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）：该提案寻求对对产生，表征以及最重要的是4个最不熟悉的细胞色素P450（及相关酶）的反应性的调查，涉及氧气的人：涉及氧的人：氧气，氧气，过氧，过氧，水质量，水透明氧和氧气，和氧气（iim）[iv）氧铁（iv）porph+]。也将研究2种类似的氧化合物II和ES。将追求3个具体目标。第一（1），快速动力学方法将通过使用具有改变近端和远端氢键特性的变化的突变体，探索氢键在形成新的“扰动”氧催化物种中的作用。氧无合酶的平行研究将使用修饰的四氢无生物蛋白探测电子转移。 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;溶剂和底物同位素对Hydroperoxo状态退火的效果将揭示有关其反应性的重要机械信息。两种目标2方法都将进一步检验P450反应性的“两个氧化剂”假设。第三个目标（3）是表征瞬态OXO CPD I（和相关）中间体的特性和反应性。具有针对P450-CAM CPD I组的优化条件，我们将使用快速冻结方法以光谱镜来表征它。这将建立其电子特性，并有助于解释其反应性。双混合止流实验将检查CPD I反应性，并阐明O原子转移的关键机理方面和P450。使用P450-CAM，这将包括CPD I与底物的第一个直接反应，并确定分子间同位素效应以进行羟基化 - 对羟基化的良好接受的“氧反弹”机制的重要测试。从这项工作中得出的知识将使对血红素酶如何激活过氧化酶和二氧化物具有对人类健康和疾病的重要医学意义，尤其是如下所述的P450所述。 P450细胞色素有超过3700个基因，是已知的最重要，无处不在的酶之一。在人类健康中，有57个P450负责类固醇，维生素D，eicosanoid以及药物代谢的无数关键转化。在人类疾病中，p450-芳香剂是乳腺癌化疗的靶标，因为其在雌激素激素生物合成中的重要作用，并且已证明几种P450是procarcinogens的激活剂，例如多环芳族芳烃和硝基胺。理解P450机制的进展将促进医疗进步以解决这些健康问题。