Masako Nagai

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.

Hemoglobin M: Effect of the proximal or distal histidine replacement on circular dichroism in the visible region

To examine the effects of a replacement of the proximal or the distal histidine on the structure of hemoglobin (Hb), absorption and circular dichroic (CD) spectra of five species of Hbs M in the visible region were measured. Four Hbs M had a characteristic but a similar absorption spectrum upon amino acid substitution, however, the proximal histidine replaced Hbs M (Hb M Iwate and Hb M Hyde Park) showed considerably different CD spectra from those of the distal histidine replaced ones (Hb M Boston and Hb M Saskatoon). The former exhibited large positive CD but the latter gave a complex CD spectrum with positive and negative extrema. On the other hand, absorption and CD spectra of Hb M Milwaukee did not changed very much from those of Hb A.

UV Resonance Raman Studies of Quaternary Structures of Hemoglobin Using Several Mutants

Hemoglobin (Hb) is an allosteric protein showing cooperativity in oxygen binding to four hemes. X-ray crystallographic studies have shown that Hb has two distinct quaternary structures named T and R, which correspond to the deoxy and ligand-bound forms, respectively. The cooperative oxygen binding has been explained in terms of a reversible transition between the T and R states upon partial ligation of four hemes. It is suggested that ligation to the α-heme causes a conformational change of the protein through the proximal His residue and induces rearrangements of subunits at the α1β2 (or α2β1) interface. However, there is still no direct information about the rearrangements of the α1β2 interface. On the other hand, recent studies have explored that excitation of Raman scattering around 230–240 nm provides resonance-enhanced Raman bands of Tyr and Trp residues. In order to elucidate structural changes at the α1β2 interface upon the ligand binding, we investigated UV resonance Raman (RR) spectra of Hb A and its mutants excited at 235 nm and revealed the vibrational spectra of single Trp-β37 or Tyr-α42 residues present at the α1β2 interface and their changes upon the quaternary structure change by using difference calculations.