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

项目摘要 本提案中概述的研究旨在进一步了解控制C-H键的因素 细胞色素P450催化的活化。在过去的几年里，我的团队取得了重大进展， 对这一领域的贡献。我们的研究结果不仅影响了人们对P450催化的看法， 通常也是金属-氧代介导的C-H键活化。我们在抓捕行动中领先， P450催化循环中关键中间体的表征，并开发了描述如何 大自然偏向于C-H键活化的酶。但仍有许多工作要做。我们理解 控制P450中C-H键活化的因素仍然不完全。重要的是，我们上次资助的结果 期间已经表明，P450可以作为一个平台，从其中攻击一些最重要的， C-H键活化领域的基本问题。目前，该领域存在一场关于 在金属-氧驱动的C-H键活化中控制反应性的因素。争论的焦点是， 状态热力学在确定反应性或是否未成对自旋密度上起主导作用。 氧代配体可以提供活化屏障的内在降低。对这一基本问题的研究 不仅由于难以测量活性高价物质的这些量， 还由于缺乏一系列的等电子和同构化合物，这些量可以被 各种各样。我们的初步数据显示，P450可以填补这一空白。上一个融资期的创新将 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），E0 I，和 pKaII），量化化合物I中的β-自由基特征的程度，重要的是，跟踪这些量如何 (and对C-H键的反应性）作为来自轴向配体的电子供给的函数而变化。的 因此，本提案中概述的实验将使用等电子和同构系统（细胞色素P450） 确定隧道效应、热力学、电子-自由基特性和强轴向电子- 捐赠促进C-H键活化。目前没有其他系统，合成或生物， 允许类似的一组测量和发现。这些实验和其他实验将评估我们的 理解化合物I的电子和几何结构以及P450的保护作用 轴向硫醇配体。我们已经提出P450的硫醇配体可以降低非- 蛋白质超结构的生产性氧化，有效地控制了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键活化。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的相互作用和化合物II的pKa的控制 通过底物定位和酶结构的质子流，仍有待验证。最新的创新 这一时间段将使我们能够验证这一假设。

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.