Metalloprotein Mechanisms of Redox Regulation and Catalysis

氧化还原调节和催化的金属蛋白机制

• 批准号: 10472758
• 负责人: Stephen Wiley Ragsdale
• 金额: $ 65.29万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10472758
• 关键词: Address; Affect; Anaerobic Bacteria; Anions; Area; Biliverdine; Binding; Binding Proteins; Biochemical; Biological; Biophysics; Biotechnology; Carbon Dioxide; Catalysis; Cells; Chemicals; Complex; Coupling; Crystallization; Environment; Enzymes; Funding; Gases; Generations; Grant; Health; Heme; Homeostasis; Human; Industrialization; Iron; Laboratories; Length; Ligation; Mercury; Metabolic; Metabolism; Metalloproteins; Metals; Methane; Methylation; Microbe; Molecular Chaperones; Molecular Conformation; Monitor; Movement; NADPH-Ferrihemoprotein Reductase; Nuclear Receptors; Oxidation-Reduction; Pathway interactions; Planet Earth; Reaction; Regulation; Research; Signaling Molecule; Structure; Sulfur; Tail; Toxic effect; Wood material; Work; career; catalyst; circadian pacemaker; cofactor; heme oxygenase-2; methyl radical; microbial; novel; protein degradation; protein protein interaction; sample fixation; sensor; success; trafficking

Abstract for Metalloprotein Mechanisms of Redox Regulation and Catalysis This proposal covers the three R01 grants funding my laboratory and aims to fill gaps in understanding the mechanisms of crucial aspects of redox regulation and catalysis by metalloproteins from microbes to humans. Successful completion of this work will reveal novel mechanisms with broad significance to human health, the environment, and biotechnology. Our research integrates a wide variety of biological, biophysical, biochemical and computational approaches. In Project Area 1, we will extend recent discoveries of novel bioinorganic and enzymatic mechanisms of anaerobic microbial CO and CO2 fixation in the Wood-Ljungdahl pathway (WLP), proposed to have fueled the origin of live on earth. We will reveal the mechanisms of these ancient enzymes: their generation and use of CO as a substrate, formation of bioorganometallic catalytic intermediates, utilization of nucleophilic and paramagnetic metal centers as catalysts, requirement of large domain movements and an interprotein CO channel and recently identified alcove for CO binding and CO2 fixation. We will define how these unique features choreograph redox activation, substrate and partner protein binding, leading to biological transformation that chemists are trying to mimic to more rapidly and efficiently accomplish chemically challenging reactions, e.g., to sequester, activate and convert CO2, methane and syngas into industrially important chemical feedstocks and fuels. While I started my career studying the WLP, I have applied the same expertise to other important evolving problems of metabolic regulation in humans by CO and metals and of mercury toxicity. In Project Area 2, we propose to deliver important discoveries on how human metabolism, metal homeostasis and the circadian clock are regulated by heme regulatory motifs (HRMs), signaling molecules (CO and NO), and cellular heme levels and redox poise. Focusing on heme oxygenase-2 (HO2), we will explore crucial conformational changes between the core and tail of HO2 and how these movements control protein turnover, protein-protein interactions, and heme conversion to CO, biliverdin and Fe. We will explore the hypothesis that HO2 serves a dual function in the cell in controlling heme trafficking and turnover. We will monitor the dynamics and interactions of full length HO2 with its redox partner cytochrome P450 reductase and with its heme donor GAPDH and define mechanisms that regulate heme-controlled HO2 turnover. Following up on our finding that the nuclear receptor Rev-Erbb uses a novel mechanism of redox- chemical coupling to serve as a CO/NO sensor, we will address how redox and gas binding affect its structure, function, activity and its interactions with partners like NCoR1 and its heme chaperone. In Project Area 3, recent successes in purifying and crystallizing the active HgcAB complex and defining its unusual thiolate- coordinated B12 cofactor, enable our proposed studies of the mechanism of microbial mercury methylation. We will determine the HgcAB structure, the redox and ligation states of the metal centers during catalysis, and whether a methyl radical or anion is used by these B12 and iron-sulfur clusters during catalysis.

金属蛋白的氧化还原调控和催化机制 这份提案涵盖了三个R 01赠款资助我的实验室，旨在填补空白，了解 从微生物到人类的氧化还原调节和金属蛋白催化的关键方面的机制。 这项工作的成功完成将揭示对人类健康具有广泛意义的新机制， 环境和生物技术。我们的研究整合了多种生物学、生物物理学、生物化学 和计算方法。在项目领域1，我们将扩大新的生物无机和 厌氧微生物在Wood-Ljungdahl途径（WLP）中固定CO和CO2的酶机制， 被认为是地球生命起源的燃料我们将揭示这些古老酶的机制： 它们的产生和使用CO作为底物，生物有机金属催化中间体的形成，利用 作为催化剂的亲核和顺磁金属中心，需要大的畴运动和 蛋白质间CO通道和最近鉴定的用于CO结合和CO2固定的凹室。我们将定义如何 这些独特的功能编排氧化还原活化，底物和伴侣蛋白结合，导致生物学 化学家们试图模仿这种转变，以更快更有效地完成化学反应。 挑战性反应，例如，将二氧化碳、甲烷和合成气封存、活化并转化为工业用的 重要的化学原料和燃料。当我开始我的职业生涯学习WLP，我已经申请了相同的 专业知识，以其他重要的不断发展的问题，代谢调节在人类的CO和金属， 汞毒性在项目领域2中，我们计划提供关于人类新陈代谢， 金属稳态和生物钟受血红素调节基序（HRM）、信号传导和信号转导的调节。 分子（CO和NO），以及细胞血红素水平和氧化还原平衡。针对血红素加氧酶-2（HO 2）， 将探索HO 2的核心和尾部之间的关键构象变化，以及这些运动如何 控制蛋白质周转、蛋白质-蛋白质相互作用和血红素转化为CO、胆绿素和Fe。我们将 探索HO 2在细胞中控制血红素运输和周转的双重功能的假设。 我们将监测全长HO 2与其氧化还原伴侣细胞色素P450的动力学和相互作用 还原酶及其血红素供体GAPDH，并确定调节血红素控制的HO 2 周转我们发现核受体Rev-Erbb使用一种新的氧化还原机制， 化学偶联作为CO/NO传感器，我们将解决氧化还原和气体结合如何影响其结构， 功能，活性及其与NCoR 1及其血红素伴侣的相互作用。在项目区3， 最近在纯化和结晶活性HgcAB复合物和确定其不寻常的硫醇盐方面取得了成功， 协调的B12辅因子，使我们能够对微生物汞甲基化机制进行拟议的研究。我们 将决定HgcAB结构，催化过程中金属中心的氧化还原和连接状态， 无论是甲基自由基还是阴离子都被这些B12和铁-硫簇在催化过程中使用。