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 该提案中概述的研究旨在进一步了解C-H债券的因素 细胞色素P450催化中的激活。在过去的几年中，我的小组变得很重要 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 金属氧气介导的C-H键激活总体上也是如此。我们已经领导了捕获的道路 P450催化循环中临界中间体的表征，并开发了理论以描述如何 自然偏见为C-H键激活。尽管如此，还有很多事情要做。我们对 在P450中控制C-H键激活的因素仍然不完整。重要的是，我们上次资金的结果 时期表明，P450可以用作攻击一些最重要的平台， C-H键激活领域的基本问题。目前，该领域有关于 控制金属氧气驱动的C-H键激活中反应性的因素。辩论以地面是否为中心 状态热力学在确定反应性或未配对的旋转密度上起主要作用 氧配体可以提供激活屏障的内在降低。对这个基本问题的检查 不仅难以测量这些数量的反应性高价值物种，还受到了阻碍 同样，由于缺乏一系列的等质和等值化合物，这些数量可以是 变化。我们的初步数据表明，P450可以填补这一空白。从上一个资金期开始的创新将 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中的oxyl-Radical特征的程度，并重要的是跟踪这些量 （以及对C-H键的反应性）随着轴向配体捐赠的电子捐赠而变化。这 因此，该提案中概述的实验将使用等元素和等值系统（细胞色素P450） 确定隧道的重要性，热力学，氧基 - 自由基特征和强轴向电子 促进C-H键激活中的捐赠。目前没有其他系统，即合成或生物学的系统 允许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 轴向硫醇酸盐配体。我们提出，P450的硫醇酸盐配体可以降低非 - 蛋白质上层建筑的生产氧化，有效地管理生产力和 非生产性氧化，将系统偏向C-H键激活。这个理论，取决于 化合物I，化合物II的PKA的单电子还原电位的相互作用和控制的控制 质子通过底物定位和酶结构的流动仍有待验证。最后的创新 资金期限将使我们能够检验这一假设。