缬氨酸61位点突变对细胞色素b5三维结构、稳定性及氧化还原电位的影响

Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5.

作者信息

Xue L L, Wang Y H, Xie Y, Yao P, Wang W H, Qian W, Huang Z X, Wu J, Xia Z X

机构信息

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, PRC.

出版信息

Biochemistry. 1999 Sep 14;38(37):11961-72. doi: 10.1021/bi990893b.

DOI:10.1021/bi990893b

PMID:10508399

Abstract

To elucidate the role played by Val61 of cytochrome b(5), this residue of the tryptic fragment of bovine liver cytochrome b(5) was chosen for replacement with tyrosine (Val61Tyr), histidine (Val61His), glutamic acid (Val61Glu), and lysine (Val61Lys) by means of site-directed mutagenesis. The mutants Val61Tyr, Val61Glu, Val61His, and Val61Lys exhibit electronic spectra identical to that of the wild type, suggesting that mutation at Val61 did not affect the overall protein structure significantly. The redox potentials determined by differential pulse voltammetry were -10 (wild type), -25 (Val61Glu), -33 (Val61Tyr), 12 (Val61His), and 17 mV (Val61Lys) versus NHE. The thermal stabilities and urea-mediated denaturation of wild-type cytochrome b(5) and its mutants were in the following order: wild type > Val61Glu > Val61Tyr > Val61His > Val61Lys. The kinetics of denaturation of cytochrome b(5) by urea was also analyzed. The first-order rate constants of heme transfer between cytochrome b(5) and apomyoglobin at 20 +/- 0.2 degrees C were 0.25 +/- 0.01 (wild type), 0.42 +/- 0.02 (Val61Tyr), 0.93 +/- 0.04 (Val61Glu), 2.88 +/- 0.01 (Val61His), and 3.88 +/- 0.02 h(-)(1) (Val61Lys). The crystal structure of Val61His was determined using the molecular replacement method and refined at 2.1 A resolution, showing that the imidazole side chain of His61 points away from the heme-binding pocket and extends into the solvent, the coordination distances from Fe to NE2 atoms of two axial ligands are approximately 0.6 A longer than the reported value, and the hydrogen bond network involving Val61, the heme propionates, and three water molecules no longer exists. We conclude that the conserved residue Val61 is located at one of the key positions, the "electrostatic potential" around the heme-exposed area and the hydrophobicity of the heme pocket are determinant factors modulating the redox potential of cytochrome b(5), and the hydrogen bond network around the exposed heme edge is also an important factor affecting the heme stability.

摘要

为阐明细胞色素b(5)的61位缬氨酸所起的作用，通过定点诱变，选择牛肝细胞色素b(5)胰蛋白酶片段中的该残基，用酪氨酸（Val61Tyr）、组氨酸（Val61His）、谷氨酸（Val61Glu）和赖氨酸（Val61Lys）进行替换。Val61Tyr、Val61Glu、Val61His和Val61Lys突变体的电子光谱与野生型相同，这表明61位缬氨酸的突变并未显著影响蛋白质的整体结构。用差分脉冲伏安法测定的相对于标准氢电极（NHE）的氧化还原电位分别为：野生型为 -10 mV，Val61Glu为 -25 mV，Val61Tyr为 -33 mV，Val61His为12 mV，Val61Lys为17 mV。野生型细胞色素b(5)及其突变体的热稳定性和尿素介导的变性情况如下：野生型>Val61Glu>Val61Tyr>Val61His>Val61Lys。还分析了尿素使细胞色素b(5)变性的动力学。在20±0.2℃下，细胞色素b(5)与脱辅基肌红蛋白之间血红素转移的一级速率常数分别为：野生型为0.25±0.01 h⁻¹，Val61Tyr为0.42±0.02 h⁻¹，Val61Glu为0.93±0.04 h⁻¹，Val61His为2.88±0.01 h⁻¹，Val61Lys为3.88±0.02 h⁻¹。采用分子置换法测定了Val61His的晶体结构，并在2.1 Å分辨率下进行了精修，结果表明61位组氨酸的咪唑侧链远离血红素结合口袋并延伸到溶剂中，两个轴向配体的铁到NE2原子的配位距离比报道值长约0.6 Å，涉及61位缬氨酸、血红素丙酸酯和三个水分子的氢键网络不再存在。我们得出结论，保守残基61位缬氨酸位于关键位置之一，血红素暴露区域周围的“静电势”和血红素口袋的疏水性是调节细胞色素b(5)氧化还原电位的决定性因素，暴露的血红素边缘周围的氢键网络也是影响血红素稳定性的重要因素。