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

项目总结/摘要 这项计划的目标是了解自然界中发生在酶中的控制良好的化学反应， 通过同时遵循蛋白质的结构动力学和催化剂的化学动力学。是 我们的目标是通过X射线晶体学和X射线光谱学来理解自然界的设计概念 使用X射线自由电子激光（XFEL）的技术。 尽管人们已经研究了酶的结构和催化位点的化学性质， 从本质上讲，对原子尺度化学的理解需要一种超越传统方法的新方法。 稳态X射线晶体学和低温下的X射线光谱学。跟随动态 在环境条件下金属酶的几何和电子结构的变化，而 克服X射线对氧化还原活性催化中心的严重损害是推导该反应的关键 机制来自X射线自由电子激光器的强超短飞秒X射线脉冲提供了 一个机会，以克服目前的限制，室温数据收集的生物样品 同步加速器X射线源fs X射线脉冲使得可以在样品被分离之前获取信号。 摧毁.本研究的目的是研究金属酶的蛋白质结构和动力学 使用晶体学以及催化配合物的化学结构和动力学（电荷， 自旋和共价）在反应过程中使用光谱学来理解电子转移过程 并阐明其机制。 我们将设计和应用一套完整的时间分辨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，以跟踪反应， 室温这将提供蛋白质和蛋白质之间的相关数据的前所未有的组合。 辅因子，所有这些都是完整理解结构和机制所必需的。 光谱学将包括发射光谱和吸收光谱，以全面了解 电子结构的时间演化，而同时室温时间分辨X射线 晶体学将提供整个蛋白质的几何结构的变化。 将用于开发这些方法的系统是一些最重要的系统， 生物学中的金属酶，利用高价铁和其他元素激活氧和C-H键。 我们将重点关注非血红素酶，如核糖核苷酸还原酶（Mn/Fe和Fe/Fe）和甲烷单 加氧酶（Fe/Fe）和含血红素的Cyt P450系统，以及血红素-铜氧化酶的功能类似物 系统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.