Anaerobic single particle cryoEM of nitrogenase

The enzyme nitrogenase catalyzes the reduction of dinitrogen to ammonia during biological nitrogen fixation through a mechanism involving the ATP dependent interaction of two component proteins adopting multiple conformational states. To date, high resolution structural information has been provided by X-ray crystallography, which restricts the states that can be accessed to those that can be crystallized. Cryo-electron microscopy (cryoEM) presents a new opportunity for structural characterization of nitrogenase solution structures, and may yield new information on the mechanism of nitrogenase by revealing structures of transient or heterogeneous states. In this study, we present single particle cryoEM structures of the MoFe-nitrogenase endogenously isolated from Azotobacter vinelandii. To maintain the fully reduced cluster states of this oxygen sensitive protein, we prepared samples within an anaerobic chamber and employed specialized conditions to minimize partial disordering of the -subunit at the air-water interface during freezing. Under these conditions, cryoEM structures of the as-isolated MoFe-protein and stabilized MoFe-protein-Fe-protein ADP-AlF4-complex were generally found to closely resemble their corresponding X-ray crystallographic structures. The cryoEM structures did reveal disordering in regions of the MoFe-protein -subunit reminiscent of that observed previously for the {Delta}nifB MoFe-protein lacking the FeMo-cofactor, suggesting that this disorder may reflect functionally relevant dynamics, as well as the possibility of asymmetric binding of the Fe-protein to the MoFe-protein in solution. The methods presented here pave the way toward the capture and interrogation of turnover-relevant nitrogenase states by cryoEM.