Electronic, Structural, and Kinetic Characterizations of Cytochrome P450 Compound

细胞色素 P450 化合物的电子、结构和动力学表征

• 批准号: 8625775
• 负责人: MICHAEL T. GREEN
• 金额: $ 25.56万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 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 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8625775
• 关键词: Biochemistry; Bioinorganic Chemistry; Chemicals; Chemistry; Chloride Peroxidase; Complex; Coupled; Coupling; Cytochrome P450; Cytochromes; Electron Nuclear Double Resonance; Electronics; Electrons; Enabling Factors; Environment; Enzymes; Event; Funding; Generations; Goals; Hemeproteins; Hour; Hydrocarbons; Hydrogen; Hydrogen Bonding; Hydroxylation; Investigation; Iron; Kinetics; Knowledge; Label; Laboratories; Ligands; Ligation; Location; Measurement; Measures; Mediating; Metabolism; Metals; Metric; Nature; Oxidation-Reduction; Oxygen; Persons; Pharmacologic Substance; Phase; Porphyrins; Process; Property; Proteins; Raman Spectrum Analysis; Reaction; Relative (related person); Relaxation; Reporting; Roentgen Rays; Role; Selenocysteine; Spectrum Analysis; Structure; Sulfur; System; Variant; absorption; abstracting; alkalinity; base; cytochrome P-450 CYP119 (Sulfolobus solfataricus); density; electronic structure; ferryl iron; insight; mutant; oxidation; protonation; quantum; research study

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.

描述（由适用提供）：生物无机化学的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定义高度反应性中间体的几何和电子结构，称为化合物I，这是这些苛刻的氧化物的原因。尽管付出了巨大的努力（在人小时和联邦资金中都衡量），但在过去四十年中，P450化合物I（P450-I）的捕获和表征是生物化学的无法实现的目标。最近，我们的实验室在这方面取得了突破，发现了如何以高收率准备P450-I。这一发现为调查打开了大门，可以为控制C-H键激活的因素提供关键的见解。具体而言，P450-I的长期电子和结构性特征以及实验（采用轴向配体突变体）也可以直接研究硫醇酸盐 - 结合在 P450介导的氧化物。据推测，P450的含硫代硫酸盐配体可以通过基本渡轮的产生促进C-H键激活。 P450介导的羟基化是通过H原子抽象引发的。有证据表明，金属氧复合物与形成的O-H键的强度抽象H量表的能力。在血红素蛋白中，该O-H键D（O-H）的强度取决于化合物I的单电子还原势（E0）和化合物II的PKA。 d（o-h）= 23.06 * e0councound I + 1.37 PKA化合物II + 57€2 kcal/mol（1） 等式。 1强调了Ferryl PKA的重要性，并提出了P450S中硫醇酸盐连接的作用：通过提高渡轮碱性，在可行化合物I降低电位上促进抽象。尽管实验已经证实了P450-II的基本性质，但是对这种碱性（以渡轮PKA的形式）的定量度量已证明是弹性的。此外，E0还没有 确定，使D（O-H）的估计值复杂化。在激动人心的事件转变中，我们确定了P450，该P450有望确定硫醇酯连接的Ferryl的PKA和还原潜力。因此，提出的实验可以访问D（O-H）的大小，从而深入了解P450如何利用氧化还原电位和渡轮碱性来激活碳氢化合物。