XAS STUDIES OF A CYTOCHROME P450 INTERMEDIATES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞色素P450酶于1966年首次被分离和报道，随后被发现在自然界中普遍存在。在人类中，大约有60种P450酶，主要参与氧化反应。肝脏中的P450是一种广谱氧化剂，可以代谢药物、前体药物和外源物质，从药理学的角度来看，它们是最重要的酶，因为它们控制着药物的剂量和清除。其他P450与疾病状态密切相关，如乳腺癌(由于制造雌二醇的P450酶芳香酶的过度产生)、前列腺癌(与P450细胞色素P17的过度产生有关)和肝病(由于可诱导的P450细胞色素P450_2E_1的过度产生)。对P450氧化剂的物理、动力学和机理研究将提供有价值的信息，但由于无法在受控条件下以高转化率产生氧化中间体，这些研究一直被排除在外。P450是含有血红素的酶，不同于其他的血红素酶，因为它们有一个硫酸盐(来自蛋白质半胱氨酸)作为铁的第五个配体。长期以来，它们被认为是通过被称为化合物I的铁(IV)-氧代卟啉自由基阳离子反应来影响氧化的，化合物I与其他含血红素的酶的化合物I衍生物有关，如过氧化物酶和过氧化氢酶。化合物I的一个电子还原物种称为化合物II。尽管人们普遍认为化合物I是P450的活性氧化剂，但追溯到1968年的快速停流混合或快速冷冻猝灭研究中并没有提供这些物种。事实上，在过去的40年里，混合方法的改进是微乎其微的，检测P450氧化剂的进展一直很缓慢，尽管据报道有数十次不成功或略有成功的尝试，人们认为，还有数十次未报告的失败。我们课题组开发了一种生产P450酶的铁氧衍生物的新方法

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The cytochrome P450 enzymes were first isolated and reported in 1966 and have subsequently been found to be ubiquitous in nature. In Man, there are about 60 P450 enzymes, involved mainly in oxidation reactions. P450s in the liver are broad spectrum oxidants that metabolize drugs, pro- drugs, and xenobiotics, making them the most important enzymes from a pharmacological perspective because they control drug dosing and clearance. Other P450s are intimately involved in disease states such as breast cancer (due to over-production of the P450 enzyme aromatase that makes estradiol), prostate cancer (linked to over-production of P450 CYP17), and liver disease (due to over-production of the inducible P450 CYP2E1). Physical, kinetic and mechanistic studies of P450 oxidants will provide valuable information, but those studies have been precluded by the inability to produce the oxidizing intermediates at high conversion under controlled conditions. P450s are heme-containing enzymes that differ from other heme enzymes in that they have a thiolate (from protein cysteine) as the fifth ligand to iron. They have long been thought to effect oxidation by reaction of an iron(IV)-oxo porphyrin radical cation known as Compound I, which is related to well characterized Compound I derivatives of other heme-containing enzymes such as peroxidases and catalases. The one electron reduced species from Compound I, an iron(IV)-oxo species with a neutral porphyrin, is termed Compound II. Despite the common belief that Compound I is the active oxidant of a P450, these species have not been available from rapid stopped-flow mixing or rapid freeze quench studies that date back to 1968. In fact, the improvements in mixing methods in the past four decades have been marginal, and progress in detecting P450 oxidants has been slow despite dozens of reported unsuccessful or marginally successful attempts and, one assumes, scores of unreported failures. Our group developed a new approach for production of the iron-oxo derivatives of P450 enzyme