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.