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: 1) the non-catalytic state of the iron-molybdenum cofactor (FeMo-co) active site that is generated by chemical oxidation of the resting-state cofactor (S=3/2) within resting MoFe (E-0); and 2) the catalytic state that has accumulated n=1 [e(-)/H+] above the resting-state level, denoted E-1(1H) (S1) 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-0 to E-1(1H), and of E-1(1H) to E-2(2H) (S=3/2). The experimental results further suggest that the E-1(1H)E-2(2H) step involves coupled delivery of a proton and an electron (PCET) to FeMo-co of E-1(H) to generate a nonequilibrium S=1/2 form E-2(2H)*. This subsequently undergoes conformational relaxation and attendant change in the FeMo-co spin state, to generate the equilibrium E-2(2H) (S=3/2) state. Unexpectedly, these experiments also reveal conformational coupling between FeMo-co and the P cluster, and between the Fe protein binding and FeMo-co, which might play a role in gated electron transfer from reduced Fe protein to FeMo-co.