Conformational Properties of Cytochromes in Disease

疾病中细胞色素的构象特性

• 批准号: 10295196
• 负责人: Ekaterina PLETNEVA
• 金额: $ 35.84万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10295196
• 关键词: Address; Affect; Affinity; Architecture; Bacteria; Behavior; Binding; Binding Proteins; Biological; Biological Assay; Catalysis; Communities; Complex; Consumption; Coupled; Cytochromes; Cytoplasmic Protein; Data; Disease; Electrochemistry; Electron Transport; Electrons; Elements; Environment; Enzymes; Exhibits; Foundations; Genetic; Growth; Health; Heme; Hemerythrin; Human; Hybrids; In Vitro; Individual; Infection; Injections; Interruption; Investigation; Kinetics; Knowledge; Label; Learning; Life; Light; Membrane; Metalloproteins; Metals; Microbe; Microbial Biofilms; Microscopic; Modeling; Molecular; Molecular Conformation; Oxidants; Oxidases; Oxidation-Reduction; Oxygen; Pathway interactions; Phenotype; Play; Process; Property; Proteins; Proteobacteria; Proton Pump; Protons; Pseudomonas; Pseudomonas aeruginosa; Reaction; Regulation; Regulatory Pathway; Respiration; Role; Shapes; Site; Testing; Theoretical Studies; Thermodynamics; Variant; Water; Work; bacterial community; bacterial fitness; base; cbb3 oxidase; design; experimental study; fitness; genetic regulatory protein; in vitro Assay; in vivo; inhibitor; insight; microbial; novel; novel therapeutics; protein function; recruit; respiratory; respiratory protein; success

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.

细菌经常遇到微氧环境，特别是在与微氧环境相关的密集社区中。 生物膜、感染部位和共生生命中。微生物已经进化到呼吸O2，即使它 虽然这些浓度很低，但微氧呼吸的机制还不十分清楚。在 许多细菌，包括铜绿假单胞菌（Pa），O2还原成水是由cbb 3末端催化的 具有与哺乳动物氧化酶不同的几个特征的氧化酶。四电子O2的成功 还原取决于多个氧化还原中心的协调作用，在微氧条件下， 管理机制来处理有限的O2供应。我们已经确定了两种金属蛋白， Pa中cbb 3氧化酶的微氧功能：电子载体细胞色素c4（cyt c4）和O2载体 hemerythrin（Hr）;这两种都是在低O2下Pa生长所必需的。Pa中的Cbb 3氧化酶和许多 其它变形菌具有六个金属氧化还原中心的延伸链，并且它们的电子载体具有两个。我们 提出了cbb 3氧化酶中cytc 4的二血红素结构和延伸的电子转移（ET）链， 与独特的构象动力学一起，用于通过这些呼吸蛋白调节ET 提供了调节氧化还原流的机会，并使得在氧化还原过程中能够进行有效的能量转换。 催化作用此外，我们假设Hr有助于通过膜结合蛋白引导O2用于呼吸。 Pa cbb 3氧化酶。在这个项目中，蛋白质组分的光谱和电化学研究， 它们的体外相互作用与体内遗传学和表型分析相结合，以（1）表征 二血红素部分在调节细胞色素c4和cbb 3氧化酶的CcoP亚基的ET特性中的作用， 建立了电子注入酶的机理;（2）阐明了电子流的机理 cbb 3氧化酶中的延伸ET链内以及构象变化与ET步骤的协调;以及 (3)建立了Hr影响Pa.拟议的研究 cbb 3氧化酶将为理解使细菌能够 在低氧环境中茁壮成长，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同性，构象门控，质子耦合过程，和调节蛋白 网络，我们的研究将解决生物氧化还原机制中的重要课题， 其他金属蛋白在人类健康中发挥作用。