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在氮酶催化中的作用的结构和机制见解。氮酶是一种复杂的含金属酶，能催化氮气转化为氨。在氮酶催化过程中，铁蛋白和钼铁蛋白以一种方式结合并解离，导致两个MgATP分子水解，并将至少一个电子转移给MoFe蛋白。铁蛋白结合解离、MgATP水解和电子转移需要多个循环才能将单个氮分子完全还原为氨。有许多方面的氮酶的结构/功能是有趣的基础研究领域。氮酶可以被认为是研究复杂金属团簇介导的催化、电子转移、复杂金属团簇组装、蛋白质-蛋白质相互作用和核苷酸依赖性信号转导的理想模型系统。此外，MgATP在氮酶催化中的作用类似于核苷酸在一大类核苷酸结合蛋白中的作用，它们将核苷酸结合和水解结合到大分子组装中转导的蛋白质构象变化中。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MgATP结合和水解的作用成为氮酶研究中最引人入胜的方面之一。我们最近已经能够确定氮酶Fe蛋白的单缺失突变体的结构，该突变体提供了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为产生有关Fe蛋白中MgATP依赖性构象变化和触发MgATP水解的初始组分蛋白相互作用的假设提供了迄今为止描述的最坚实的基础。拟议的研究采用结合的方法，包括x射线衍射方法和位点特异性氨基酸替代实验的结构测定，以深入了解核苷酸依赖的构象变化，大分子复合物的形成，以及在复合物形成时发生的启动氮酶中MgATP水解的特定蛋白质-蛋白质相互作用。

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.