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 d_π(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.

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