XAS STUDIES OF A CYTOCHROME P450 INTERMEDIATES

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 细胞色素P450酶于1966年首次被分离和报道，随后发现其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是代谢药物、前药和外源性物质的广谱氧化剂，使其成为从药理学角度来看最重要的酶，因为它们控制药物给药和清除。 其他P450与疾病状态密切相关，如乳腺癌（由于产生雌二醇的P450芳香酶的过度产生），前列腺癌（与P450 CYP 17的过度产生有关）和肝病（由于诱导型P450 CYP 2 E1的过度产生）。 P450氧化剂的物理、动力学和机理研究将提供有价值的信息，但这些研究由于不能在受控条件下以高转化率产生氧化中间体而被排除。P450是含血红素的酶，与其他血红素酶的不同之处在于它们具有硫醇盐（来自蛋白质半胱氨酸）作为铁的第五配体。 它们长期以来被认为通过称为化合物I的铁（IV）-氧代卟啉自由基阳离子的反应来实现氧化，所述化合物I与其它含血红素的酶（例如过氧化物酶和过氧化氢酶）的充分表征的化合物I衍生物有关。 来自化合物I的单电子还原物质，具有中性卟啉的铁（IV）-氧代物质，称为化合物II。 尽管普遍认为化合物I是P450的活性氧化剂，但这些物质还没有从可追溯到1968年的快速停流混合或快速冷冻淬灭研究中获得。 事实上，在过去的四十年里，混合方法的改进是微不足道的，尽管有几十次报道不成功或勉强成功的尝试，人们认为，还有几十次未报道的失败，但检测P450氧化剂的进展一直很缓慢。本课题组开发了一种新的P450酶铁氧衍生物的制备方法

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