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

Abstract

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.

High Affinity Electrostatic Interactions Support the Formation of CdS Quantum Dot:Nitrogenase MoFe Protein Complexes

“…In this Letter, we describe studies using MST to comparatively examine the binding events of the native Fe protein–MoFe protein and the 3-MPA capped CdS QD–MoFe protein complexes. We compare the interaction of MoFe protein and Fe protein to the interactions between MoFe protein and 3-MPA capped CdS QDs that we have employed in N 2 reduction catalysis in previous work, which have diameters of 3.7 nm. , Furthermore, we examine the impact of the reaction solution ionic strength and characterize the binding interactions of MoFe protein with QDs of different sizes and observe that the binding strength can vary significantly across this range of parameters.…”