Thermodynamic, Electronic, Structural, and Kinetic Characterizations of Cytochrome P450 Compounds I & II

细胞色素 P450 化合物 I 的热力学、电子、结构和动力学表征

• 批准号: 9918762
• 负责人: MICHAEL T. GREEN
• 金额: $ 32.77万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918762
• 关键词: Address; Architecture; Area; Biological; Catalysis; Chemistry; Cytochrome P450; Cytochromes; Data; Electrons; Enzymes; Equation; Funding; Heme; Histidine; Hydrogen Bonding; Hydroxides; Investigation; Kinetics; Label; Ligands; Ligation; Measurement; Measures; Mediating; Metabolism; Metals; Nature; Neutron Diffraction; Oxygen; Pathway interactions; Peroxidases; Pharmacologic Substance; Phase; Play; Positioning Attribute; Process; Proteins; Protons; Reporting; Research Project Summaries; Role; Series; Synthesis Chemistry; System; Techniques; Testing; Thermodynamics; Variant; alkalinity; ascorbate; catalyst; density; design; driving force; electronic structure; experimental study; ferryl iron; geometric structure; innovation; insight; oxidation; protonation; theories

Project Summary The research outlined in this proposal seeks to further our understanding of the factors that govern C-H bond activation in cytochrome P450 catalysis. Over the past several years my group has had made significant contributions to this area. Our results have impacted not only the way people think about P450 catalysis but also metal-oxo mediated C-H bond activation in general. We have led the way in the capture and characterization of critical intermediates in the P450 catalytic cycle and developed theories to describe how Nature biases enzymes for C-H bond activation. Still, much remains to be done. Our understanding of the factors that govern C-H bond activation in P450s remains incomplete. Importantly, results from our last funding period have shown that P450 can serve as a platform from which to attack some of the most important and fundamental questions in the field of C-H bond activation. There is currently a debate in the field about the factors that govern reactivity in metal-oxo driven C-H bond activation. The debate centers on whether ground state thermodynamics play the dominant role in determining reactivity or whether unpaired spin-density on the oxo ligand can provide an intrinsic lowering of the activation barrier. The examination of this fundamental issue has been hindered not only by the difficulty of measuring these quantities for reactive high-valent species but also by the lack of a series of isoelectronic and isostructural compounds over which these quantities can be varied. Our preliminary data show that P450 can fill this void. Innovations, from the last funding period, will allow us use P450 to measure the ground state thermodynamics of C-H bond activation (i.e. D(O-H), E0I, and pKaII), quantify the degree of oxyl-radical character in compound I, and, importantly, track how these quantities (and the reactivity towards C-H bonds) change as a function of electron donation from the axial ligand. The experiments outlined in this proposal will thus use an isoelectronic and isostructural system (cytochrome P450) to determine the importance of tunneling, thermodynamics, oxyl-radical character, and strong axial electron- donation in promoting C-H bond activation. There is currently no other system, synthetic or biological, that allows for a similar set of measurements and discovery. These experiments and others will evaluate our understanding of the electronic and geometric structures of compound I as well as the protective role of P450's axial thiolate ligand. We have proposed that P450's thiolate ligand can decrease the driving force for non- productive oxidations of the protein superstructure, effectively governing the partition between productive and non-productive oxidations, biasing the system towards C-H bond activation. This theory, which depends on the interplay of the one-electron reduction potential of compound I, the pKa of compound II, and the control of proton flow via substrate positioning and enzyme architecture, remains to be verified. Innovations from the last funding period will allow us to test this hypothesis.

项目概要 本提案中概述的研究旨在进一步了解控制 C-H 债券的因素 细胞色素 P450 催化的激活。在过去的几年里，我的团队取得了重大成就 对该领域的贡献。我们的结果不仅影响了人们对 P450 催化的看法，而且影响了 一般而言，还有金属-氧介导的C-H键活化。我们在捕获和捕获方面处于领先地位 P450 催化循环中关键中间体的表征并开发了描述如何进行的理论 大自然偏爱酶来激活 C-H 键。尽管如此，仍有许多工作要做。我们的理解 控制 P450 中 C-H 键激活的因素仍然不完整。重要的是，我们上次融资的结果 时期已经表明，P450 可以作为一个平台来攻击一些最重要和最重要的 C-H键活化领域的基本问题。目前业界对此存在争论 控制金属-氧驱动的 C-H 键活化反应性的因素。争论的焦点是是否地面 状态热力学在确定反应性或是否存在不成对的自旋密度方面起着主导作用 氧代配体可以提供内在的活化势垒降低。对这个基本问题的考察 不仅因为难以测量活性高价物质的这些量，而且 还由于缺乏一系列等电子和等结构化合物，这些量可以通过这些化合物来表示 多种多样。我们的初步数据表明P450可以填补这一空白。上一个资助期的创新将 允许我们使用 P450 测量 C-H 键激活的基态热力学（即 D(O-H)、E0I 和 pKaII)，量化化合物 I 中氧自由基特征的程度，并且重要的是，跟踪这些量如何 （以及对 C-H 键的反应性）随着轴向配体的电子供给而变化。这 因此，本提案中概述的实验将使用等电子和等结构系统（细胞色素 P450） 确定隧道效应、热力学、氧自由基特征和强轴向电子的重要性 捐赠促进C-H键活化。目前还没有其他合成或生物系统可以 允许进行一组类似的测量和发现。这些实验和其他实验将评估我们的 了解化合物 I 的电子和几何结构以及 P450 的保护作用 轴向硫醇配体。我们提出P450的硫醇配体可以降低非-的驱动力 蛋白质上层结构的生产性氧化，有效控制生产性和生产性之间的分配 非生产性氧化，使系统偏向 C-H 键激活。这个理论取决于 化合物 I 的单电子还原电位、化合物 II 的 pKa 以及控制的相互作用 通过底物定位和酶结构的质子流仍有待验证。过去的创新 资助期将使我们能够检验这一假设。