Taking Snapshots of the Water-Oxidation Reaction by the Mn4Ca Oxygen-Evolving Complex Using X-ray Crystallography and Spectroscopy

项目总结/摘要 具有氧化还原活性作用的含锰金属蛋白参与多种生理学过程， 分子氧代谢的重要反应。也许最复杂的是Mn4CaO5簇， 在光系统II（PS II）中将水氧化为分子氧，PS II是一种~500 kDa的多亚基膜蛋白， 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， 通过光诱导氧化的逐步方式，产生氧分子。PS II和Mn4CaO5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一种 在生物圈中如此大规模地发生的最重要的化学过程。 虽然PS II的结构和催化位点的化学性质已经被深入研究， 要理解从光吸收到水氧化的原子级化学过程， 一种超越传统稳态X射线晶体学和X射线光谱学的新方法， 低温。随着PS Ⅱ和Mn_4CaO_5团簇结构的动态变化， 在生理温度下的环境条件下，同时克服了严重的X射线对氧化还原的损害， 活性中心是衍生机理的关键。强超短飞秒X射线脉冲 X射线自由电子激光（XFEL）提供了一个机会，以克服目前的限制，在房间里， 在传统的X射线源上收集生物样品的温度数据。fs x射线脉冲让我们能够 在样品被破坏之前获取信号，从而使得本文提出的光致快拍研究成为可能 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Mn4CaO5团簇的化学结构和变化（电荷和自旋密度， 共价）与X射线光谱学在光驱动过程中的PS II。我们将使用XFEL设施， 斯坦福大学和其他地方收集X射线衍射和发射光谱同时，和X射线吸收 在室温下以时间分辨的方式在其天然和中间状态下的Mn簇的光谱， 捕获短暂的中间体和包括O-O键形成的步骤。我们还开始 研究Mn团簇的组装过程作为PS Ⅱ的修复和组装是PS Ⅱ的重要组成部分 在自然界中。 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， PS II蛋白、辅因子和Mn4CaO5簇，提供了几何和电子结构以及 在催化循环过程中发生的变化，所有这些都是必要的一个完整的理解， 水的氧化机制。 !

Project Summary/Abstract Metalloproteins containing manganese in a redox-active role are involved in a variety of physiologically important reactions of dioxygen metabolism. Perhaps the most complex is the Mn4CaO5 cluster that is involved in the oxidation of water to dioxygen in photosystem II (PS II), an ~500 kDa 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 intense and 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 proposed here 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 process of assembly of the Mn cluster as repair and assembly of PS II is an essential component in nature. These studies have the potential to provide an unprecedented combination of correlated data between the PS II protein, the co-factors, and the Mn4CaO5 cluster, 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 water oxidation. !