Accumulation of 55Fe-labeled precursors of the iron-molybdenum cofactor of nitrogenase on NifH and NifX of Azotobacter vinelandii

Iron-molybdenum cofactor (FeMo-co) biosynthesis involves the participation of several proteins. We have used Fe-55-labeled NifB-co, the specific iron and sulfur donor to FeMo-co, to investigate the accumulation of protein-bound precursors of FeMo-co. The Fe-55 label from radiolabeled NifB-co became associated with two major protein bands when the in vitro FeMo-co synthesis reaction was carried out with the extract of an Azotobacter vinelandii mutant lacking apodinitrogenase. One of the bands, termed Fe-55-labeled upper band, was purified and shown to be NifH by immunoblot analysis. The Fe-55-labeled lower band was identified as NifX by N-terminal sequencing, NifX purified from an A. vinelandii nifB strain showed a different electrophoretic mobility on anoxic native gels than did NifX with the FeMo-co precursor bound.