Reactivity of Transient Cyt P450 Oxygen Intermediates

描述（由申请方提供）：本提案旨在支持对4种最不了解的细胞色素P450（和相关酶）瞬时中间体的生成、表征以及最重要的反应性进行研究-这些中间体涉及氧：氧合亚铁、过氧铁、氢过氧铁和氧合铁（IV）[化合物I，化合物I，氧合铁（IV）porph+]。还将检查2种类似的含氧物质，化合物II和ES。将实现三个具体目标。首先（1），快速动力学方法将通过使用具有改变的近端和远端氢键性质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。氧化NO合酶的平行研究将使用修饰的四氢生物蝶呤探测电子转移。第二，将使用T252 A P450-CAM测试过氧和氢过氧P450可作为替代氧化剂的机制，T252 A P450-CAM是形成这些物质但几乎不形成主要氧化剂Cpd I的突变体。氧P450-CAM的单电子冷冻还原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状态退火的影响将揭示其反应性的重要机理信息。这两种目标2方法将进一步测试P450反应性的“两种氧化剂”假设。第三个目标（3）是表征瞬时氧代化合物I（和相关）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化合物I的反应性，并澄清过氧化物酶和P450的O原子转移的关键机制方面。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分子间同位素效应的测定--这是对广为人知的羟基化“氧反弹”机制的关键测试。从这项工作中获得的知识将导致对血红素酶如何激活过氧化物和双氧的更完整的理解，对人类健康和疾病具有重要的医学意义，特别是如下文所述的P450。P450细胞色素基因组包含超过3700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，类花生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，因为它在雌激素生物合成中起着重要作用，并且已经证明几种P450是前致癌物如多环芳烃和亚硝胺的活化剂。了解P450机制的进展将促进医学进步以解决这些健康问题。

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.