Chloe Garcia
Analyzing Restored Nitrogen Fixation Through CowN in G. diazotrophicus
Overview: My research pertains to nitrogen fixation. In soil, there are bacteria that hold the protein nitrogenase, responsible for the nitrogen fixation process. However, this process is inhibited by carbon monoxide. CowN is another protein produced in the bacteria, which prevents the inhibition of nitrogenase. My research looks into the mechanism of CowN. Abstract: Nitrogen fixation occurs when atmospheric dinitrogen gas is reduced to ammonia. In certain bacteria, such as Gluconacetobacter diazotrophicus, nitrogen fixation is catalyzed by the enzyme nitrogenase, a multisubunit protein composed of an iron protein subunit (FeP) and a molybdenum-iron protein subunit (MoFeP). Nitrogenase is inhibited by carbon monoxide (CO). In certain soil conditions, CO levels are sufficiently high to inhibit nitrogenase activity and prevent nitrogen fixation. However, G. diazotrophicus expresses a protein called CowN that prevents the inhibition of nitrogenase by CO. This current research attempts to understand the mechanism of how CowN prevents inhibition of nitrogenase to restore nitrogen fixation. We have expressed G. diazotrophicus CowN heterologously in E. coli and purified the protein to homogeneity. In vitro studies with FeP, MoFeP, and CowN in the presence of CO showed that CowN effectively restores nitrogenase activity for CO concentrations up to 0.1 atm. The activity of CowN exhibits Michaelis-Menten-like kinetics with a Km of approximately 8 µM. Our experiments further show that CowN, which exists in both a monomeric and oligomeric state, is only active as a monomer. Further studies have partially elucidated how CowN binds to nitrogenase and if the prevention of inhibition is due to CowN directly preventing CO access to the active site of nitrogenase.
Zoom Link