Probing the role of the active-site Cysteine of Azurin by site-directed mutagenesis
Probing the role of the active-site Cysteine of Azurin by site-directed mutagenesis
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通过定点诱变探讨天青蛋白活性位点半胱氨酸的作用
DOI：
10.7907/4vtz-pe72
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
T. Mizoguchi
中科院分区：
文献类型：
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作者：
T. Mizoguchi
研究了铜绿假单胞菌单核铜电子转移蛋白天青蛋白的单位点突变体的配位化学和电子转移性质。在位置112的活性位点半胱氨酸被替换为天冬氨酸（Cys 112 Asp），以直接评估该配体对天青蛋白的结构-功能特性的重要性。虽然突变蛋白保留了高亲和力的铜结合活性位点，但Cu[上标II] Cys 112 Asp天青蛋白的吸收光谱和EPR光谱与野生型蛋白的吸收光谱和EPR光谱完全不同，表明存在正常的（2型）铜中心。通过细胞色素c[下标551]的氧化还原滴定实验，得到Cu[上标II/I]还原电位为180 mV（相对于NHE（pH 7.0））。合成了Cys 112 Asp天青蛋白的Co[上标II]衍生物，发现其具有顺磁核磁共振谱。结合电子吸收数据，核磁共振数据被用来生成Co[上标II]活性中心结构的计算机模型，其中金属被两个组氨酸（His 46和His 117）、一个多肽骨架羰基氧（Gly 45）和一个平面外的、不对称结合的二齿天冬氨酸（Asp 112）配位，呈整体扭曲的四方锥形几何结构。报道了Cu[上标II] Cys 112 Asp天青蛋白的2.4-A分辨率的X射线晶体结构，证实了配体集合和几何构型。也制备了Ni[上标II]取代的Cys 112 Asp天青蛋白，但与Cu[上标II]和Co[上标II]不同，Ni[上标II]离子与蛋白质的结合不那么紧密。Ni[上标II] Cys 112 Asp天青的电子光谱表明其与Met 121的硫醚硫原子之间存在一定的成键作用。与含Cys 112的天青相比，激光诱导的分子内电子转移反应从含Asp 112的天青的还原铜中心到表面组氨酸结合的双（联吡啶1）（咪唑）钌（III）标签不能观察到。然而，Cys 112 Asp和野生型天青蛋白之间的分子间蛋白质电子转移的动力学记录通过停流分光光度法。双分子动力学数据的分析表明，Cys-天冬氨酸突变已显着减少了铜和钌中心之间的分子内电子转移系统的电子耦合。
The coordination chemistry and electron-transfer properties of a single-site mutant of the mononuclear copper electron-transfer protein azurin from Pseudomonas aeruginosa have been studied. The active-site cysteine at position 112 was replaced by an aspartate (Cys112Asp) to assess directly the importance of this ligand to the structure-function properties of azurin. Although the mutant protein retains a high-affinity copper-binding active site, the absorption and EPR spectra of Cu[superscript II]Cys112Asp azurin are quite distinct from those of the wild-type protein and indicate the presence of a normal (type 2) copper center. A Cu[superscript II/I] reduction potential of 180 mV vs. NHE (pH 7.0) was obtained through a redox titration experiment with cytochrome c[subscript 551]. The Co[superscript II] derivative of Cys112Asp azurin was prepared and found to be amenable to paramagnetic NMR spectroscopy. In conjunction with electronic absorption data, the NMR data were used to generate a computer model of the Co[superscript II] active-site structure in which the metal is coordinated by two histidines (His46 and His 117), a polypeptide backbone carbonyl oxygen (of Gly 45), and an out-of-plane, asymmetrically-bound bidentate aspartate (Asp 112) in an overall distorted square pyramidal geometry. The 2.4-A resolution X-ray crystal structure of Cu[superscript II] Cys112Asp azurin is reported and confirms this ligand set and geometry. Ni[superscript II]-substituted Cys112Asp azurin was also made, but unlike Cu[superscript II] and Co[superscript II], the Ni[superscript II] ion is much less tightly bound by the protein. The electronic spectroscopy of Ni[superscript II] Cys112Asp azurin suggests the existence of some bonding interaction with the thioether sulfur atom of Met121. In contrast to Cys112-containing azurins, laser-induced intramolecular electron-transfer reactions from the reduced copper center of Asp 112-containing azurins to surface histidine-bound bis(bipyridy1)(imidazole) ruthenium(III) labels could not be observed. However, the kinetics of intermolecular protein-protein electron transfer between Cys112Asp and wild-type azurins were recorded by stopped-flow spectrophotometry. Analysis of the bimolecular kinetic data suggests that the Cys-to-Asp mutation has diminished significantly the electronic coupling between the copper and ruthenium centers in the intramolecular electron-transfer systems.