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激活惰性C-H键的因素。这些努力的核心是试图确定高活性中间体的几何和电子结构，称为化合物I，它负责这些苛刻的氧化。尽管付出了巨大的努力（以人力和联邦资金来衡量），P450化合物I （P450-I）的捕获和表征在过去的四十年中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，发现了如何高产地制备P450-I。这一发现为研究打开了大门，可以为控制C-H键激活的因素提供关键见解。具体来说，长期寻求的P450-I的电子和结构特征是触手可及的，正如实验（采用轴向配体突变体）直接检查硫酸盐连接的作用

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.