MOLECULAR GENETICS OF FLAVIN AND FE-S CONTAINING ENZYMES

这个研究项目的目的是为了更清楚地了解 黄素和含[Fe-S]酶的结构和功能， 参与真核细胞和 原核生物 主要的努力集中在细菌模型系统上 包括富马酸还原酶复合物（Frd）和琥珀酸还原酶复合物， 脱氢酶复合物（Sdh）。 两种细菌 酶是研究这类蛋白质 酶相互转化富马酸盐和琥珀酸盐，并发挥协同作用 与细胞电子传递机制一起完成ATP合成 通过氧化磷酸化过程。 细菌的酶是 在结构上非常相似，尽管每个都有独特的不同， 就其辅基氧化还原性质和醌的类型而言， 采用 自从大肠杆菌的分子和生物化学工具 Frd和Sdh系统非常发达，他们的持续 本申请应有助于阐明该装置的组装和结构。 一类膜结合的氧化还原酶复合物，其共价地含有 连接黄素和多个非血红素铁中心。 我们将评估 特定氨基酸在提供结构和功能中的作用 FrdB的[2Fe-2S]中心、[3Fe-4S]中心和[4Fe-4S]中心， FrdA的活性位点和膜中的醌相互作用位点 FrdCD亚基联合。 这些研究的预测将是 用E.杆菌 Frd和Sdh嵌合复合物 将通过使用蛋白质结构域交换方法产生，以测试是否 涉及特定酶性质的决定簇可以被定位。 从这些研究中获得的知识应该提供一个详细的分子 这类膜结合蛋白的结构和功能的描述 内切酶 我们正在使用非动物系统进行基础研究， 以具有成本效益和及时的方式解决人类问题， 等效实验系统尚未开发。

The objective of this research project is to gain a clearer understanding of the structure and function of flavin and [Fe-S] containing enzymes that participate in electron transfer processes in both eucaryotic and procaryotic organisms. Major efforts center on the bacterial model systems including the fumarate reductase enzyme complex (Frd) and the succinate dehydrogenase enzyme complex (Sdh) of Escherichia coli. The two bacterial enzymes are powerful models to examine questions regarding how this class of enzymes inter-convert fumarate and succinate, and function in concert with the cellular electron transport machinery to accomplish ATP synthesis via oxidative phosphorylation processes. The bacterial enzymes are remarkably similar structurally although each is uniquely different with respect to their prosthetic group redox properties and the type of quinone used. Since the molecular and biochemical tools for the Escherichia coli Frd and Sdh systems are extremely well developed, their continued application should aid in elucidating the assembly and structure of this class of membrane bound oxidoreductase complexes that contain covalently linked flavin and multiple non-heme iron centers. We will evaluate the roles of specific amino acids in providing structaure and function at the [2Fe-2S] center, the [3Fe-4S] center, and the [4Fe-4S] centers of FrdB, the active site of FrdA, and the quinone interaction sites in the membrane association FrdCD subunits. Predictions from these studies will be evaluated with the Sdh complex of E. coli. Chimeric Frd and Sdh complexes will be generated by use of protein domain swapping methods to test whether determinants referring specific enzyme properties can be localized. Knowledge gained from these studies should provide a detailed molecular description of the structure and function of this class of membrane bound enzymes. We are using a non-animal system to perform basic research in a cost effective and timely manner to address human problems for which no equivalent experimental system has yet been development.