Structural characterization of the P 1+ intermediate state of the P-cluster of nitrogenase.

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N ₂ ) to ammonia (NH ₃ ) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N ₂ is reduced to NH ₃ The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (P ^N ) state to the two-electron oxidized P ²⁺ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P ²⁺ , P ¹⁺ , and P ^N ) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P ¹⁺ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P ²⁺ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.