Characterization, Structure And Mechanism Of Sulfide:Quinone Oxidoreductase From Acidithiobacillus Ferrooxidans

Sulfide:quinone oxidoreductase (SQR) is a ubiquitous ancient flavoprotein, which is involved in the electron transfer from sulfide to quinone via flavin adenine dinucleotide. The enzymatic reactions catalyzed by SQR include the oxidation of sulfide to soluble polysulfide chains or to elemental sulfur in the form of octasulfur rings; this oxidation is coupled to the reduction of quinone. In bioleaching bacteria Acidithiobacillus ferrooxidans, sulfur oxidation is catalyzed by the sulfide:quinone oxidoreductase (SQR) system and the initial step is the oxidation of sulfide to sulfur by SQR.In this study, we report the heterologous expression, purification, enzymology and biochemical characteristics, phylogenetic analysis, crystal structure and the sulfide oxidation mechanism of SQR from Acidithiobacillus ferrooxidans ATCC23270.The purified SQR is a48kDa monomer with sulfide:quinone redox activity and has a bright yellow color due to the cofactor FAD (1mol per mol protein, non-covalently bound). The recombinant SQR showed maximum activity at30℃and pH7.0, the Km values for sulfide and for decylubiquinone were determined to be6.3μM and14.6μM respectively. SQR was stable when incubated at0℃,23℃or30℃with80-90%of the activity remaining after2.5hrs. Purified SQR lost activity at45℃with a half-life of approximately1hr. The maximum visible absorption of the protein was at375and450nm, which is typical for proteins containing FAD as a cofactor. The subcellular localization study showed SQR was a peripheral membrane protein binding to the inner membrane by hydrophobic interaction.In order to investigate the role of the highly conserved cysteine residues that might be critical for SQR activity, site-directed mutagenesis was used for making variants. The Cys128Ser variant lost all the DUQ reduction activity, and the Cys128Ala had only30-50%DUQ reduction activity left. Both of Cys128Ser and Cys128Ala variants exhibited full activity in the FAD reduction assay. The Cys160Ala variant lost all the DUQ reduction activity, but still exhibited10%of FAD reduction activity. The Cys356Ala variant showed complete loss of activity in both DUQ reduction assay and FAD reduction assay.This study first determed the complete structures of At. ferrooxidans SQR and its cysteine variants. The overall structure of At. ferrooxidans SQR comprises two tandem Rossmann fold domains and a very flexible C-terminal domain containing two amphipathic helices that are thought to provide for membrane binding; there also is one noncovalently bound flavin adenine dinucleotide (FAD) cofactor in the first tandem Rossmann fold domain. Several wide channels connect the FAD cofactor to the exterior of the protein molecule; some of the channels would provide access to the membrane. The ubiquinone molecule is bound in one of these channels; its benzoquinone ring is stacked between the aromatic rings of two conserved Phe residues, and it closely approaches the isoalloxazine moiety of the FAD cofactor. Two active-site cysteine residues (Cys160and Cys356) situated on the re side of the FAD cofactor form a branched polysulfide bridge.Based on both the enzymology and biochemical characteristics and structal information, we proposed a sulfide oxidation mechanism of SQR from At. ferrooxidans, which involves Cys356disulfide acts as a nucleophile that attacks the C4A atom of the FAD cofactor in electron transfer reaction. The polysulfide chain grows on Cys160. The third essential cysteine Cys128(most likely in the form of a disulfide) is confined to the release of the polysulfur product.