Nucleotide-Dependent Energy Transduction in Nitrogenase

该项目的长期目标是明确金属酶复合氮酶中能量耦合（如ATP水解）与N2还原的机制。固定形式的氮（如氨）的可用性对所有生物都是必不可少的，它被用来制造蛋白质、DNA和一系列其他生物分子。N2从空气还原为氨是生物圈中最大的生物可利用氮输入，其中生物固氮占N2还原的大部分。生物固氮发生在大量可称为重氮营养体的微生物中，由一种高度保守的称为氮酶的金属酶催化。本研究计划的具体目标是解决关于氮酶机制的一个重要未知数，即了解MgATP结合和水解如何与N2还原耦合。近年来的进展表明，核苷酸结合和水解控制着氮酶机制的几个步骤，包括从Fe蛋白组分到MoFe蛋白组分的电子转移，MoFe蛋白上的底物还原以及每次电子转移事件后两组分蛋白的解离。重要的是，很明显，核苷酸通过远距离蛋白质构象变化在远处发挥其影响。在这个项目中，将考虑三个关于核苷酸如何参与氮酶机制的突出问题：1)铁蛋白内的两个肽延伸（称为开关I和II）在核苷酸结合位点的通信中对MoFe蛋白的影响有什么贡献？2)核苷酸如何调节电子从Fe蛋白到MoFe蛋白的转移？3)两个MgATP结合和水解事件对整个氮酶机制的单独贡献是什么？将采用多学科方法，利用x射线结构信息，结合定点诱变、生化和光谱方法，揭示核苷酸机制的细节。预计本项目的结果将有助于详细了解核苷酸在氮酶机制中的功能，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.