Assembly and Function of the Cofactors of Fumarate Reductasefrom Escherichia Coli

这项研究的目的是扩大知识， 铁-硫簇和共价黄素组装， 在膜结合蛋白复合物中的功能 电子传输和能量守恒。 这些研究 旨在回答现有的问题， 三个铁硫团簇之间的相互关系 已知存在于富马酸还原酶中以及是否存在 是"黄素连接"酶催化 形成共价的黄素蛋白复合物。 的 研究还将解决有关这种关系的问题 铁硫簇结构和蛋白质序列之间的关系 以及单个团簇在电子 运输 用于实现这些目标的方法包括 富马酸还原酶的遗传修饰 抗坏血酸定向诱变和化学诱变方法。 然后将通过生物化学方法分析修饰的蛋白质。 为了确定催化剂的催化活性 修饰的酶及其细胞定位（膜结合或 细胞质）。 此外，我们将使用生物物理 例如EPR和磁性圆二色性的方法， 监测铁硫的功能和结构 酶的簇。 这项研究将有助于我们 了解复杂的黄素蛋白氧化还原酶 组装成功能性蛋白质复合物， 阐明了电子传输的路径， proteins.

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.