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

拟议的项目是在NSF项目DMB 8715560下进行的研究的直接延续，建立和利用了该调查的发现。来自大肠杆菌的富马酸还原酶将被用作研究蛋白质假体基组装和功能的模型系统。这种酶复合物特别适合这些研究，因为它很容易遗传操纵，复合物稳定，可以扩增到高水平用于分离和分析，并且它在结构和功能上与琥珀酸脱氢酶相似，琥珀酸脱氢酶是线粒体电子传递链的关键组成部分。特别是研究将提供关于共价结合黄素的组装和功能的信息，酶的不同铁硫簇(?2Fe-2S！, 4 fe-4s !， ？3Fe-4S!)和与醌相互作用的膜内结构域的位点。我们将使用生物化学、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诱变，分析产生的突变酶复合物的酶功能，并通过EPR、磁性CD、ENDOR和穆斯堡尔光谱的组合来监测铁硫簇的组装。这项研究将有助于我们理解复杂的黄蛋白氧化还原酶如何组装成功能性的膜蛋白复合物，从而有助于阐明电子通过这些蛋白质的传递途径。

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.