CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide

Michael S Medina,Kevin O Bretzing,Richard A Aviles,Kiersten M Chong,Alejandro Espinoza,Chloe Nicole G Garcia,Benjamin B Katz,Ruchita N Kharwa,Andrea Hernandez,Justin L Lee,Terrence M Lee,Christine Lo Verde,Max W Strul,Emily Y Wong,Cedric P Owens

doi:10.1016/j.jbc.2021.100501

Michael S Medina, Kevin O Bretzing + Show 13 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100501

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Abstract

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). 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.

Highlights

Background on carbon monoxide (CO) binding to molybdenum–iron protein (MoFeP)We present a brief summary of the mechanism of CO binding to MoFeP since this will help contextualize results that are presented in the subsequent sections
CooA expression in G. diazotrophicus is likely dependent on RpoN, a σ54 factor commonly associated with nitrogen fixation [36]
We caution against trying to assign KIa and KIb based on a particular CO-bound structure since the number of known CO-bound forms of MoFeP exceeds the number of inhibition constants and we do not know which CO bound structure is relevant for a particular inhibition mode

Summary

Results and discussion

We chose to study CowN in the diazotroph Gluconacetobacter diazotrophicus, an agriculturally relevant organism with. In G. diazotrophicus, CowN is part of a three-gene cluster consisting of algH, cooA, and cowN (Fig. 1D). Previous studies in diazotrophs that have both Mo- and alternative nitrogenases indicate that CowN expression is turned on by CO and, to a lesser extent, nitrogen fixing conditions [20]. Dynamic light scattering (DLS) experiments reveal that CowN has a hydrodynamic radius of 1.96 ± 0.25 nm, from which a 15 to 18 kDa molecular weight can be calculated (Fig. 3D) These experiments suggest CowN is most likely monomeric. Refolding the protein in the presence of DTT and performing gel filtration chromatography under reducing conditions does not increase the amount of CowN monomer, nor does it change the retention time of the monomer peak or CowN’s secondary structure

B Expression changes due to CO

Background on CO binding to MoFeP

Conclusions

Experimental procedures