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.

项目总结/摘要 含有Mn和Fe的金属蛋白具有氧化还原活性作用，参与多种生理学过程。 分子氧代谢和活化的重要反应。也许最复杂的是Mn 4CaO 5簇 参与光系统II（PS II）中水氧化为分子氧的过程，PS II是一种多亚基膜蛋白 复杂. PS II中的水氧化反应涉及从两个水分子中移除四个电子， 通过光诱导氧化以逐步的方式产生氧分子。PS II和Mn 4CaO 5簇合物 产生几乎所有的双氧，支持有氧生活，它是丰富的大气中，因为 它通过水的氧化不断再生。光诱导水氧化成分子氧是光诱导水氧化的一个重要途径。 最重要的化学过程在生物圈中如此大规模地发生。 虽然PS II的结构和催化位点的化学性质已经被深入研究， 理解从光吸收到水氧化的原子尺度的化学顺序需要一个 超低温下常规稳态X射线晶体学和X射线光谱学的新方法 温度随着PS Ⅱ和Mn_4CaO_5团簇结构在室温下的动态变化， 在生理温度条件下，同时克服了对氧化还原活性中心的严重X射线损伤 是衍生机制的关键。来自无X射线的超短飞秒X射线脉冲 电子激光器（XFEL）提供了一个机会，以克服目前的限制，在室温数据 在传统的X射线源上收集生物样品。飞秒X射线脉冲让我们可以获取信号 在样品被破坏之前，从而使光致快照研究成为可能。 本研究的目的是利用X射线衍射研究PS II的蛋白质结构和动力学， 以及Mn 4CaO 5簇的化学结构和变化（电荷和自旋密度，以及共价） 在PS II的光驱动过程中使用X射线光谱学。我们将使用斯坦福大学的XFEL设施， 在其他地方同时收集X射线衍射和发射光谱，和X射线吸收光谱的 Mn团簇在其自然和中间状态在室温下在时间分辨的方式，以捕获 短寿命的中间体和包括O-O键形成的步骤。我们亦已开始研究 其他含Mn/Fe/Ni的重要金属酶的化学，如甲烷单加氧酶， 核糖核苷酸还原酶、异青霉素N合酶和其它相关酶。 这些研究有可能提供一个前所未有的组合之间的相关数据， 蛋白质和金属辅因子，提供几何和电子结构以及发生的变化 在催化循环过程中，所有这些对于完全理解催化剂的作用机理都是必要的。 酶促反应