Nucleotide-Dependent Energy Transduction in Nitrogenase

该项目的长期目标是确定金属酶复合体固氮酶中能量偶联(例如，ATP水解)与氮还原的机制。固定形态的氮(例如氨)的可用性对所有生物来说都是必不可少的，在这些生物中，固定形态的氮被用来制造蛋白质、DNA和一系列其他生物分子。氮气从空气还原为氨是生物圈中生物有效氮的最大输入，生物固氮占氮气减少的大部分。生物固氮发生在大量可称为重氮菌的微生物中，由一种被称为固氮酶的高度保守的金属酶催化。这项研究计划的具体目标是解决关于固氮酶机制的一个重要未知问题，即了解镁ATP结合和水解是如何与氮气还原耦合的。近年来的研究进展表明，核苷酸结合和水解控制着固氮酶机制中的几个步骤，包括从铁蛋白组分到MoFe蛋白组分的电子转移，MoFe蛋白上底物的还原，以及每一次电子转移事件后两个组分蛋白质的解离。重要的是，很明显，核苷酸通过长距离的蛋白质构象变化来发挥其影响。在这个项目中，关于核苷酸如何参与固氮酶机制的三个悬而未决的问题将被考虑：1)铁蛋白内的两个肽伸展(称为开关I和II)在与核苷酸结合部位的通信中对影响MoFe蛋白的贡献是什么？2)核苷酸如何调节从Fe蛋白到MoFe蛋白的电子转移？3)两个MgATP结合和水解事件对整个固氮酶机制的单独贡献是什么？将使用一种多学科的方法，利用X射线结构信息，结合定点突变以及生化和光谱方法，来揭开核苷酸机制的细节。本项目的研究结果将有助于更详细地理解核苷酸在固氮酶机制中的作用，并将对理解其他核苷酸偶联能量转导蛋白的机制具有更广泛的意义。

The long-term goal of this project is to define the mechanism of energy coupling (e.g., ATP hydrolysis) to N2 reduction in the metalloenzyme complex nitrogenase. The availability of fixed forms of nitrogen (e.g., ammonia) is essential to all living organisms, where it is used to make proteins, DNA, and a range of other biomolecules. The reduction of N2 from air to ammonia represents the largest input of bioavailable nitrogen in the biosphere, with biological nitrogen fixation accounting for most of the N2 reduction. Biological nitrogen fixation occurs in a large number of microorganisms that can be called diazotrophs, being catalyzed by a highly conserved metalloenzyme called nitrogenase. The specific objective of this research program is to address one of the significant unknowns about the nitrogenase mechanism, namely understanding how MgATP binding and hydrolysis are coupled to N2 reduction. Progress over recent years has revealed that nucleotide binding and hydrolysis control several steps in the nitrogenase mechanism including electron transfer from the Fe protein component to the MoFe protein component, substrate reduction on the MoFe protein, and dissociation of the two component proteins following each electron transfer event. Importantly, it is clear that nucleotides exert their influence at a distance through long-range protein conformational changes. In this project, three outstanding questions about how nucleotides participate in the nitrogenase mechanism will be considered: 1) What are the contributions of two peptide stretches within the Fe protein (termed switches I and II) in communicating from the nucleotide binding sites to influence the MoFe protein? 2) How do nucleotides regulate electron transfer from the Fe protein to the MoFe protein? 3) What are the individual contributions of two MgATP binding and hydrolysis events to the overall nitrogenase mechanism? A multidisciplinary approach will be used that utilizes x-ray structural information, in conjunction with site-directed mutagenesis and biochemical and spectroscopic methods, to unravel details of the nucleotide mechanisms. It is expected that the results from this project will contribute to a detailed understanding of the functions of nucleotides in the nitrogenase mechanism and will have broader implications in understanding the mechanisms of other nucleotide-coupled energy transduction proteins.