INVESTIGATION OF HEME DISTORTIONS AND HEME PROTEIN COUPLING IN THE ISOLATED SUBUNITS OF OXYGENATED HUMAN HEMOGLOBIN BY RESONANCE RAMAN DISPERSION SPECTROSCOPY

To probe the distortions of the heme groups resulting from heme-apoprotein interaction in the isolated subunits of oxygenated human hemoglobin (i.e., alpha(SH)-oxyHbA and beta(SH)-oxyHbA), the dispersion of the depolarization ratio of the Raman lines at 1375 cm-1 (nu-4) and 1638 cm-1 (nu-10) was measured at various pHs. The data were analyzed in terms of vibronic coupling parameters which depend on symmetry-classified normal distortions of the heme groups. In the alpha-chain the nu-10 mode is not affected by symmetry-lowering distortions. In the beta-chain, however, this mode is significantly influenced by asymmetric B1g and B2g distortions. This was interpreted in terms of different interactions between the peripheral substituents and the porphyrin macrocycle in the respective chains. The nu-4 mode of both chains is subject to B1g (B2g) and A2g distortions, which are more pronounced in beta(SH)-oxyHbA. This is most probably due to differences in the repulsive interactions between the proximal imidazole and the pyrrole. While the depolarization ratio of both lines investigated is pH-independent in alpha(SH)-oxyHbA, it exhibits a significant pH dependence in beta(SH)-oxyHbA. This parallels the finding that the isolated beta-chains exhibit a Bohr effect whereas the alpha-chains do not. Consequently, the pH dependence of the coupling parameters and the Bohr effect of beta(SH)-oxyHbA could be rationalized in terms of the very same proton binding processes. Moreover, the Raman data correlate with low-temperature kinetic measurements by DeIorio et al. [(1990) Biophys. J. 59, 1-13] which reveal that the beta(SH)-Hb exhibits a larger structural heterogenity than alpha(SH)-Hb. This indicates that the normal distortions monitored by vibronic coupling matrix elements are provided by different conformational substates of the porphyrin macrocycle.