FLAVIN-PROTEIN INTERACTIONS IN FLAVOENZYME CATALYSIS

电子转移反应在广泛的碱性反应中起着关键作用 生物现象，包括呼吸作用、光合作用、氮素 类固醇和DNA的固定和生物合成，以及更多 与健康直接相关的领域，如化学致癌、药物 解毒、自由基损伤、衰老。这个项目有其独特的优势 长期目标--阐明分子间的关系 参与这些过程的蛋白质的结构和它们的 生化和生物物理特性。目前的提案重点是 这个问题的两个方面：一、发生的电子转移反应 在瞬时蛋白质-蛋白质和蛋白质-膜复合体内； 多氧化还原中心蛋白质中的分子内电子转移。在……里面 第一，电子在两个小分子之间转移的动力学 氧化还原蛋白、铁氧还蛋白和黄还蛋白及其生理电子 供体和受体，将用激光闪光光解方法进行研究。 这项技术允许直接测量关联度 形成有效的电子转移络合物的步骤，以及 复合体内电子转移步骤，在结构未改变的体系中。 作为供体，膜结合植物光系统I和可溶性人类 将利用线粒体铁氧还蛋白还原酶；受体包括 植物可溶性铁还蛋白-NADP+还原酶与膜相关 线粒体细胞色素P450scc系统。基因的定点突变 铁氧还蛋白、黄还蛋白和还原酶将被用来探索 特殊带电氨基酸和芳香族氨基酸侧链的作用 在络合物的形成和电子转移过程中。在该地区 在多中心氧化还原蛋白中，人们的注意力将集中在 荷电和芳香氨基酸侧链，再次使用激光光解 和定点突变技术，在分子内转移中 酵母黄色素b2的黄素和血红素辅因子之间的电子 (乳酸脱氢酶)，以及b2血红素之间的电子转移 和生理受体细胞色素c。此外，对 第一类铜与第二类、第三类铜之间的分子内电子转移 将开展抗坏血酸氧化酶和漆酶中心的研究。它是 预计这些研究将为以下方面提供新的见解 蛋白质结构在控制分子识别、特异性和 生物电子转移过程中的速率。

Electron transfer reactions play key roles in a wide range of basic biological phenomena, including respiration, photosynthesis, nitrogen fixation and the biosynthesis of steroids and of DNA, as well as in more directly health related areas such as chemical carcinogenesis, drug detoxification, free radical damage and aging. This project has as its long-range goal the elucidation of the relationships between the molecular structures of the proteins involved in these processes and their biochemical and biophysical properties. The current proposal focusses on two aspects of this problem: i. electron transfer reactions occurring within transient protein-protein and protein-membrane complexes; ii. intramolecular electron transfer within multi-redox center proteins. In the first of these, the kinetics of electron transfer between two small redox proteins, ferredoxin and flavodoxin, and their physiological electron donors and acceptors, will be studied using laser flash photolysis methods. This technique allows direct measurements of the rates of the association step to form a productive electron transfer complex, and of the intracomplex electron transfer step, in structurally unmodified systems. As donors, the membrane-bound plant Photosystem I and the soluble human mitochondrial ferredoxin reductase will be utilized; acceptors include the soluble plant ferredoxin-NADP+ reductase and the membrane-associated mitochondrial cytochrome P450scc system. Site-specific mutagenesis of ferredoxins, flavodoxin and the reductases will be used to explore the roles of specific electrically charged and aromatic amino acid side chains in the processes of complex formation and electron transfer. In the area of multicenter redox proteins, attention will be focussed on the roles of charged and aromatic amino acid side chains, again using laser photolysis and site-specific mutagenesis techniques, in the intramolecular transfer of electrons between the flavin and heme cofactors of yeast flavocytochrome b2 (lactate dehydrogenase), and in the electron transfer between the b2 heme and the physiological acceptor cytochrome c. Additionally, studies of intramolecular electron transfer between the type I and type II,III copper centers of ascorbate oxidase and laccase will be carried out. It is anticipated that these studies will provide new insights into the roles of protein structure in controlling molecular recognition, specificity and rates in biological electron transfer processes.