Reactivity of Transient Cyt P450 Oxygen Intermediates

瞬时 Cyt P450 氧中间体的反应性

• 批准号: 7144188
• 负责人: JOHN H DAWSON
• 金额: $ 27.53万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7144188
• 关键词: Mossbauer spectrometry; X ray crystallography; bridged cyclic compound; chemical kinetics; cryoscience; cytochrome P450; cytochrome c peroxidase; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; electron transport; enzyme mechanism; enzyme substrate; hemoprotein; horseradish peroxidase; hydrogen bond; hydrogen peroxide; iron; mutant; nitric oxide synthase; nonradiation isotope effect; oxidation reduction reaction; oxidizing agents; oxygen compounds; peroxidases; peroxides; solvents; stop flow technique; tetrahydrobiopterin

DESCRIPTION (provided by applicant): This proposal seeks support for investigations into the generation, characterization and, most importantly, reactivity of the 4 least well-understood cytochrome P450 (and related enzyme) transient intermediates - those involving oxygen: oxy-ferrous, peroxo-ferric, hydroperoxo-ferric, and oxo-iron(IV) [compound I, Cpd I, oxo-iron(IV)porph+]. 2 similar oxo species, compounds II and ES, will also be examined. 3 specific aims will be pursued. First (1), rapid kinetics methods will explore the role of hydrogen bonding in formation of a newly-observed "perturbed" oxy catalytic species by use of mutants with altered proximal and distal hydrogen bonding properties. Parallel studies of oxy NO synthase will use modified tetrahydrobiopterins to probe electron transfer. Second (2), the mechanism whereby peroxo and hydroperoxo P450 may serve as alternate oxidants will be tested using T252A P450-CAM, a mutant that forms those species but almost none of the primary oxidant, Cpd I. One-electron cryoreduction of oxy P450-CAM provides another way to study the peroxo and hydroperoxo states; solvent and substrate isotope effects on the annealing of the hydroperoxo state will reveal important mechanistic information about its reactivity. Both Aim 2 approaches will further test the "two oxidant" hypothesis of P450 reactivity. The third goal (3) is to characterize the properties and reactivities of transient oxo Cpd I (and related) intermediates. Having optimized conditions for P450-CAM Cpd I formation, we will use rapid freeze-quench methods to characterize it spectroscopically. This will establish its electronic properties, and help explain its reactivity. Double-mix stopped-flow experiments will examine Cpd I reactivity and clarify key mechanistic aspects of O atom transfer by peroxidases and P450. With P450-CAM, this will include the first direct reactions of Cpd I with substrates and determination of the intermolecular isotope effect for hydroxylation - a crucial test of the well-accepted "oxygen rebound" mechanism of hydroxylation. The knowledge derived from this work will lead to a more complete understanding of how heme enzymes activate peroxide and dioxygen with important medical implications for human health and disease, especially as described below for P450,. With over 3700 genes, P450 cytochromes are among the most essential and ubiquitous enzymes known. In human health, 57 P450s are responsible for countless critical transformations in steroid, vitamin D, eicosanoid, as well as drug metabolism. In human disease, P450-aromatase is a target for breast cancer chemotherapy owing to its vital role in estrogen hormone biosynthesis and several P450s have been shown to be activators of procarcinogens such as polycyclic aromatic hydrocarbons and nitrosamines. Progress in comprehending the P450 mechanism will promote medical advances to address these health issues.

描述（由申请人提供）：本提案寻求支持对 4 种最不为人所知的细胞色素 P450（及相关酶）瞬时中间体的生成、表征以及最重要的反应性的研究 - 涉及氧的中间体：含氧亚铁、过氧化氢-三价铁、氢过氧化铁和氧化铁 (IV) [化合物 I、Cpd I、氧化铁 (IV)porph+]。还将检查 2 种类似的含氧化合物，即化合物 II 和 ES。将追求3个具体目标。首先（1），快速动力学方法将通过使用近端和远端氢键特性改变的突变体来探索氢键在新观察到的“扰动”氧催化物种形成中的作用。氧一氧化氮合酶的平行研究将使用修饰的四氢生物蝶呤来探测电子转移。第二 (2)，将使用 T252A P450-CAM 测试过氧和氢过氧 P450 作为替代氧化剂的机制，T252A P450-CAM 是一种突变体，可形成这些物种，但几乎不形成主要氧化剂 Cpd I。氧 P450 的单电子冷冻还原-CAM提供了另一种研究过氧和氢过氧状态的方法；溶剂和底物同位素对氢过氧化态退火的影响将揭示有关其反应性的重要机制信息。两种 Aim 2 方法都将进一步测试 P450 反应性的“双氧化剂”假设。第三个目标 (3) 是表征瞬态氧化 Cpd I（及相关）中间体的性质和反应性。在优化 P450-CAM Cpd I 形成条件后，我们将使用快速冷冻猝灭方法对其进行光谱表征。这将确定其电子特性，并有助于解释其反应性。双混合停流实验将检查 Cpd I 反应性并阐明过氧化物酶和 P450 进行 O 原子转移的关键机制。借助 P450-CAM，这将包括 Cpd I 与底物的首次直接反应以及羟基化分子间同位素效应的测定——这是对公认的羟基化“氧反弹”机制的关键测试。从这项工作中获得的知识将有助于更全面地了解血红素酶如何激活过氧化物和双氧，对人类健康和疾病具有重要的医学意义，特别是如下所述的 P450。 P450 细胞色素包含 3700 多个基因，是已知最重要、最普遍的酶之一。在人类健康中，57 P450 负责类固醇、维生素 D、类二十烷酸以及药物代谢的无数关键转变。在人类疾病中，P450-芳香酶因其在雌激素生物合成中的重要作用而成为乳腺癌化疗的靶标，并且多种 P450 已被证明是多环芳烃和亚硝胺等致癌物质的激活剂。对 P450 机制的理解取得进展将促进解决这些健康问题的医学进步。