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: 8254044
• 负责人: Rosalie Tran
• 金额: $ 4.92万
• 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/8254044
• 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

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.

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，并且通常参与多种重要的生理过程。特别是，已经出现了几种在以下方面具有功能性作用的含锰金属蛋白： 自从锰被鉴定为生物氧化还原催化中的必需金属以来，O{2} 代谢。其中包括线粒体锰超氧化物歧化酶 (SOD2)，可将超氧自由基解毒成 O{2} 和过氧化物；一种非血红素含锰假过氧化氢酶，催化过氧化物分解成H{2}O和O{2}；以及光系统 II (PSII) 中的析氧复合物 (OEC)，它可能是最重要的，因为它在光合作用过程中 H{2}O 氧化为 O{2} 过程中发挥着关键作用。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。尽管已知 OEC 是由异核 Mn4CaOx 团簇组成，其中四个电子逐步从两个 H{2}O 分子中提取，产生一个 O{2} 分子，但这一过程如何发生的详细结构和机制没有被很好地理解。此外，传统的 X 射线晶体学和光谱学方法受到氧化还原活性金属络合物对光还原辐射损伤的敏感性的限制。然而，最近开发的高强度X射线自由电子激光器（X-FEL）和“先收集后销毁”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标是确定完整 OEC 的结构，并通过绘制 Mn{4}CaO{x 的时间演化图来阐明 H{2}O 氧化为 O{2} 的催化机制。 } 使用这种新的 X-FEL 技术的集群。具体来说，将使用飞秒 X-FEL 脉冲从 PSII 微晶的连续流中同时测量 X 射线衍射 (XRD) 和 X 射线发射光谱 (XES)，以便确定 Mn{ 的电子和几何结构。 4}CaO{x}簇，也是完整的金属配合物。对于理解光合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) 配体环境和 Mn{4}CaO{x} 簇循环时的结构动力学催化步骤。为了解决这些问题并实时绘制光诱导化学步骤，实验装置中将结合具有可变时间延迟的激光激发“泵”和 X-FEL“探针”。这项研究不仅有助于了解其机制 H{2}O 氧化形成 O{2}，但这里开发的方法也应该具有广泛的应用，作为使用 X-FEL 确定其他生理上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，这些膜蛋白和氧化还原活性金属酶是容易受到X射线辐射损伤。