Low-frequency resonance Raman study of modified hemoglobins
Abstract
Resonance Raman spectroscopy is now well established as a powerful structural probe of heme proteins. In our present study we employed RR spectroscopy to obtain an insight into the cooperative binding of oxygen by monitoring the low frequency region of native and modified hemoglobins. The strength of the Fe-His(N$\sb{\epsilon}$) bonding has been suggested as an essential structural parameter that controls the ligand binding properties. Although the Fe-His stretching frequency has been assigned for the deoxy form, the same has not been reported for any ligated forms of Hb. In the low frequency region RR spectra of oxyHb reported here, the H$\sb2$O/D$\sb2$O buffer exchange permitted the assignment of Fe-His stretching frequency to a band around 230cm$\sp{-1}$. The Fe-O-O bending mode is also identified here in the low frequency region by isotopic substitution studies. Hemoglobin and its isolated subunits were reconstituted with meso and deuterohemes in order to study the effect of heme peripheral substituent modification on heme-protein interaction. Hybrid hemoglobins containing proto/meso and proto/deutero hemes were prepared in the met hybrid form as well as in the fully reduced form. The native and modified subunits of these hybrids in the deoxy form were selectively probed by using two different excitation lines. The Fe-His stretching frequencies of each subunit were obtained from the spectra. Despite functional differences, the Fe-His stretching frequencies of neither the modified nor the partner subunits were significantly altered from that of native hemoglobin. The inadequacy of two state model to explain the cooperative binding of Hb along with the possibility of a three state model are discussed briefly with reference to our present results, recent RR results, NMR results and molecular energetic calculations. Finally a selective deuteriation study was carried out on deoxy hemoglobins reconstituted with mesoheme to assign the other low frequency modes, which displayed a significant change in the intensity pattern when going from the subunits to the tetramer.
Recommended Citation
Jeyarajah, Shanthini, "Low-frequency resonance Raman study of modified hemoglobins" (1990). Dissertations (1962 - 2010) Access via Proquest Digital Dissertations. AAI9117349.
https://epublications.marquette.edu/dissertations/AAI9117349