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

Abstract

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.