The Role of MgATP in Nitrogenase Catalysis

描述（由申请人提供）：该提案的广泛，长期目标是获得MgATP在固氮酶催化中的作用的结构和机制见解。固氮酶是一种复杂的含金属酶，催化氮气转化为氨。在固氮酶催化过程中，铁蛋白和铼-铁蛋白以导致两个MgATP分子水解和至少一个电子转移到MoFe蛋白的方式缔合和解离。铁蛋白结合和解离、MgATP水解和电子转移的多个循环是将单个氮分子完全还原成氨所必需的。固氮酶结构/功能的许多方面是基础研究的有趣领域。固氮酶是研究络合金属簇催化、电子传递、络合金属簇组装、蛋白质-蛋白质相互作用以及核苷酸依赖的信号转导的理想模型体系。此外，参与固氮酶催化的MgATP是类似的作用，在一个大类的核苷酸结合蛋白，耦合核苷酸的结合和水解蛋白质的构象变化内转导的大分子组装的核苷酸。该类成员包括G蛋白，Ras p21，RecA，延伸因子Tu，肌球蛋白和转导蛋白，使得MgATP结合和水解的作用成为固氮酶研究中最吸引人的方面之一。我们最近已经能够确定固氮酶Fe蛋白的单一缺失突变体的结构，其提供了MgATP结合状态的结构模拟。该提案的初步结果部分中描述的结构见解提供了迄今为止所描述的最坚实的基础，用于生成有关MgATP依赖的Fe蛋白质构象变化和触发MgATP水解的初始组分蛋白质相互作用的假设。拟议的研究采用了一种组合的方法，包括结构测定的X-射线衍射方法和位点特异性氨基酸取代实验，以深入了解核苷酸依赖的构象变化，大分子复合物的形成，和特定的蛋白质-蛋白质相互作用后发生的复杂的形成，启动MgATP水解固氮酶。

DESCRIPTION (provided by applicant): The broad, long term goal of the proposal is to gain structural and mechanistic insights into the role of MgATP in nitrogenase catalysis. Nitrogenase is a complex metal-containing enzyme that catalyzes the conversion of nitrogen gas to ammonia. During nitrogenase catalysis, the iron protein and molybdenum-iron protein associate and dissociate in a manner resulting in the hydrolysis of two molecules of MgATP and the transfer of at least one electron to the MoFe protein. Multiple cycles of iron protein association and dissociation, MgATP hydrolysis, and electron transfer are required for the complete reduction of a single molecule of nitrogen to ammonia. There are a number of aspects of nitrogenase structure/function that are interesting areas of fundamental research. Nitrogenase can be considered an ideal model system for the study of the complex metal cluster mediated catalysis, electron transfer, complex metal cluster assembly, protein-protein interactions, and nucleotide dependent signal transduction. In addition, the involvement of MgATP in nitrogenase catalysis is similar to the role of nucleotides in a large class of nucleotide binding proteins that couple nucleotide binding and hydrolysis to protein conformational changes transduced within a macromolecular assembly. Members of the class include G proteins, Ras p21, RecA, elongation factor Tu, myosin, and transducin, making the role of MgATP binding and hydrolysis one of the most fascinating aspects of nitrogenase research. We have recently been able to determine the structure of a single deletion mutant of the nitrogenase Fe protein that provides a structural mimic of the MgATP bound state. The structural insights described in the preliminary results section of the proposal provide the firmest foundation described to date for generating hypotheses concerning MgATP dependent conformational change in the Fe protein and the initial component protein interactions that trigger MgATP hydrolysis. The proposed studies apply a combined approach involving structure determination by x-ray diffraction methods and site-specific amino acid substitution experiments to gain insights into nucleotide dependent conformational change, macromolecular complex formation, and the specific protein-protein interactions occurring upon complex formation that initiate MgATP hydrolysis in nitrogenase.