Expression, Purification And Interaction Of α, β Subunit Of The Sulfite Reductase From Acidithiobacillus Ferrooxidans

Sulfite Reductase(SiR)is a principal component in the sulfur oxidation electron transport chain of Acidithiobacillus ferrooxidans(A. ferrooxidans).It is a large and soluble enzyme which catalyzes the transfer of six electrons from sulfite to produce sulfide and plays a crucial role in the sulfate-reducing bacteria.We took a Acidithiobacillus ferrooxidans ATCC23270 genome team as a template,expressed through the gene recombination technology in Escherichia coli,purified the SiR withαsubunit(flavoprotein component, SiR-FP,the gene cysJ code)andβsubunit(hemoprotein component, SiR-HP,the gene cysI code)with high density and purity by one-step affinity chromatography.Then we did some assays to revealed its character and activity.To the purified Flavoprotein(SiR-FP),the molecular mass was 66 kDa measured by SDS-PAGE.UV-vis scanning showed absorption maxima at 405 and 580 nm,suggesting the existence of FAD and FMN. A 303.74%-fold purification was achieved and the recovery rate was 12.15%in the active assay.The activity is high.The sequences alignment and the molecular modeling of the Flavoprotein suggested that the existence of FAD and FMN while NADPH connects FAD and FMN connects hemoprotein.To the Hemoprotein,the molecular mass was 60 kDa measured by SDS-PAGE.UV-vis scanning showed absorption maxima at 388 and 598 nm,suggesting the existence of the[Fe₄S₄]cluster and the ferrisiroheme. EPR showed the presence of the[Fe₄S₄]cluster.The sequences alignment and the molecular modeling of the Hemoprotein suggest that Cys427, Cys433,Cys472 and Cys476 are four conserved cycteines for the[Fe₄S₄] cluster binding of the protein.Then Site-directed mutagenesis results strongly revealed that the four conserved cycteines of Cys427,Cys433, Cys472 and Cys476 were crucial residues for iron-sulfur cluster binding, and removal of the sulfhydryl group of the cysteines resulted in[Fe₄S₄] cluster cluster loss.Then carry on the interaction union according to 1:1 to produce artificial SiR.The recovery rate of the artificial SiR was 164.46% comparing with the Flavoprotein in the active assay.The enzyme is set up to arrange the redox cofactors in a FAD-FMN-Fe₄S₄-Heme sequence to make an electron pathway between NADPH and A.ferrooxidans was cloned,overexpressed purified and activity assayed successfully in Escherichia coli,which provides the foundation for further studies of the function and application of SiR,and for discussing its function and mechanism in the sulfur oxidation.