Spectroscopy Investigations of Metalloenzyme Mechanisms

金属酶机理的光谱研究

• 批准号: 10378679
• 负责人: R David Britt
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10378679
• 关键词: Active Sites; Anabolism; Biochemistry; Catalysis; Cell Nucleus; Cells; Complex; Dangerousness; Disease; Drug Metabolic Detoxication; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Electrons; Enzymes; Goals; Health; Heart; Homeostasis; Human; Hydrogen; Investigation; Ions; Isotopes; Knowledge; Laboratories; Magnetism; Metals; Methods; Nature; Nitrogenase; Nuclear; Oxygen; Peptides; Play; Post-Translational Protein Processing; Process; Protons; Reaction; Research Project Grants; Research Support; Role; Signal Transduction; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Techniques; United States National Institutes of Health; Work; base; cofactor; experimental study; iron hydrogenase; member; metalloenzyme; oxidation; vitamin biosynthesis

Enzymes using metal centers and/or organic radicals play many crucial roles in the fundamental biochemistry of human health, with deficiencies in their bioassembly or enzymatic functions associated with various diseases. The R. David Britt laboratory is using advanced spectroscopic techniques such as multifrequency electron paramagnetic resonance (EPR) to understand the assembly and catalytic mechanism of a number of such metal and radical centers. Many important enzymes involved in multielectron oxidation or reduction reactions employ metal clusters in their catalysis. The Britt laboratory is studying how such clusters are assembled by identifying and interrogating assembly intermediates with their spectroscopic methods. For example the [Fe-Fe] hydrogenase enzyme uses a complex multinuclear Fe-S “H-cluster”, containing organometallic Fe-CO and Fe-CN components, to catalyze reversible interconversion of H2 with protons and electrons. How does nature safely assemble such a center involving potentially dangerous CO and CN- species? The Britt laboratory is exploring how a radical reaction catalyzed by HydG, a member of the “radical SAM” superfamily of enzymes, safely forms Fe(CO)x(CN)y organometallic synthons at the earliest stage of cluster synthesis and how these are processed by other maturase enzymes in the synthesis of the catalytic cluster. Magnetic nuclear isotopes (e.g. 13C, 15N, 57Fe) provide important magnetic interactions with paramagnetic forms of such clusters, as observed by EPR-based methods such as electron-nuclear double resonance (ENDOR). These and other nuclei provide handles for other spectroscopic probes of FeS cluster assembly such as Mössbauer, NMR, and FTIR. Their ability to assemble the active site enzymatically via cell-free synthesis allows the Britt laboratory to isotope-edit the assembled H-cluster and probe intermediates in hydrogen catalysis with isotope specific spectroscopies. Parallel experiments are unraveling the biosynthesis of the complex Fe-S “M-cluster” at the heart of the nitrogenase enzyme, which can incorporate Mo or V or an additional Fe in its active site. The Britt lab is studying other members of the radical SAM superfamily that can carry out a wide variety of reactions such as organic cofactor and vitamin biosynthesis and post translational modifications of short peptides. Further investigations with EPR and other spectroscopies will probe metalloenzymes and radical enzymes with diverse functions, including NO and Ca2+ signaling, organohalide detoxification, metal ion sequestration and homeostasis, and substrate oxygen insertion. In such projects the Britt laboratory works closely with a wide variety of collaborators working on numerous NIH-supported research projects.

使用金属中心和/或有机自由基的酶在基本原理中发挥着许多关键作用。 人类健康的生物化学，其生物组装或酶功能存在缺陷 R. David Britt 实验室正在使用先进的光谱技术。 多频电子顺磁共振（EPR）等技术来了解 许多此类金属和自由基中心的组装和催化机制。 参与多电子氧化或还原反应的重要酶采用金属 布里特实验室正在研究这些簇是如何在其催化作用下组装的。 用光谱方法识别和询问组装中间体。 例如 [Fe-Fe] 氢化酶使用复杂的多核 Fe-S“H 簇”， 含有有机金属 Fe-CO 和 Fe-CN 组分，催化可逆相互转化 H2 与质子和电子的结合。大自然如何安全地组装这样一个中心？ 潜在危险的 CO 和 CN- 物种？ Britt 实验室正在探索如何发生自由基反应 在“自由基 SAM”酶超家族成员 HydG 的催化下，安全地形成 Fe(CO)x(CN)y 有机金属合成子在簇合成的最早阶段以及它们如何 在磁性催化簇的合成中由其他成熟酶加工。 核同位素（例如 13C、15N、57Fe）提供与顺磁性的重要磁相互作用 通过基于 EPR 的方法（例如电子-核双核）观察到此类团簇的形式 这些原子核和其他原子核为其他光谱探针提供了手柄。 FeS 簇组装，例如穆斯堡尔、NMR 和 FTIR 组装活性的能力。 通过无细胞合成的酶促位点允许布里特实验室对组装的同位素进行编辑 具有同位素特异性光谱的氢催化中的 H 簇和探针中间体。 平行实验正在揭示复杂的 Fe-S“M 簇”在心脏的生物合成 固氮酶，它可以在其活性位点掺入 Mo 或 V 或额外的 Fe。 Britt 实验室正在研究激进 SAM 超家族的其他成员，这些成员可以进行广泛的研究。 各种反应，例如有机辅因子和维生素生物合成以及翻译后 短肽的修饰将通过 EPR 和其他光谱进行进一步研究。 探测具有多种功能的金属酶和自由基酶，包括 NO 和 Ca2+ 信号传导、有机卤化物解毒、金属离子螯合和稳态以及底物 在此类项目中，布里特实验室与各种领域密切合作。 合作者致力于许多 NIH 支持的研究项目。