【作者】 唐琳；

【导师】 曾嘉；

【作者基本信息】 湖南大学 ， 生物医学工程， 2012， 硕士

【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。更多还原

【Abstract】 Rusticyanin is a member of type I copper protein, which considered as the majorcomponent in the iron respiratory electron transport chain of A. ferrooxidans. The ioncopper is coordinated in a distorted tetrahedral geometry with three strongly ligandsCys138, His85, His143and a relatively weaker ligand Met148. Both His85andCys138residues have been proved essential to binding with ion copper in Rusticyanin.The axial ligand of Met148have been extensively studied and it was revealed at someextent the distance between cooper atom and protein determined the level of the redoxpotential of Rusticyanin, but this is not the most important factor. Meanwhile, to dateonly a few studies have been done toward residue His143, but it is likely that residueHis143plays a key residue as a “redox switch”, nevertheless no report found.In this research, the gene of rusticyanin was cloned and constructed the plasmids,then expressed in Escherichia coli competent cells, and later purified using one-stepaffinity chromatography. The absorption spectra study results confirmed that therusticyanin have reaction with IRO. These results confirmed that the electron transferbetween rusticyanin and IRO is conducted in the iron respiratory electron transportchain of A. ferrooxidans.We furthermore constructed the mutant expression plasmids of residue His143using site-directed mutagenesis. Mutant proteins were expressed in E.coli and purifiedwith a nickel metal affinity column. The absorption spectra study showed that His143is a key residue for the high reduction potential of Rusticyanin. Removal of theimidazole group of His143residue resulted in copper reduction and loss ofactivity.We found recovery of activity when the mutant protein143Gly was incubatedin imidazole solution. Molecular structure modeling showed that mutation ofimidazole group caused exposure of copper ions to environment solution, the solventoutside can easily go inside and caused the copper ions structure collapsed, thuselectron transferred activity disrupted. Hence, His143residue is a key component ofelectron transfer in Rusticyanin.更多还原