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

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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.