Expression, Purification And Site-directed Mutagenesis The High Potential Iron-sulfur Protein And Iron Sulphur Cluster Protein A From Acidithiobacillus Ferrooxidans, Iron-sulfur Cluster Assembly Of The In Vitro

The high potential iron-sulfur protein(HiPIP)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 HiPIP 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.Molecular modelling for the protein revealed that Cys25,Cys28,Cys37 and Cys50 were in ligating with the iron-sulfur cluster.Molecular modelling for the protein and UV-Vis scanning and EPR spectrum results revealed that Cys25,Cys28,Cys37 and Cys50were in ligating with the iron-sulfur cluster.Three proteins of IscS(a cysteine desulfurase),IscU(a scaffold protein)and IscA(an iron chaperon)encoded by the operon iscSUA were proposed to be involved in the iron-sulphur cluster assembly in A. ferrooxidans,but the mechanism is still not clear so far.The gene of IscA from A.ferrooxidans ATCC 23270 was cloned,expressed and purified by one-step affinity chromatography.The optical and EPR spectra results of the recombinant IscA confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein,which was capable of recruiting intracellular iron and sulfide and hosted a stable[Fe₂S₂]cluster.The [Fe₂S₂]cluster can be assembled in apoIscA with ferrous iron and sulfide in vitro.We constructed the mutant expression plasmid Prus(D97A, E103A)of the protein using site-directed mutagenesis.Molecular modelling for the protein and UV-Vis scanning and EPR spectrum results revealed that D97 were in ligating with the iron-sulfur cluster and Cys35 may be not involved in the cluster binding.The IscA from A. ferrooxidans may function as a scaffold protein for the pre-assembly of Fe-S cluster and then transfer it to target proteins,which is required for maturation of other cellular Fe-S proteins such as HiPIP in A. ferrooxidans.The high potential iron-sulfur protein(HiPIP)has been proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans,which contains a[Fe₄S₄]cluster.We report here the assembly of[Fe₄S₄]cluster in HiPIP from A.ferrooxidans ATCC 23270 in vitro in the presence of Fe²⁺and sulfide.The spectra and MALDI-TOF MS results of the holoHiPIP confirmed that the iron-sulfur cluster was correctly assembled into the protein.