EVIDENCE FOR THE DIRECT INTERACTION OF THE NIFW GENE-PRODUCT WITH THEMOFE PROTEIN

The Azotobacter vinelandii nifW gene, under control of the nifH promot er, was subcloned into the broad host range multicopy plasmid pKT230 f or overexpression in both wild-type and Delta nifW strains of A. vinel andii, Unlike the parent Delta nifW strain, which grows slowly relativ e 60 wild-type under N-2-fixing conditions, both overproduction strain s grow at the same rate, showing that the overexpressed nifW product i s functional in vivo. The similar to 40-fold overexpressed protein was purified, and sequence analysis confirmed its identity, During purifi cation it was observed that NifW in crude extracts ran above the predi cted 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 demons trate that NifW is a homomultimer, most likely a trimer, Native gel el ectrophoresis analysis shows that the behavior of wild-type and overex pressed NifW are identical and that when extracts are prepared anaerob ically only the homomultimeric forms of NifW are observed. When extrac ts are exposed to oxygen, however, NifW becomes part of a very high mo lecular weight complex, Immunoprecipitation with NifW antibodies demon strate that under those conditions NifW specifically associates with t he MoFe protein, These data are consistent with a model whereby NifW i s not involved in the initial assembly of an active MoFe protein but r ather is part of a system design to protect the MoFe protein from O-2 damage.