Characterization,Structure And Function Of Sulfide Quinone Oxidoreductase From Extremphle Acidithiobacillus Caldus

Sulfide quinone oxidoreductase（SQR）is an ancient riboflavin protein,which belongs to the disulfide oxidoreductase family（DSR）,and its main function is to oxidize sulfide to elemental sulfur.And transfer electrons to quinone to complete the respiratory chain.Acidithiobacillus caldus is dominant microorganism in the bioleaching process.Its main energy source is sulfur metabolism,and SQR is the first key enzyme in the sulfur oxidation reaction pathway,which plays an important role in sulfur metabolism.In this study,the sulfide quinone oxidoreductase SQR^Ac derived from A.caldus was used as the object,and a series of studies were carried out on the properties,functions and catalytic mechanism of SQR^Ac by using molecular biology and bioinformatics techniques.（1）Through the construction of the phylogenetic tree of SQR homologous sequences and the study of subcellular localization,it was identified that SQR^Ac belongs to TypeⅠof SQR proteins among the six type proteins,and has three conserved cysteine sites（Cys128,Cys160,Cys356）.It was determined that the SQR^Ac protein is an internal membrane protein and there is no signal peptide and transmembrane structure inside the protein.（2）The Escherichia coli protein expression strain BL21-p ET28a:sqr was constructed by homologous recombination,and the intracellular soluble expression of SQR^Ac in E.coli BL21（DE3）was realized.The molecular mass of recombinant SQR^Acis approximately 47.7 k Da.The kinetic parameters of the enzymatic reaction of SQR^Acwere determined:the K_{m（sulfide）}was 23.5μM,and the V_{max（sulfide）}was 0.51±0.05μmol·min^-1·mg^-1,K_m（DUQ）is 17.4μM,V_max（DUQ）is 0.60±0.05μmol·min^-1·mg^-1.By measuring the change of characteristic absorption peaks of SQR^Ac protein at 375nm and 450nm,it is determined that 1mol of SQR^Ac protein contains about 1mol of FAD.（3）The 3D structure model of SQR^Ac was obtained through Alpha Fold protein structure prediction.The protein structure of SQR^Ac conforms to the characteristics of disulfide bond oxidoreductase family proteins,and consists of two tandem Rossmann fold domains and a very flexible C-terminal domain composition.A single Rothmann fold domain consists of six parallelβ-sheets and aβ-α-β-αtopology formed by two pairs ofα-helices.Comparing the structure of SQR^Ac with the SQR^Af structure of acidic Acidithiobacillus ferrooxidans,it is found that the C-terminal conformations of the two proteins are significantly different.It is speculated that the possible reason is that the C-terminalα-helix of SQR^Af is extremely flexible,which makes the conformations of the two proteins significantly different.（4）Five key amino acid sites（Cys128,Cys160,Cys356,His132,and His198）were screened out by simulated alanine mutation scanning and homologous sequence comparison analysis.After mutating the five sites into alanine,five expression strains of Escherichia coli were constructed.Five mutant proteins were isolated and purified,and the in vitro sulfide quinone redox reaction experiments were performed.And it was found that the mutations of Cys128,Cys160 and Cys356 would greatly reduce the enzyme activity of the protein,while the mutations of His132 and His198 have no significant effect on enzyme activity.Combining the three-dimensional structural features of SQR^Ac and the in vitro sulfide quinone redox reaction experiments of mutant proteins,a sulfur redox mechanism model suitable for Acidithiobacillus caldus was proposed.