Cryo-annealing of Photoreduced CdS Quantum Dot-Nitrogenase MoFe Protein Complexes Reveals the Kinetic Stability of the E 4 (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.