Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N 2 ) to ammonia (NH 3 ). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN’s protection mechanism involves decreasing the binding affinity of CO to nitrogenase’s active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.

Gluconacetobacter diazotrophicus is a nitrogen fixing bacterium that associates with plants and plays a crucial part in providing fixed nitrogen to many crops such as sugar cane. The enzyme responsible for reducing atmospheric nitrogen to ammonia is nitrogenase. The presence of carbon monoxide gas will inhibit nitrogen fixation by nitrogenase. Many diazotrophs have a mechanism of protecting nitrogenase in vivo. In G. diazotrophicus, a protein called CowN protects nitrogenase from inhibition by carbon monoxide. Here, we present structural and biophysical characterization of CowN. Following heterologous expression of CowN in Escherichia coli and purification by affinity and size exclusion chromatography, CowN is found in two different oligomeric states; monomer and large soluble aggregate. Dynamic light scattering indicates the monomer has a radius of 1.9 nm, whereas the aggregate is larger than 5 nm. While the monomer protects nitrogenase from carbon monoxide inhibition, the aggregate does not. To determine what causes CowN aggregation and if it represents a physiologically relevant process, we investigated the mechanism of CowN aggregation. We discovered that high salt and protein concentration increase the propensity of CowN to aggregate. We further determined that under reducing conditions the monomeric state is preferred, suggesting that oxidation of CowN’s single cysteine residue plays a role in aggregation. Together, the data suggests that CowN may be most active under oxidizing conditions and, further, that aggregation might represent a mechanism to inactivate the enzyme at high concentration. 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).

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 Km 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).

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 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).