Oxidation of Sulfur and Inorganic Sulfur Compounds in the Thermoacidophilic Archaeon Acidianus ambivalens²

嗜热性古细菌Acidianus bivalens在氧气气氛下生长时，将元素硫氧化为硫酸作为能源。该生物体是火山温泉化学自养生活方式和生物浸出的重要模式器官。硫氧化途径中的一个关键酶是可溶性硫加氧酶还原酶(SOR)，它能将元素硫转化为硫化物和亚硫酸盐。我们已经测定了双壳拟青霉和嗜盐嗜盐细菌的SOR的三维结构。我们还对SOR突变体进行了研究，得出了到目前为止的假设反应机制和底物和产物路径的模型。这一建议的目的是在实验上重新评估我们的假设，并更详细地阐明SORS的反应机理。我们想确定在底物周转过程中，活性部位铁是否发生氧化还原变化，以及为什么氧化和还原反应产物的比例随着温度、pH和突变体的分析而变化。我们希望确定活性部位中的反应中间体和抑制剂加合物，并生成该酶不可或缺的残基图(S)。为了实现我们的目标，我们将结合使用更多的诱变试验、还原电位测定、各种光谱技术(Möbie Bauer、电子顺磁共振、振动光谱)、计算机模拟、快速反应动力学、抑制剂研究和蛋白质衍生物的X射线结晶学。

The thermoacidophilic Archaeon Acidianus ambivalens oxidizes elemental sulfur to sulfuric acid as energy source when growing under oxygen atmosphere. The organism is an important model organ-ism of the chemolithoautotrophic lifestyle in volcanic hot springs and in bioleaching. A key enzyme in the sulfur oxidation pathway is a soluble sulfur oxygenase reductase (SOR), which dismutates ele-mental sulfur to sulfide and sulfite. We had determined the three-dimensional structure of the SOR of A. ambivalens and of the mesophilic bacterium Halothiobacillus neapolitanus. We had also conducted studies with SOR mutants, which resulted in a so far hypothetical reaction mechanism and a model of substrate and product pathways. The goals of this proposal are to reappraise our hypotheses experi-mentally and to elucidate the reaction mechanism of the SORs in more detail. We want to determine whether a redox change occurs at the active site iron during substrate turnover and why the ratio of oxidized and reduced reaction products changes with temperature, pH and mutants analyzed. We want to identify reaction intermediates and inhibitor adducts in the active site and to generate a map of indispensable residues of the enzyme(s). To achieve our goals, we will use a combination of additional mutagenesis trials, reduction potential determination, various spectroscopic techniques (Mößbauer, electron paramagnetic resonance, vibrational spectroscopy), computer simulations, rapid reaction kinetics, inhibitor studies, and X-ray crystallography of protein derivatives.