MgATP in Nitrogenase Catalysis

描述(由申请人提供)：该提案的广泛的、长期的目标是获得对镁ATP在固氮酶催化中的作用的结构性和机械性的见解。固氮酶是一种复杂的金属酶，催化氮气转化为氨。在固氮酶催化过程中，铁蛋白和钼-铁蛋白以一种方式结合和解离，导致两个分子的镁三磷酸腺苷的水解和至少一个电子转移到MoFe蛋白。要将单个氮分子完全还原为氨，需要多个循环的铁蛋白结合和解离、镁-三磷酸腺苷的水解和电子转移。固氮酶的结构/功能有许多方面是基础研究的有趣领域。固氮酶可以被认为是研究复杂金属簇介导的催化、电子转移、复杂金属簇组装、蛋白质-蛋白质相互作用和核苷酸依赖的信号转导的理想模型体系。此外，镁三磷酸腺苷在固氮酶催化中的作用类似于核苷酸结合蛋白中核苷酸的作用，核苷酸结合蛋白将核苷酸结合和水解与大分子组装内转导的蛋白质构象变化相结合。这一类的成员包括G蛋白、Ras p21、RecA、延伸因子Tu、肌球蛋白和转导蛋白，这使得镁ATP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蛋白的单个缺失突变体的结构，该突变体提供了对镁ATP结合状态的结构模拟。在该提案的初步结果部分中描述的结构洞察为产生关于依赖于镁ATP的铁蛋白的构象变化和触发镁ATP水解的初始组分蛋白质相互作用的假说提供了迄今为止描述的最坚实的基础。本研究采用X射线衍射法结构测定和定点氨基酸替代实验相结合的方法，深入了解核苷酸依赖的构象变化、大分子复合体的形成，以及在复合体形成过程中引发固氮酶中镁三磷酸腺苷水解的特定蛋白质-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.