Incorporation of Iron and Sulfur from NifB Cofactor into the Iron-Molybdenum Cofactor of Dinitrogenase

Abstract

NifB-co is an iron- and sulfur-containing precursor to the iron-molybdenum cofactor (FeMo-co) of dinitrogenase. The synthesis of NifB-co requires at least the product of the nifB gene. Incorporation of ⁵⁵Fe and ³⁵S from NifB-co into FeMo-co was observed only when all components of the in vitro FeMo-co synthesis system were present. Incorporation of iron and sulfur from NifB-co into dinitrogenase was not observed in control experiments in which the apodinitrogenase (lacking FeMo-co) was initially activated with purified, unlabeled FeMo-co or in assays where NifB-co was oxygen-inactivated prior to addition to the synthesis system. These data clearly demonstrate that iron and sulfur from active NifB-co are specifically incorporated into FeMo-co of dinitrogenase and provide direct biochemical identification of an iron-sulfur precursor of FeMo-co. Under different in vitro FeMo-co synthesis conditions, iron and sulfur from NifB-co were associated with at least two other proteins (NIFNE and gamma) that are involved in the formation of active dinitrogenase. The results presented here suggest that multiple FeMo-co processing steps might occur on NIFNE and that FeMo-co synthesis is most likely completed prior to the association of FeMo-co with gamma.