Structure/Function of Complex II Oxidoreductases

本研究旨在探讨膜结合呼吸复合物II电子传递过程中发生的整合事件。 使用两种模型系统，来自大肠杆菌的琥珀酸-泛醌氧化还原酶（SQR，琥珀酸脱氢酶）和甲萘醌-延胡索酸氧化还原酶（QFR，延胡索酸还原酶）。 两者都是研究复合物II功能的极好模型系统，复合物II在线粒体中发生的代谢过程中起重要作用，因此对细胞产生能量很重要。 这些酶在结构上类似于它们的真核生物对应物，但更容易进行遗传操作，并且易于产生大量的蛋白质，这些蛋白质可以通过生物化学和生物物理方法进行研究。 这些酶复合物似乎是从一个共同的进化前体进化而来的，并且在结构和功能上非常相似。 富马酸还原酶通常在无氧环境中发挥作用，而SQR是克雷布斯循环的一个组成部分，在有氧代谢过程中发挥作用。在功能上，酶可以进行将琥珀酸氧化为富马酸和将富马酸还原为琥珀酸的相同反应，然而，QFR在两种反应中比体内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。 QFR最近的高分辨率结构允许设计特定的定点突变，这将解决两种酶之间围绕催化位点和共价黄素辅因子的差异。 在这些研究中，将获得高分辨率的结构与抑制剂结合在醌结合位点，将参与质子化/去质子化与醌的氨基酸的身份。 进一步的研究将探讨如何在QFR和SQR的醌和血红素辅因子之间的电子传递发生。 本研究将有助于理解呼吸复合物中醌结合位点的结构和功能，以及在复合物II中蛋白质稳定的半醌上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.