CHARACTERIZATION OF VNFG, THE DELTA-SUBUNIT OF THE VNF-ENCODED APODINITROGENASE FROM AZOTOBACTER-VINELANDII - IMPLICATIONS FOR ITS ROLE IN THE FORMATION OF FUNCTIONAL DINITROGENASE-2

The vnf-encoded apodinitrogenase (apodinitrogenase 2) from Azotobacter vinelandii is an alpha(2) beta(2) delta(2) hexamer. The delta subunit (the VNFG protein) has been characterized in order to further delinea te its function in the nitrogenase 2 enzyme system. Two species of VNF G were observed in cell-free extracts resolved on anoxic native gels; one is composed of VNFG; associated with the VNFDK polypeptides, and t he other is a homodimer of the VNFG protein. Both species of VNFG are observed in extracts of A. vinelandii strains that accumulate dinitrog enase 2, whereas extracts of strains impaired in the biosynthetic path way of the iron-vanadium cofactor (FeV-co) that accumulate apodinitrog enase 2 (a catalytically inactive form of dinitrogenase 2 that lacks F eV-co) exhibit only the VNFG dimer on native gels, FeV-co and nucleoti de are required for the stable association of VNFG with the VNFDK poly peptides; this stable association can be correlated with the formation of active dinitrogenase 2. The iron-molybdenum cofactor was unable to replace FeV-co in promoting the stable association of VNFG with VNFDK , FeV-co specifically associates with the VNFG dimer in vitro to form a complex of unknown stoichiometry; combination of this VNFG-FeV-co sp ecies with apodinitrogenase 2 results in its reconstitution to dinitro genase 2. The results presented here suggest that VNFG is required for processing apodinitrogenase 2 to functional dinitrogenase 2.