Maurus R, Bogumil R, Luo Y, Tang H L, Smith M, Mauk A G, Brayer G D
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1994 Apr 29;269(17):12606-10. doi: 10.2210/pdb1yma/pdb.
A site-specific mutant of horse heart myoglobin has been prepared in which the distal heme pocket residue, His64, is replaced by tyrosine. The structure of this myoglobin variant has been determined to 2.0-A resolution using x-ray diffraction techniques and refined to a final crystallographic R-factor of 16.9%. The polypeptide backbone conformation of the His64-->Tyr variant of myoglobin is very similar to that of the wild-type protein. However, in the variant the water normally found coordinated to the heme iron atom and hydrogen-bonded to His64 has been displaced by the hydroxyl oxygen of the Tyr64 side chain. The tyrosine oxygen atom is directly coordinated to the heme iron atom with a bond length of 2.18 A. Distortion of heme planarity and changes in the packing of the Leu29 and Leu104 side chains are related to this mutation. The ligand environment of the ferric iron has been studied by electron paramagnetic resonance (EPR) spectroscopy using crystalline material and protein in solution. The protein in solution exhibits a rhombically split ferric high spin EPR spectrum with g values of 6.64, 5.34, and 1.98. The EPR spectrum of the crystalline sample consists of two different ferric high spin signals. The main signal is similar to the signal observed in solution and is assigned to His93-Fe(III)-Tyr64 coordination. The relatively high rhombicity of this signal can be explained as arising from distortions of the heme plane seen in the crystal structure. The second, more axial high spin signal found in the crystalline state can be tentatively assigned to another form of iron ligation with a different iron-tyrosine bond length and a less distorted heme plane.
已制备出一种马心脏肌红蛋白的位点特异性突变体，其中血红素远端口袋残基His64被酪氨酸取代。利用X射线衍射技术确定了这种肌红蛋白变体的结构，分辨率达到2.0埃，并将其精修至最终晶体学R因子为16.9%。肌红蛋白His64→Tyr变体的多肽主链构象与野生型蛋白非常相似。然而，在该变体中，通常发现与血红素铁原子配位并与His64形成氢键的水已被Tyr64侧链的羟基氧取代。酪氨酸氧原子直接与血红素铁原子配位，键长为2.18埃。血红素平面度的扭曲以及Leu29和Leu104侧链堆积的变化与该突变有关。利用晶体材料和溶液中的蛋白质，通过电子顺磁共振（EPR）光谱研究了三价铁的配体环境。溶液中的蛋白质呈现出菱形分裂的三价铁高自旋EPR光谱，g值为6.64、5.34和1.98。晶体样品的EPR光谱由两种不同的三价铁高自旋信号组成。主要信号与溶液中观察到的信号相似，归因于His93-Fe(III)-Tyr64配位。该信号相对较高的菱形度可解释为源于晶体结构中血红素平面的扭曲。在晶体状态下发现的第二个更轴向的高自旋信号可初步归因于另一种铁配位形式，其铁-酪氨酸键长不同，血红素平面扭曲较小。
