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: 10204329
• 负责人: Stephen Wiley Ragsdale
• 金额: $ 34.57万
• 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/10204329
• 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.

金属蛋白对氧化还原的调节和催化作用机制 这份提案涵盖了资助我的实验室的三笔R01赠款，旨在填补在理解 从微生物到人类的金属蛋白对氧化还原调节和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。我们的研究综合了广泛的生物学、生物物理、生化 和计算方法。在项目区域1，我们将推广最近发现的新型生物无机物和 伍德-永达尔途径(WLP)厌氧微生物固定CO和CO2的酶机制， 被认为促进了地球上生命的起源。我们将揭示这些古老酶的机制： 它们产生和使用CO作为底物，形成生物有机金属催化中间体，利用 亲核金属中心和顺磁金属中心作为催化剂，需要大的磁区移动和 蛋白间CO通道和最近发现的用于CO结合和CO2固定的凹槽。我们将定义如何 这些独特的功能编排了氧化还原激活，底物和伴侣蛋白结合，导致生物 化学家们试图模拟的转变，以更快、更有效地完成化学转化 具有挑战性的反应，例如，隔离、活化和将二氧化碳、甲烷和合成气转化为工业气体 重要的化学原料和燃料。虽然我的职业生涯是从学习WLP开始的，但我已经应用了同样的 一氧化碳和金属对人体代谢调节的其他重要进化问题的专门知识 汞中毒。在项目区域2，我们建议提供关于人类新陈代谢的重要发现， 金属动态平衡和生物钟由血红素调节基序(HRMS)调节，信号转导 分子(CO和NO)，以及细胞内的血红素水平和氧化还原平衡。聚焦于血红素加氧酶-2(HO2)，我们 将探索HO2核心和尾部之间的关键构象变化以及这些运动是如何 控制蛋白质周转，蛋白质-蛋白质相互作用，以及将血红素转化为一氧化碳、胆绿素和铁。我们会 探索HO2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HO2与其氧化还原伙伴细胞色素P450的动力学和相互作用 还原酶及其亚铁血红素供体GAPDH及其调控机制的研究 营业额。继我们的发现之后，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，我们将讨论氧化还原和气体结合如何影响其结构， 功能、活性及其与NCoR1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络合物并确定其不同寻常的硫酸盐- 协调的B12辅因子，使我们提出的微生物汞甲基化机制的研究成为可能。我们 将决定HgcAB的结构，金属中心在催化过程中的氧化还原和连接状态，以及 在催化过程中，这些B12和铁硫团簇是否使用甲基自由基或阴离子。