Oxidation of Sulfur and Inorganic Sulfur Compounds in the Thermoacidophilic Archaeon Acidianus ambivalens

嗜热嗜酸的古细菌在需氧生长时将单质硫氧化为硫酸作为电子供体和能量来源。我们最近纯化了几种氧化途径的酶，包括可溶性硫氧化酶还原酶（SOR），膜结合硫代硫酸盐：醌氧化还原酶（TQO）和可溶性四硫酸水解酶（TTH）。此外，我们确定了SOR的三维结构。本文的目的是在分子细节上阐明这三种酶的反应机制。对于SOR，我们计划结合位点定向诱变、光谱学、反应中间体的快速反应动力学和突变蛋白的x射线晶体学。通过TQO，我们希望建立基因表达，进行系统的诱变调查，并尝试解决三维结构。利用TTH我们想要鉴定基因，建立基因表达并做彻底的生化表征。对于这三种酶，我们将尝试识别未知的抑制剂作为活性位点结构的探针。

The thermophilic and acidophilic Archaeon Acidianus ambivalens oxidizes elemental sulfur to sulfuric acid as electron donor and energy source when growing aerobically. We purified several enzymes of the oxidation pathway recently including a soluble sulfur oxygenase reductase (SOR), a membrane-bound thiosulfate:quinone oxidoreductase (TQO) and a soluble tetrathionate hydrolase (TTH). In addition, we determined the 3D structure of the SOR. The goal of this proposal is to elucidate the reaction mechanisms of each of the three enzymes in molecular detail. With the SOR we plan to use a combination of site-directed mutagenesis, spectroscopy, rapid reaction kinetics of reaction intermediates, and X-ray crystallography of mutant proteins. With the TQO we want to establish gene expression, to do a systematic mutagenesis survey, and try to solve the 3D structure. With the TTH we want to identify the gene, establish gene expression and do a thorough biochemical characterization. With all three enzymes we will try to identify yet unknown inhibitors as probes for the active site structure.