Kiersten Chong
Protection of Nitrogen Fixation by CowN in G. diazotrophicus
Overview: Nitrogen fixation is the reduction of atmospheric dinitrogen to ammonia, a process that occurs naturally in certain bacteria via the enzyme nitrogenase which is inhibited by carbon monoxide. This research attempts to understand the protective effects of the protein CowN on carbon monoxide inhibition of nitrogenase.Abstract: 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). In some soil conditions, CO levels are sufficient to inhibit nitrogen fixation. However, nitrogenase inhibition is prevented by CowN, a protein expressed by G. diazotrophicus. This research attempts to understand the mechanism behind CowN-mediated protection of nitrogenase from CO inhibition. We expressed G. diazotrophicus CowN heterologously in E. coli and purified the protein to homogeneity. In vitro, in presence of CowN, nitrogenase is able to tolerate CO concentrations up to 0.1 atm, a concentration that completely inhibits the enzyme in absence of CowN. 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. Future work will aim to elucidate if/how CowN binds to nitrogenase and if CowN protects nitrogenase by directly preventing CO access to the active site or if it engenders CO reduction abilities to nitrogenase.
Zoom Link