title: The sulfhydryl group of Cys138 of rusticyanin from Acidithiobacillus ferrooxidans is crucial for copper binding.
abstract: 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.
authors: Department of Bioengineering, School of Resources Processing and Bioengineering, Central South University, Changsha, PR China.
authors: Jia
authors: J
authors: Zeng
authors: Meimei
authors: M
authors: Geng
authors: Yuandong
authors: Y
authors: Liu
authors: Lexian
authors: L
authors: Xia
authors: Jianshe
authors: J
authors: Liu
authors: Guanzhou
authors: G
authors: Qiu
mesh_terms: D042763:Acidithiobacillus / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013054:Spectrophotometry, Atomic
chemical_list: C012543:rusticyanin; D001400:Azurin; D003300:Copper; D003545:Cysteine