Structure/Function of Complex II Oxidoreductases

本项目旨在研究膜结合呼吸复合体II在电子传递过程中发生的完整事件。使用了两个模型系统，琥珀酸-泛醌氧化还原酶(SQR，琥珀酸脱氢酶)和来自大肠杆菌的尿喹酚-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发挥重要作用，因此对细胞产生能量很重要。这些酶在结构上与真核生物的同类酶相似，但更容易在基因上进行操作，并易于生产大量可通过生化和生物物理方法研究的蛋白质。这些酶复合体似乎是从共同的进化前体进化而来的，在结构和功能上都非常相似。富马酸还原酶通常在厌氧环境中发挥作用，而Sqr是Krebs循环中的一个组成部分，在有氧代谢中发挥作用。在功能上，这两种酶可以执行氧化琥珀酸成富马酸和还原富马酸成琥珀酸的相同反应，但在体内，QFR在这两个反应中的效率都比SQR高得多。这些研究旨在确定这两种酶催化效率不同的结构和功能原因。最近QFR的高分辨率结构允许设计特定的定点突变，以解决两种酶在催化位点和共价黄素辅因子方面的差异。在这些研究过程中，将获得高分辨结构，其抑制剂结合在醌结合部位，以及参与与醌的质子化/去质子化的氨基酸的同一性。其他研究将调查QFR和SQR中的苯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体中的苯醌结合部位的结构和功能，以及在复合体II中蛋白质稳定的半喹酮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.