Purification and characterization of the nifN and nifE gene products from Azotobacter vinelandii mutant UW45.
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 greater than 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 as 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.
Paustian
T D
TD
Department of Bacteriology, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Shah
V K
VK
Roberts
G P
GP
D001395:Azotobacter / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D005288:Ferredoxins / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000235:genetics; D008970:Molecular Weight; D008983:Molybdoferredoxin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D009586:Nitrogen Fixation / Q000235:genetics*; D013053:Spectrophotometry
D004269:DNA, Bacterial; D005288:Ferredoxins; D008983:Molybdoferredoxin