Metalloprotein Mechanisms of Redox Regulation and Catalysis

氧化还原调节和催化的金属蛋白机制

基本信息

批准号：
10643866
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
项目类别：
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

项目摘要

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.

金属蛋白氧化还原调节和催化机制摘要该提案涵盖了资助我的实验室的三项 R01 赠款，旨在填补理解方面的空白从微生物到人类的金属蛋白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。我们的研究综合了多种生物学、生物物理、生物化学和计算方法。在项目领域 1，我们将扩展新型生物无机和 Wood-Ljungdahl 途径 (WLP) 中厌氧微生物 CO 和 CO2 固定的酶机制，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：它们以CO为底物的产生和使用、生物有机金属催化中间体的形成、利用亲核和顺磁性金属中心作为催化剂，大域运动的要求和蛋白间 CO 通道和最近发现的用于 CO 结合和 CO2 固定的凹室。我们将定义如何这些独特的功能精心设计了氧化还原激活、底物和伴侣蛋白结合，从而导致生物化学家试图模仿以更快速、更有效地完成化学转化具有挑战性的反应，例如，隔离、活化二氧化碳、甲烷和合成气并将其转化为工业用重要的化工原料和燃料。当我开始学习 WLP 的职业生涯时，我也应用了同样的方法二氧化碳和金属对人类代谢调节的其他重要不断发展的问题的专业知识以及汞的毒性。在项目领域 2 中，我们建议就人类新陈代谢如何、金属稳态和生物钟由血红素调节基序 (HRM) 调节，信号传导分子（CO 和 NO）、细胞血红素水平和氧化还原平衡。专注于血红素加氧酶-2 (HO2)，我们将探索 HO2 核心和尾部之间的重要构象变化以及这些运动如何控制蛋白质周转、蛋白质-蛋白质相互作用以及血红素转化为 CO、胆绿素和 Fe。我们将探索 HO2 在细胞中具有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血红素供体 GAPDH 并定义了调节血红素控制的 HO2 的机制周转。继我们发现核受体 Rev-Erbb 使用一种新的氧化还原机制后，化学耦合作为 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 NCoR1 及其血红素伴侣等伙伴的相互作用。在项目3区，最近在纯化和结晶活性 HgcAB 复合物以及定义其不寻常的硫醇盐方面取得了成功协调的 B12 辅因子，使我们能够研究微生物汞甲基化的机制。我们将确定 HgcAB 结构、催化过程中金属中心的氧化还原和连接状态，以及这些 B12 和铁硫簇在催化过程中是否使用甲基自由基或阴离子。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Heme delivery to heme oxygenase-2 involves glyceraldehyde-3-phosphate dehydrogenase.

血红素向血红素加氧酶 2 的输送涉及 3-磷酸甘油醛脱氢酶。

DOI：
发表时间：
2022-11-25
期刊：
影响因子：
3.7
作者：
Dai, Yue;Fleischhacker, Angela S;Liu, Liu;Fayad, Sara;Gunawan, Amanda L;Stuehr, Dennis J;Ragsdale, Stephen W
通讯作者：
Ragsdale, Stephen W

Characterization of Methyl- and Acetyl-Ni Intermediates in Acetyl CoA Synthase Formed during Anaerobic CO2 and CO Fixation.

厌氧 CO2 和 CO 固定过程中形成的乙酰辅酶 A 合酶中甲基和乙酰基 Ni 中间体的表征。

DOI：
发表时间：
2023-06-28
期刊：
影响因子：
15
作者：
Can, Mehmet;Abernathy, Macon J;Wiley, Seth;Griffith, Claire;James, Christopher D;Xiong, Jin;Guo, Yisong;Hoffman, Brian M;Ragsdale, Stephen W;Sarangi, Ritimukta
通讯作者：
Sarangi, Ritimukta

Regulation of protein function and degradation by heme, heme responsive motifs, and CO.

血红素、血红素反应基序和 CO 对蛋白质功能和降解的调节。