CAREER:The Role of ATP Hydrolysis in Biological Nitrogen Fixation

还原形式的分子氮（二氮）对于化肥和无数工业化学品的生产以及氨基酸和核酸的生物合成至关重要。通过Haber-Bosch工艺将二氮还原为氨（固氮）需要高温和高压，每年消耗超过1000亿瓦的电力。生物二氮固定不需要如此极端的条件；事实上，在环境条件下，酶氮酶催化氨合成。然而，经过几十年的密集研究，人们仍然不知道氮酶是如何激活二氮的。氮酶催化与其他多电子/多质子催化反应不同的是，尽管有良好的热力学驱动力，但每个转化反应需要16个ATP分子。本项目旨在阐明ATP水解在生物固氮过程中为何以及如何被需要。最近的结构研究表明，氮酶的两种组分，fe -蛋白（电子供体/ atp酶）和mofe -蛋白（催化组分），根据atp水解状态的不同，可以假设至少三种对接几何形状。通过开发和利用几种强大的化学和生物物理工具，本项目将探索多种fe -蛋白：mofe -蛋白对接模式是否在功能上很重要，以及它们是否参与了电子和质子连续转移到催化金属簇（FeMoco）的时间。同时，光化学方法将用于研究在不需要ATP水解的情况下驱动FeMoco底物还原的可能性。氮酶光活化的证明将为研究其机制开辟新的途径，进而可能导致氨和氢生产生物催化系统的发展。更广泛的影响：生物固氮的复杂性需要一个多学科的攻击计划。该项目结合了多种实验方法，将为研究生和本科生提供广阔的训练场地。生物固氮维持了世界上很大一部分（40%）的人口，而工业哈伯-博世工艺造成了相当数量的能源消耗和温室气体排放。深入了解氮酶的作用机理，可以设计出清洁高效的合成氨生物催化剂，这将产生巨大的经济和环境影响。固氮也为这个项目的教育目标提供了一个渠道，该目标是提高学生对全球能源问题的认识，并培养能源生物科学方面的科学家。这些目标将在几个方面得到解决，包括a)在实验室对研究生和本科生进行跨学科培训，b)重组生物无机化学高级课程，重点关注涉及全球碳、氮、氧和硫循环的氧化还原催化过程，以及设计全球能源问题和替代能源研究的多学科课程。c)向在当地特许学校就读的来自弱势群体的低收入学生伸出援手，并通过研讨会招募他们。

Reduced forms of molecular nitrogen (dinitrogen) are essential for the production of fertilizers and countless industrial chemicals, as well as the biosynthesis of amino and nucleic acids. The reduction of dinitrogen to ammonia (nitrogen fixation) by the Haber-Bosch process requires high temperatures and pressures, consuming over 100 billion watts of power every year. Biological dinitrogen fixation does not require such extreme conditions; indeed, the enzyme nitrogenase catalyzes ammonia synthesis under ambient conditions. After decades of intense research efforts, however, it is still not known how nitrogenase activates dinitrogen. Nitrogenase catalysis is distinct from other multielectron/multiproton catalytic reactions in its requirement of 16 ATP molecules per turnover reaction despite a favorable thermodynamic driving force. This project aims to elucidate why and how ATP hydrolysis is required in biological nitrogen fixation. Recent structural studies show that the two constituents of nitrogenase, the Fe-protein (electron donor/ATPase) and the MoFe-protein (catalytic component), can assume at least three docking geometries, depending on the ATP-hydrolysis state. By developing and utilizing several powerful chemical and biophysical tools, this project will probe whether multiple Fe-protein:MoFe-protein docking modes are functionally important, and if they are involved in timing the successive electron and proton transfers into the catalytic metal cluster (FeMoco). In parallel, photochemical methods will be utilized to investigate the possibility of driving substrate reduction at FeMoco without requiring ATP hydrolysis. The demonstration of photoactivation of nitrogenase will open new avenues for studying its mechanism that in turn could lead to the development of biocatalytic systems for ammonia and hydrogen production.Broader Impacts: The complexity of biological nitrogen fixation requires a multidisciplinary plan of attack. This project combines a multitude of experimental approaches that will provide an expansive training ground for graduate and undergraduate students. Biological nitrogen fixation sustains a large fraction (40%) of the world's population, and the industrial Haber-Bosch process is responsible for considerable amounts of energy consumption and greenhouse gas emissions. A thorough understanding of nitrogenase mechanism could lead to the design of clean and efficient biocatalysts for ammonia production, which would have an immense economic and environmental impact. Nitrogen fixation also provides a conduit into the education goals of this project, which is to raise the awareness of students about the global energy problem, and to train scientists in energy biosciences. These goals will be addressed on several fronts, including a) the interdisciplinary training of graduate and undergraduate students in the laboratory, b) restructuring of an advanced course in Bioinorganic Chemistry to focus on redox-catalytic processes involved in global carbon, nitrogen, oxygen, and sulfur cycles, as well as the design of a multidisciplinary course on Global Energy Problem and Alternative Energy Research, and c) outreach to low-income students from underrepresented groups attending a local charter school, and their recruitment through seminars.