Spectroscopy Investigations of Metalloenzyme Mechanisms

使用金属中心和/或有机自由基的酶在基础酶中起着许多关键作用。 人体健康的生物化学，其生物组装或酶功能缺陷 与各种疾病有关。中华民国大卫布里特实验室正在使用先进的光谱 技术，如多频电子顺磁共振（EPR），以了解 组装和催化机制的数量这样的金属和自由基中心。许多 参与多电子氧化或还原反应的重要酶使用金属 催化剂中的簇。布里特实验室正在研究这些星团是如何通过 用光谱方法鉴定和询问组装中间体。为 实施例[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，用于催化可逆互变 质子和电子的混合物。大自然如何安全地聚集这样一个中心， 具有潜在危险的CO和CN物质？布里特实验室正在探索 由HydG催化，HydG是酶的“自由基SAM”超家族的成员， Fe（CO）x（CN）y有机金属离子在簇合成的最早阶段，以及这些 在催化簇的合成中被其它成熟酶加工。磁 核同位素（例如13 C、15 N、57 Fe）提供重要的与顺磁性的磁性相互作用。 这种集群的形式，如通过EPR为基础的方法，如电子-核双 共振（ENDOR）。这些原子核和其他原子核为其他光谱探针提供了把手， FeS簇组装，如穆斯堡尔谱、NMR和FTIR。他们聚集活跃分子的能力 通过无细胞合成的酶促位点允许布里特实验室对组装的 氢催化中氢簇和探针中间体的同位素特异光谱学。 平行实验正在解开复杂的Fe-S“M-簇”在心脏的生物合成 固氮酶，它可以纳入钼或钒或额外的铁在其活性部位。 布里特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 各种反应，如有机辅因子和维生素的生物合成和翻译后 短肽的修饰。EPR和其他光谱学的进一步研究将 具有多种功能的探针金属酶和自由基酶，包括NO和Ca 2 + 信号传导、有机卤化物解毒、金属离子螯合和体内平衡以及底物 氧气注入在这些项目中，布里特实验室与各种各样的 在许多NIH支持的研究项目中工作的合作者。

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.