Investigation of pH-induced symmetry distortions of the prosthetic group in oxyhaemoglobin by resonance Raman scattering

Abstract

The depolarisation ratio and the excitation profiles of some prominent Raman lines of the oxyhaemoglobin spectrum (1,375 cm^-1, 1,583 cm^-1, 1,638 cm^-1) have been measured as functions of the exciting laser frequency. The depolarisation ratio shows a complicated minimum-maximum structure in the preresonant region between Soret- and β-band of the optical spectrum, which depends on the pH-value of the solution. These dispersion curves are interpreted by fifth-order Loudon theory of the polarisability tensor including static distortions of the haem group, which lower its symmetry from the ideal D _4h-symmetry, and enhancement by a second, non-Raman-active phonon. The fitting constants needed to fit the experimental data are related to static distortions of A _1g, B _1g, B _2g, and A _2g` symmetry types and thus give information on the symmetry lowering from D _4h. The variation of the fitting constants with the pH-value of the solution is interpreted to be caused by protonation/deprotonation processes of titrable amino acid groups contributing to the alkaline and acid Bohr effect. The protonation changes the electrostatic interaction energies in the globular protein and destabilises the salt bridge between His(HC3)β and Asp(FG1)β in the R-state. These processes induce distortions of the haem group via haem-apoprotein interactions. Our results give no indication for a dominant role of the covalent Fe²⁺-N[His(F8)] bond in this process. They are in agreement, however, with the allosteric model of Hopfield, which assumes all interactions to be evenly distributed all over the protein molecule.