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 VFe-O2 to the porphyrin pi-pi* transitions is explained on the basis of competition between the pi* orbitals of porphyrin and O2 for Fe d pi 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 approximately 270 cm-1, which shifts down by approximately 3 cm-1 upon 18O2 substitution, and is suggested to contain the Fe-ImH stretching mode. The weakness of VFe-ImH, relative to VFe-O2, is attributable to the lack of ImH involvement in the heme pi bonding.