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石碑 加氧酶 肺、肝、肾和上皮组织的同种型 长期以来一直被认为在致癌物活化、药物 和异生物质解毒以及类固醇和前列腺素代谢。 现在，一氧化氮合酶（NOS）已被证明是细胞色素P-450 这也为这些酶的研究揭开了新的篇章。 的 该计划的目标是阐明有机和无机化学 由细胞色素P-450介导的过程。 我们的双管齐下 一直是（i）设计细胞色素P-450和模型铁的底物 卟啉络合物，可以揭示看不见的中间体的性质 和（ii）观察模型系统中的活性中间体， 作为这些过程的化学范例。 具体活动 下一个补助期的建议是：A。机理模型研究 NO合成酶 氧代和过氧铁卟啉的反应性 与精氨酸和N-羟基精氨酸相关的化合物的复合物， 将检测一氧化氮合酶（NOS）的天然底物。 的 目的是：（1）阐明NOS的可能机制 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衍生物，可以允许研究每个 一个基本步骤的建议催化循环和（2）寻求新的， 可能异常的氧化过程，可能有助于合理的 NOS抑制剂的设计可能具有许多重要的 药理活性 B。过氧化铁的亲核反应 用来解释不寻常的碳-碳键的复合物 细胞色素P-450芳香化酶和 N-羟基精氨酸通过NO合酶转化为瓜氨酸和一氧化氮， 研究模型复杂的设计，以阐明的程度和性质， 这样的过程。 C.过氧亚硝基阴离子与水溶性 铁、锰和铜的络合物将在存在下进行检查。 DNA作为金属介导损伤的检测器。 实验将是 目的是确定形成了什么反应性中间体以及它们的 生物目标可能是。 对这些过程的阐述 将促进用于催化的金属络合物的设计， 过氧亚硝酸盐的分解。 这些代理人可能是重要的 用于控制组织损伤的药理学意义 过氧亚硝酸盐与病理状态有关， 缺血-再灌注和自身免疫性疾病。 D.的发展 合成的和半合成的磷脂组件来建模， 了解P-450作用中的电子转移事件将得到扩展。 具体地，区分物体的空间取向的方法 膜结合卟啉和相关的氧化还原组分， 将探索对矢量过程的一般理解， 最近的研究发现， 空间分隔的合成的泡状组装体的氧化还原组分 提供有关氧化还原中心之间距离的信息将被扩展。

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.