Electronic, Structural, and Kinetic Characterizations of Cytochrome P450 Compound

描述（由申请人提供）：生物无机化学的一个主要目标是阐明使细胞色素P450能够激活惰性C-H键的因素。这些努力的核心是试图定义高度反应性中间体（称为化合物I）的几何和电子结构，该中间体负责这些苛刻的氧化反应。尽管付出了相当大的努力（以工时和联邦基金衡量），但在过去的四十年里，P450化合物I（P450-I）的捕获和表征被证明是生物化学中无法实现的目标。最近，我们的实验室在这方面取得了突破，发现了如何以高产率制备P450-I。这一发现打开了研究的大门，可以为控制C-H键活化的因素提供关键的见解。具体来说，长期寻求的P450-I的电子和结构表征是触手可及的，因为实验（采用轴向配体突变体）直接检查硫醇连接在P450-I中的作用。 P450介导的氧化。据推测，P450的给电子硫醇盐配体通过产生碱性ferryl促进C-H键活化。P450介导的羟基化由H原子提取引发。有证据表明，金属-氧代络合物提取H的能力 与所形成的O-H键的强度成比例。在血红素蛋白中，这种O-H键的强度D（O-H）由化合物I的单电子还原电位（E0）和化合物II的pKa决定。 D（O-H）= 23.06 * E0化合物I + 1.37 pKa化合物II + 57 <$2 kcal/mol（1） 当量1突出了铁基pKa的重要性，并表明P450中硫醇连接的作用：通过增加铁基碱性在可行的化合物I还原电位下促进氢提取。虽然实验已经证实了P450-II的基本性质，但这种碱性的定量测量（以铁基pKa的形式）已被证明是难以捉摸的。此外，E0没有 确定，使D（O-H）的估计复杂化。在一个令人兴奋的转折事件中，我们已经确定了一个P450，承诺允许测定硫醇连接的铁的pKa和还原电位。因此，拟议的实验提供了D（O-H）的大小，允许深入了解P450如何利用氧化还原电位和铁基碱度来活化烃。

DESCRIPTION (provided by applicant): A major goal of bioinorganic chemistry has been to elucidate factors that enable cytochrome P450 to activate inert C-H bonds. Central to these efforts have been attempts to define the geometric and electronic structures of the highly reactive intermediate, termed compound I, that is responsible for these demanding oxidations. Despite considerable effort (measured in both person hours and federal funds) the capture and characterization of P450 compound I (P450-I) proved to be an unattainable goal in biological chemistry for the last forty years. Recently, our laboratory made a breakthrough on this front, discovering how to prepare P450-I in high yield. This discovery opens the door for investigations that could provide key insights into the factors that govern C-H bond activation. Specifically, long-sought electronic and structural characterizations of P450-I are within reach, as are experiments (employing axial-ligand mutants) that directly examine the role of thiolate-ligation in P450 mediated oxidations. It has been hypothesized that P450's electron-donating thiolate ligand promotes C-H bond activation through the generation of basic ferryls. P450-mediated hydroxylations are initiated by H-atom abstraction. Evidence suggests that the ability of metal-oxo complexes to abstract H scales with the strength of the O-H bond formed. In heme proteins, the strength of this O-H bond, D(O-H), is determined by the one-electron reduction potential (E0) of compound I and the pKa of compound II. D(O-H) = 23.06 * E0compound I + 1.37 pKa compound II + 57 ¿ 2 kcal/mol (1) Eq. 1 highlights the importance of the ferryl pKa and suggests a role for thiolate ligation in P450s: to promote hydrogen abstraction at viable compound I reduction potentials by increasing ferryl basicity. Although experiments have confirmed the basic nature of P450-II, a quantitative measure of this basicity (in the form of a ferryl pKa) has proven elusive. Additionally, E0 has not been determined, complicating estimates of D(O-H). In an exciting turn of events, we have identified a P450 that promises to allow for the determination of a thiolate-ligated ferryl's pKa and reduction potential. Proposed experiments thus provide access to the magnitude of D(O-H), allowing insight into how P450s leverage redox potential and ferryl basicity to activate hydrocarbons.