Azotobacter vinelandii glutaredoxin D delivers the core [Fe₂S₂] cluster to nitrogenase cofactor scaffold protein NifU

The scaffold protein NifU plays a central role in assembling the precursor [Fe₄S₄] clusters required for nitrogenase to function. The synthesis of these precursors depends on a catalytic [Fe₂S₂] group within NifU core ferredoxin domain. Here, we show that the monothiol glutaredoxin GrxD is one of the proteins delivering this cluster to the NifU scaffold protein. Consistently, grxD mutants have reduced nitrogenase activity, the result of altered iron allocation to this enzyme due to a suboptimal [Fe₂S₂] cluster occupancy of the core NifU domain in the cell. These results also indicate the existence of additional pathways to provide NifU with its core [Fe₂S₂] group. Biochemical assays show that GrxD unidirectionally transfers [Fe₂S₂] to NifU through protein-protein interaction. This allows GrxD to restore apo-NifU functionality, enabling proper [Fe₄S₄] synthesis and NifH activation. These findings are crucial to understand how iron is allocated to nitrogenase for biological nitrogen fixation.