Dynamic protein structures. Effects of pH on conformer stabilities at the ligand-binding site of bovine heart myoglobin carbonyl.

The single ligand-binding site of bovine myoglobin carbonyl exists in four discrete conformations as shown by four C--O stretch bands for the carbonyl ligand. Both this infrared spectrum and the visible spectrum are altered by changes in pH from 4.7 to 8.2 at 20 degrees C. The spectral changes can be related to monoprotonation or monodeprotonation at a protein residue with a pKa = 6.0 +/- 0.2. Below pH 5.5, an additional proton-coupled infrared spectral change is evident. Histidine is an appropriate site for pKa approximately 6; there are 13 histidines in the protein. However, the nature of the pH effects on infrared spectra indicates that neither the proximal nor the distal histidine is a likely site. The state of protonation of the protein has a marked effect on the relative stabilities of the four conformers but appears to have little effect on the discrete conformer structures per se in that C--O stretch band frequencies and shapes are nearly insensitive to changes in pH. The sum of the four integrated infrared band intensities is similarly insensitive. These findings provide strong support for the presence of four conformers of significantly different structure at the heme ligand-binding site and for rapid interconversions among these structures.