Functional analysis of the nitrogenase protective protein CowN

Abstract

Nitrogenase is the only enzyme that can catalyze ATP‐dependent nitrogen fixation in bacteria, an important biological process that reduces atmospheric nitrogen gas into ammonia. The bacterium Gluconacetobacter diazotrophicus expresses nitrogenase and is agriculturally relevant since it provides ammonia to commercial crops such as sugarcane and pineapple. The environmental gas carbon monoxide tightly inhibits nitrogenase. To prevent this inhibition, diazotrophs have evolved a small protein, CowN, which interacts with nitroganse and lowers the affinity of CO binding. In doing so, CowN allows nitrogenase to stay active despite the presence of CO Although we know that CowN protects nitrogenase from CO, we do not know how it achieves its protective effect. Both CowN’s mechanism and structure remain undetermined. This poster presents a structure‐function analysis of CowN. The C‐terminus of CowN contains a conserved Cys (C90 in G. diazotrophicus) and a semi‐conserved second S‐containing residue, either Cys or Met. Due to this conservation, we hypothesize that the C‐terminus of CowN is crucial to its function. To test this hypothesis, we made a series of C‐terminal mutations, expecting that mutations to C90 would abolish activity. Surprisingly, we discovered that the conserved Cys is not required for CowN function. A C90S mutant was almost fully active, suggesting that either a thiol or a hydroxyl group at the CowN C‐terminus is sufficient for activity. In contrast, mutations to the second S‐containing residue, M91 in G. diazotrophcius, and to both C90 and M91 fully abolish CowN activity and destabilize the structure. Together, the results confirm our hypothesis that CowN’s C‐terminus is required for activity, however, they also reveal that the presence of the conserved Cys is not necessary for function. Future work will aim to solve the CowN structure and to elucidate the role of residue C90.