Conformational Properties of Cytochromes in Disease

疾病中细胞色素的构象特性

基本信息

批准号：
10526430
负责人：
Ekaterina PLETNEVA
金额：
$ 35.84万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10526430
关键词：
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 Hemeproteins Hemerythrin Human Hybrids In Vitro Individual Infection Injections Interruption Investigation Kinetics Knowledge Label Learning Life 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 Rationalization Reaction Regulation Regulatory Pathway Respiration Role Shapes Site Testing Theoretical Studies Thermodynamics Variant Water Work bacterial community bacterial fitness cbb3 oxidase design energy efficiency experimental study fitness genetic regulatory protein in vitro Assay in vivo inhibitor insight microbial novel novel therapeutics protein function recruit respiratory respiratory protein success

项目摘要

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.

细菌经常遇到微毒性环境，尤其是在与之相关的密集社区生物膜，感染部位和共生生活。微生物即使在浓度较低，但尚不清楚微氧呼吸的基础机制。在许多细菌，包括铜绿假单胞菌（PA），O2降低到水为CBB3末端催化具有与哺乳动物氧化酶不同的几种特征的氧化酶。四电子O2的成功还原取决于多个氧化还原中心的协调游戏，在微氧气条件下也在处理有限的O2供应的监管机制。我们已经确定了两种对于至关重要的金属蛋白 PA中CBB3氧化酶的微毒素功能：电子载体细胞色素C4（Cyt C4）和O2载体 hemerythrin（HR）；它们俩都是在低O2处强大的PA生长所必需的。 PA中的CBB3氧化酶，许多其他蛋白质细菌具有六个金属氧化还原中心的延长链，其电子载体有两个。我们提出Cyt C4的二蓝色结构和CBB3氧化酶中的扩展电子转移（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 为调节氧化还原流量提供机会，并在期间实现有效的能量转换催化。此外，我们假设人力资源部有助于指导O2用于通过膜结合的呼吸 PA CBB3氧化酶。在这个项目中，蛋白质成分和的光谱和电化学研究它们在体外的相互作用与体内的遗传学和表型分析结合在一起，以（1）表征 Diheme部分在调节CBB3氧化酶和CCOP亚基的ET特性中的作用建立向酶注入电子的机制；（2）阐明电子流的机理在CBB3氧化酶的扩展ET链中，以及与ET步骤的构象变化的配位；和（3）建立HR对PA中CBB3氧化酶功能的影响的机理。 CBB3氧化酶将为理解能够使细菌的分子机制提供基础在低O2环境中壮成长，并确定可能破坏这些环境中细菌生长的策略。我们专注于氧化还原协作，构象门控，质子耦合过程和调节蛋白网络，我们的研究将讨论与许多许多功能相关的生物氧化还原机制的重要主题其他在人类健康中发挥作用的金属蛋白。