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

项目摘要/摘要 含锰的金属蛋白具有氧化还原活性，参与多种生理过程。 氧代谢的重要反应。也许最复杂的是涉及到的Mn4CaO5簇 在光系统II(PS II)中水氧化为氧气的过程中，~500 kDa的多亚单位膜蛋白 很复杂。PS II中的水氧化反应包括从两个水分子中移除四个电子，在 通过光诱导氧化的一种循序渐进的方式，产生氧分子。PS II和Mn4CaO5簇合物 产生几乎所有支持有氧生命的氧气，它在大气中非常丰富，因为 它通过水的氧化不断地再生。光诱导水氧化成氧气是其中之一 生物圈中发生的如此大规模的最重要的化学过程。 虽然对PS II的结构和催化部位的化学进行了深入的研究， 在原子尺度上理解从光吸收到水氧化的化学顺序需要 一种超越传统稳态X射线结晶学和X射线能谱的新方法 低温温度。随着PS II和Mn4CaO5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X射线对氧化还原的严重损害 主动中心是推导这一机制的关键。高强度超短飞秒(Fs)X射线脉冲 来自X射线的自由电子激光(XFEL)提供了一个克服目前房间限制的机会 在传统X射线源上采集生物样品的温度数据。飞秒X射线脉冲使我们能够 在样品被破坏之前获取信号，从而使本文提出的光诱导快照研究 有可能。 本研究的目的是用X射线衍射法研究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(电荷密度和自旋密度，以及 在PS II的光驱动过程中，我们将使用XFEL设备 斯坦福大学和其他地方同时收集X射线衍射和发射光谱，以及X射线吸收 以时间分辨的方式在室温下以其自然态和中间态的Mn团簇的光谱， 以捕捉短暂的中间体和包括O-O键形成的步骤。我们也已经开始 研究作为PS II修复和组装的MN簇的组装过程是必不可少的组成部分 在自然界中。 这些研究有可能提供史无前例的相关数据组合， PS II蛋白、辅助因子和Mn4CaO5簇，提供了几何和电子结构以及 在催化循环中发生的变化，所有这些都是完全理解 水的氧化机理。 好了！

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. !