Abstract
Iron-molybdenum cofactor (FeMo-co) biosynthesis involves the participation of several proteins. We have used (55)Fe-labeled NifB-co, the specific iron and sulfur donor to FeMo-co, to investigate the accumulation of protein-bound precursors of FeMo-co. The (55)Fe 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 (55)Fe-labeled upper band, was purified and shown to be NifH by immunoblot analysis. The (55)Fe-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.
Highlights
Nitrogenase, the enzyme responsible for the conversion of dinitrogen to ammonium, is composed of two oxygen-labile metalloproteins: dinitrogenase and dinitrogenase reductase [1, 2]
Incorporation of 55Fe-Label from 55Fe-NifB-co into Extracts of Various A. vinelandii Mutants—FeMo-co is unstable in aqueous solutions; it is likely that precursors of FeMo-co accumulate on proteins during the course of cofactor biosynthesis
Though use of the in vitro FeMo-co biosynthesis system has yielded considerable information regarding the biosynthesis of FeMo-co, not much is known about the proteins that are associated with precursors of the cofactor
Summary
NifKD, dinitrogenase ( called MoFe protein); FeMo-co, iron-molybdenum cofactor; NifB-co, NifB-cofactor; DTH, sodium dithionite; PAGE, polyacrylamide gel electrophoresis. An in vitro reaction for the biosynthesis of FeMo-co has been described that requires the presence of an ATP-regenerating system, MoO42Ϫ, homocitrate, NifB-cofactor (NifB-co), and the gene products of nifH, N, and E [11]. Previous studies have identified the metabolic product of NifB, NifB-co, as a potential iron and sulfur source for FeMo-co biosynthesis [17]. Studies with purified 55Fe-labeled NifB-co have conclusively shown that NifB-co serves as the specific iron and sulfur donor to FeMo-co [18]. To identify proteins that play a role in FeMo-co biosynthesis, we have used 55Fe-labeled NifB-co in the in vitro FeMo-co synthesis reaction. We report the accumulation of a radiolabeled species on NifH under conditions in which FeMo-co synthesis is allowed to take place Another protein, NifX, accumulates the radiolabel from 55Fe-labeled NifB-co under similar conditions
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