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

描述(申请人提供)：许多含有氧化还原活性金属中心的酶在细胞功能中发挥重要作用，并经常参与各种重要的生理过程。特别是，自从锰被确定为生物氧化还原催化中的必需金属以来，已经出现了几种在氧代谢中起功能作用的含锰金属蛋白。其中包括线粒体锰超氧化物歧化酶(SOD2)，它将超氧阴离子自由基解毒为O2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为H2O和O2；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H2O氧化为O2。几乎所有支持有氧生命的大气O2都是由OEC通过H2O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，其中四个电子以一种逐步的方式从两个H2O分子中提取出来，产生一个O2分子，但这个过程如何发生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Mn4CaOx簇的时间演化图来阐明H2O被氧化为O2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射线衍射谱和X射线发射谱，以确定Mn4CaOx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电子结构的时间演变；(Ii)配体环境和Mn4CaOx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”。这项研究不仅将有助于理解H2O氧化生成O2的机制，而且所开发的方法也将作为使用X-FELS来确定其他生理重要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。

DESCRIPTION (provided by applicant): 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 O2 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 O2 and peroxide; a non-heme Mn-containing pseudocatalase that catalyzes the decomposition of peroxide into H2O and O2; 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 H2O to O2 during photosynthesis. Nearly all of the atmospheric O2 that supports aerobic life is produced and replenished by the OEC through H2O 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 H2O molecules to produce one O2 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 H2O is oxidized to O2 by mapping the time evolution of the Mn4CaOx 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 Mn4CaOx 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 Mn4CaOx 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 H2O oxidation to form O2, 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.