X-RAY-ANALYSIS AND SPECTROSCOPIC CHARACTERIZATION OF M121Q AZURIN - A COPPER SITE MODEL FOR STELLACYANIN
X-RAY-ANALYSIS AND SPECTROSCOPIC CHARACTERIZATION OF M121Q AZURIN - A COPPER SITE MODEL FOR STELLACYANIN
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DOI：
10.1006/jmbi.1993.1101
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发表时间：
1993-02-20
影响因子：
5.6
通讯作者：
CANTERS, GW
中科院分区：
文献类型：
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作者：
ROMERO, A;HOITINK, CWG;CANTERS, GW
通过定点诱变测试了天青蓝铜位点对位置121处的轴向配体的性质的依赖性。这个残基被谷氨酰胺取代，谷氨酰胺是相关蛋白质星花青苷中的第四个铜配体。从大肠杆菌中分离纯化了M121 Q天青蛋白，并对其进行了光谱表征。突变铜网站具有紫外-维斯和电子顺磁共振（EPR）的一个I型网站的特点，但光谱的细节显着不同的野生型（WT）天青蛋白。M121 Q天青蛋白的X带和S带EPR谱可以很好地用星花青蛋白的参数来模拟，这表明两种蛋白在氧化态下的铜位点是相似的。M121 Q的中点电位比wt天青蛋白低263 mV。通过核磁共振光谱法测量电子自交换速率来探测突变体的反应性。该速率为8 × 103 mol-1 s-1，比wt天青蛋白的值（5 × 105 mol-1 s-1）低近两个数量级。M121 Q Cu（II）-位点的详细结构信息通过M121 Q天青晶体的X射线分析在1.9 μ m分辨率下获得。组氨酸和半胱氨酸铜配体的距离和角度在赤道平面周围的铜是非常相似的野生型蛋白质。Gln 121通过其侧链氧原子以单齿方式配位，距离为2·26 π。Cu与Gly-45的羰基之间的距离从3.13 μ m（wt）增加到3.37 μ m，导致四面体N_2SO铜配位畸变。可能的意义，这一结果的结构的铜网站的stellacyanin，唯一的小蓝色铜蛋白缺乏甲硫氨酸配体，进行了讨论。在二级结构元件之间的一些连接环、突变位点和β链2a中观察到相对于wt天青蛋白的构象变化。His 117周围疏水斑块中涉及的侧链在其构象中发生了很大的变化。与wt天青蛋白相反，M121 Q天青蛋白中的铜位点在还原时经历显著的结构变化。Cu-Oε Gin 121和Nδ His 117键长分别增加2.73 π和2.68 π，Sγ Cys 112-Cu（I）-Nδ His 46夹角为156°，产生几乎线性的Cu（I）位点，具有两个强键，Cys 112的硫醇硫原子（2.09 π）和His 46的咪唑氮原子（1.98 A），与Oε Gln 121和Nδ His 117有两个弱相互作用。氧化态和还原态M121 Q天青蛋白的结构差异可能对电子自交换速率和中点电位有显著影响。
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 fromEscherichia coliand 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 protein in the oxidized state are similar. The midpoint potential of M121Q is 263 mV lower than for wt azurin. The reactivity of the mutants was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy. The rate was 8 × 103mol-1s-1, almost two orders of magnitude lower than the value for wt azurin (5 × 105mol-1s-1). Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q azurin crystals at 1·9 Å 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 fashionviaits side-chain oxygen atom at a distance of 2·26 Å. The distance between copper and the carbonyl group of Gly-45 is increased from 3·13 Å (wt) to 3·37 Å resulting in a distorted tetrahedral N2SO copper co-ordination. The possible significance of this 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 β-strand 2a. The side-chains involved in the hydrophobic patch surrounding His 117 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. An increase of the Cu-OεGin121 and NδHis117 bond lengths of 2·73 Å and 2·68 Å, respectively, with an Sγ Cys112-Cu(I)-Nδ His46 angle of 156° produces an almost linear Cu(I) site with two strong bonds, of the thiolate sulphur atom of Cys112 (2·09 Å) and the imidazole nitrogen atom of His46 (1·98 A), and two weak interactions with the Oε Gln121 and the Nδ His117. The differences in structure between oxidized and reduced M121Q azurin probably influence significantly the electron self exchange rate and the midpoint potential.