Bogumil R, Maurus R, Hildebrand D P, Brayer G D, Mauk A G
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Biochemistry. 1995 Aug 22;34(33):10483-90. doi: 10.1021/bi00033a021.
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of the electronic and EPR spectra of two variants of horse heart myoglobin (Mb) in which the distal His64 ligand has been replaced by either Thr or Ile has been studied.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.
对马心脏肌红蛋白（Mb）的两种变体进行了研究，这两种变体中远端的His64配体分别被Thr或Ile取代，研究了它们的电子光谱和电子顺磁共振（EPR）光谱对pH值的依赖性。这两种变体的光谱都随pH值发生变化，表明在两种三价高自旋形式之间存在转变，His64Thr变体的pKa为9.49，His64Ile变体的pKa为9.26，这与野生型Mb远端水合配体滴定相关的pH依赖性光谱变化明显不同。在所研究的所有pH值下，两种变体的电子光谱和EPR光谱都与五配位血红素铁中心一致。对于His64Thr变体，高分辨率（1.9 Å）结构测定表明不存在远端水合配体，并证明三价铁向近端组氨酸发生平面外移动，同时Fe-His键长缩短。通过EPR光谱对这种与pH相关的平衡进行研究，发现在pH 7和11时都有菱形分裂的高自旋信号，碱性物种表现出更大程度的菱形度。我们提出，这些远端组氨酸变体所表现出的与pH相关的光谱转变是由于近端His93残基去质子化，在碱性pH下产生咪唑配位所致。
