Assembly and Function of the Cofactors of Fumarate Reductasefrom Escherichia Coli

提出的研究旨在扩大对铁硫簇和共价黄素如何在电子传递和能量守恒所需的膜结合蛋白复合物中组装和功能的了解。这些研究旨在回答有关已知存在于富马酸还原酶中的三个铁硫簇之间的相互关系以及是否需要“黄素连接”酶来催化形成共价黄素蛋白复合物的现有问题。该研究还将解决有关铁硫簇结构与蛋白质序列之间的关系以及单个簇在电子传递中的作用的问题。用于实现这些目标的方法包括通过寡核苷酸定向和化学诱变方法对富马酸还原酶进行遗传修饰。然后通过生化分析来分析修饰后的蛋白质，以确定修饰酶的催化活性及其细胞位置（膜结合或细胞质）。此外，我们将使用生物物理方法，如EPR和磁圆二色性来监测酶的铁硫簇的功能和结构。这项研究将有助于我们理解复杂的黄蛋白氧化还原酶是如何组装成功能性蛋白质复合物的，从而有助于阐明电子通过这些蛋白质的传递途径。

The research proposed is intended to expand knowledge on how iron-sulfer clusters and covalent flavins assemble and function in membrane-bound protein complexes required for electron transport and energy conservation. The studies are aimed at answering the existing questions relating to the interrelationship between the three iron-sulfur clusters known to be present in fumarate reductase and whether there is a requirement for a "flavin-ligating" enzyme to catalyze the formation of covalent flavin protein complexes. The research will also address questions about the relationship between iron-sulfur cluster structure and protein sequence and the role of the individual clusters in electron transport. Approaches used to meet these goals include genetic modification of fumarate reductase by both oligonucleotide-directed and chemical mutagenesis methods. The modified proteins will then be analyzed by biochemical assays in order to define the catalytic activity of the modified enzyme and its cellular location (membrane-bound or cytoplasmic). In addition we will use biophysical approaches such as EPR and magnetic circular dichroism to monitor the function and structure of the iron-sulfur clusters of the enzyme. This research will contribute to our understanding of how complex flavoprotein oxidoreductases assemble into functional protein complexes and thus help elucidate the path of electron transport through such proteins.