Structure of reaction intermediates in the bc1 complex

描述（由申请人提供）：在本提案中，我们将使用高分辨率EPR光谱来探索被困在顺磁中间体结合状态的bc1配合物的催化结构域。细胞色素bc1复合物家族在生物圈的能量代谢中起着至关重要的作用。三个催化亚基，cyt b， cyt c1和Rieske铁硫蛋白（ISP），包含了该机制。cytb中的两个催化位点参与了泛醌的氧化或还原。氧化和还原反应与水相中质子的释放或吸收的结合，使络合物能够将质子泵过膜。为了理解这一机制，我们需要关于局部反应环境的详细信息，包括蛋白质结构、氢键和距离，以提供控制速率和电子分配到不同途径的参数。bc1络合物中的几个部分反应涉及顺磁中间体。EPR方法非常适合解剖这些，因为脉冲EPR技术可以探测中间和局部磁核的电子自旋之间的相互作用。因此，它们提供了关于中间体和直接的蛋白质和溶剂环境的空间和电子结构的直接信息。该提案的重点物种是参与qo位点泛氢醌（醌，QH2）氧化的ISP的还原[2Fe-2S]簇；半醌（SQ）参与醌还原qi位点的反应。这些位点的催化编排在很大程度上是由适应不同醌形式的结合要求所需的局部结构的变化控制的，SQ的EPR提供了一个直接的工具。这项工作将在动力学和机械方面的单独资助研究的背景下进行，以及生物物理，生化和分子工程协议的广泛清单，使我们能够将顺磁物种的数据与突变菌株的功能和结构变化联系起来。要解决的主要问题是蛋白质环境如何改变中间体的空间和电子结构以适应生理功能，这是反应机制理论中更广泛感兴趣的问题。bc1复合体在许多缺陷导致病理的医学场景中发挥核心作用，其中包括通过ros介导的损伤、线粒体肌病和凋亡导致的细胞死亡，以及药物靶向。

DESCRIPTION (provided by applicant): In this proposal we will use high-resolution EPR spectroscopy to explore the catalytic domains of the bc1 complex trapped in states with paramagnetic intermediates bound. The cytochrome bc1 complex family plays an essential role in the energy metabolism of the biosphere. Three catalytic subunits, cyt b, cyt c1 and the Rieske iron sulfur protein (ISP), house the mechanism. Two catalytic sites in cyt b are involved in oxi-dation or reduction of ubiquinone. The integration of the oxidation and reduction reactions with the release or uptake of protons in the aqueous phases, allows the complex to pump protons across the membrane. To understand the mechanism, we need detailed information about the local reaction environment, including protein structure, hydrogen bonding, and distances, to provide the parameters that control rates, and partitioning of electrons to different pathways. Several partial reactions in the bc1 complex involve paramagnetic intermediates. The EPR approach is well suited to dissecting these, because pulsed-EPR techniques can probe interactions between the electron spin of the intermediate and local magnetic nuclei. They thus pro- vide direct information about spatial and electronic structure of the intermediate and the immediate protein and solvent environment. The species at the focus of the proposal are the reduced [2Fe-2S] cluster of the ISP participating in ubihydroquinone (quinol, QH2) oxidation at the Qo-site; and the semiquinone (SQ) involved in the reactions of the quinone-reducing Qi-site. The choreography of catalysis at these sites is largely controlled by the changes in local configuration needed to accommodate the binding requirements of the different quinone forms, and EPR of the SQ provides a direct tool. This work will be pursued in the context of separately funded studies of kinetic and mechanistic aspects, and an extensive inventory of biophysical, biochemical, and molecular engineering protocols allowing us to correlate data for paramagnetic species, and functional and structural changes in mutant strains. The main question to be addressed is that of how the protein environment modifies the spatial and electronic structure of the intermediates to fit the physiological function, a question of much wider interest in reaction mechanism theory. The central role of bc1 complex plays out in many medical scenarios in which defects lead to pathology, among them cellular death through ROS-mediated damage, mitochondrial myopathies, and apoptosis, and in drug targeting.