Assembly and Function of the Cofactors of Fumarate Reductase from Escherichia coli

拟议的项目是已进行的研究的直接延续 根据NSF项目DMB 8715560，建立和利用研究结果， 参与调查 来自大肠杆菌的富马酸还原酶 将被用作研究装配和功能的模型系统 蛋白质辅基。 该酶复合物特别是 非常适合这些研究，因为它很容易遗传 操纵，复合物是稳定的，可以放大到高 用于隔离和分析的水平，其结构类似于 并对琥珀酸脱氢酶起作用， 线粒体电子传递链。 特别是 调查将提供关于组装和功能的信息 共价结合的黄素，不同的铁硫簇， 酶（？2Fe-2S！，? 4Fe-4S！? 3Fe-4S！）以及位于 与醌类相互作用的膜内结构域。 的 生物化学、分子生物学和生物物理学的方法将 可以用来研究这些问题。 定点诱变 将进行特定氨基酸残基的分析， 分析突变酶复合物的酶功能，和 铁硫团簇的组装将由一个组合来监测。 EPR、磁性CD、ENDOR和穆斯堡尔谱。 这 研究将有助于我们理解 黄素蛋白氧化还原酶组装成功能膜 蛋白质复合物，从而有助于阐明电子的路径 通过这些蛋白质运输。

The proposed project is a direct continuation of studies conducted under NSF project DMB 8715560, building on and exploiting findings in that investigation. Fumarate reductase from Escherichia coli will be used as a model system for studies on assembly and function of protein prosthetic groups. This enzyme complex is particularly well suited for these studies in that it is easily genetically manipulated, the complex is stable and can be amplified to high levels for isolation and analysis, and it is similar in structure and function to succinate dehydrogenase which is a key component of the mitochondrial electron transport chain. In particular the investigations will provide information on assembly and function of covalently bound flavins, the different iron-sulfur clusters of the enzyme (?2Fe-2S!, ?4Fe-4S!, ?3Fe-4S!) and the sites in the membrane-intrinsic domain that interact with quinones. The approaches of biochemistry, molecular biology, and biophysics will be used to investigate these questions. Site-directed mutagenesis of specific amino acid residues will be performed and the resulting mutant enzyme complexes analyzed for enzymatic function, and assembly of iron-sulfur clusters will be monitored by a combination of EPR, magnetic CD, ENDOR, and Mossbauer spectroscopies. This research will contribute to our understanding of how complex flavoprotein oxidoreductases assemble into functional membrane protein complexes and thus help elucidate the path of electron transport through such proteins.