamin and cob(II)alamin. Each cofactor was investigated at 100 K in a water/glycerol or water/trehalose glass, both as unbound free species and bound to the protein components of the enzyme glutamate mutase. XAS data were collected for each sample around the cobalt absorption edge before and after exhaustive (10 min) irradiation with X-rays of energy 7.76 keV. While XAS spectra for cob(II)alamin, methylcobalamin and 5H -desoxyadenosylcobalamin were the same (within experimental error) before and after irradiation, both in the free and protein-bound state, the spectra of samples with aquocobalamin and cyanocobalamin changed substantially upon irradiation. The spectra of the irradiated samples resembled each other and were similar ± but not identical ± to the spectrum of the reduced cob(II)alamin. The implications of these observations for the interpretation of the`long' axial CoÐN bonds observed crystallographically in B 12 proteins are discussed.

We describe the studies of new copper complexes [MeSPY2]CuPF(6), 2, and [MeSPY2]Cu(ClO(4))(2).CH(3)CN, 3, as models for the Cu(B) center of dopamine beta-hydroxylase and peptidylglycine alpha-hydroxylating monooxygenase. The structure of [MeSPY2]Cu(ClO(4))(2).CH(3)CN, 3, has been established by X-ray crystallography. The copper coordination exhibits a square pyramidal geometry where the equatorial plane is occupied by the SCH(3) group and three nitrogen atoms (tertiary amine, one pyridine, and acetonitrile solvent), whereas the axial position binds the second pyridine. Using FEFF calculations and multiscattering interaction, EXAFS refinements show that the SMe group lies in the coordination sphere of copper complexes [MeSPY2]CuPF(6), 2, and [MeSPY2]Cu(ClO(4))(2).CH(3)CN, 3. While [MeSPY2]CuPF(6), 2, reacts with dioxygen in dichloromethane without oxidation of the ligand, we observed an oxidation of the sulfide ligand when [MeSPY2]Cu(ClO(4))(2).CH(3)CN, 3, reacts with hydrogen peroxide in methanol. Considering results, we propose that Met(314), crucial for DBH and PHM activity, could be the site of the H(2)O(2) (or ascorbate) inactivation by oxidation to the sulfoxide group.

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 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. Soc. 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-B12 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 B12 cofactor, with the exception of the axial cobalt−nitrogen distance, which was varied between 1.7 and 2.9 Å. The resulting optimization profiles show the deepest minimums between 2.1 and 2.2 Å, close to the value observed for methylcobalamin. This is also true for 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 Å. In conclusion, our EXAFS evidence suggests a “normal” axial Co−N bond in the two coenzyme B12-dependent enzymes glutamate mutase and 2-methyleneglutarate mutase. Although a very long (2.6−2.8 Å) Co−N bond would also be compatible with...