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Reactivity of Transient Cyt P450 Oxygen Intermediates

瞬时 Cyt P450 氧中间体的反应性

基本信息

批准号：
7644332
负责人：
JOHN H DAWSON
金额：
$ 27.26万
依托单位：
UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
1979
资助国家：
美国
起止时间：
1979-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7644332
关键词：
Address Affect Alkanes Alkenes Amino Acids Aromatase Aromatic Polycyclic Hydrocarbons Biological Camphor Catalysis Cell Respiration Chemistry Chemotherapy-Oncologic Procedure Chloride Peroxidase Complex Cytochrome P450 Dioxygen Disease Distal Eicosanoids Electron Transport Electronics Electrons Enzymatic Biochemistry Enzymes Estrogens Freezing Generations Genes Goals Health Heme Heme Iron Horseradish Peroxidase Human Hydrogen Bonding Hydroxylation Imidazole Investigation Isotopes Kinetics Knowledge Lead Length Life Ligands Measures Medical Methods Modeling Myoglobin Nature Nitric Oxide Synthase Nitrosamines Oxidants Oximes Oxygen Peroxidases Peroxides Play Preparation Property Reaction Reporting Research Research Personnel Role Scaffolding Protein Scanning Series Solvents Spectrum Analysis Steroids Stretching Structure System Testing Time Vitamin D Work adduct cofactor design drug metabolism electronic structure ferryl iron hormone biosynthesis human disease malignant breast neoplasm mutant programs research study tetrahydrobiopterin theories thioether

项目摘要

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活性的研究，这些中间体涉及氧：氧亚铁、过氧铁、氢过氧铁和氧合铁(IV)[化合物I，CPD I，氧合铁(IV)Porph+]。2个类似的氧物种，化合物II和ES，也将被检查。将追求3个具体目标。首先(1)，快速动力学方法将利用近端和远端氢键性质改变的突变体来探索氢键在形成新观察到的“扰动”氧催化物种中的作用。对一氧化氮合酶的平行研究将使用改良的四氢生物蝶呤来探测电子转移。其次(2)，过氧基和氢过氧基P450可能作为替代氧化剂的机制将使用T252AP450-CAM来测试，T252AP450-CAM是形成这些物种但几乎不是主要氧化剂的突变体，CPD I。氧P450-CAM的单电子冷冻还原为研究过氧基和氢过氧基状态提供了另一种方法；溶剂和底物对氢过氧基状态的退火的同位素效应将揭示有关其反应性的重要机理信息。两种Aim 2方法都将进一步检验P450反应性的“两种氧化剂”假说。第三个目标(3)是表征瞬时氧代CPD I(和相关的)中间体的性质和反应活性。在优化了形成P450-CAM CPD I的条件后，我们将使用快速冷冻-淬灭方法对其进行光谱表征。这将确定其电子性质，并有助于解释其反应性。双混合停流实验将检查CPD I的反应活性，并阐明过氧化物酶和P450进行O原子转移的关键机制。有了P450-CAM，这将包括CPD I与底物的第一次直接反应，以及羟化分子间同位素效应的测定-这是对公认的羟化“氧反弹”机制的关键测试。从这项工作中获得的知识将导致更完整地了解血红素酶如何激活过氧化氢和二氧化氧，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细胞色素有3700多个基因，是已知的最基本和最普遍的酶之一。在人类健康中，57个P450负责类固醇、维生素D、二十烷类化合物以及药物代谢的无数关键转变。在人类疾病中，P450-芳香酶是乳腺癌化疗的靶点，因为它在雌激素的生物合成中起着至关重要的作用，一些P450被证明是多环芳烃和亚硝胺等前致癌物的激活剂。理解P450机制的进展将促进医学进步，以解决这些健康问题。

项目成果

期刊论文数量（39）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Spectroscopic characterization of the ferric states of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase: His-ligated peroxidases with globin-like proximal and distal properties.

Amphitrite ornata 脱卤过氧化物酶和 Notomastus lobatus 氯过氧化物酶的铁态光谱表征：具有珠蛋白样近端和远端特性的 His 连接过氧化物酶。

DOI：
10.1016/j.jinorgbio.2006.02.008
发表时间：
2006
期刊：
Journal of inorganic biochemistry.
影响因子：
0
作者：
Osborne,RobertL;Sumithran,Suganya;Coggins,MichaelK;Chen,Yung-Pin;Lincoln,DavidE;Dawson,JohnH
通讯作者：
Dawson,JohnH

Mono- and bis-phosphine-ligated H93G myoglobin: spectral models for ferrous-phosphine and ferrous-CO cytochrome P450.

单膦和双膦连接的 H93G 肌红蛋白：亚铁膦和亚铁 CO 细胞色素 P450 的光谱模型。