Structure/Function of Complex II Oxidoreductases

本项目旨在研究膜结合呼吸复合体II中电子传递过程中发生的综合事件。采用了两种模型体系，分别是来自大肠杆菌的琥珀酸-泛醌氧化还原酶（SQR，琥珀酸脱氢酶）和甲基萘酚-富马酸氧化还原酶（QFR，富马酸还原酶）。两者都是研究复合体II功能的优秀模型系统，复合体II在线粒体的代谢过程中起着重要作用，因此对细胞产生能量很重要。这些酶在结构上与真核酶相似，但更容易在基因上进行操作，也更容易生产大量可通过生化和生物物理方法进行研究的蛋白质。酶复合物似乎是从一个共同的进化前体进化而来的，在结构和功能上非常相似。富马酸还原酶通常在厌氧环境中起作用，而作为克雷布斯循环组成部分的SQR在有氧代谢中起作用。在功能上，这些酶可以进行同样的反应，即将琥珀酸氧化为富马酸和将富马酸还原为琥珀酸，但在体内，QFR在这两种反应中都比SQR更有效。这些研究旨在确定两种酶催化效率差异的结构和功能原因。最近的高分辨率QFR结构允许设计特定位点定向突变，这将解决催化位点和共价黄素辅助因子周围两种酶之间的差异。在这些研究中，将获得在醌结合位点结合抑制剂的高分辨率结构，以及与醌进行质子化/去质子化的氨基酸的身份。进一步的研究将探讨QFR和SQR中醌和血红素辅助因子之间的电子传递是如何发生的。这项研究将有助于了解呼吸复合物中醌结合位点的结构和功能，以及复合物II中蛋白质稳定的半醌发生的电子转移反应。

This project is intended to investigate the integrated events that occur during electron transport in membrane-bound respiratory Complex II. Two model systems are used, succinate- ubiquinone oxidoreductase (SQR, succinate dehydrogenase) and menaquinol-fumarate oxidoreductase (QFR, fumarate reductase) from Escherichia coli. Both are excellent model systems for investigating the function of Complex II that plays an important role in the metabolic processes that occur in mitochondria and is thus important for energy generation by the cell. These enzymes are structurally like their eukaryotic counterparts but much easier to manipulate genetically and for ease of production of large quantities of the proteins that can be studied by biochemical and biophysical methods. The enzyme complexes appear to have evolved from a common evolutionary precursor and are structurally and functionally very similar. Fumarate reductase usually functions in an anaerobic environment whereas, SQR a component of the Krebs cycle functions during aerobic metabolism. Functionally the enzymes can carry out the same reactions of oxidizing succinate to fumarate and reducing fumarate to succinate, however, QFR is much more efficient in both reactions than is SQR in vivo. These studies are intended to determine the structural and functional reasons for the differences in catalytic efficiency of the two enzymes. A recent high resolution structure of QFR allows design of specific site- directed mutations that will address the differences between the two enzymes around the catalytic site and covalent flavin cofactor. High resolution structures with inhibitors bound at the quinone binding sites will be obtained during these studies, as will the identity of amino acids involved in protonation/deprotonation with quinones. Additional studies will investigate how electron transport between the quinone and heme cofactors in QFR and SQR occurs. This research will help to understand the structure and function of quinone binding sites in respiratory complexes and the electron transfer reactions that take place at protein stabilized semiquinones in Complex II.