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

同时进行时间分辨 X 射线光谱和晶体学：一种机制

• 批准号: 8417793
• 负责人: Rosalie Tran
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8417793
• 关键词: Address; Aerobic; Biological; Biomedical Research; Catalysis; Cell physiology; Chemicals; Chemistry; Collection; Complex; Conflict (Psychology); Coupled; Couples; Crystallography; Cyanobacterium; Data; Development; Diagnostic radiologic examination; Electronics; Electrons; Ensure; Environment; Enzymes; Event; Evolution; Generations; Goals; Green Algae; In Situ; Laboratories; Lasers; Lead; Life; Ligands; Light; Lighting; Manganese Superoxide Dismutase; Maps; Measures; Membrane Proteins; Metabolism; Metalloproteins; Metals; Methodology; Mitochondria; Nature; Noise; Oxidation-Reduction; Oxides; Oxygen; Peroxides; Photochemistry; Photons; Photosynthesis; Physiologic pulse; Pigments; Play; Population; Positioning Attribute; Process; Pump; Radiation; Reaction; Research; Resolution; Respiration; Roentgen Rays; Role; SOD2 gene; Sampling; Shapes; Signal Transduction; Source; Spectrum Analysis; Stream; Structural Chemistry; Structure; Study models; Superoxides; Suspension substance; Suspensions; Synchrotrons; System; Technology; Time; Vascular Plant; Water; X ray diffraction analysis; X ray spectroscopy; X-Ray Crystallography; X-Ray Diffraction; Xray Emission Spectroscopy; biological systems; chemical reaction; cytochrome c oxidase; electronic structure; insight; metal complex; metalloenzyme; novel; novel strategies; oxidation; photosystem II; planetary Atmosphere; protein complex

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.

描述（由申请人提供）：许多含有氧化还原活性金属中心的酶在细胞功能中起着重要作用，并且通常参与各种重要的重要过程。特别是，自从Mn鉴定为生物氧化还原催化中的必需金属以来，几种含MN的金属蛋白在O2代谢中具有功能。其中包括将超氧化物自由基解毒为O2和过氧化物的线粒体MN-塞氧化物歧化酶（SOD2）。一种非血红素含Mn的假催化酶，将过氧化物的分解催化为H2O和O2；以及光系统II（PSII）中的氧气进化复合物（OEC），这可能是最重要的，这是由于其在光合作用过程中H2O到O2的氧化中的关键作用。几乎所有支持有氧寿命的大气O2都是通过H2O氧化产生和补充的。因此，这种光引起的反应是自然界中最重要的生物氧化还原过程之一。尽管众所周知，OEC由异核MN4CAOX群集组成，其中四个电子是从两个H2O分子中逐步提取的，以产生一个O2分子，但该过程如何发生的详细结构和机制尚不很好地理解。此外，常规的X射线晶体学和光谱方法受到氧化还原活性金属络合物对光降低辐射损伤的敏感性的限制。但是，最近强烈的X射线无电子激光器（X-FEL）以及“销毁之前的收集”方法的应用为克服这一障碍提供了可行的选择。因此，该提案的一个关键目的是确定完整的OEC结构，并通过绘制使用这种新的X-FEL技术的MN4CAOX群集的时间演化来阐明H2O氧化为O2的催化机制。具体而言，将同时从具有飞秒X-Fel脉冲的PSII微晶体的连续流中同时测量X射线衍射（XRD）和X射线发射光谱（XES），以确定MN4CAOX群集的电子和几何结构，还确定金属复合物的完整性。对了解光合作用水氧化至关重要的两个基本点包括：（i）OEC电子结构的时间演化，以及（ii）配体环境中的结构动力学和MN4CAOX群集，因为它循环逐渐通过催化步骤。为了解决这些点并实时绘制光引起的化学步骤，将结合使用时间延迟的激光激发“泵”和X-FEL“探针”，将纳入实验设置中。这项研究不仅会导致对H2O氧化形成O2的机制的理解，而且此处开发的方法也应具有广泛的应用，作为使用X-Fels来确定其他重要具有生理上重要的膜蛋白的结构和动力学的模型研究，并且是X射线辐射损坏的X射线辐射损伤。