Cryo-annealing of Photoreduced CdS Quantum Dot–Nitrogenase MoFe Protein Complexes Reveals the Kinetic Stability of the E(4)(2N2H) Intermediate

[Image: see text] 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.