jp001231v_si_001.pdf (1.01 MB)
Resonance Raman Studies of Heme Axial Ligation in H93G Myoglobin
journal contribution
posted on 2000-10-12, 00:00 authored by Stefan Franzen, Steven G. Boxer, R. Brian Dyer, William H. WoodruffThe resonant Raman active mode identified in numerous studies as the heme iron−histidine stretch has been
systematically investigated in the Raman spectrum of 15 exogenous ligands to the heme iron in the myoglobin
proximal cavity mutant H93G. Mutation of the native histidine 93 of myoglobin to glycine (H93G) creates
a cavity at the heme iron that can be filled with exogenous ligands. Substituted imidazoles and pyridines
were introduced into the cavity at the heme iron of the deoxy ferrous H93G myoglobin by dialysis. Raman
bands attributed to in-plane modes of the heme are unaffected by the change 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
dependent on the identity of the axial ligand. Information on the normal mode was obtained using isotopically
labeled imidazole, pyridine, and 1-methyl and 2-methyl imidazole. Relatively small isotope effects are observed
for the heme−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 calculations of the FG matrix. These models indicate
that the potential energy distribution of the axial-ligand out-of-plane normal mode observed in the region
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. Using the normal coordinate analysis to correct for differences in ligand
mass, we have compared the frequencies of the ligands as a function of their basicity. Although the iron−ligand force constant is linearly proportional to ligand basicity at pKa > 5, the correlation is less pronounced
at lower pKa.