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

拟议的项目是在国家科学基金会项目DMB 8715560下进行的研究的直接继续，以该调查的结果为基础并加以利用。大肠杆菌富马酸还原酶将作为研究蛋白质修复基团组装和功能的模型系统。这种酶复合体特别适合于这些研究，因为它易于遗传操作，复合体稳定，可以扩增到高水平用于分离和分析，而且它在结构和功能上与线粒体电子传输链的关键组成部分琥珀酸脱氢酶相似。特别是，这些研究将提供共价结合黄素的组装和功能信息，即酶(？2Fe-2S！，？4Fe-4S！，？3Fe-4S！)的不同铁-硫簇。以及与苯二酚相互作用的膜固有结构域中的位置。我们将运用生物化学、分子生物学和生物物理学的方法来研究这些问题。将对特定氨基酸残基进行定点突变，并分析所产生的突变酶复合体的酶功能，并将结合EPR、磁CD、Endor和穆斯堡尔光谱监测铁-硫簇的组装。这项研究将有助于我们理解复杂的黄素蛋白氧化还原酶如何组装成具有功能的膜蛋白复合体，从而有助于阐明电子通过此类蛋白的传递途径。

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.