Ultraviolet resonance Raman studies of hemoglobin quaternary structure using a tyrosine-α42 mutant: changes in the α1β2 subunit interface upon the T → R transition

Abstract

Quaternary structure changes between T (tense) and R (relaxed) states of human hemoglobin A (Hb A) and its α42Tyr mutant, obtained through site-directed mutagenesis, were investigated by ultraviolet resonance Raman (UVRR) spectroscopy using 235-nm excitation. Raman excitation at 235 nm enabled us to detect bands of tryptophan (Trp) and tyrosine (Tyr) residues. The UVRR spectral contribution of α42Tyr, which is located in the “switch” region of the α 1 β 2 interface and forms an H-bond with the carboxylate side chain of β99Asp only in the T state, was deduced for each of the deoxy-and CO-forms by subtracting the spectra of Hb αY42H from those of Hb A under a certain assumption. This suggested that α42Tyr is responsible for the frequency shifts of Y8a (1619 cm −1) and Y9a (1179 cm −1) of the Tyr RR bands of Hb A, but that other Tyr residues are involved in intensity changes. The ligand-induced intensity changes of Trp UVRR bands were similar for Hb A and Hb αY42H, indicating that the conformation changes of Trp residues of Hb A and Hb αY42H upon quaternary structure change are alike. In order to get an insight into implications of these changes of the Tyr UVRR bands of Hb A between the R and T states, UVRR spectra of tyrosine and p-cresol in various solvents were examined with 235-nm excitation. The UVRR spectrum of Tyr residues in Hb A was similar to that of tyrosine in an aqueous solution, but distinct from that of tyrosine crystalline powder. The ν 8 a band of p-cresol was upshifted and intensified in H-bond-forming solvents, irrespective of the H-bond donor or acceptor, compared with that in a non-H-bonding solvent. Accordingly, the present results are compatible with the statement that the frequency shifts of Y8a and Y9a of Hb A upon the T → R transition are caused by the H-bond formation of α42Tyr in the T state.