Crystallographic Studies of Electron Tranfer Proteins

描述（由申请人提供）：固氮，即由固氮酶催化的大气二氮还原为氨，是补充细胞材料生物合成中所用氮的唯一生物过程。该酶系统由两种金属蛋白组成，Fe蛋白和MoFe蛋白，它们介导ATP水解与底物还原的偶联。固氮酶是一种典型的酶，具有多种不同的铁硫簇，参与电子转移和底物还原，以及提供了一个很好的模型，ATP水解的能量转导。我们将利用晶体学、生物化学和光谱学的方法来研究固氮酶的酶促组装和酶簇组装机制。特别强调将放在建立我们最近观察到的FeMo-辅因子的中心的光原子配体的原子身份和机械意义，并在评估的结构框架的核苷酸介导的门控固氮酶中的电子转移过程。为了实现这些目标，我们将致力于： 1.固氮酶蛋白质的高分辨率（<1.2A）结构，特别是相关的蛋白质簇，在确定的氧化态。这将不仅包括建立集群的度量参数，而且还包括使用基于衍射的方法来分配每个集群内的各个金属站点的氧化态，并确定相关EPR g张量相对于集群结构的取向。中间体的FeMo-辅因子纳入的钼铁蛋白质也将进行研究，以解决固氮酶的生物合成机制的方面。 2.铁蛋白与MgATP的相互作用以及铁氧还蛋白和黄素氧还蛋白与生理电子供体的结合位点。 3.在转换条件下，底物和抑制剂与固氮酶结合的方式，最初通过光谱学方法，最终通过晶体学方法。 从这些研究中，我们试图建立固氮酶蛋白质的机械相关状态的分子细节，这对于定义底物还原的化学明确机制至关重要。更广泛的影响将解决核苷酸依赖的转导过程的基础上的铁蛋白和其他核苷酸开关蛋白参与信号和能量转导过程之间的相似性。

DESCRIPTION (provided by applicant): Nitrogen fixation, the reduction of atmospheric dinitrogen to ammonia catalyzed by the enzyme nitrogenase, is the sole biological process for replenishing the nitrogen that is used in the biosynthesis of cellular materials. This enzyme system consists of two metalloproteins, the Fe-protein and MoFe-protein, that mediate the coupling of ATP hydrolysis to substrate reduction. Nitrogenase is a prototypic example of an enzyme with multiple and varied iron-sulfur clusters that participate in electron transfer and substrate reduction, as well as providing an excellent model for energy transduction of ATP hydrolysis. We will utilize crystallographic, biochemical and spectroscopic approaches to investigate the enzymatic and metallocluster assembly mechanisms of nitrogenase. Special emphasis will be placed on establishing the atomic identity and mechanistic significance of the light-atom ligand we recently observed in the center of the FeMo-cofactor, and in assessing the structural framework for the nucleotide-mediated gating of electron transfer processes in nitrogenase. Towards these objectives, we will address: 1. Very high resolution (<1.2A) structures of the nitrogenase proteins, and particularly the associated metalloclusters, in defined oxidation states. This will include not only establishing the metric parameters of the clusters, but also the use of diffraction-based methods to assign the oxidation states to individual metal sites within each cluster, and to determine the orientation of the relevant EPR g-tensors with respect to the metallocluster structures. Intermediates in the incorporation of the FeMo-cofactor into the MoFe-protein will also be studied to address aspects of the biosynthetic mechanism of the nitrogenase metalloclusters. 2. The interactions of Fe-protein with MgATP and the site(s) of binding to the physiological electron donors for the reduction of substrates, ferredoxin and flavodoxin. 3. The mode of substrate and inhibitor binding to nitrogenase under turnover conditions, initially through spectroscopic methods and ultimately by crystallography. From these studies, we seek to establish molecular details of mechanistically relevant states of the nitrogenase proteins that are essential to defining a chemically explicit mechanism for substrate reduction. Broader implications will address nucleotide dependent transduction processes based on the similarities between Fe-protein and other nucleotide switch proteins involved in signal and energy transduction processes.