Zinc-substituted hemoglobins: alpha- and beta-chain differences monitored by high-resolution emission spectroscopy.
The absorption and emission properties of hybrid Zn-substituted human hemoglobin (Hb) were used to monitor differences in interaction between the porphyrin and the polypeptide chain for the two subunits. Although alpha-substituted (alpha-ZnHb), beta-substituted (beta-ZnHb), or totally substituted Hb all show optical properties characteristic of Zn porphyrins, the spectra are also indicative of specific interactions between the polypeptide chain and the porphyrin. The Q0,0 absorption band of alpha-ZnHb at 5 K shows a splitting of approximately 300 cm-1, comparable to the largest split ever reported for a heme protein. This value is approximately 140 cm-1 for beta-ZnHb. The possible origin of the split is discussed in terms of the local electric field imposed by the amino acids of the respective heme pockets, different configurations of the porphyrin, and/or influences of the liganding histidine. The Zn derivatives show quasiline spectra under fluorescence line narrowing conditions, and the resolved excitation spectrum reveals differences in the vibrational levels of the Zn porphyrin in the two subunits. Broad underlying emission in the fluorescence line-narrowed emission spectrum can be accounted for, in part, by the existence of the two closely spaced electronic origins and also by the extent of phonon coupling between the porphyrin and the protein matrix.