Cryo-annealing of Photoreduced CdS Quantum Dot-Nitrogenase MoFe Protein Complexes Reveals the Kinetic Stability of the E₄(2N2H) Intermediate

A critical step in the mechanism of N-2 reduction to 2NH(3) catalyzed by the enzyme nitrogenase is the reaction of the four-electron/four-proton reduced intermediate state of the active-site FeMo-cofactor (E-4(4H)). This state is a junction in the catalytic mechanism, either relaxing by the reaction of a metal bound Fe-hydride with a proton forming H-2 or going forward with N-2 binding coupled to the reductive elimination (re) of two Fe-hydrides as H-2 to form the E-4(2N2H) state. E-4(2N2H) can relax to E-4(4H) by the oxidative addition (oa) of H-2 and release of N-2 or can be further reduced in a series of catalytic steps to release 2NH(3). If the H-2 re/oa mechanism is correct, it requires that oa of H-2 be associative with E-4(2N2H). In this report, we have taken advantage of CdS quantum dots in complex with MoFe protein to achieve photodriven electron delivery in the frozen state, with cryo-annealing in the dark, to reveal details of the E-state species and to test the stability of E-4(2N2H). Illumination of frozen CdS:MoFe protein complexes led to formation of a population of reduced intermediates. Electron paramagnetic resonance spectroscopy identified E-state signals including E-2 and E-4(2N2H), as well as signals suggesting the formation of E-6 or E-8. It is shown that in the frozen state when pN(2) is much greater than pH(2), the E-4(2N2H) state is kinetically stable, with very limited forward or reverse reaction rates. These results establish that the oa of H-2 to the E-4(2N2H) state follows an associative reaction mechanism.