Expression, Purification Of Cytochrome C Oxidase And Site-directed Mutagenesis Of Iron Sulphur Cluster Regulator From Acidithiobacillus Ferrooxidans

The cytochrome c oxidase from Acidithiobacillus ferrooxidans was proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans, but its exact role is still unclear so far. The gene of cytochrome c oxidase from A. ferrooxidans ATCC 23270 was cloned and successfully expressed in Escherichia coli, finally purified by one-step affinity chromatography to homogeneity. The protein is thought to be a principal component in the iron respiratory electron transport chain of A.ferrooxidans. Though the interaction between cytochrome c oxidase and cytochrome c, the activity of cytochrome c oxidase was measured and the four subunits were found to maintain the bioactivity together.The iron - sulfur cluster regulator (IscR) was reported to be a repressor of the iscSUA operon. In vitro transcription reactions revealed that the IscR had a repression effect on the iscR promoter. The IscR contains a [Fe2S2] cluster per each monomer, and three highly conserved cysteines were identified to ligate the [Fe2S2] cluster. It was proposed that a non-cysteine residue might be the fourth ligand for the [Fe2S2] cluster. In this study, using site-directed mutagenesis, Glu43 was found to be the fourth residue that coordinates the [Fe2S2] cluster of IscR.