JBC, Vol. 250, Issue 18, 7461-7472, Sep, 1975
Nuclear magnetic resonance titration curves of histidine ring protons. Human metmyoglobin and the effects of azide on human, horse, and sperm whale metmyoglobins
M. B. Hayes, H. Hagenmaier and J. S. Cohen
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.
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