Simultaneous Time-Resolved X-ray Spectroscopy and Crystallography: A Mechanistic

许多含有氧化还原活性金属中心的酶在细胞功能中发挥着重要作用，并经常参与各种重要的生理过程。特别是，一些含锰的金属蛋白已经出现，它们在 自确认锰是生物氧化还原催化中的必需金属以来，O2代谢。它们包括线粒体锰超氧化物歧化酶(SOD2)，它能将超氧阴离子自由基解毒为O{2}和过氧化氢；含非血红素锰的假催化酶，它催化过氧化氢分解成H{2}O和O{2}；以及光系统II(PSII)中的放氧复合体(OEC)，它可能是最重要的，因为它在光合作用过程中将H{2}O氧化为O{2}。几乎所有支持有氧生命的大气O{2}都是由OEC通过H{2}O氧化产生和补充的；因此，这种光诱导反应是自然界中发现的最重要的生物氧化还原过程之一。虽然已经知道OEC是由一个异核的Mn4CaOx团簇组成的，其中四个电子以分步方式从两个H{2}O分子中提取出来，从而产生一个O{2}分子，但这个过程的详细结构和机理还不是很清楚。此外，传统的X射线结晶学和光谱学方法受到氧化还原活性金属络合物对光还原造成的辐射损伤的敏感性的限制。然而，最近强X射线自由电子激光(X-FEL)的发展和“先收集后销毁”方法的应用为克服这一障碍提供了一个可行的选择。因此，这项建议的一个关键目标是确定完整的OEC的结构，并通过使用这种新的X-FEL技术绘制Mn{4}CaO{x}团簇的时间演化图来阐明H{2}O被氧化为O{2}的催化机理。具体地说，我们将用飞秒X-FEL脉冲同时测量PSII微晶的X射线衍射(XRD)和X射线发射光谱(XES)，以确定Mn4}CaO{x}团簇的电子结构和几何结构，以及金属络合物的完整性。理解光合作用水氧化的两个基本要点包括：(I)OEC电子结构的时间演化；(Ii)配体环境和Mn4}CaO{x}团簇在催化步骤中循环时的结构动力学。为了解决这些问题并实时绘制光诱导的化学步骤图，将在实验装置中结合具有可变时间延迟的激光激发“泵浦”和X-FEL“探头”。这项研究不仅将使我们了解心力衰竭的发病机制 H{2}O氧化生成O{2}，但该方法也可作为利用X-FELS测定其他具有生理意义的膜蛋白和氧化还原活性金属酶的结构和动力学的模型研究，具有广泛的应用前景。

Many enzymes containing redox-active metal centers play significant roles in cellular function, and are often involved in a variety of physiologically important processes. In particular, several Mn-containing metalloproteins have emerged with functional roles in O{2} metabolism since the identification of Mn as an essential metal in biological redox catalysis. These include a mitochondrial Mn-superoxide dismutase (SOD2) that detoxifies superoxide radicals into O{2} and peroxide; a non-heme Mn-containing pseudocatalase that catalyzes the decomposition of peroxide into H{2}O and O{2}; and the oxygen-evolving complex (OEC) in photosystem II (PSII), which is possibly the most important due to its key role in the oxidation of H{2}O to O{2} during photosynthesis. Nearly all of the atmospheric O{2} that supports aerobic life is produced and replenished by the OEC through H{2}O oxidation; hence, this light-induced reaction is one of the most important biological redox processes found in nature. Although it is known that the OEC is composed of a heteronuclear Mn4CaOx cluster where four electrons are extracted in a stepwise manner from two H{2}O molecules to produce one O{2} molecule, the detailed structure and mechanism of how this process occurs are not well understood. Furthermore, conventional X-ray crystallography and spectroscopy approaches are limited by the sensitivity of the redox-active metal complex to radiation damage by photoreduction. However, the recent development of the powerfully intense X-ray free electron laser (X-FEL) and application of the "collect before destroy" approach provide a viable option for overcoming this obstacle. Thus, a key objective of this proposal is to determine the structure of the intact OEC and elucidate the catalytic mechanism by which H{2}O is oxidized to O{2} by mapping the time evolution of the Mn{4}CaO{x} cluster using this new X-FEL technology. Specifically, X-ray diffraction (XRD) and X-ray emission spectra (XES) will be simultaneously measured from a continuous stream of PSII microcrystals with femtosecond X-FEL pulses in order to determine not only the electronic and geometric structure of the Mn{4}CaO{x} cluster, but also the integrity of the metal complex. Two fundamental points that are central to understanding photosynthetic water oxidation include: (i) the temporal evolution of the OEC electronic structure, and (ii) the structural dynamics in the ligand environment and Mn{4}CaO{x} cluster as it cycles through the catalytic steps. To address these points and map the light-induced chemical steps in real time, a combined laser excitation 'pump' and X-FEL 'probe' with variable time delays will be incorporated into the experimental setup. Not only will this study lead to an understanding of the mechanism of H{2}O oxidation to form O{2}, but the methodology developed here should also have broad applications as a model study for using X-FELs to determine structure and dynamics in other physiologically important membrane proteins and redox- active metalloenzymes that are prone to X-ray radiation damage.