Structure/Function of Complex ll Oxidoreductases

描述(由申请人提供)： 本研究项目涉及膜结合呼吸蛋白琥珀酸酯：泛醌氧化还原酶(复合体II)及其细菌同系物的研究。电子从共价结合的黄素辅因子，通过一系列的铁-硫簇，到蛋白质结合的醌和血红素的电子转移机制是研究的重点。研究表明，复合物II的苯醌和血红素结合域的突变与真核生物的过早衰老以及人类副神经节瘤和嗜铬细胞瘤等高度血管化的肿瘤的形成有关。建议的研究将利用最近描述的来自大肠杆菌的复合体II的X射线晶体结构，这是一个很好的哺乳动物酶模型系统。将构建模拟真核酶中类似保守残基的位点特异性氨基酸替换。将对野生型和突变型酶进行生化和生物物理分析，以剖析对电子转移和催化活性的影响。活性和形成活性氧物种的能力的改变将与在真核细胞和哺乳动物突变中发现的表型相关。这些研究的重点将是络合物II的苯醌和血红素结合域。研究旨在探讨b血红素辅助因子是否直接参与电子传递途径，或者它是否作为电子接收器，可能只在反向电子转移中发挥作用，如在缺氧或缺血期间可能发生的那样。还将研究可能稳定络合物II催化循环中生成的半喹酮物种的氨基酸残基。还将研究酶的铁-硫簇如何控制电子转移速率，以及这种门电子是否进入/离开苯二酚。在这些研究中，将开发复合体II的野生型和突变体的额外结构，以帮助进行结构分析，并帮助描述导致肿瘤形成的复合体II的生化缺陷。

DESCRIPTION (provided by applicant): This research project involves the study of the membrane-bound respiratory protein succinate:ubiquinone oxidoreductase (Complex II) and its bacterial homologs. The mechanism of electron transfer from the covalently bound flavin cofactor, through the series of iron-sulfur clusters and to a protein bound quinone and heme are a focus of the studies. Mutations in the quinone and heme-binding domains of Complex II have been shown to be linked to premature aging in eukaryotes and the formation of highly vascularized tumors such as paragangliomas and pheochromocytomas in humans. The proposed studies will take advantage of the recently described x-ray crystal structure for Complex II from Escherichia coli which is an excellent model system for the mammalian enzyme. Site-specific amino acid substitutions will be constructed that mimic the similarly conserved residues in the eukaryotic enzymes. Biochemical and biophysical analysis of wild type and mutant enzymes will be done to dissect the effects on electron transfer and catalytic activity. Alteration in activity and the ability to form reactive oxygen species will be correlated with the phenotype found in the eukaryotic and mammalian mutations. The particular focus of these studies will be the quinone and heme-binding domains of Complex II. Studies are designed to investigate if the b heme cofactor is directly involved in the electron transport pathway or if it serves as an electron sink which may only function in reverse electron transfer such as could occur during periods of hypoxia or ischemia. Studies will also be done on the amino acid residues which may stabilize the semiquinone species generated during the catalytic cycle of Complex II. How the iron-sulfur clusters of the enzyme control the electron transfer rate and if this gates electron entry to/from the quinone will also be investigated. During these investigations additional structures of wild-type and mutants of Complex II will be developed to aid in structural analysis and to assist in describing the biochemical defects of Complex II that lead to tumor formation.