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

Abstract

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 (i) 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 (E0), and (ii) the catalytic state that has accumulated n =1 [e-/H+] above the resting-state level, denoted E1(1H) (S ≥ 1) 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 E0 to E1(1H), and of E1(1H) to E2(2H) (n = 2; S = 3/2). The experimental results further suggest that the E1(1H) → E2(2H) step involves coupled delivery of a proton and electron (PCET) to FeMo-co of E1(H) to generate a non-equilibrium S = ½ form E2(2H)*. This subsequently undergoes conformational relaxation and attendant change in FeMo-co spin state, to generate the equilibrium E2(2H) (S = 3/2) 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.