Purification and characterization of the nifN and nifE gene products from Azotobacter vinelandii mutant UW45.

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Abstract
The nifN and -E gene products are involved in the synthesis of the iron-molybdenum cofactor of dinitrogenase, the enzyme responsible for the reduction of dinitrogen to ammonia. By using the in vitro iron-molybdenum cofactor biosynthesis assay, we have followed the purification of these gene products 450-fold to > 95% purity. An overall recovery of 20% was obtained with the purified protein having a specific activity of 6900 units/mg of protein. The protein (hereafter referred to a NIFNE) was found to contain equimolar amounts of the nifN and -E gene products and have a native molecular mass of 200 .+-. 10 kDa, which indicates an .alpha.2.beta.2 structure. NIFNE was oxygen labile with a half-life of 1 min in air. A UV-visible spectrum of the dye-oxidized protein showed an absorption maximum at 425 nm that could be bleached by reduction of NIFNE with sodium dithionite, suggesting the presence of an Fe center in NIFNE.