Novel routes to catalytic intermediates in the cytochrome P450 catalytic cycle

被称为细胞色素P450（P450）的蛋白质在所有生命形式的生理学中是必不可少的。它们是血红素结合蛋白，与携带氧气的血液蛋白血红蛋白结合相同的血红素辅因子。像血红蛋白一样，P450也结合分子氧（O2）。然而，与血红蛋白不同，它们通过从伴侣蛋白传递到血红素的电子还原结合氧，并且最终来自细胞辅酶NADPH。这使得P450能够将氧分子分裂成其组成原子。两个原子中的一个用于形成水（H2O），而另一个用于与P450结合的有机底物分子结合，靠近其血红素铁。通常，羟基化（引入OH基团）被催化。在人类中，P450的活性对于类固醇激素的产生是必不可少的，并且还对于产生许多对于体内信号传导（例如用于激活免疫系统）必不可少的脂质分子是必不可少的。然而，人类有57种不同的P450，它们最著名的作用是解毒和清除体内的药物和其他外源性物质，主要由肝脏P450执行。在细菌和低等真核生物中，P450在允许不寻常分子（例如樟脑）用于提供生长能量的途径中具有重要作用，并且对于抗生素（例如红霉素）等分子的产生至关重要。P450酶在有机分子的特定位置引入氧原子的能力也引起了有机化学家的极大关注，他们正在寻找更清洁和更环保的路线来合成药物和其他重要分子。对P450结构和活性的基本了解对于了解它们如何实现其生物学功能以及如何将其应用于生物技术角色至关重要。此外，人们对了解处方药物如何与单个P450结合（以及生物技术感兴趣的分子如何与相关P450结合）非常感兴趣，因为这可以准确预测单个P450如何作用于这些分子，它们在体内的寿命以及如何通过改变药物结构来改变这些参数。确定底物/药物与P450的结合模式的常用方法是形成P450和药物之间形成的复合物的晶体，然后使用X射线衍射技术获得晶体结构。在这项提案中，我们试图解决与P450如何“激活”氧和催化羟基化反应有关的基本问题。具体来说，我们将使用现代动力学技术（包括激光闪光光解），以提供证据，形成瞬态反应血红素物种被认为是至关重要的氧化化学。此外，我们将使用这些方法来回答一个关键问题，即在P450反应“循环”中是否形成了两种不同的反应物质，以及这些物质是否具有可以利用生物技术的不同类型的活性。此外，我们将解决严重的问题有关的相关性的结合模式，在不同的P450 X射线结构的基板。我们将使用一个模型系统（P450 BM 3），以建立一个观察到的底物结合模式是否与P450中的催化有关，并挑战假设，表明底物重新定位为P450减少，或是否热效应是关键的，使基板重新定位。总的来说，这项工作将回答关于P450催化的性质和过程中不同活性中间体的相关性的基本问题。此外，它将定义在一个关键模型P450的基板结合模式和基板搬迁的相关性，与合理化基板如何结合到生物医学相关的P450的重要分支。因此，该研究对理解哺乳动物生理学中的P450活性和生物技术应用具有广泛的相关性。

The proteins known as cytochromes P450 (P450s) are essential in physiology of all life forms. They are heme-binding proteins, and bind the same heme cofactor as does the oxygen carrying blood protein hemoglobin. Like hemoglobin, P450s also bind molecular oxygen (O2). However, unlike hemoglobin they reduce bound oxygen with electrons delivered to the heme from partner proteins, and which ultimately are derived from the cell coenzyme NADPH. This enables P450s to split the oxygen molecule into its component atoms. One of the two atoms is used to form water (H2O), while the other is used to oxygenate an organic substrate molecule bound by the P450 close to its heme iron. Frequently, hydroxylation (introduction of an OH group) is catalysed. In humans, activity of P450s is essential for production of steroid hormones, and also for creation of many lipid molecules essential for signalling within the body (e.g. for activation of the immune system). However, humans have 57 different P450s, and their most famous roles are in detoxification and removal of drugs and other xenobiotics from the body / performed mainly by hepatic P450s. In bacteria and lower eukaryotes, the P450s have important roles in pathways that allow unusual molecules (e.g. camphor) to be used to provide energy for growth, and are essential for production of molecules such as antibiotics (e.g. erythromycin). The ability of P450 enzymes to introduce oxygen atoms at defined positions in organic molecules has also attracted much attention from organic chemists, who are looking for cleaner and more environmentally friendly routes to synthesis of drugs and other important molecules. A fundamental understanding of P450 structure and activity is essential to understand how they achieve their biological functions, and how they can be applied for biotechnological roles. Also, there is enormous interest in understanding how prescribed drugs bind to individual P450s (and how molecules of biotechnological interest bind to the relevant P450s), since this can lead to accurate predictions of how individual P450s act on these molecules, their lifetimes in the body and how these parameters can be changed by altering the drug structure. The usual way of determining binding modes of substrates/drugs to P450s is to form crystals of the complex made between the P450 and the drug, and then use the technique of x-ray diffraction to obtain the crystal structure. In this proposal, we seek to address fundamental questions relating to how P450s 'activate' oxygen and catalyse hydroxylation reactions. Specifically, we will use modern kinetic techniques (including laser flash photolysis) to provide evidence for formation of transient reactive heme species that are considered critical for oxygenation chemistry. Also, we will use these methods to answer a critical question relating to whether two different reactive species are formed in the P450 reaction 'cycle' and if these have differing types of activities that could be exploited biotechnologically. In addition, we will address serious issues relating to the relevance of binding modes seen for substrates in different P450 x-ray structures. We will use a model system (P450 BM3) to establish whether an observed substrate binding mode is relevant to catalysis in the P450 and to challenge hypotheses suggesting that the substrate re-positions as the P450 is reduced, or whether thermal effects are critical for causing substrate to relocate. Collectively, this work will answer fundamental questions on the nature of P450 catalysis and the relevance of distinct reactive intermediates in the process. Also, it will define the relevance of substrate binding mode and substrate relocation in a key model P450, with important ramifications for rationalising how substrates bind to biomedically relevant P450s. Thus, the study proposed has wide ranging relevance to understanding P450 activity in mammalian physiology and for biotechnological applications.