P97 Characterization, structure and mechanism of sulfide:quinone oxidoreductase (SQR) from acidithiobacillus ferrooxidans

Abstract

A key enzyme in maintaining sulfide homeostasis is the membrane-associated flavoenzyme sulfide:quinone oxidoreductase (SQR) found in all domains of life (except plants). SQR maintains a critical equilibrium between sulfide (H2S, HS− and S2−) and elemental sulfur (S0), coupling the oxidation of sulfide to the reduction of ubiquinone via a non-covalent FAD cofactor. SQR interacts with the membrane via two amphipathic helices and the Q-pool through a conserved hydrophobic domain. The active site of SQR includes three cysteines (Cys160, Cys356 and Cys128) and the FAD in close juxtaposition to the ubiquinone binding site. To understand the sulfide oxidation mechanism, we expressed the wild-type sqr gene from Acidithiobacillus ferrooxidans, as well as several variants of conserved, catalytically important amino acids in E. coli BL21(DE3) and purified soluble, active, His-tagged SQR. The purified wild-type SQR and the variants were subjected to extensive kinetic (stopped-flow spectrophotometry) and structural analysis (X-ray crystallography). We also monitored SQR activity in vivo by detecting the H2S produced by growing E. coli transformed with wild-type and variant SQR. Our catalytic activity analysis and structural determination led us to propose two alternative mechanisms: (1) A nucleophilic attack mechanism that involves Cys356–S–S− as a nucleophile which attacks the C4A atom of FAD; or (2) A radical mechanism of direct electron transfer from Cys356 disulfide to FAD. The growing polysulfide is held between Cys160 and Cys356. The role of Cys128 (most likely in the form of a disulfide) is confined to the release of the polysulfur product.