Origin of the pH-dependent spectroscopic properties of pentacoordinate metmyoglobin variants.

The pH dependence of the electronic and EPR spectra of two variants of horse heart myoglobin (Mb) in which the distal His64 ligand has been replaced by either Thr or Ile has been studied. Both of these variants exhibit spectroscopic changes with pH that are indicative of a transition between two ferric high-spin forms that occurs with a pKa of 9.49 for the His64Thr variant and 9.26 for the His64Ile variant and that is distinctly different from the pH-dependent spectroscopic changes related to titration of the distal aquo ligand of wild-type Mb. The electronic and EPR spectra of both variants at all values of pH studied are consistent with the presence of a pentacoordinate heme iron center. For the His64Thr variant, a high-resolution (1.9 A) structure determination establishes the lack of the distal aquo ligand and demonstrates an out-of-plane movement of the ferric iron toward the proximal histidine together with a decrease of the Fe-His bond length. Investigation of this pH-linked equilibrium by EPR spectroscopy reveals rhombically split high-spin signals at both pH 7 and 11 with a greater degree of rhombicity exhibited by the alkaline species. We propose that the pH-linked spectroscopic transition exhibited by these distal histidine variants results from the deprotonation of the proximal His93 residue to produce imidazolate ligation at alkaline pH.