Enzymology of Sulfide Oxidation

描述（由申请人提供）：硫化氢（H2S）是一种产生深远生理效应的信号分子，是哺乳动物硫代谢的产物，合成速率相对较高。由于H2S具有高毒性，细胞通过位于线粒体中并与产生能量的电子传递链偶联的有效氧化途径来避免其积聚。组成蛋白质包括硫化物-醌氧化还原酶（SQR）、过硫化物双加氧酶（ETHE 1）和罗丹酸盐，以及研究充分的亚硫酸盐氧化酶，其在途径的末端步骤中催化亚硫酸盐转化为硫酸盐。在 另一方面，红细胞显示出强大的H2S生产能力，但缺乏线粒体，因此缺乏硫化物氧化的经典途径，硫化物清除的机制尚不清楚。在这项研究中，我们建议解决根本的差距，在我们的理解的氧依赖性步骤催化SQR和ETHE 1，联合收割机，以保持低稳态水平的硫化氢（在10-30纳米范围内，在大多数组织），并阐明的作用，硫氧化途径中的铑。我们还建议阐明高铁血红蛋白在红细胞中硫化物去除的潜在作用。这些目标将通过实现以下具体目标来实现。(i)使用光谱和动力学方法相结合，我们将阐明人类SQR和ETHE 1的反应机制。(ii)我们将评估的相对催化效率与rhodanese产生谷胱甘肽过硫化物，硫代硫酸盐和H2S，并阐明两种常见的多态rhodanese变体之间的生化差异。(iii)我们将阐明高铁血红蛋白依赖性硫化物氧化的机制，并评估其在生理相关浓度的硫化氢和高铁血红蛋白的硫化物清除的贡献。我们的研究将解决一些基本问题，如硫化物氧化途径是如何连接的，阐明关键酶的反应机制，这些酶是调节H2S水平的潜在重要药物靶点，并评估人类血红蛋白在维持低血浆H2S浓度方面的新的和以前未探索的作用。

DESCRIPTION (provided by applicant): Hydrogen sulfide (H2S), a signaling molecule that elicits profound physiological effects, is a product of mammalian sulfur metabolism and is synthesized at relatively high rates. Since H2S is highly toxic, cells avoid its build-up by an efficient oxidation pathway that is housed in mitochondria and coupled to the energy-generating electron transport chain. The constituent proteins include sulfide-quinone oxidoreductase (SQR), a persulfide dioxygenase (ETHE1) and rhodanese in addition to the well-studied sulfite oxidase that catalyzes the conversion of sulfite to sulfate in the terminal step in the pathway. In red blood cells on the other hand, which exhibit robust H2S production capacity but lack mitochondria and hence the classical route for sulfide oxidation, the mechanism of sulfide clearance is unknown. In this study, we propose to address fundamental gaps in our understanding of the oxygen-dependent steps catalyzed by SQR and ETHE1, which combine to maintain low steady-state levels of H2S (in the 10-30 nM range in most tissues) and to elucidate the role of rhodanese in the sulfide oxidation pathway. We also propose to elucidate the potential role of methemoglobin in sulfide removal in red blood cells. These goals will be realized by addressing the following specific aims. (i) Using a combination of spectroscopic and kinetic methods we will elucidate the reaction mechanisms of human SQR and ETHE1. (ii) We will assess the relative catalytic efficiencies with which rhodanese generates glutathione persulfide, thiosulfate and H2S and elucidate the biochemical differences between two common polymorphic rhodanese variants. (iii) We will elucidate the mechanism of methemoglobin-dependent sulfide oxidation and assess its contribution to sulfide clearance at physiologically relevant concentrations of H2S and methemoglobin. Our studies will address fundamental questions such as how the sulfide oxidation pathway is wired, elucidate the reaction mechanisms of key enzymes that are potentially important pharmaceutical targets for modulating H2S levels and assess a novel and previously unexplored role for human hemoglobin in maintaining low plasma H2S concentrations.