Romero A, Hoitink C W, Nar H, Huber R, Messerschmidt A, Canters G W
Max-Planck Institut für Biochemie, Abteilung Strukurforschung, Martinsried bei München, Germany.
J Mol Biol. 1993 Feb 20;229(4):1007-21. doi: 10.1006/jmbi.1993.1101.
The dependence of the properties of the azurin blue copper site on the nature of the axial ligand at position 121 was tested by site-directed mutagenesis. This residue was substituted for a glutamine, the purported fourth copper ligand in the related protein stellacyanin. M121Q azurin was isolated and purified from Escherichia coli and characterized by spectroscopic methods. The mutant copper site has the ultra-violet-vis and electron paramagnetic resonance (EPR) characteristics of a type I site, but the spectroscopic details differ significantly from wild-type (wt) azurin. The X and S-band EPR spectra of M121Q azurin can be well stimulated with the parameters for stellacyanin, indicating that the copper sites of both proteins in the oxidized state are similar. The midpoint potential of M121Q is 263 mV, 25 mV lower than for wt azurin. The reactivity of the mutant was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy. The rate was 8 x 10(3) mol-1 s-1, almost two orders of magnitude lower than the value for wt azurin (5 x 10(5) mol-1 s-1). Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q azurin crystals at 1.9 A resolution. The histidine and cysteine copper ligand distances and angles in the equatorial plane around the copper are very similar to the wt protein. Gln121 is co-ordinated in a monodentate fashion via its side-chain oxygen atom at a distance of 2.26 A. The distance between copper and the carbonyl group of Gly45 is increased from 3.13 A (wt) to 3.37 A resulting in a distorted tetrahedral N2SO copper co-ordination. The possible significance of these results for the structure of the copper site of stellacyanin, the only small blue copper protein lacking a methionine ligand, is discussed. Conformational changes with respect to the wt azurin are seen in some of the connecting loops between secondary structure elements, in the mutation site and in the beta-strand 2a. The side-chains involved in the hydrophobic patch surrounding His117 are subject to large changes in their conformations. In contrast to wt azurin, the copper site in M121Q azurin undergoes significant structural changes on reduction.(ABSTRACT TRUNCATED AT 400 WORDS)
通过定点诱变来测试天青蛋白蓝色铜位点的性质对第121位轴向配体性质的依赖性。该残基被替换为谷氨酰胺，谷氨酰胺被认为是相关蛋白星蓝蛋白中的第四个铜配体。从大肠杆菌中分离并纯化了M121Q天青蛋白，并通过光谱方法对其进行了表征。突变型铜位点具有I型位点的紫外可见和电子顺磁共振（EPR）特征，但光谱细节与野生型（wt）天青蛋白有显著差异。M121Q天青蛋白的X波段和S波段EPR光谱可以用星蓝蛋白的参数很好地模拟，这表明两种蛋白质在氧化态下的铜位点相似。M121Q的中点电位为263 mV，比wt天青蛋白低25 mV。通过核磁共振光谱测量电子自交换速率来探究突变体的反应性。该速率为8×10³ mol⁻¹ s⁻¹，几乎比wt天青蛋白的值（5×10⁵ mol⁻¹ s⁻¹）低两个数量级。通过对分辨率为1.9 Å的M121Q天青蛋白晶体进行X射线分析，获得了关于M121Q Cu(II)位点的详细结构信息。铜周围赤道平面内的组氨酸和半胱氨酸铜配体距离和角度与wt蛋白非常相似。Gln121通过其侧链氧原子以单齿方式配位，距离为2.26 Å。铜与Gly45羰基之间的距离从3.13 Å（wt）增加到3.37 Å，导致四面体N₂SO铜配位扭曲。讨论了这些结果对于星蓝蛋白铜位点结构的可能意义，星蓝蛋白是唯一一种缺乏甲硫氨酸配体的小蓝色铜蛋白。在二级结构元件之间的一些连接环、突变位点和β链2a中可以看到相对于wt天青蛋白的构象变化。围绕His117的疏水区域中涉及的侧链构象发生了很大变化。与wt天青蛋白不同，M121Q天青蛋白中的铜位点在还原时会发生显著的结构变化。（摘要截断于400字）
