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.