Regulation of Substrate Binding in the bc1 Complex

摘要 泛喹啉-细胞色素c氧化还原酶（bc1复合物，复合物III）是一种关键的膜酶 参与呼吸。它被认为是活性氧的主要生产者之一 (ROS)在线粒体中。我们的总体假设是，至少有三个 忽视了涉及细胞色素（cyt）c1的bc1调节机制。具体来说，我们 目的I是使用计算和实验技术的组合来测试 bc1复合物中的反合作底物结合。这种效应是基于 可用的X射线晶体结构，但没有实验测试。我们初步的分子 动力学（MD）模拟为我们提供了一个可测试的结构机制，我们将测试 实验性的我们的目的II是确定天然存在的Lys-77三甲基化的作用 由酵母中独特的特异性Cyt-C赖氨酸甲基转移酶（Ctm1）引起。我们假设 翻译后修饰调节cyt-Lys-77之间阳离子-π相互作用的强度 c和普遍保守的物种与Ctm1p Phe-132在细胞色素c1。最后，我们的目标三是 重点是测试一个假设，即脂质膜组成和脂质电荷可以调节 BCl复合物中的底物结合亲和力。该项目将采用多管齐下的方法 结合计算和实验技术预测分子水平的bc1调控 机制，并进行实验测试。我们将使用bc1的长时间全原子MD模拟， 不同的脂质环境，以预测与不同的占据相关的结构变化， 底物结合位点，并指导我们的实验工作，洗涤剂增溶， 嵌入纳米盘的BC1.我们将使用等温量热法（ITC）来测试底物结合， 体外，并测量结合化学计量，缔合常数，和热力学 参数作为离子强度和脂质电荷的函数。我们会用小角度X光 通过SAXS独立验证ITC结果，确认基板位置 结合位点，并构建酶-底物的低分辨率溶液状态结构 配合物激光诱导时间分辨光谱学将用于测量变化 在响应于脂质环境和底物结合的变化的电荷转移速率中， 调控最后，我们将使用动力学光谱来研究脂质膜的作用， bc1复合物二聚体内的单体间相互作用对催化周转率和 ROS的产生。总的来说，这种跨学科的方法将促进对细胞色素T的理解 BC 1调节，并将测试三种预测的调节机制。此外，该项目 每年将直接支持4名感兴趣的本科生的研究培训， 追求生物医学事业

Abstract Ubiquinol-cytochrome c oxidoreductase (bc1 complex, complex III) is a key membrane enzyme involved in respiration. It is known to be one of the major producers of reactive oxygen species (ROS) in mitochondria. Our overarching hypothesis is that there are at least three overlooked bc1 regulations mechanisms involving cytochrome (cyt) c1. Specifically, our Aim I is to use a combination of computational and experimental techniques to test anticooperative substrate binding in the bc1 complex. This effect was suggested based on available X-ray crystal structures but was not experimentally tested. Our preliminary molecular dynamics (MD) simulations provide us with a testable structural mechanism which we will test experimentally. Our Aim II is to establish the role of naturally occurring trimethylation of Lys-77 by a unique and specific cyt c lysine methylatransferase (Ctm1) in yeast. Our hypothesis that this posttranslation modification regulates the strength of cation-pi interaction between Lys-77 of cyt c and universally conserved in species with Ctm1p Phe-132 in cyt c1. Finally, our Aim III is focused on testing a hypothesis that lipid membrane composition and lipid charge can regulate substrate binding affinity in the bc1 complex. This project will use a multi-pronged approached combining computational and experimental techniques to predict molecular level bc1 regulation mechanisms and to test them experimentally. We will use long all-atom MD simulations of bc1 in different lipid environments to predict structural changes associated with different occupancy of the substrate binding sites and to guide our experimental work on detergent-solubilized and nanodisc-embedded bc1. We will use isothermal calorimetry (ITC) to test substrate binding in vitro, and to measure binding stoichiometries, association constants, and thermodynamic parameters as a function of ionic strength and lipid charge. We will use small-angle X-ray scattering (SAXS) to independently verify the ITC results, to confirm the locations of substrate binding sites, and to construct low-resolution solution-state structures of the enzyme-substrate complexes. Laser-induced time-resolved optical spectroscopy will be used to measure changes in the charge transfer rates as response to changes in lipid environment and substrate binding regulation. Finally, we will use kinetic spectroscopy to study the roles of lipid membranes and intermonomer interactions within the bc1 complex dimer on the catalytic turnover rates and the rate of ROS production. Overall, this interdisciplinary approach will advance understanding of cyt bc1 regulation and will test the three predicted regulation mechanisms. In addition, this project will directly support each year research training of 4 undergraduate students interested in pursuing biomedical careers.