The sulfhydryl group of Cys138 of rusticyanin from Acidithiobacillus ferrooxidans is crucial for copper binding.
The sulfhydryl group of Cys138 of rusticyanin from Acidithiobacillus ferrooxidans is crucial for copper binding.
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DOI：
10.1016/j.bbapap.2007.02.008
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发表时间：
2007-04
期刊：
影响因子：
--
通讯作者：
J. Zeng;M. Geng;Yuandong Liu;Lexian Xia;Jianshe Liu;G. Qiu
中科院分区：
文献类型：
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作者：
J. Zeng;M. Geng;Yuandong Liu;Lexian Xia;Jianshe Liu;G. Qiu
绿锈青苷是一种从嗜酸氧化亚铁硫杆菌中分离出来的蓝色小分子铜蛋白，具有极强的酸稳定性和氧化还原电位。该蛋白被认为是该微生物中铁呼吸电子传递链的主要成分，但其在电子传递中的确切作用仍有争议。克隆并在大肠杆菌中高效表达了粗花青苷基因，经一步亲和层析纯化得到了粗花青苷可溶性蛋白。Cys138、His85和His143是铜离子结合的重要残基，但Cys138的意义尚未得到证实。我们利用定点突变技术构建了这三个残基的突变表达质粒。突变蛋白在E.大肠杆菌表达，用镍金属亲和柱纯化。EPR和原子吸收光谱的结果证实，Cys138是至关重要的铜结合。Cys138的巯基的去除导致铜损失。His85和His143的突变对铜结合几乎没有影响。
Rusticyanin is a small blue copper protein isolated from Acidithiobacillus ferrooxidans with extreme acid stability and redox potential. The protein is thought to be a principal component in the iron respiratory electron transport chain in this microorganism, but its exact role in electron transfer remains controversial. The gene of rusticyanin was cloned then overexpressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. It was reported that Cys138, His85 and His143 were important residues for copper binding, but the significance of Cys138 was not verified so far. We constructed the mutant expression plasmids of these three residues using site-directed mutagenesis. Mutant proteins were expressed in E. coli and purified with a nickel metal affinity column. The EPR and atomic absorption spectroscopy results confirmed that Cys138 was crucial for copper binding. Removal of the sulfhydryl group of Cys138 resulted in copper loss. Mutations of His85 and His143 showed little effect on copper binding.