Synchrotron Spectroscopy of Nitrogenase and Hydrogenase

描述(由申请人提供)：这项提案的广泛目标是更好地了解含有金属的酶的工作原理。更具体的目标包括表征两类具有“不寻常”活性部位的铁基金属酶--固氮酶和氢酶。我们希望回答的问题围绕着分子结构(原子在哪里)、动力学(原子如何运动)和电子结构(电子如何在各种原子中分布)。为了实现这一知识，我们安排了微生物学家、生物化学家和光谱学家之间的密切合作，包括哈伍德、牛顿和克雷默小组之间的长期联合努力，以及许多其他实验室的重大贡献。在这个项目的过程中，我们将开发或增强各种光谱‘探测器’，使我们能够回答有关酶中间体结构的问题。其中包括许多基于现代同步辐射源(XAFS、NRVS、SRPACS和NFS)的X射线和核技术。我们将用一种新的激光技术-飞秒泵浦-探测光谱(FPPS)来补充这些技术。最后，我们将平衡这些‘大设施’的方法和校园的光谱，包括远红外吸收，振动圆二色谱，和共振拉曼。对于固氮酶和氢酶，我们计划解决的问题是：在催化循环过程中结构是如何变化的？底物和抑制剂结合在哪里？什么是未定义的轻原子？我们的光谱技术将使我们能够监测酶的活性部位，重点是某些区域。我们将利用这一能力专注于蛋白质结晶学无法触及的结构和动态问题。细菌酶的研究与公众健康有何关系？很简单，固氮酶和生物固氮是氮循环的重要组成部分，这个过程负责我们吃的一半以上的食物。对于氢酶来说，对其催化机理的更好理解为未来无污染的“氢经济”提供了希望。除了关于这两种酶的专门知识外，在这项研究过程中开发的技术将适用于参与人类新陈代谢的数百种金属蛋白。

DESCRIPTION (provided by applicant): The broad objective of this proposal is a better understanding of how metal-containing enzymes work. The more specific aims involve characterization of two classes of Fe-based metalloenzymes with 'unusual' active sites - nitrogenase and hydrogenase. The questions that we hope to answer revolve around molecular structure (what atoms are where) dynamics (how the atoms move), and electronic structure (how are electrons distributed among the various atoms). To achieve this knowledge, we have arranged a close collaboration between microbiologists, biochemists, and spectroscopists, including a long-term joint effort between the Harwood, Newton, and Cramer groups, as well as major contributions from many other labs. In the course of this project we will develop or enhance a variety of spectroscopic 'probes' that allow us to answer questions about the structure of enzyme intermediates. These include numerous x-ray and nuclear techniques based on modern synchrotron radiation sources - (XAFS, NRVS, SRPACS, and NFS). We will complement these with a novel laser technique - femtosecond pump-probe spectroscopy (FPPS). Finally, we will balance these 'large facility' methods with campus-based spectroscopies including far-infrared absorption, vibrational circular dichroism, and resonance Raman. For nitrogenase and hydrogenase, the questions that we plan to address are: How does structure change during the course of the catalytic cycle? Where do substrates and inhibitors bind? What are the undefined light atoms? Our spectroscopic techniques will allow us to monitor the enzyme active sites with emphasis on certain regions. We will use this capability to focus on structural and dynamic issues that are beyond the reach of protein crystallography. How is research on bacterial enzymes related to public health? Quite simply, nitrogenase and biological nitrogen fixation are essential components of the nitrogen cycle, a process responsible for more than half of the food we eat. For hydrogenase, a better understanding of its catalytic mechanism offers the promise of a future pollution-free 'hydrogen economy'. Apart from specific knowledge about these two enzymes, the techniques developed in the course of this study will be applicable to the hundreds of metalloproteins involved in human metabolism.