MOLECULAR GENETICS OF FLAVIN AND FE-S CONTAINING ENZYMES

这项研究项目的目的是为了更清楚地了解 黄素和[Fe-S]含有的酶的结构和功能 参与真核生物和生物体内的电子转移过程 原核生物。主要工作集中在细菌模型系统上 包括富马酸还原酶复合体(Frd)和琥珀酸 大肠杆菌脱氢酶复合体(SDH)。这两种细菌 酶是一种强大的模型，可以用来检查关于这门课如何 相互转化富马酸和琥珀酸的酶，并协同发挥作用 利用细胞电子传递机制完成三磷酸腺苷合成 通过氧化磷酸化过程。细菌酶是 在结构上非常相似，尽管每个都与 关于它们的假体基团的氧化还原性质和苯二酚的类型 使用。由于大肠杆菌的分子和生化工具 FRD和SDH系统非常发达，他们继续 申请应有助于阐明该组件的组装和结构 一类膜结合的氧化还原酶复合体，含有共价 连接黄素和多个非血红素铁中心。我们将评估 特定的氨基酸在提供结构和功能方面的作用 FrdB的[2Fe-2S]中心、[3Fe-4S]中心和[4Fe-4S]中心， FrdA的活性部位和膜中的苯二酚相互作用部位 协会FrdCD亚基。这些研究的预测将是 用大肠杆菌的SDH复合体进行评价。嵌合Frd和SDH复合体 将通过使用蛋白质结构域交换方法来测试是否会产生 涉及特定酶性质的决定因素可以被定位。 从这些研究中获得的知识应该提供详细的分子 这类膜边界的结构和功能的描述 酵素。我们正在使用一个非动物系统来进行基础研究 以经济高效和及时的方式解决人类问题，而不是 等效实验系统尚未研制成功。

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.