MECHANISMS INVOLVED IN FLAVIN-LINKED OXYGEN METABOLISM

在吡啶中出现了一种新的黄素蛋白质类别 核苷酸：二硫键氧化还原酶家族。到目前为止有两种黄素蛋白 过氧化氢还原酶、NADH过氧化物酶和NADH氧化酶 粪链球菌，已被描述。两者之间的主要区别 这些酶和二硫键还原酶，如谷胱甘肽还原酶(GR) 位于不寻常的半胱氨酸-磺酸(Cys-SOH)氧化还原中心，它 取代了GR中发现的具有氧化还原活性的二硫键。这样做的一个主要目标是 建议书将涉及结构、功能和 用于阐明a)Cys-SOH结构的遗传学方法 氧化还原中心和蛋白质结构中有助于 它的稳定性，b)功能发散的结构基础 在NADH过氧化物酶(过氧化氢产生2H2O)和NADH氧化物酶(产生O2)之间 2H2O)，以及c)那些蛋白质结构的模块元素， 区分二硫化物还原酶和过氧化氢还原酶。动能 并对NADH过氧化物酶和NADH氧化酶的作用机制进行了探讨。 进一步通过稳态和快速反应的组合 技巧。在这种情况下，NADH氧化酶的氧反应性 为我们的 对黄素蛋白氧反应性的理解。 我们还将重点关注链球菌-阿尔法- 甘油磷酸酶，它似乎代表了仅有的两种 黄素蛋白氧化酶(O2产生过氧化氢)通过氢化物转移发挥作用 底物脱氢的机理。一个主要目标将是 确定两个明确定义的氧化酶的结构关系 类(α-羟基和α-氨基酸氧化酶)和 大肠杆菌需氧α-甘油磷酸脱氢酶。这个 要获得的信息不仅对 理解黄素蛋白中氢化物与碳负离子的作用机制 催化作用，但也区分黄素蛋白氧化酶和 脱氢酶起作用。 链球菌是缺乏血红素的兼性厌氧菌，它依赖于 完全依赖于底物水平的磷酸化来合成ATP。角色 这些黄素连接酶在链球菌氧化代谢中的作用，以及 它们对链球菌增殖的贡献，本质上是 与这些生物体参与人类疾病状态有关 如风湿热和心内膜炎。

A new flavoprotein class has emerged within the pyridine nucleotide:disulfide oxidoreductase family. To date two flavoprotein peroxide reductases, the NADH peroxidase and NADH oxidase from Streptococcus faecalis, have been described. The key distinction between these enzymes and disulfide reductases such as glutathione reductase (GR) lies in the unusual cysteine-sulfenic acid (Cys-SOH) redox center, which replaces the redox-active disulfide found in GR. A major goal of this proposal will involve the application of structural, functional, and genetic approaches in order to elucidate a) the structure of the Cys-SOH redox center and those elements of protein structure which contribute to its stabilization, b) the structural basis for the functional divergence between NADH peroxidase (H2O2 yields 2H2O) and NADH oxidase (O2 yields 2H2O), and c) those modular elements of protein structure which distinguish the disulfide and peroxide reductases. The kinetic mechanisms of NADH peroxidase and NADH oxidase will also be probed further through a combination of steady-state and rapid-reaction techniques. In this light, the oxygen reactivity of NADH oxidase provides the potential for significant, new contributions to our understanding of flavoprotein oxygen reactivity. We will also focus our efforts on the streptococcal alpha- glycerophosphate oxidase, which appears to represent one of only two flavoprotein oxidases (O2 yields H2O2) functioning via a hydride transfer mechanism in substrate dehydrogenation. A major goal will be to determine the structural relationship to the two well-defined oxidase classes (the alpha-hydroxy- and alpha-amino acid oxidases) and to the aerobic alpha-glycerophosphate dehydrogenase of Escherichia coli. The information to be gained should be significant not only to the understanding of hydride versus carbanion mechanisms in flavoprotein catalysis, but also to the distinction between flavoprotein oxidase and dehydrogenase functions. The streptococci are heme-deficient facultative anaerobes which rely entirely on substrate-level phosphorylation for ATP synthesis. The roles of these flavin-linked enzymes in streptococcal oxidative metabolism, and their contributions to streptococcal proliferation, are intrinsically linked to the involvement of these organisms in human disease states such as rheumatic fever and endocarditis.