MECHANISMS INVOLVED IN FLAVIN-LINKED OXYGEN METABOLISM

本研究的长期目标是了解 三种独特的链球菌黄素蛋白 催化通常归因于血红素蛋白或血红素的反应， 依赖过程。 研究两者的催化作用 黄素和非黄素氧化还原中心将是必不可少的， 关键结构-功能关系的总体评价 在三种酶中起作用， 链球菌氧代谢 重组DNA方法将应用于FAD的研究， 含有NADH过氧化物酶：需要解决的具体问题 包括所提出的氧化还原的半胱氨酸肽序列， 活性二硫键和过氧化物酶之间的总体相关性 和黄素蛋白二硫键还原酶。 在吾等 机制的研究，两个不同的复合物的作用， 还原过氧化物酶与吡啶核苷酸在两个过氧化物 还原和转氢将通过静态 滴定和快速反应技术，将具体的作用 半胱氨酸硫醇在过氧化物还原中的作用。 对不寻常的含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， 酶和这个中心与黄素的相互作用， 氧的全部四电子还原。 可能发挥的作用 催化机制中的氧化黄素中间体将 还将通过停流法进行研究，包括 用修饰的FAD类似物重构的酶的研究。 的 NADH氧化酶可能的结构和功能关系 将通过蛋白质化学检查NADH过氧化物酶， 分子遗传学方法 关于甲型链球菌有两个主要问题- 甘油磷酸氧化酶 氢化物和 碳负离子介导的底物降解是重要的一种， 和天然底物的卤代类似物将用于 更详细地探讨这个问题。 第二，比较 这种黄素蛋白和含有FAD的厌氧α- 甘油磷酸脱氢酶;大肠杆菌应该提供有价值的 有关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.