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

描述（由申请人提供）：本提案的科学目的是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，了解酶中发生的自然界良好控制的化学反应。我们的目标是利用X射线晶体学和X射线光谱学技术，使用X射线自由电子激光（XFEL）来理解这些来自自然界的设计概念。尽管酶的结构和催化位点的化学反应已经得到了深入的研究，但对原子尺度化学的理解需要一种超越低温下传统稳态X射线晶体学和X射线光谱学的新方法。遵循金属酶在环境条件下几何结构和电子结构的动态变化，同时克服对氧化还原活性催化中心的严重X射线损伤，是推导反应机理的关键。LCLS（直线加速器相干光源）X射线自由电子激光器的强烈和超短飞秒（fs）X射线脉冲提供了一个机会，以克服目前的限制，在常规同步加速器X射线源的生物样品的室温数据收集。fs X射线脉冲使得在样品被破坏之前获取信号成为可能。该提案的目的是使用晶体学研究金属酶的蛋白质结构和动力学，以及在反应期间使用光谱学研究催化复合物的化学结构和动力学（电荷，自旋和共价），以了解电子转移过程并阐明机制。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 光谱方法在室温下跟踪反应，这将提供蛋白质，辅因子之间的相关数据的前所未有的组合，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K边发射和L边吸收光谱，以全面了解电子结构的时间演变，同时室温时间分辨X射线晶体学将提供整个蛋白质复合物几何结构的变化。该提案还将侧重于同时跟踪生物系统中多个地点发生的化学反应。这将使我们能够遵循 在不同的时间尺度和水平上，金属蛋白质中多个位点之间的电子转移;在一个位点内，在一个分子中的两个位点之间或在两个不同的分子中。这些系统将 用于开发这些方法的是生物学中一些最重要的金属酶;细胞色素c氧化酶（Fe，Cu）、核糖核苷酸还原酶（Mn，Fe）、固氮酶（Mo，Fe）和血红素酶（Fe）、细胞色素c过氧化物酶和一氧化氮合酶。

DESCRIPTION (provided by applicant): The scientific aim 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 these 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 of the LCLS (Linac Coherent Light Source) X-ray free electron laser provide an opportunity to overcome the current limitations of room temperature data collection for biological samples at regular 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 to follow the reaction at room temperature, that will provide an unprecedented combination of correlated data between the protein, the co-factors, all of which are necessary for a complete understanding of structure and mechanism. Spectroscopy will include both K-edge-emission and L-edge 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 complex. The proposal will also focus on simultaneously following the chemistry that occurs at multiple sites in biological systems. This will allow us to follow the electron transfer between the multiple sites in metalloproteins at various time-scales and levels; within a site, between two sites in a molecule or in two different molecules. The systems that will be used for developing these methodologies are some of the most important metallo-enzymes in biology; cytochrome c oxidase (Fe, Cu), ribonucleotide reductase (Mn, Fe), nitrogenase (Mo, Fe) and heme enzymes (Fe), cyctochrome c peroxidase and nitric oxide synthase.