Resonance Raman studies of heme axial ligation in H93G myoglobin

The resonant Raman active mode identified in numerous studies as the heme i ron-histidine stretch has been systematically investigated in the Raman spe ctrum of 15 exogenous ligands to the heme iron in the myoglobin proximal ca vity mutant H93G. Mutation of the native histidine 93 of myoglobin to glyci ne (H93G) creates a cavity at the heme iron that can be filled with exogeno us ligands. Substituted imidazoles and pyridines were introduced into the c avity at the heme iron of the deoxy ferrous H93G myoglobin by dialysis. Ram an bands attributed to in-plane modes of the heme are unaffected by the cha nge in heme-iron ligation. However, the Raman active band in the 180-250 cm (-1) region ascribed to an iron-ligand out-of-plane mode is highly dependen t on the identity of the axial ligand. Information on the normal mode was o btained using isotopically labeled imidazole, pyridine, and I-methyl and 2- methyl imidazole. Relatively small isotope effects ate observed for the hem e-iron ligands imidazole H93G(Im) and pyridine H93G(Pyr). We have examined models for the normal mode based on three-body, six-body, and 38-body calcu lations of the FG matrix. These models indicate that the potential energy d istribution of the axial-ligand out-of-plane normal mode observed in the re gion from 180 to 245 cm(-1) has a significant contribution from iron-ligand (Fe-L) stretching (50-70%) but also from significant iron-heme doming. Usi ng the normal coordinate analysis to correct for differences in ligand mass , we have compared the frequencies of the ligands as a function of their ba sicity. Although the iron-ligand force constant is linearly proportional to ligand basicity at pK(a) > 5, the correlation is less pronounced at lower pK(a).