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Keywords:

  • B12;
  • X-ray absorption spectroscopy;
  • XANES;
  • glutamate mutase;
  • cobalamin;
  • enzyme mechanisms.

Crystal structures of several proteins with a B12 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, 5′-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 5′-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 Co—N bonds observed crystallographically in B12 proteins are discussed.