Ultraviolet resonance Raman studies of quaternary structure of hemoglobin using a tryptophan beta 37 mutant.

Environmental changes of tyrosine and tryptophan residues of hemoglobin (Hb) upon its T to R transition of quaternary structure were investigated with ultraviolet resonance Raman (UVRR) spectroscopy excited at 235 nm. DeoxyHb A (T-form) showed a UVRR spectrum distinctly different from those of the ligated Hbs (R-form) including oxyHb, COHb, and metHb A, whereas the ligated Hbs exhibited similar UVRR spectra irrespective of the ligand species and the oxidation state of the heme. To characterize the spectral change of Trp-beta 37 at the alpha 1 beta 2 interface due to the quaternary structure transition, the UVRR spectra of Hb A were compared with the corresponding spectra of Hb Hirose (Trp-beta 37-->Ser). A difference spectrum between deoxyHb A and deoxyHb Hirose showed only Trp resonance Raman (RR) bands, which were reasonably ascribed to Trp-beta 37 in deoxyHb A. RR bands at 873 cm-1 (W17) and at 1360 and 1343 cm-1 (W7, Fermi doublet) indicated that the indole ring of Trp-beta 37 in deoxyHb A formed a strong hydrogen bond at the N1H site in hydrophobic environments. Tyr residues in deoxyHb Hirose seemed to be in the same environments as those of deoxyHb A. In contrast, the difference spectrum between Hb A and Hb Hirose in the ligated state displayed peaks for RR bands of both Trp and Tyr. The difference spectra were unaltered by the addition of 5 mM inositol hexaphosphate. This means that the differences were not caused by the tetramer to dimer dissociation but by a conformation change within a tetramer. Comparison of the Hb A-Hb Hirose difference spectra in the oxy and deoxy states revealed that the oxygenation-induced changes of Trp RR bands arose mostly from Trp-beta 37 with the small portion of remaining changes coming from Trp-beta 15, demonstrating that Trp-beta 37 plays a pivotal role in the quaternary structural change in Hb A.