Proton NMR study of structural perturbation in sperm whale myoglobin due to pentaammineruthenium(III) groups appended to surface histidyl imidazoles

Abstract

Ruthenium-labeled sperm whale myoglobin, [a5Ru]3Mb (a = NH3), which exhibits novel catalytic properties, and its model compound [a5RuImH]Cl3·2H2O (ImH = imidazole) have been investigated by 360-MHz 1H NMR. The model compound in 2H2O exhibits three nonlabile single-proton peaks corresponding to 2′-H, 5′-H, and 4′-H of the coordinated imidazole at -31.2, -2.5, and near 4.7 ppm at 25°C and pH 6.1, respectively. A pH titration gives a single pK 8.4 for deprotonation of the imidazole, with resulting increases of imidazole contact shifts. Ruthenium-labeled Mb and Ru-labeled apo-Mb also exhibit peaks at -36 ppm, which are assigned to the 2′-H of the imidazoles of His-12, His-81, and His-113, Integration of these 2′-H peaks relative to a heme methyl allows determination of the extent of reaction of surface histidines with pentaammineruthenium(III). Detailed comparison of the hyperfine shifted resonances for metaquo, methydroxy, metcyano, metazide, and deoxy forms of the proteins show negligible influence of the Ru chromophores on shifts, indicating protein folding essentially unaltered from that in the native protein. The difference in the high-spin/low-spin separation in the metazide form for the native and Ru-labeled proteins is estimated at only 24 cal, indicating a slightly weaker axial ligand field for the latter derivative. The decreased proton spin-lattice relaxation times and increased line widths of Ru-labeled Mb relative to native Mb complexes indicate the presence of metal-metal interactions which influence the electron-spin relaxation of the iron center. © 1984 American Chemical Society.