Characterization of the Azotobacter vinelandii nitrogenase complex expressed in Escherichia coli toward further activity improvement

We previously constructed an Escherichia coli strain expressing 16 nitrogen fixation (nif) and 2 nif-related genes from Azotobacter vinelandii and improved nitrogenase activity to some extent by enhancing NifH-related functions. In the present study, we analyzed the formation of dinitrogenase, a heterotetrameric NifD₂K₂, produced in E. coli, using gel-filtration chromatography and blue native PAGE to gain insight into further increases in nitrogenase activity. A certain proportion of NifD and NifK proteins produced in E. coli were present as the complete NifD₂K₂ component, but some remained in the intermediate stages of maturation. Overexpression of nafY, which is involved in holo-NifD₂K₂ formation, effectively increased nitrogenase activity.