Expression,Purification And Site-directed Mutagenesis Of The HdrC Subunit From Heterodisulfide Reductase In Acidithiobacillus Ferrooxidans

Several biological pathways for RISCs oxidation have been identified including the phylogenetically widespread sulfur oxidizing (sox) pathway and the archaeal type sulfur oxygenase reductase (sor) system.However,Sulfur oxidation in a variety of different A.ferrooxidans strains has been shown to have a novel sulfur oxidation pathway to require glutathione and the sulfane sulfur of persulfides is the actual substrate of the sulfur-oxidizing enzymes system.A heterodisulfide reductase complex HdrABC catalyze the reversible reduction of the disulfide bond X-S-S-X coupled with energy conservation in methanogenic archaea and sulfate reducing archaea and bacteria. The gene cluster of this complex have been detected in the genome of A.ferrooxidans.The predicted A.ferrooxidans heterodisulfide reductase complex has the new feature that catalytic sulfane sulfate (GSSH) to form sulphurous acid in the sulfur oxidation pathway.In our researches,we report the cloning,expression,purification, renaturation of two genes(hdrCl and hdrC2) of the HdrC subunit from A.ferrooxidans ATCC23270.We analyzed the structure and the function of the proteins by site-directed mutagenesis and bioinformatics technology.This HdrCl was expressed in inclusion bodies in all conditions tested. the purified protein HdrCl displayed brown color, the molecular weight is27KDa.There were two major absorption peaks between300-500nm,corresponding to330nm and410nm respectively by UV Scanning.EPR spectra results show a clear signal (the g factor is2.0173),these results indicated that the protein contained an iron-sulfur cluster.This HdrC owned two identical motifs for iron-sulfur cluster binding.But the sitedirected mutagenesis results revealed that HdrCl bound only one cluster,and the cluster was ligated by Cys63,Cys73,Cys76and Cys82.Molecular modeling results also supported the above results.This HdrC2also was expressed in inclusion bodies and the color is brown.The molecular weight is28KDa.There was one major absorption peaks between300-500nm,corresponding to415nm respectively by UV Scanning.EPR spectra results show a clear signal(the g factor is 2.0192),these results also indicated that the protein contained an iron-sulfur cluster.The sitedirected mutagenesis results revealed that HdrC2bound only one cluster,not two clusters.The cluster was ligated by Cys109,Cys112,Cys115and Cys119.Molecular modeling results also supported the above results.Compared with HdrCl and HdrC2,we found they have the same color,and all of them have one iron-sulfur cluster.But the ultraviolet absorption curve and the EPR signals are not the same.The reason is that the iron-sulfur cluster which these proteins connected are different.The HdrCl bounds an [Fe4S4] cluster, the HdrC2bounds an [Fe3S4] cluster. These conclusions demonstrated that this HdrC bound only one cluster, and it might be responsible for causing the HdrABC in A.ferrooxidans working in reverse.The two genes were cloned,overexpressed,purified and activity ass-ayed successfully in Escherichia coli,which provides the foundation for further studies of the function and application of HDR and for discussing its function and mechanism in the sulfur oxidation.