Synchrotron NRVS and Campus Spectroscopy of Multinuclear Fe Proteins: N2ase, H2as

描述（由申请人提供）：本提案的广泛目标是更好地了解含金属酶的工作原理。更具体的目标涉及三个类别的铁基金属酶与“不寻常的”活性位点的表征-固氮酶（N2酶），氢化酶（H2酶），和甲烷单加氧酶（MMO）。我们还试图了解来自分枝杆菌和链霉菌细菌的特殊Fe-S簇（“Wbl”）蛋白如何在其环境中感知NO和O2。我们希望回答的问题围绕着分子结构（什么原子在哪里）和动力学（原子如何运动）。为了获得这些知识，我们安排了化学家，生物化学家和光谱学家之间的密切合作。我们将开发或增强光谱“探针”，使我们能够回答有关酶中间体结构的问题。其中包括同步辐射实验室的X射线和核技术- XAFS和NRVS。我们将平衡这些“大型设施”的方法与校园为基础的光谱学，包括EPR，FT-IR和穆斯堡尔谱，无论是作为直接探针，并结合紫外可见光光解方法。对于N2酶，H2酶和MMO，我们计划解决的问题是：在催化循环过程中结构如何变化？底物和抑制剂在哪里结合？什么是未定义的轻原子？同样的问题也适用于NO和O2与Wbl蛋白的相互作用，除了反应涉及传感小分子而不是催化。我们的光谱技术将使我们能够监测酶的活性位点。我们将利用这种能力，专注于结构和动力学的问题，是超出了蛋白质晶体学的范围。

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 three classes of Fe-based metalloenzymes with 'unusual' active sites -- nitrogenase (N2ase), hydrogenase (H2ase), and methane monooxygenase (MMO). We also seek to understand how special Fe-S cluster ('Wbl') proteins from Mycobacteria and Streptomyces bacteria sense NO and O2 in their environment. The questions that we hope to answer revolve around molecular structure (what atoms are where) and dynamics (how the atoms move). To achieve this knowledge, we have arranged close collaborations between chemists, biochemists, and spectroscopists. We will develop or enhance spectroscopic 'probes' that allow us to answer questions about the structure of enzyme intermediates. These include x-ray and nuclear techniques at synchrotron radiation labs -- XAFS and NRVS. We will balance these 'large facility' methods with campus-based spectroscopies including EPR, FT-IR, and M"ssbauer, both as direct probes and combined with UV-visible photolysis methods. For N2ase, H2ase, and MMO, 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? The same questions apply to the interactions of NO and O2 with Wbl proteins, except that the reactions involve sensing small molecules as opposed to catalysis. Our spectroscopic techniques will allow us to monitor the enzyme active sites. We will use this capability to focus on structural and dynamic issues that are beyond the reach of protein crystallography.