RUI: Nitrogenase and friends: Uncovering how diazotrophs regulate and maintain nitrogenase activity under unfavorable environmental conditions

固氮酶是在某些细菌中发现的一种酶，称为固氮菌，它将氮气转化为氨，氨是植物生长的必需营养素，因此固氮酶对农业至关重要。有了这个奖项，化学部的生命过程化学计划正在资助查普曼大学的塞德里克·欧文斯博士，以确定固氮生物如何在通常预期会破坏酶的环境条件下保持固氮酶的活性。固氮酶被一氧化碳（一种代谢产物）和氧气（许多固氮生物有氧呼吸的必需气体）抑制。本计画研究固氮菌为保护固氮酶免于一氧化碳的失活所采取的对策，以及固氮酶的活性如何受细胞内氧和氧化性化合物的调节。从这个项目的结果将提高固氮生物固氮酶活性的理解，并可能通知合成固氮系统的设计，更耐环境失活。该项目扩大了查普曼大学（四年制学院）和圣安娜学院（两年制少数民族学院）本科生参与研究的范围。此外，来自圣安娜学院的一名教员通过夏季讲习班参加研究活动，并与欧文斯博士一起开发生物化学实验，向一年级普通化学学生教授基本化学概念。在这个项目中正在研究的两种固氮蛋白是CowN，它响应于一氧化碳（CO），和α-变形菌NifA，它响应于细胞氧和/或氧化还原（氧化还原）水平。虽然CO是含钼固氮酶（Mo-固氮酶，最常见的固氮酶）的有效抑制剂，但它是通过替代的含钒固氮酶（V-固氮酶）还原为短链烃的底物。产生不同效果的机制原因尚不清楚，也无法用酶的不同金属成分来解释。有趣的是，Mo-固氮酶耐受CO的存在下，一个小的蛋白质，CowN，这是一个CO依赖性的方式表达。该项目的第一个目的是应用酶学，生物物理和结构的方法来揭示CowN保护钼固氮酶免受CO抑制的机制，并确定CowN是否改变钼固氮酶的反应性。NifA是固氮酶的主要转录调节因子，可检测氧化还原水平、氧水平或两者。第二个目的是使用结构生物学，光谱和生物物理工具，以测试氧化还原和/或氧水平通过以前未表征的铁硫簇通过非典型的环境传感机制感测的假设。该项目的结果预计将产生固氮酶表达所需的细胞条件的关键信息。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

Nitrogenase is an enzyme found in certain bacteria, called diazotrophs, that converts nitrogen gas into ammonia, an essential nutrient for plant growth and, thus, nitrogenase is critical to agriculture. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Cedric Owens of Chapman University to determine how diazotrophs maintain nitrogenase activity under environmental conditions that would typically be expected to inactivate the enzyme. Nitrogenase is inhibited by both carbon monoxide, a metabolic product, and oxygen, an essential gas for aerobic respiration in many diazotrophs. This project studies the countermeasures diazotrophs have taken to protect nitrogenase from inactivation by carbon monoxide, and how nitrogenase activity is regulated by cellular levels of oxygen and oxidizing compounds. Results from this project will enhance the understanding of nitrogenase activity in diazotrophs and potentially inform the design of synthetic nitrogen fixation systems that are more resistant to environmental inactivation. This project broadens participation of undergraduate students in research at Chapman University (a 4-year college) and at Santa Ana College (a 2-year minority serving college). Furthermore, a faculty member from Santa Ana College participates in research activities through a summer workshop and, together with the Dr. Owens, develops biochemistry experiments to teach fundamental chemical concepts to first-year general chemistry students. The two nitrogen fixing proteins being studied in this project are CowN, which responds to carbon monoxide (CO), and alpha-proteobacterial NifA, which responds to cellular oxygen and/or oxidation-reduction (redox) levels. While CO is a potent inhibitor of molybdenum containing nitrogenase (Mo-nitrogenase, the most common nitrogenase), it is a substrate that is reduced to short-chain hydrocarbons by the alternative vanadium-containing nitrogenase (V-nitrogenase). The mechanistic reason for the differing effects is unknown and is not explained by the enzymes' dissimilar metal compositions. Interestingly, Mo-nitrogenase tolerates CO in the presence of a small protein, CowN, which is expressed in a CO-dependent manner. The first aim of this project is to apply enzymological, biophysical and structural approaches to uncover the mechanism by which CowN protects Mo-nitrogenase from CO inhibition and determine if CowN alters Mo-nitrogenase reactivity. NifA is the main transcriptional regulator of nitrogenase, sensing either redox levels, oxygen levels, or both. The second aim is to use structural biology, spectroscopic and biophysical tools, in order to test the hypothesis that redox and/or oxygen levels are sensed through a previously uncharacterized iron-sulfur cluster via a non-canonical environmental sensing mechanism. The results of the project are expected to yield critical information on the cellular conditions required for nitrogenase expression.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.