Taking Snapshots of Enzymatic Reactions Using X-ray Crystallography and Spectroscopy

使用 X 射线晶体学和光谱学拍摄酶反应快照

• 批准号: 10623717
• 负责人: VITTAL YACHANDRA
• 金额: $ 67.43万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623717
• 关键词: Aerobic; Atmosphere; Biochemical Reaction; Biological; Calcium; Catalytic Domain; Charge; Chemical Structure; Chemicals; Chemistry; Complex; Data Collection; Data Correlations; Dioxygen; Electronics; Electrons; Enzymes; Evolution; Excision; Life; Light; Manganese; Mediating; Membrane Proteins; Metabolism; Metalloproteins; Metals; Methane; Natural regeneration; Nature; Oxidation-Reduction; Oxygen; Physiologic pulse; Physiological; Process; Protein Dynamics; Proteins; Reaction; Ribonucleotide Reductase; Roentgen Rays; Role; Sampling; Signal Transduction; Source; Spectrum Analysis; Structure; Temperature; Time; Water; X ray diffraction analysis; X ray spectroscopy; X-Ray Crystallography; absorption; cofactor; cryogenics; density; electronic structure; geometric structure; isopenicillin N; metalloenzyme; novel strategies; optical spectra; oxidation; photosystem II; protein complex; protein structure; x-ray free-electron laser

Project Summary/Abstract Metalloproteins containing Mn and Fe in a redox-active role are involved in a variety of physiologically important reactions of dioxygen metabolism and activation. Perhaps the most complex is the Mn4CaO5 cluster that is involved in the oxidation of water to dioxygen in photosystem II (PS II), a multi-subunit membrane protein complex. The water-oxidation reaction in PS II involves removal of four electrons from two water molecules, in a stepwise manner by light-induced oxidation, to produce a molecule of oxygen. PS II and the Mn4CaO5 cluster generate almost all of the dioxygen that supports aerobic life, and it is abundant in the atmosphere because of its constant regeneration by the oxidation of water. The light-induced oxidation of water to dioxygen is one of the most important chemical processes occurring on such a large scale in the biosphere. Although the structure of PS II and the chemistry at the catalytic site have been studied intensively, understanding the sequence in the chemistry at atomic-scale from light absorption to water-oxidation requires a new approach beyond the conventional steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the structure of PS II and the Mn4CaO5 cluster at ambient conditions at physiological temperatures, while overcoming the severe X-ray damage to the redox active center is key for deriving the mechanism. The very intense, ultra-short femtosecond (fs) X-ray pulses from a X-ray free electron laser (XFEL) provide an opportunity to overcome the current limitations in room temperature data collection for biological samples at traditional X-ray sources. The fs X-ray pulses allow us to acquire the signal before the sample is destroyed, thus making the light-induced snapshot study possible. The objective of this proposal is to study the protein structure and dynamics of PS II with X-ray diffraction, as well as the chemical structure and changes in the Mn4CaO5 cluster (charge and spin density, and covalency) with X-ray spectroscopy during the light-driven process of PS II. We will use the XFEL facilities at Stanford and elsewhere to collect X-ray diffraction and emission spectra simultaneously, and X-ray absorption spectra of the Mn cluster in its native and intermediates states at room temperature in a time-resolved manner, to capture short-lived intermediates and the step that includes the O-O bond formation. We have also started studying the chemistry of other Mn/Fe/Ni containing metalloenzymes of importance such as methane monooxgygenase, ribonucleotide reductase, isopenicillin N synthase and other related enzymes. These studies have the potential to provide an unprecedented combination of correlated data between the proteins and the metal co-factors, providing the geometric and electronic structure and the changes that occur during the catalytic cycle, all of which are necessary for a complete understanding of the mechanism of the enzymatic reactions.

项目摘要/摘要 在氧化还原活性作用中含有Mn和Fe的金属蛋白参与多种生理学 二氧化代谢和激活的重要反应。也许最复杂的是MN4CAO5群集 在光系统II（PS II）中氧化为二氧化物的氧化（一种多生育膜蛋白） 复杂的。 PS II中的水氧化反应涉及从两个水分子中去除四个电子 通过光诱导的氧化逐步方式，产生氧分子。 PS II和MN4CAO5群集 生成几乎所有支持有氧生活的二氧化物，并且在大气中很丰富 它通过氧化水的恒定再生。向二氧化物的光引起的光引起的氧化是 最重要的化学过程发生在生物圈中的如此大规模上。 尽管已经对PS II的结构和催化位点的化学结构进行了深入研究，但 了解从光吸收到水氧化的原子级化学序列需要 除了低温时，除常规稳态X射线晶体学和X射线光谱以外的新方法 温度。遵循PS II和MN4CAO5群集在环境上的动态变化 生理温度下的条件，同时克服了对氧化还原活动中心的严重X射线损害 是得出该机制的关键。来自X射线的非常激烈的超短距离飞秒（FS）X射线脉冲 电子激光器（XFEL）提供了克服室温数据当前限制的机会 传统X射线源的生物样品收集。 FS X射线脉冲使我们能够获取信号 在样品被破坏之前，从而使光引起的快照研究成为可能。 该建议的目的是研究PS II的蛋白质结构和动力学，并具有X射线衍射，如 以及MN4CAO5簇的化学结构和变化（电荷和旋转密度以及共价） 在PS II的轻驱动过程中，带有X射线光谱。我们将在斯坦福大学使用XFEL设施 在其他地方同时收集X射线衍射和发射光谱，以及X射线吸收光谱 Mn簇在其天然中，并以时间分辨的方式在室温下中间状态，以捕获 短暂的中间体和包括O-O键形成的步骤。我们也开始研究 其他Mn/Fe/ni的化学含有重要性的金属酶，例如甲烷单氧化物酶， 核糖核苷酸还原酶，亚匹热蛋白N合酶和其他相关酶。 这些研究有可能提供相关数据的前所未有的组合 蛋白质和金属共同因素，提供几何和电子结构以及发生的变化 在催化循环中，所有这些都是完全理解机制的必要条件 酶促反应。