XAS spectroscopy reveals X-ray-induced photoreduction of free and protein-bound B₁₂cofactors

Abstract

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.

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

“…Although care was taken to expose the enzyme and the crystal to as little light as possible, the I690C/G743C MetH(649-1227) is more susceptible to photolysis than the WT enzyme. The demethylation was possibly due to photoreduction during preparation of the crystals and/or photoreduction due to exposure to high-energy x-ray to form cob(II)alamin, as previously observed in other determinations of the structures of cobalamin-containing enzymes (16)(17)(18)(19). Magnetic circular dichroism studies of the cob(II)alamin formed by photolysis of methylated I690C/G743C MetH(649-1227) in a rigid glass at 77 K has established that the cobalamin is fourcoordinate (unpublished data obtained in collaboration with Matthew Liptak and Thomas Brunold, University of Wisconsin, Madison).…”

Structural basis for mammalian vitamin B 12 transport by transcobalamin

“…8, which is published as supporting information on the PNAS web site) and the greater distance (2.8 Å) between the N nitrogen atom of His-173 and the Co ion suggest the presence of a mix of conformations with and without His-Co coordination (within the limits of resolution of our crystallographic data; see Table 1, which is published as supporting information on the PNAS web site). Reduction of hexacoordinate Co(III) to pentacoordinate Co(II) may have been induced by x-rays during data collection, as observed in a similar case of the Cbl-dependent enzyme glutamate mutase (24), and may have led to the loss of His coordination in our case. We hypothesize that only human TC was affected, because the loop preceding His-173 in human TC is three residues shorter than that in bovine TC (Fig.…”

Crystal structure of human intrinsic factor: Cobalamin complex at 2.6-Å resolution

“…The five-coordinate nature of the cobalt ligation in IF suggests that the cobalt ion is in the 2 oxidation state. The reduction of Co(III) to Co(II) might have been induced by extensive x-ray exposure during data collection, as observed in human TC (18) and glutamate mutase (24). Helix 7 of IF (residues 128-145) is displaced along its axis by Ϸ2.5 Å compared with TC, and the following loop, from 146 to 150, is oriented differently from the corresponding loop in TC.…”