Expression, purification and characterization of a high potential iron-sulfur protein from Acidithiobacillus ferrooxidans

• Published in: Biotechnology letters Vol. 30; no. 5; pp. 905 - 910
• Main Authors: Zeng, Jia, Jiang, Huidan, Liu, Yuandong, Liu, Jianshe, Qiu, Guanzhou
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01-05-2008; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Acidithiobacillus - chemistry; Acidithiobacillus - genetics; Acidithiobacillus ferrooxidans; Amino Acid Sequence; Applied Microbiology; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Binding Sites; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Chromatography, Affinity; Cloning, Molecular; Cysteine - chemistry; E coli; Electron Spin Resonance Spectroscopy; Escherichia coli; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Gene expression; Iron; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - isolation & purification; Life Sciences; Microbiology; Models, Molecular; Molecular biology; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Original Research Paper; Photosynthetic Reaction Center Complex Proteins - chemistry; Photosynthetic Reaction Center Complex Proteins - isolation & purification; Plasmids; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfur; High potential iron–sulfur protein; Molecular modelling; Iron–sulfur cluster; Characterization; Acidithiobacillus ferrooxidans; Purification; High potential iron-sulfur protein,Iron-sulfur cluster, Molecular modelling; Iron; Sulfur; Modeling; Protein
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-007-9612-2

The high potential iron-sulfur protein (HiPIP) is involved in the iron respiratory electron transport chain of Acidithiobacillus ferrooxidans but its exact role is unclear. The gene of HiPIP from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, and the protein then purified by one-step affinity chromatography to homogeneity. The molecular mass of the HiPIP monomer was 7250.43 Da by MALDI-TOF MS, indicating the presence of the [Fe₄S₄] cluster. The optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys25, Cys28, Cys37 and Cys50 were involved in ligating to the iron-sulfur cluster.