MOLECULAR PROBES OF THE MECHANISM OF CYTOCHROME P450

细胞色素P-450已成为含血红素的罗塞塔之石 加氧酶。肺、肝、肾和上皮组织的亚型 长期以来一直被认为在致癌物激活、药物 异物解毒、类固醇和前列腺素代谢。 目前，一氧化氮合酶(NOS)已被证明是一种细胞色素P-450 从而揭开了这些酶研究的新篇章。这个 本课程的目标是阐明有机化学和无机化学。 在细胞色素P-450介导的过程中。我们的双管齐下 (I)设计细胞色素P-450和模型铁的底物 可以揭示看不见的中间体性质的卟啉络合物 以及(Ii)观察模型体系中的活性中间体，该模型体系可以 作为这些过程的化学范例。具体活动 为下一个赠款期间提出的建议如下：a.机制模型研究 没有合酶。氧代和过氧铁卟啉的反应性 与精氨酸和N-羟基精氨酸有关的化合物的配合物， 我们将研究一氧化氮合酶(NOS)的天然底物。这个 目标将是：(1)阐明一氧化氮合酶的可能机制的范围 通过观察铁卟啉介导的这些精氨酸的氧化 在定义良好的模型系统中的导数，可以研究每个 建议的催化循环的基本步骤和(2)寻找新的和 可能是异常的氧化过程，这可能有助于 一氧化氮合酶抑制剂的设计可能具有许多重要的 药理活性。B.过氧铁(III)的亲核反应 被引用来解释这种不寻常的碳-碳键的络合物 细胞色素P-450芳香酶及其氧化中的断裂事件 N-羟基精氨酸通过一氧化氮合酶转化为瓜氨酸和一氧化氮 在模型复合体中进行研究，旨在阐明 这样的过程。C.过氧亚硝酸根与水溶性的反应 铁、锰和铜的络合物将在现场进行检查 作为金属介导性损伤的探测器。这些实验将是 旨在确定形成哪些活性中间体以及它们的 生物目标很可能是。对这些过程的详细说明 将有助于设计用于催化的金属络合物 过氧亚硝酸盐的分解。这样的代理人可能是重要的 控制组织损伤的药理学意义 过氧亚硝酸根与病理状态有关，如 缺血再灌注和自身免疫性疾病。D.发展 合成和半合成磷脂组件以模拟和 了解电子转移事件在P-450中的作用是否会延长。 具体来说，区分空间方向的方法 膜结合的卟啉和相关的氧化还原组分 本课程将探索对向量过程的一般理解， 最近的研究发现，电子在两个或两个之间的转移速度 泡状组件的空间分割的合成氧化还原组分 提供有关氧化还原中心之间距离的信息将延长。

Cytochrome P-450 has become the Rosetta Stone of the heme-containing oxygenases. The isoforms of lung, liver, kidney, and epithelial tissue have been long known to play a central role in carcinogen activation, drug and xenobiotic detoxification and steroid and prostaglandin metabolism. Now, nitric oxide synthase (NOS) has been shown to be a cytochrome P-450 as well, thus opening a new chapter in the study of these enzymes. The goals of this program are to elucidate the organic and inorganic chemistry of the processes mediated by cytochrome P-450. Our two-pronged approach has been (i) to design substrates for cytochrome P-450 and model iron porphyrin complexes which can reveal the nature of unseen intermediates and (ii) to observe reactive intermediates in model systems which can serve as chemical paradigms for these processes. Specific activities proposed for the next grant period are: A. Model Studies of the Mechanism of NO Synthase. The reactivity of oxo- and peroxo-iron porphyrin complexes with compounds related to arginine and N-hydroxy arginine, the natural substrates for nitric oxide synthase (NOS) will be examined. The goals will be: (1) to illuminate the range of possible mechanisms for NOS by looking at iron porphyrin mediated oxidation of these arginine derivatives in well-defined model systems which can allow study of each elementary step of a proposed catalytic cycle and (2) to seek out new and possibly aberrant oxidation processes that may help with the rational design of NOS inhibitors which could have a number of important pharmacological activities. B. Nucleophilic reactions of iron(III) peroxo complexes that have been invoked to explain the unusual carbon-carbon bond scission event in the cytochrome P-450 aromatase and in the oxidation of N-hydroxyarginine to citrulline and nitric oxide by NO synthase will be studied in model complexes designed to illuminate the extent and nature of such processes. C. The reactions of peroxynitrite with water soluble complexes of iron, manganese and copper will be examined in the presence of DNA as a detector for metal-mediated damage. The experiments will be aimed at determining what reactive intermediates are formed and what their biological targets are likely to be. The elaboration of these processes will facilitate the design of metal complexes for the catalytic decomposition of peroxynitrite. Such agents could be of important pharmacological interest for the control of tissue damage from peroxynitrite which have been implicated in such pathological states as ischemia-reperfusion and autoimmune diseases. D. The development of synthetic and semi-synthetic phospholipid assemblies to model and understand the electron transfer events in P-450 action will be extended. Specifically, methods of differentiating the spatial orientation of membrane bound porphyrins and related redox components toward an understanding of vectorial processes in general will be explored and the very recent finding that rates of electron transfer between and among spatially partitioned synthetic redox components of vesicular assemblies afford information about distance between redox centers will be extended.