Biological Spectroscopy and Crystallography Using an X-ray Free Electron Laser

项目摘要/摘要 这项提议的目标是了解自然中发生的受到良好控制的化学物质 通过开发必要的工具并应用它们来跟踪金属酶的结构动力学 蛋白质和金属的化学动力学--催化剂。为此，我们将使用X射线结晶学和 X射线自由电子激光器(XFELs)的X射线能谱技术。 虽然已经研究了酶的结构和催化部位的化学 深入地说，对原子尺度化学的理解需要一种超越传统方法的新方法 低温下的稳态X射线结晶学和X射线能谱。遵循动态 金属酶的几何和电子结构在常温下的变化，而 克服X射线对氧化还原活性催化中心的严重破坏是产生反应的关键 机制。来自XFELs的高强度和超短飞秒(Fs)X射线脉冲提供了一个机会 克服目前同步加速器X-X生物样品室温数据采集的局限性 射线源。通过逐个样品替换的飞秒X射线脉冲，可以获取信号 在损坏发生和样品被销毁之前。 我们将设计和应用一套时间分辨X射线衍射和X射线吸收/发射 利用XFEL光束的独特性质的光谱方法。这将提供一个 前所未有的蛋白质和辅助因子之间的相关数据组合，所有这些都是 必须了解蛋白质的几何结构和电子结构之间的相互作用 金属络合物，以及功能后果。发射和吸收光谱学 光谱学，将提供电子结构的时间演化，而同时室温 X射线结晶学将帮助我们可视化整个蛋白质几何结构的变化。 我们将使用这些方法来研究生物学中一些最重要的金属酶 深入了解催化机理，包括单核和双核体系 异质金属中心。这些系统的一个典型例子是用于氧键和C-H键的铁酶 高价铁参与的激活，例如含有细胞色素P450的血红素 系统、非血红素酶、核糖核酸还原酶(Mn/Fe和Fe/Fe)和甲烷单加氧酶 (铁/铁)。我们还将重点介绍与氢气生成和甲烷代谢相关的镍和镍/铁酶，以及 重要的一类血红素-铜氧化酶系统的功能类似物，被设计成更简单的蛋白质。

Project Summary/Abstract The goal of this proposal is to understand nature’s well-controlled chemistry that occurs in metalloenzymes, by developing the necessary tools and applying them to follow the structural dynamics of the protein and chemical dynamics of the metal-catalyst. For this purpose, we will use X-ray crystallography and X-ray spectroscopy techniques at X-ray Free Electron Lasers (XFELs). Although the structure of enzymes and the chemistry at the catalytic sites have been studied intensively, an understanding of the atomic-scale chemistry 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 geometric and electronic structure of metalloenzymes at ambient temperature, while overcoming the severe X-ray damage to the redox active catalytic center, is key for deriving the reaction mechanism. The intense and ultra-short femtosecond (fs) X-ray pulses from XFELs provide an opportunity to overcome the current limitations of room temperature data collection for biological samples at synchrotron X- ray sources. The fs X-ray pulses with shot-by-shot sample replacement make it possible to acquire the signal before damage occurs and the sample is destroyed. We will design and apply a suite of time-resolved X-ray diffraction and X-ray absorption/emission spectroscopy methods, that make use of the unique properties of the XFEL beam. This will provide an unprecedented combination of correlated data between the protein and the co-factors, all of which are necessary to understand the interplay between the geometric structure of the protein and electronic structure of the metal complex, and the functional consequences. Spectroscopy, both emission and absorption spectroscopy, will provide the time-evolution of the electronic structure, while simultaneous room temperature X-ray crystallography will help us visualize the changes in the geometric structure of the overall protein. We will use these methodologies to study some of the most important metalloenzymes in biology to gain insights into the catalytic mechanisms, including mono, and dinuclear systems both with homo- and hetero-metallic centers. A representative example of these systems are Fe enzymes for oxygen and C-H bond activation where the involvement of high-valent Fe are proposed, such as the heme containing Cyt P450 systems, non-heme enzymes ribonucleotide reductase (Mn/Fe and Fe/Fe), and methane mono oxygenase (Fe/Fe). We will also focus on Ni and Ni/Fe enzymes relevant to H2 generation and methane metabolism, and functional analogs of the important class of heme-copper oxidase systems engineered into simpler proteins.