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：
9886350
负责人：
Ekaterina PLETNEVA
金额：
$ 30.38万
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 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9886350
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/antagonist 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.

细菌经常遇到微氧环境，特别是在与微氧相关的密集群落中生物膜、感染部位和共生生活。微生物已经进化到可以呼吸氧气，即使是在氧气存在的情况下浓度较低，但微氧呼吸的机制尚不清楚。在许多细菌，包括铜绿假单胞菌 (Pa)，O2 还原成水是由 cbb3 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，并且在微氧条件下，还取决于应对氧气供应有限的监管机制。我们已经鉴定出两种对于 Pa 中 cbb3 氧化酶的微氧功能：电子载体细胞色素 c4 (cyt c4) 和 O2 载体血红蛋白（Hr）；它们都是在低 O2 条件下 Pa 的强劲生长所必需的。 Pa 和许多中的 Cbb3 氧化酶其他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有两个。我们提出 cyt c4 的二血红素结构和 cbb3 氧化酶中延伸的电子转移 (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 为氧化还原流的调节提供机会，并在过程中实现高效的能量转换催化。此外，我们假设 Hr 有助于通过膜结合引导 O2 用于呼吸。 Pa cbb3 氧化酶。在这个项目中，蛋白质成分和蛋白质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相结合，以（1）表征二血红素部分在调节细胞色素 c4 和 cbb3 氧化酶的 CcoP 亚基的 ET 特性中的作用建立电子注入酶的机制； (2)阐明电子流动机理 cbb3氧化酶中延伸的ET链内以及构象变化与ET步骤的协调；和 (3)建立Hr对Pa中cbb3氧化酶功能的影响机制。 cbb3 氧化酶将为理解使细菌能够在低氧气环境中茁壮成长，并确定可能破坏这些环境中细菌生长的策略。我们专注于氧化还原协同性、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发挥作用的其他金属蛋白。