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 N₂ reduction to 2NH₃ catalyzed by the enzyme nitrogenase is the reaction of the four-electron/four-proton reduced intermediate state of the active-site FeMo-cofactor (E₄(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 H₂ or going forward with N₂ binding coupled to the reductive elimination (re) of two Fe-hydrides as H₂ to form the E₄(2N2H) state. E₄(2N2H) can relax to E₄(4H) by the oxidative addition (oa) of H₂ and release of N₂ or can be further reduced in a series of catalytic steps to release 2NH₃. If the H₂ re/oa mechanism is correct, it requires that oa of H₂ be associative with E₄(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 E₄(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 E₂ and E₄(2N2H), as well as signals suggesting the formation of E₆ or E₈. It is shown that in the frozen state when pN₂ is much greater than pH₂, the E₄(2N2H) state is kinetically stable, with very limited forward or reverse reaction rates. These results establish that the oa of H₂ to the E₄(2N2H) state follows an associative reaction mechanism.