MECHANISMS INVOLVED IN FLAVIN-LINKED OXYGEN METABOLISM

这项研究的长期目标是了解 三种独特的链球菌黄素蛋白的作用机制 催化通常归因于血红素蛋白或血红素的反应- 从属进程。两者催化作用的研究 黄素和非黄素氧化还原中心将是必不可少的 全面评价关键结构--功能关系 在三种酶中起作用，这三种酶在 链球菌的氧代谢。 重组DNA方法将应用于FAD的研究-- 含NADH过氧化物酶：需要解决的具体问题 包括所建议的氧化还原的半胱氨酸肽序列- 活性二硫化物与过氧化物酶的总体相关性 以及黄素蛋白二硫键还原酶。在我们的背景下 力学研究，两个不同的复合体的作用 过氧化氢中含有吡啶核苷酸的还原过氧化物酶 还原和转氢反应将通过静态研究 滴定和快速反应技术，将发挥特定的作用 在过氧化氢还原中的半胱氨酸硫醇(S)。 对这种不寻常的含有FAD的NADH氧化酶的研究将集中在 第二个非黄素氧化还原中心的鉴定 酶及其与黄素中心的相互作用 氧的整体四电子还原。可能起到的作用 含氧黄素中间体在催化机理中会 还将通过停流法进行调查，将包括 改良型FAD类似物重组酶的研究。这个 NADH氧化酶可能的结构和功能关系 NADH过氧化物酶将通过蛋白质化学和 分子遗传学方法。 关于甲型链球菌有两个主要问题- 甘油磷酸酶。氢化物和氢化物之间的区别 碳负离子介导的底物脱氢反应是一种重要的反应。 以及天然底物的卤化类似物将被用于 更详细地探讨这个问题。第二，比较 在这种黄素蛋白和含有FAD的厌氧阿尔法之间- 大肠杆菌的甘油磷酸脱氢酶应提供有价值的 关于共享的结构和机械特征的信息 两个都是。

The long-term objectives of this study are to understand the mechanisms by which three unique streptococcal flavoproteins catalyze reactions normally ascribed to hemeproteins or to heme- dependent processes. Investigation of the catalytic roles of both flavin and non-flavin redox centers will be essential to the overall evaluation of the key structure-function relationships operative in the three enzymes, which are important in streptococcal oxygen metabolism. Recombinant DNA approaches will be applied to the study of the FAD- containing NADH peroxidase: the specific questions to be addressed include the cysteinyl peptide sequences of the proposed redox- active disulfide and the overall relatedness between the peroxidase and the flavoprotein disulfide reductases. In the context of our mechanistic studies, the roles of two distinct complexes of the reduced peroxidase with pyridine nucleotides in both peroxide reduction and in transhydrogenation will be investigated by static titration and rapid-reaction techniques, as will the specific role of cysteine thiol(s) in peroxide reduction. Studies of the unusual FAD-containing NADH oxidase will focus on the identification of the second non-flavin redox center in this enzyme and on the interaction of this center with the flavin in the overall four-electron reduction of oxygen. The possible role of oxygenated flavin intermediates in the catalytic mechanism will also be investigated by stopped-flow methods and will include studies of the enzyme reconstituted with modified FAD analogs. The possible structural and functional relationship of the NADH oxidase and the NADH peroxidase will be examined by protein chemistry and molecular genetic approaches. There are two major questions concerning the streptococcal alpha- glycerophosphate oxidase. The distinction between hydride- and carbanion-mediated substrate dehydrogenations is an important one, and halogenated analogs of the natural substrate will be used to probe this question in more detail. Secondly, the comparison between this flavoprotein and the FAD-containing anaerobic alpha- glycerophosphate dehydrogenase of E. coli should provide valuable information concerning structural and mechanistic features shared by both.