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.

项目摘要 这项建议中概述的研究旨在加深我们对支配C-H键的因素的理解 细胞色素P450催化中的激活。在过去的几年里，我的团队已经取得了重要的成就 对这一领域的贡献。我们的结果不仅影响了人们对P450催化的看法，而且 一般情况下，金属氧基也介导了C-H键的活化。我们在抓捕行动中处于领先地位 P450催化循环中关键中间体的表征和描述如何 自然界偏向于激活C-H键的酶。尽管如此，仍有许多工作要做。我们对这一现象的理解 控制P450中C-H键激活的因素仍然不完整。重要的是，我们上一次资助的结果 期间已经表明，P450可以作为攻击一些最重要和 C-H键活化领域的基本问题。目前在这一领域有一场关于 在金属氧基驱动的C-H键活化中控制反应性的因素。争论的中心是是否有理由 状态热力学在决定反应性或不成对的自旋密度方面起着主导作用 氧代配体可以提供一种内在的降低激活势垒。对这一根本问题的审视 不仅因为难以测量活性高价物种的这些数量，而且 也由于缺乏一系列等电子和等结构的化合物，这些量可以在这些化合物上 多种多样。我们的初步数据显示，P450可以填补这一空白。来自上一个资助期的创新将 允许我们使用P450来测量C-H键活化的基态热力学(即D(O-H)、E0I和 PKaII)，量化化合物I中氧自由基性质的程度，并且重要的是，跟踪这些量如何 (以及对C-H键的反应性)随轴向配体的电子供给量而变化。这个 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)。 为了确定隧道效应、热力学、氧自由基特性和强轴电子的重要性- 捐赠促进C-H键活化。目前还没有其他系统，无论是合成的还是生物的 允许进行类似的测量和发现。这些实验和其他实验将评估我们的 了解化合物I的电子结构和几何结构以及P450‘S的保护作用 轴向硫酸盐配体。我们提出了P450‘S硫代配体可以降低非核糖核酸的驱动力。 蛋白质超结构的产生性氧化，有效地控制产生性和 非生产性氧化，使体系偏向C-H键活化。这一理论依赖于 化合物I的单电子还原电势与化合物II的pKa的相互作用及其调控 质子流通过底物定位和酶结构，仍有待验证。来自过去的创新 资金期限将允许我们检验这一假设。