Resonance Raman investigation of carbon monoxide bonding in (carbon monoxy)hemoglobin and -myoglobin: detection of iron-carbon monoxide stretching and iron-carbon-oxygen bending vibrations and influence of the quaternary structure change

We report for the first time the direct identification of the iron-carbon bond in (carbon monoxy)hemoglobins (HbCO) and -myoglobin (MbCO) by resonance Raman spectroscopy. The Fe-CO stretching, Fe-C-O bending, and bound C-O stretching vibrations have been detected at 507 (512), 578 (577), and 1951 (1944) cm−1, respectively, in human (carbon monoxy)HbA (sperm whale MbCO) upon excitation at 406.7 nm within the Soret band. These assignments were made on the basis of frequency shifts with the isotopes 13C16O, 12C18O, and 13C18O. Calculated isotope shifts according to the model Im-Fe-C-O (but not Im-Fe-O-C) agree well with the observed data. The possible mechanisms of resonance Raman enhancement of these vibrations are discussed in terms of the dπ(Fe)−π*(CO) interaction. Careful examination of the Fe-CO stretching mode at 507 cm−1 (ρ = 0.055) in both (carbon monoxy)HbA and (carbon monoxy)Hb Kansas with and without inositol hexaphosphate (IHP) reveals no changes in frequency and intensity. This implies that no significant change in the Fe-C bond energy is induced by switching the quaternary structure from the R to the T form in ligated (carbon monoxy)Hb Kansas. The absence of bond tension between the iron atom and the proximal histidine is suggested, as it has been demonstrated that the v(Fe-CO) frequency is sensitive to a change from 1-methylimidazole to 1,2-dimethylimidazole (as fifth ligand) in model heme-CO complexes. However, the resonance Raman spectrum of carp (carbon monoxy)Hb exhibits a broadening of the Fe-CO stretching mode on the lower energy side upon R → T conversion with IHP, suggesting the presence of a new conformer (or conformers) with a weaker Fe-CO bond or a somewhat different CO distortion.