Spectroscopy Investigations of Metalloenzyme Mechanisms

使用金属中心和/或有机自由基的酶在基础上扮演着许多重要的角色 人体健康的生物化学，缺乏生物组装或酶功能 与各种疾病相关的。R.大卫·布里特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 多个此类金属和自由基中心的组装和催化机理。许多 参与多电子氧化或还原反应的重要酶利用金属 在他们的催化作用下形成的团簇。布里特实验室正在研究这样的星团是如何通过 用它们的光谱方法鉴定和询问组装中间体。为 实施例[Fe-Fe]氢酶使用复杂的多核Fe-S“H-簇”， 含有机金属Fe-CO和Fe-CN组分，用于催化可逆的相互转化 氢气中含有质子和电子。大自然如何安全地聚集这样一个中心，包括 具有潜在危险的CO和CN-物种？布里特实验室正在探索一种激进的反应 在HydG的催化下，安全地形成 团簇合成初期的Fe(CO)x(CN)y有机金属合成子及其如何 在催化簇的合成过程中被其他成熟酶处理。磁性 核同位素(如13C、15N、57Fe)提供与顺磁的重要磁相互作用 用基于EPR的方法观察到的这种团簇的形式，如电子-核双核 共振(Endor)。这些原子核和其他原子核为其他光谱探测器提供了句柄 FeS团簇组装，如穆斯堡尔、核磁共振和FTIR。他们组装现役人员的能力 通过无细胞合成的酶切位点允许Britt实验室对组装的 用同位素特定光谱研究氢催化中的氢团簇和探针中间体。 平行实验正在解开复杂的Fe-S“M-星团”的核心生物合成 固氮酶的活性部位可以结合钼、钒或额外的铁。 布里特实验室正在研究激进的SAM超家族的其他成员，这些成员可以进行广泛的 各种反应，如有机辅因子、维生素生物合成和翻译后 短肽的修饰。对EPR和其他光谱分析的进一步研究将 具有多种功能的探针金属酶和自由基酶，包括NO和钙离子 信号、有机卤化物解毒、金属离子隔离和动态平衡以及底物 氧气注入。在这样的项目中，布里特实验室与各种各样的 美国国立卫生研究院资助的众多研究项目的合作者。

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.