文摘
Myoglobins reconstituted with aqua- and cyano-Co(III) tetradehydrocorrins, rMb(CoIII(OH2)(TDHC)) and rMb(CoIII(CN)(TDHC)), respectively, were prepared and investigated as models of a cobalamin-dependent enzyme. The former protein was obtained by oxidation of rMb(CoII(TDHC)) with K3[Fe(CN)6]. The cyanide-coordinated Co(III) species in the latter protein was prepared by ligand exchange of rMb(CoIII(OH2)(TDHC)) with exogenous cyanide upon addition of KCN. The X-ray crystallographic study reveals the hexacoordinated structures of rMb(CoIII(OH)(TDHC)) and rMb(CoIII(CN)(TDHC)) at 1.20 and 1.40 ? resolution, respectively. The 13C NMR chemical shifts of the cyanide in rMb(CoIII(CN)(TDHC)) were determined to be 108.6 and 110.6 ppm. IR measurements show that the cyanide of rMb(CoIII(CN)(TDHC)) has a stretching frequency peak at 2151 cm–1 which is higher than that of cyanocobalamin. The 13C NMR and IR measurements indicate weaker coordination of the cyanide to CoIII(TDHC) relative to cobalamin, a vitamin B12 derivative. Thus, the extent of π-back-donation from the cobalt ion to the cyanide ion is lower in rMb(CoIII(CN)(TDHC)). Furthermore, the pK1/2 values of rMb(CoIII(OH2)(TDHC)) and rMb(CoIII(CN)(TDHC)) were determined by a pH titration experiment to be 3.2 and 5.5, respectively, indicating that the cyanide ligation weakens the Co–N(His93) bond. Theoretical calculations also demonstrate that the axial ligand exchange from water to cyanide elongates the Co–N(axial) bond with a decrease in the bond dissociation energy. Taken together, the cyano-Co(III) tetradehydrocorrin in myoglobin is appropriate for investigation as a structural analogue of methylcobalamin, a key intermediate in methionine synthase reaction.