Characterizing the Interaction Between CowN and Nitrogenase

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Nitrogenase catalyzes the conversion of atmospheric dinitrogen into ammonia. The enzyme produces ammonia under ambient conditions using the free energy of ATP to drive dinitrogen’s reduction. The most common form of nitrogenase is molybdenum nitrogenase (Mo‐nitrogenase). Mo‐nitrogenase is inhibited by the ubiquitous pollutant carbon monoxide (CO). To prevent inhibition of Mo‐nitrogenase by CO, nitrogen‐fixing bacteria produce the protein CowN and in its presence, Mo‐nitrogenase avoids inhibition by CO and remains active. However, the mechanism by which CowN protects Mo‐nitrogenase is unknown. Enzymatic assays suggest that CowN and Mo‐nitrogenase interact with a K‐d of approximately 5–10μM. Here, we present data from crosslinking and pulldown assays that were used to determine how CowN interacts with Mo‐nitrogenase. Crosslinking assays using the heterobifunctional crosslinker EDC showed no binding between CowN and Mo‐nitrogenase in the presence and absence of CO gas. Pulldown assays using Ni‐NTA resin and a His‐tagged CowN bait also did not show significant binding between CowN and Mo‐nitrogenase. These assays indicate that CowN may not have a strong binding affinity to Mo‐nitrogenase to be detected by crosslinking or pulldown assays. Future work will investigate potential nitrogenase‐CowN interaction using fluorescence anisotropy.Support or Funding InformationThis 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).