Resonance Raman and absorption spectroscopic detection of distal histidine-fluoride interactions in human methemoglobin fluoride and sperm whale metmyoglobin fluoride: measurements of distal histidine ionization constants

Abstract

The pH dependence of the resonance Raman and absorption spectra of human methemoglobin fluoride (HbIIIF) and sperm whale metmyoglobin fluoride (MbIIIF) was examined. Both the Raman and absorption spectra of HbIIIF and MBIIIF indicate the existence at alkaline pH of an equilibrium between the hydroxide and fluoride complexes. The absorption maxima of HbIIIF and MbIIIF solutions shift to longer wavelengths as the pH is decreased from neutrality. The Raman data show a corresponding shift of the 461 and 468 cm-1 Fe-F vibrational stretching peaks at pH 7.0 to 399 and 407 cm-1 at acid pH in MbIIIF, and HBIIIF, respectively. These shifts are interpreted to result from protonation of the distal histidine and the formation of a H-bond to the F- ligand. Measurements of the pH dependence of the absorption and resonance Raman spectra give distal histidine ionization constants (apparent) corresponding to pK = 5.1 (.+-.0.1) for HbIIIF and pK = 5.5 (.+-.0.1) for MbIIIF. An examination of the distal histidine pK values and the frequency of the H-bonded Fe-F stretching vibration at pH 5.0 of HbIIIF with and without inositol hexaphosphate indicates little difference in the distal histidine-heme distance between the so-called R and T quaternary forms of HbIIIF. These results indicate that the changes in the electronic spectrum of HbIIIF that occur upon switching from the R to the T form do not result from alterations in the Fe-F- bond distance, the Fe out-of-heme plane distance, or the distal histidine-F- distance.