Resonance Raman Spectroscopic Studies of Axial Ligation in Oxyhemoglobin, Oxymyoglobin, and Nitrosylmyoglobin

Raman intensity measurements for the Fe-O₂ stretching band of HbO₂ (Hb = hemoglobin) have been used to construct an excitation profile, which shows that resonance enhancement occurs mainly via the B and Q transition; no contribution is detectable from an out-of-plane charge-transfer transition. Direct coupling of [formula omitted] to the porphyrin π−π* transitions is explained on the basis of competition between the π* orbitals of porphyrin and O₂ for Fe d_π electrons. The RR spectrum of MbNO (Mb = myoglobin) at pH 8.4 is due solely to six-coordinate heme-NO, but lowering the pH to 5.8 converts the RR spectrum to one characteristic of five-coordinate heme-NO, consistent with Fe-ImH (ImH = imidazole) dissociation via protonation. The Fe-NO stretching frequencies are at 553 and 596 cm⁻¹ for the high- and low-pH forms, as expected, but the low-pH form shows an additional ¹⁵NO-sensitive band, at 573 cm⁻¹, which is assigned to Fe-N-O bending in the five-coordinate complex. The RR spectrum of MbO₂ shows a shoulder at −270 cm⁻¹, which shifts down by −3 cm⁻¹ upon ¹⁸O₂ substitution, and is suggested to contain the Fe-ImH stretching mode. The weakness of v_Fe-imH, relative to [formula omitted], is attributable to the lack of ImH involvement in the heme π bonding.