Spectroscopy Investigations of Metalloenzyme Mechanisms

金属酶机理的光谱研究

• 批准号: 9903396
• 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 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9903396
• 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中起许多至关重要的作用 人类健康的生物化学，其生物组装或酶促功能缺陷 与各种疾病有关。 R. David Britt实验室正在使用高级光谱学 诸如多频电子顺磁共振（EPR）之类的技术以了解 许多此类金属和激进中心的组装和催化机制。许多 参与多电体氧化或还原反应的重要酶员工金属 集群在催化中。布里特实验室正在研究如何通过 识别和审问组装中间体的光谱方法。为了 示例[Fe-Fe]氢化酶使用复杂的多核Fe-S“ H-Cluster”， 包含有机Fe-CO和Fe-CN组件，以催化可逆互转换 带有质子和电子的H2。自然如何安全地组装这样的中心 潜在的危险CO和CN种类？布里特实验室正在探索如何产生根本性反应 由酶的“激进SAM”超家族的成员Hydg催化，安全形式 Fe（CO）X（CN）Y有机合成子在簇合成的最早阶段以及如何 在催化簇的合成中，其他成熟酶处理。磁的 核同位素（例如13C，15N，57FE）提供了与顺磁性的重要磁相互作用 这种簇的形式，如通过EPR基于电子核双重的方法所观察到的 共鸣（Endor）。这些和其他核为其他光谱问题提供了手柄 FES群集组件，例如Mössbauer，NMR和FTIR。他们组装活跃的能力 通过无细胞合成酶促的位点使Britt实验室可以同位素编辑组装 与同位素特异性光谱法中的H簇和探测中间体在氢催化中。 平行实验正在阐明复杂的Fe-s“ M-Cluster”的生物合成 氮酶的酶，可以在其活性位点掺入MO或V或其他Fe。 布里特实验室正在研究激进的SAM超家族的其他成员，该成员可以进行宽阔的 各种反应，例如有机辅助因子和维生素生物合成和翻译后 短肽的修饰。对EPR和其他光谱学的进一步研究将 探针金属酶和具有不同功能的自由基酶，包括NO和Ca2+ 信号传导，有机甲酯解毒，金属离子隔离和稳态以及底物 氧气插入。在这样的项目中，布里特实验室与各种各样的 合作者从事众多NIH支持的研究项目。