Exploring Electron/Proton Transfer and Conformational Changes in the Nitrogenase MoFe Protein and FeMo-cofactor Through Cryoreduction/EPR Measurements.

We combine cryoreduction/annealing/EPR measurements of nitrogenase MoFe protein with results of earlier investigations to provide a detailed view of the electron/proton transfer events and conformational changes that occur during early stages of [e/H] accumulation by the MoFe protein. This includes reduction of the non-catalytic state of the iron-molybdenum cofactor (FeMo-co) active site that is generated by chemical oxidation of the resting-state cofactor ()) within resting MoFe (E), and the catalytic state that has accumulated [e/H] above the resting-state level, denoted E(1H) () in the Lowe-Thorneley kinetic scheme. FeMo-co does not undergo a major change of conformation during reduction of oxidized FeMo-co. In contrast, FeMo-co undergoes substantial conformational changes during the reduction of E to E(1H), and of E(1H) to E(2H) (; ). The experimental results further suggest that the E(1H) → E(2H) step involves coupled delivery of a proton and electron (PCET) to FeMo-co of E(H) to generate a non-equilibrium form E(2H)*. This subsequently undergoes conformational relaxation and attendant change in FeMo-co spin state, to generate the equilibrium E(2H) () state. Unexpectedly, these experiments also reveal conformational coupling between FeMo-co and P-cluster, and between Fe protein binding and FeMo-co, which might play a role in gated ET from reduced Fe protein to FeMo-co.