Reactivity of Transient Cyt P450 Oxygen Intermediates

瞬时 Cyt P450 氧中间体的反应性

• 批准号: 7247138
• 负责人: JOHN H DAWSON
• 金额: $ 27.33万
• 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/7247138
• 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; Condition; 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; Object Attachment; Oxidants; Oximes; Oxygen; Peroxidase; 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; ferryl iron; hormone biosynthesis; human disease; malignant breast neoplasm; mutant; programs; research study; tetrahydrobiopterin; theories; thioether

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）：本提案寻求对4种最不为人所知的细胞色素P450（及相关酶）瞬态中间体的生成、表征和最重要的反应性的研究支持，这些中间体涉及氧：氧亚铁、过氧铁、氢过氧铁和氧铁（IV）[化合物I、Cpd I、氧铁（IV）卟啉+]。2个类似的含氧化合物，化合物II和ES，也将被研究。将追求3个具体目标。首先，快速动力学方法将利用改变近端和远端氢键性质的突变体，探索氢键在新观察到的“扰动”氧催化物种形成中的作用。氧NO合成酶的平行研究将使用修饰的四氢生物蛋白来探测电子转移。其次，将使用T252A P450- cam来测试过氧和氢过氧P450作为替代氧化剂的机制，T252A P450- cam是一种突变体，它形成了这些物种，但几乎没有形成主氧化剂cppd。溶剂和底物同位素对氢过氧态退火的影响将揭示其反应性的重要机理信息。两种Aim 2方法都将进一步验证P450反应性的“双氧化剂”假说。第三个目标(3)是表征瞬态氧化cppd I（及相关）中间体的性质和反应性。在优化了P450-CAM Cpd I形成的条件后，我们将使用快速冷冻淬火方法对其进行光谱表征。这将确定它的电子性质，并有助于解释它的反应性。双混合停流实验将检验cppd I的反应性，并阐明O原子通过过氧化物酶和P450转移的关键机制。使用P450-CAM，这将包括Cpd I与底物的首次直接反应，以及羟基化的分子间同位素效应的测定——羟基化的“氧反弹”机制的关键测试。从这项工作中获得的知识将使我们更全面地了解血红素酶如何激活过氧化物和双氧，这对人类健康和疾病具有重要的医学意义，特别是下文对P450的描述。P450细胞色素拥有超过3700个基因，是已知的最重要和最普遍的酶之一。在人类健康中，57个p450负责类固醇、维生素D、类二十烷酸以及药物代谢的无数关键转化。在人类疾病中，p450 -芳香化酶是乳腺癌化疗的靶点，因为它在雌激素激素的生物合成中起着至关重要的作用，并且几种p450已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。