Azotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe₃S₄] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe₄-S₄] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.