Resonance Raman examination of axial ligand bonding and spin-state equilibriums in metmyoglobin hydroxide and other heme derivatives

Abstract

Resonance Raman spectra and excitation profiles were obtained within the 5700-6300 .ANG. absorption band of purified sperm whale metmyoglobin hydroxide (MbIIIOH) solutions. A large enhancement occurs for a Raman peak at 490 cm-1 which is shown by isotopic substitution of 18O for 16O to be almost purely an Fe-O stretch. The Fe-O vibration in MbIIIOH occurs 5 cm-1 to lower energy than the corresponding vibration at 495 cm-1 in human methemoglobin hydroxide (HbIIIOH), reflecting differences in ligand bonding between Mb(III) and Hb(III). A larger frequency difference (10 cm-1) exists between MbIIIF and HbIIIF for the Fe-F stretch. Separate Fe-O or Fe-F stretches from the .alpha. and .beta. chains of either HbIIIOH or HbIIIF were not observed. Excitation profile measurements for MbIIIOH indicate that the 5700-6300 .ANG. absorption band is composed of 2 separate absorption bands which result from a high- and a low-spin form of MbIIIOH. The spin-state-sensitive Raman band at 1608 cm-1 reflects the high-spin species and has an excitation profile maximum at about 6000 .ANG. while the low-spin Raman band occurs at 1644 cm-1 and shows an excitation profile maximum at 5800 .ANG.. The Fe-O stretch at 490 cm-1 has an excitation profile maximum at about 6000 .ANG.. Differences in frequency and Raman cross section between the Fe-X vibrations in MbIIIX and HbIIIX (X = OH-, F-) can be related to increases in the out-of-plane Fe distance for the high-spin species of MbIIIX. The shift in the 1644 cm-1 MbIIIOH low-spin-state Raman band indicative of the heme core size to 1636 cm-1 in HbIIIOH indicates a larger heme core size in HbIIIOH. Raman frequency shifts are used to estimate differences in bond strain energies between MbIIIX and HbIIIX (X = OH-, F-). Previous resonance Raman excitation profile data can be interpreted in terms of separate contributions from different spin-state species.