Conformational Properties of Cytochromes in Disease

细菌经常遇到微氧环境，特别是在与 生物膜、感染部位和共生生命。微生物已经进化成呼吸氧气，即使当它的 浓度很低，但微氧呼吸的机制还不是很清楚。在……里面 许多细菌，包括铜绿假单胞菌(PA)，氧还原为水是由CBB3末端催化的 具有与哺乳动物的氧化酶不同的几个特征的氧化酶。四电子O2的成功 还原依赖于多个氧化还原中心的协调作用，在微氧条件下，还取决于 应对氧气供应有限的调控机制。我们已经确定了两种关键的金属蛋白 铜绿假单胞菌中CBB3氧化酶的微氧功能：电子载体细胞色素C4和O2载体 高氯氰菊酯(HR)；两者都是在低氧条件下强劲的PA生长所必需的。PA和MULT中的Cbb3氧化酶 其他蛋白质细菌有六个金属氧化还原中心的延伸链，它们的电子载体有两个。我们 提出了CytC4的双希姆结构和CBB3氧化酶中扩展的电子转移(ET)链， 与独特的构象动力学一起，通过这些呼吸蛋白来调节ET 提供了调节氧化还原流动的机会，并能够在 催化作用。此外，我们假设，HR通过膜结合帮助指导O2用于呼吸。 PaCBB3加氧酶。在这个项目中，蛋白质组分的光谱和电化学研究以及 它们在体外的相互作用与遗传学和体内表型分析相结合，以(1)表征 Diheme部分在调节CytC4和CBB3氧化酶CcoP亚单位ET性质中的作用 确定了电子注入酶的机理；(2)阐明了电子流的机理 在CBB3氧化酶中延伸的ET链内，构象变化与ET步骤的协调； (3)建立HR影响PA中CBB3氧化酶功能的机制。建议进行的研究 Cbb3氧化酶将为理解使细菌能够 在低氧气环境中茁壮成长，并确定可能在这些环境中破坏细菌生长的策略。 我们的重点是氧化还原协作性、构象门控、质子耦合过程和调节蛋白质 网络，我们的研究将解决与许多功能相关的生物氧化还原机制的重要主题 对人类健康起作用的其他金属蛋白。

Bacteria frequently encounter microoxic environments, particularly in dense communities associated with biofilms, sites of infection, and in symbiotic life. Microbes have evolved to respire O2 even when its concentrations are low but the mechanisms that underlie microoxic respiration are not well understood. In many bacteria, including Pseudomonas aeruginosa (Pa), O2 reduction to water is catalyzed by cbb3 terminal oxidases that have several features distinct from those of mammalian oxidases. Success of four-electron O2 reduction depends on the coordinated play of multiple redox centers and, under microoxic conditions, also on regulatory mechanisms to deal with the limited supply of O2. We have identified two metalloproteins critical for the microoxic function of cbb3 oxidases in Pa: the electron carrier cytochrome c4 (cyt c4) and the O2 carrier hemerythrin (Hr); both of them are required for robust Pa growth at low O2. Cbb3 oxidases in Pa and many other proteobacteria have an extended chain of six metal redox centers and their electron carrier has two. We propose that diheme architecture of cyt c4 and the extended electron-transfer (ET) chain in cbb3 oxidases, together with unique conformational dynamics, are used to modulate ET through these respiratory proteins providing opportunities for regulation of the redox flow and enabling efficient energy conversion during catalysis. Furthermore, we hypothesize that Hr aids in directing O2 for use in respiration by membrane-bound Pa cbb3 oxidases. In this project, the spectroscopic and electrochemistry studies of protein components and their interactions in vitro are combined with genetics and phenotypic analyses in vivo to (1) characterize the role of diheme moieties in regulating ET properties of cyt c4 and of the CcoP subunit of cbb3 oxidase and establish the mechanism of the electron injection to the enzyme; (2) elucidate the mechanism of electron flow within the extended ET chain in cbb3 oxidase and coordination of conformational changes with ET steps; and (3) establish the mechanism of effects of Hr on the function of cbb3 oxidase in Pa. The proposed studies of cbb3 oxidases will provide a foundation for understanding the molecular mechanisms that enable bacteria to thrive in low-O2 environments and identifying strategies that may disrupt bacterial growth in these settings. With our focus on redox cooperativity, conformational gating, proton-coupled processes, and regulatory protein networks, our studies will address important topics in biological redox mechanisms relevant to function of many other metalloproteins that play a role in human health.