Nuclear magnetic resonance titration curves of histidine ring protons. Human metmyoglobin and the effects of azide on human, horse, and sperm whale metmyoglobins.
Four titrating histidine ring C2 and C4 proton resonances are observed in 220 MHz proton NMR spectra of human metmyoglobin as a function of pH. Values of ionization constants determined from the NMR titration data using an equation describing a simple proton association-dissociation equilibrium are curves (1) 6.6, (2) 7.0, (3) 5.8, and (4) 7.4. Four histidine residues have also been found to be solvent-accessible in human metmyoglobin by carboxymethylation studies (Harris, C.M., and Hill, R.L. (1969) J. Biol. Chem. 244, 2195-2203). Two of the titration curves (3 and 4) deviate significantly from the chemical shift values normally observed for histidine C2 proton resonances. Curve 3, with a low pKa, is shifted downfield at high values of pH and also exhibits a second minor inflection with a pKa value of 8.8. On the other hand, the high pKa curve, 4, is shifted upfield at all values of pH. The characteristics of the NMR titration curves with the lowest and highest pKa values (3 and4) are very similar to curves observed previously with sperm whale and horse metmyoglobins (Cohen, J.S., Hagenmaier, H., Pollard, H., and Schechter, A.N. (1972) J. Mol. Biol. 71, 513-519). These results indicate that the histidine residues from which these curves are derived have unusual and characteristic environments in this series of homologous proteins. The NMR spectra of all three metmyoglobins are changed extensively as a result of azide ion binding, indicating conformational changes affecting the environments of several imidazole side chains. The presence of azide ion causes a selective downfield chemical shift for the low pKa curve and a selective upfield chemical shift for the high pKa curve in all three proteins. Azide also abolishes the second inflection seen in the low pKa curve at high pH. In addition to these effects, the presence of azide ion permits the observation of two additional titrating proton resonances for all three metmyoglobins. Increasing the azide to protein ratio at several fixed values of pH yields results which show that a slow exchange process is occurring with each of the metmyoglobins. In the azide titration studies the maximum changes in the NMR spectra occurred at approximately equimolar concentrations. The NMR results for these proteins in the absence and presence of azide ion are related to x-ray crystallographic studies of sperm whale metmyoglobin and the known alkylation properties of the histidine residues. Tentative assignments of the titrating resonances observed are suggested.