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

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 beta-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 polarizability tensor including static distortions of the haem group, which lower its symmetry from the ideal D4h-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 A1g, B1g, B2g, and A2g symmetry types and thus give information on the symmetry lowering from D4h. 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 destabilizes the salt bridge between His(HC3)beta and Asp(FG1)beta 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.