Evidence for the direct interaction of the nifW gene product with the MoFe protein.
The Azotobacter vinelandii nifW gene, under control of the nifH promoter, was subcloned into the broad host range multicopy plasmid pKT230 for overexpression in both wild-type and delta nifW strains of A. vinelandii. Unlike the parent delta nifW strain, which grows slowly relative to wild-type under N2-fixing conditions, both overproduction strains grow at the same rate, showing that the overexpressed nifW product is functional in vivo. The approximately 40-fold overexpressed protein was purified, and sequence analysis confirmed its identity. During purification it was observed that NifW in crude extracts ran above the predicted molecular weight on denaturing gels and that as the purification proceeded lower molecular weight forms appeared. Mass spectrometry and studies with protease inhibitors revealed that this abnormal behavior was due to proteolysis. Native molecular weight determinations demonstrate that NifW is a homomultimer, most likely a trimer. Native gel electrophoresis analysis shows that the behavior of wild-type and overexpressed NifW are identical and that when extracts are prepared anaerobically only the homomultimeric forms of NifW are observed. When extracts are exposed to oxygen, however, NifW becomes part of a very high molecular weight complex. Immunoprecipitation with NifW antibodies demonstrate that under those conditions NifW specifically associates with the MoFe protein. These data are consistent with a model whereby NifW is not involved in the initial assembly of an active MoFe protein but rather is part of a system design to protect the MoFe protein from O2 damage.
Kim
S
S
Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92717-3900, USA.
Burgess
B K
BK
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008983:Molybdoferredoxin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D001426:Bacterial Proteins; D008983:Molybdoferredoxin; D011994:Recombinant Proteins; C068360:nitrogenase stabilizing-protective protein, Bacteria