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

项目摘要/摘要 这一提议的目标是了解自然界中酶类中发生的、受到很好控制的化学物质， 通过同时跟踪蛋白质的结构动力学和催化剂的化学动力学。它是 我们的目标是通过X射线结晶学和X射线光谱学了解来自大自然的设计概念 使用X射线自由电子激光(XFELs)的技术。 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 在环境条件下金属酶的几何结构和电子结构的变化，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X射线自由电子激光的高强度和超短飞秒(FS)X射线脉冲提供 克服目前生物样品室温数据收集的局限性的机会 在同步辐射X射线源上。飞秒X射线脉冲使得有可能在样品 被毁了。这项建议的目的是研究金属酶的蛋白质结构和动力学。 使用结晶学以及催化络合物的化学结构和动力学(电荷， 自旋和共价性)在反应过程中使用光谱来理解电子转移过程 并阐明其作用机制。 我们将设计和应用全套时间分辨X射线衍射和X射线吸收/发射 光谱学方法，利用XFEL光束的独特性质，跟踪 常温的。这将提供前所未有的蛋白质和蛋白质之间相关数据的组合 共同因素，所有这些都是完整理解结构和机制所必需的。 光谱学包括发射光谱学和吸收光谱学，以全面了解 电子结构的时间演化，同时进行室温时间分辨X射线 结晶学将提供整个蛋白质几何结构的变化。 将用于开发这些方法的系统是一些最重要的 生物中利用高价铁和其他元素进行氧和C-H键活化的金属酶。 我们将专注于非血红素酶，如核糖核苷酸还原酶(锰/铁和铁/铁)和甲烷单核苷酸 加氧酶(Fe/Fe)，含细胞色素P450的血红素系统，以及血红素-铜氧化酶的功能类似物 系统被改造成更简单的蛋白质。

Project Summary/Abstract The goal of this proposal is to understand nature's well-controlled chemistry that occurs in enzymes, by following the structural dynamics of the protein and chemical dynamics of the catalyst simultaneously. It is our goal to understand the design concepts from nature with X-ray crystallography and X-ray spectroscopy techniques using 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 metallo-enzymes at ambient conditions, 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 X-ray free electron lasers 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 make it possible to acquire the signal before the sample is destroyed. The objective of this proposal is to study the protein structure and dynamics of metallo-enzymes using crystallography, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism. We will design and apply a full 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, to follow the reaction at room temperature. This will provide an unprecedented combination of correlated data between the protein and the co-factors, all of which are necessary for a complete understanding of structure and mechanism. Spectroscopy will include both emission and absorption spectroscopy to get a complete understanding of the time-evolution of the electronic structure, while simultaneous room temperature time-resolved X-ray crystallography would provide the changes in the geometric structure of the overall protein. The systems that will be used for developing these methodologies are some of the most important metallo-enzymes in biology that use high-valent Fe and other elements for oxygen and C-H bond activation. We will focus on non-heme enzymes such as ribonucleotide reductase (Mn/Fe and Fe/Fe) and methane mono oxygenase (Fe/Fe), and heme containing Cyt P450 systems, and functional analogs of heme-copper oxidase systems engineered into simpler proteins.