Enzymology of Sulfide Oxidation

描述(申请人提供)：硫化氢(H_2S)是一种能引起深刻生理效应的信号分子，是哺乳动物硫代谢的产物，合成速度相对较快。由于硫化氢是剧毒的，细胞通过一条有效的氧化途径来避免其积聚，该途径位于线粒体中，并与产生能量的电子传输链相连。组成蛋白包括硫代苯醌氧化还原酶(SQR)、过硫代双加氧酶(ETHE1)和罗丹宁，以及在该途径的最后一步催化亚硫酸盐转化为硫酸盐的亚硫酸盐氧化酶。在……里面 另一方面，红细胞表现出强大的硫化氢产生能力，但缺乏线粒体，因此是硫化物氧化的经典途径，其清除硫化物的机制尚不清楚。在这项研究中，我们建议解决我们对SQR和ETHE1催化的氧依赖步骤的理解上的根本空白，这两个步骤结合在一起可以维持较低的稳态H_2S水平(在大多数组织中在10-30 nm范围内)，并阐明罗丹在硫化物氧化途径中的作用。我们还建议阐明去除红细胞中高铁血红蛋白硫化物的潜在作用。这些目标将通过解决以下具体目标来实现。(I)利用光谱和动力学相结合的方法，我们将阐明人SQR和ETHE1的反应机理。(Ii)我们将评估氧化铁产生谷胱甘肽过硫化物、硫代硫酸盐和硫化氢的相对催化效率，并阐明两种常见的多态氧化铁变种之间的生化差异。(Iii)我们将阐明依赖高铁血红蛋白的硫化物氧化的机制，并评估其在生理上相关的硫化氢和高铁血红蛋白浓度下对硫化物清除的贡献。我们的研究将解决基本问题，如硫化物氧化途径是如何连接的，阐明关键酶的反应机制，这些酶可能是调节硫化氢水平的重要药物靶标，并评估人类血红蛋白在维持低血浆硫化氢浓度方面的新的和以前未被探索的作用。

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.