蓝铜蛋白分子内电子传递机理研究

发布时间：2019-05-28 18:23

【摘要】：蓝铜蛋白(Rusticyanin)是第I类铜蛋白家族成员，是氧化亚铁硫杆菌中铁呼吸电子链中重要的组分。该蛋白分子内的铜原子与Cys138，His85、His143以强健结合，与Met148以较弱的键结合，，形成四面体结构。Cys138和His85只是维持铜与蛋白结合的关键位点，Met148位点轴向配体对于氧化还原电子传递有一定的影响，但并不是控制氧化还原反应的关键位点，His143位点目前对其的研究还很少，很可能是氧化还原的开关，但目前没有相关文献报道过。本论文从嗜酸亚铁氧化硫杆菌中克隆出蓝铜蛋白基因，构建了蓝铜蛋白重组质粒，并将其在大肠杆菌载体中表达，用亲和一步层析方法纯化。光谱学数据表明其能够和IRO蛋白发生反应，证明了目前亚铁氧化系统电子传递的途径：IRO和蓝铜蛋白之间发生了电子传递。本论文构建了His143位点的定点突变质粒，并将其在大肠杆菌中表达用镍柱纯化。通过聚丙烯酰胺凝胶电泳，光谱学数据确定143位点是蓝铜蛋白与外界电子传递的关键位点，移除其结构上与铜连接的咪唑基团导致结合的铜减少，若继续在咪唑溶液中孵育则蓝铜突变蛋白活性有所增加。分子结构模型证明若将咪唑基团突变后，铜离子就暴露下溶液环境中，外面的溶剂很容易进去到四面棱形结构里导致铜离子结构瓦解，电子不发生传递。因此His143位点是蓝铜蛋白电子传递的关键部位。
[Abstract]:Blue copper protein (Rusticyanin) is a member of class I copper protein family and an important component of iron respiratory electron chain in thiobacillus ferrooxidans. The copper atoms in the protein molecule bind strongly to Cys138,His85,His143 and weak bond to Met148 to form tetrahedral structure. Cys138 and His85 are only the key sites to maintain copper binding to protein. The axial ligand of Met148 site has a certain effect on redox electron transfer, but it is not the key site to control the redox reaction. At present, there is little research on it at His143 site, which may be the switch of redox. However, no relevant literature has been reported at present. In this paper, the blue copper protein gene was cloned from thiobacillus ferrooxidans, and the recombinant plasmid of blue copper protein was constructed and expressed in E. coli vector. The recombinant plasmid was purified by affinity one step chromatography. Spectroscopic data show that it can react with IRO protein, which proves that electron transfer takes place between IRO and blue copper protein in ferrous oxidation system at present. In this paper, the site-directed mutant plasmid of His143 site was constructed and purified by nickel column. Through polyacrylamide gel electrophoresis, the 143 site was determined to be the key site for the electron transfer between blue copper protein and the outside world, and the removal of imidazole groups linked to copper resulted in the decrease of copper binding. If incubated in imidazole solution, the activity of blue copper mutant protein increased. The molecular structure model shows that if the imidazole group is mutated, the copper ion will be exposed to the solution environment, and the external solvent can easily go into the tetrahedral structure and lead to the collapse of the copper ion structure, and the electron does not transfer. Therefore, His143 site is the key site of electron transmission of blue copper protein.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

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