Protection of Nitrogen Fixation from CO by CowN in G. diazotrophicus

Nitrogen fixation is the process in which atmospheric dinitrogen is reduced to ammonia. Nitrogen fixation occurs naturally in certain bacteria, such as Gluconacetobacter diazotrophicus , via the enzyme nitrogenase, a multisubunit protein with an iron protein subunit (FeP) and a molybdenum‐iron protein subunit (MoFeP). Nitrogenase is inhibited by carbon monoxide (CO). Under some soil conditions, CO levels are sufficiently high to inhibit nitrogenase and prevent nitrogen fixation. However, G. diazotrophicus expresses a protein called CowN that prevents the inhibition of nitrogenase by CO. This research attempts to understand the mechanism of how CowN prevents inhibition of nitrogenase. We 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 K m of approximately 8 μM. Our experiments further show that CowN, which exists in a monomeric and oligomeric state, is only active as a monomer. Further work will aim to elucidate how CowN binds to nitrogenase and if CowN protects nitrogenase by directly preventing CO access to the active site. Support or Funding Information This research was supported by the Chapman Center for Undergraduate Excellence, USDA‐NIFA (Grant no. 2015‐67012‐22895) and the National Science Foundation (Grant no. 1905399).