FLAVIN-PROTEIN INTERACTIONS IN FLAVOENZYME CATALYSIS

电子转移反应在广泛的基础化学中起着关键作用。 生物现象，包括呼吸作用、光合作用、氮 固定和类固醇和DNA的生物合成，以及在更多 直接与健康相关的领域，如化学致癌，药物 解毒、自由基损伤和衰老。 该项目作为其 长期目标：阐明分子之间的关系 参与这些过程的蛋白质的结构及其 生物化学和生物物理特性。 目前的建议侧重于 这个问题的两个方面：一，发生电子转移反应 在瞬时蛋白质-蛋白质和蛋白质-膜复合物内; ii. 多氧化还原中心蛋白质内的分子内电子转移。 在 其中第一个，两个小分子之间的电子转移动力学 氧化还原蛋白、铁氧还蛋白和黄素氧还蛋白及其生理电子 将使用激光闪光光解方法研究施主和受主。 该技术允许直接测量关联的速率 形成生产性电子转移复合物的步骤，以及 复合物内电子转移步骤，在结构未改性的系统。 作为供体，膜结合的植物光系统I和可溶性的人 将利用线粒体铁氧还蛋白还原酶;受体包括 可溶性植物铁氧还蛋白-NADP+还原酶和膜相关的 线粒体细胞色素P450SCC系统。 的位点特异性诱变 铁氧还蛋白，黄素氧还蛋白和还原酶将被用来探索 特定带电和芳香族氨基酸侧链的作用 在络合物形成和电子转移的过程中。 该地区 的多中心氧化还原蛋白，注意力将集中在作用， 带电荷和芳香族氨基酸侧链，同样使用激光光解 和位点特异性诱变技术，在分子内转移 酵母黄细胞色素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，研究 I型和II、III型铜之间的分子内电子转移 抗坏血酸氧化酶和漆酶的中心将进行。 是 预计这些研究将提供新的见解的作用 蛋白质结构在控制分子识别、特异性和 生物电子传递过程的速率。

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.