Purification and characterization of nitrogenase from a delta nifW strain of Azotobacter vinelandii.
Deletion of the nifW gene in Azotobacter vinelandii yields a strain (DJ224) that grows poorly under N2 fixing conditions (Jacobson, M. R., Cash, V. L., Weiss, M. C., Laird, N. F., Newton, W. E., and Dean, D. R. (1989) Mol. & Gen. Genet. 219, 49-57). Here we report the purification of nitrogenase from DJ224. The purified Fe protein was indistinguishable from wild-type. The MoFe protein was indistinguishable from the wild-type MoFe protein by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, native gel electrophoresis, two-dimensional gel electrophoresis, metal analysis, UV/visible and EPR spectroscopies. It was different by the criteria of CD spectroscopy and specific activities. At a 5:1 molar ratio of Fe protein to MoFe protein, H2 evolution under argon was identical to wild-type, C2H2 reduction was inhibited by 27%, N2 reduction was inhibited by 38%, and CO inhibited H2 evolution by 17%. The above data show that the nifW gene product is not required for: 1) detectable alteration of the polypeptide; 2) the synthesis of the metal portion of FeMo cofactor; or 3) FeMo cofactor insertion. The MoFe protein synthesized in the absence of NifW appears to have an alteration near the FeMo cofactor site, possibly at homocitrate, which causes differential inhibition of different substrates.
Kim
S
S
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Burgess
B K
BK
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000502:physiology*; D002384:Catalysis; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D017353:Gene Deletion; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics; D009591:Nitrogenase / Q000302:isolation & purification* / Q000378:metabolism; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D008983:Molybdoferredoxin; C068360:nitrogenase stabilizing-protective protein, Bacteria; D009591:Nitrogenase