Crystallographic Studies of Electron Tranfer Proteins

说明(申请人提供)：固氮，由固氮酶催化将大气中的氮素还原为氨的过程，是补充用于细胞材料生物合成的氮的唯一生物过程。该酶系统由两种金属蛋白组成，即铁蛋白和钼铁蛋白，它们介导了ATP水解酶与底物还原的偶联。固氮酶是一个典型的具有多个不同的铁-硫簇的酶，参与电子转移和底物还原，并为ATP水解酶的能量传递提供了一个很好的模型。我们将利用结晶学、生化和光谱学方法来研究固氮酶的酶和金属簇组装机制。我们将特别强调建立我们最近在FEMO辅因子中心观察到的轻原子配体的原子同一性和机制意义，以及评估固氮酶中核苷酸介导的电子转移过程的门控的结构框架。为达致这些目标，我们会采取以下措施： 1.在限定的氧化状态下，固氮酶蛋白质，特别是相关金属簇的极高分辨率(&lt；1.2a)结构。这不仅包括建立团簇的计量参数，而且还包括使用基于衍射的方法将氧化态分配给每个团簇中的单个金属位置，并确定相关EPR g张量相对于金属团簇结构的取向。还将研究将FeMo辅因子结合到MoFe蛋白中的中间体，以解决固氮酶金属簇生物合成机制的各个方面。 2.铁蛋白与三磷酸镁的相互作用及与底物铁氧还蛋白和黄褐还蛋白的结合部位(S)。 3.底物和抑制剂在周转条件下与固氮酶的结合方式，最初是通过光谱方法，最终是通过结晶学。 从这些研究中，我们试图建立固氮酶蛋白的机械相关状态的分子细节，这对于定义底物还原的化学显性机制是必不可少的。更广泛的影响将解决核苷酸依赖的转导过程，基于铁蛋白和其他参与信号和能量转导过程的核苷酸开关蛋白之间的相似性。

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.