Dissecting Electronic-Structural Transitions in the Nitrogenase MoFe Protein P-Cluster during Reduction

[Image: see text] The [8Fe-7S] P-cluster of nitrogenase MoFe protein mediates electron transfer from nitrogenase Fe protein during the catalytic production of ammonia. The P-cluster transitions between three oxidation states, P(N), P(+), P(2+) of which P(N)↔P(+) is critical to electron exchange in the nitrogenase complex during turnover. To dissect the steps in formation of P(+) during electron transfer, photochemical reduction of MoFe protein at 231–263 K was used to trap formation of P(+) intermediates for analysis by EPR. In complexes with CdS nanocrystals, illumination of MoFe protein led to reduction of the P-cluster P(2+) that was coincident with formation of three distinct EPR signals: S = 1/2 axial and rhombic signals, and a high-spin S = 7/2 signal. Under dark annealing the axial and high-spin signal intensities declined, which coincided with an increase in the rhombic signal intensity. A fit of the time-dependent changes of the axial and high-spin signals to a reaction model demonstrates they are intermediates in the formation of the P-cluster P(+) resting state and defines how spin-state transitions are coupled to changes in P-cluster oxidation state in MoFe protein during electron transfer.