Expression, Purification And Site-directed Mutagenesis Iron Oxidase, Rusticyanin And Iron Sulphur Cluster Protein A From Acidithiobacillus Ferrooxidans

Rusticyanin is a small blue copper protein with extreme acidstability and redox potential. The protein is thought to be a principalcomponent in the iron respiratory electron transport chain of A.ferrooxidans, and its function is proposed to mediate electron transferbetween a cytochrome c and the terminal cytochrome c oxidase. The geneof rusticyanin from A. ferrooxidans was cloned,overexpressed andpurified by one-step affinity chromatography. We constructed the mutantexpression plasmid Prus (C138A, C138D, C138S,H85A,H143A) of theprotein using site-directed mutagenesis. The UV-Vis scanning and EPRspectrum results confirmed that residue C138 is an important residue forbinding with copper atom.The Iro protein was proposed to be involved in the iron respiratoryelectron transport chain of A. ferrooxidans, it is a member of HiPIPfamily with the iron-sulfur cluster for electron transfer. The gene of Iroprotein from A. ferrooxidans Fe-1 was cloned, expressed and purified byone-step affinity chromatography. The recombinant protein was observedto be dimmer. The molecular mass of a monomer of the Iro protein was6860.6 by MALDI-TOF MS. The optical and EPR spectra results of therecombinant protein confirmed that the iron-sulfur cluster was correctlyinserted into the active site of the protein. Molecular modeling for theprotein revealed that Cys20, Cys23, Cys32 and Cys45 were in ligatingwith the iron-sulfur cluster, and Tyr10 was important for the stability ofthe [Fe₄S₄] cluster. As we know, this is the first report of expression inE.coli of the Iro protein from A.ferrooxidans Fe-1.Three proteins of IscS (a cysteine desulfurase), IscU (a scaffoldprotein) and IscA (an iron chaperon) encoded by the operon iscSUA wereproposed to be involved in the iron-sulphur cluster assembly in A.ferrooxidans, but the mechanism is still not clear so far. The gene of IscAfrom A.ferrooxidans ATCC 23270 was cloned, expressed and purified byone-step affinity chromatography. The optical and EPR spectra results ofthe recombinant IscA confirmed that the iron-sulfur cluster was correctlyinserted into the active site of the protein, which was capable of recruiting intracellular iron and sulfide and hosted a stable [Fe₄S₄] cluster. The[Fe₄S₄] cluster can be assembled in apoIscA with ferrous iron and sulfidein vitro.We constructed the mutant expression plasmid Prus (C35A,C99A,C101A) of the protein using site-directed mutagenesis. Molecularmodelling for the protein and UV-Vis scanning and EPR spectrum resultsrevealed that Cys99 and Cys101 were in ligating with the iron-sulfurcluster, and Cys35 may be involved in the cluster binding. The IscA fromA. ferrooxidans may function as a scaffold protein for the pre-assembly ofFe-S cluster and then transfer it to target proteins, which is required formaturation of other cellular Fe-S proteins such as HiPIP in A.ferrooxidans.