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

Gregory Vansuch, David Mulder, Bryant Chica, Jesse Ruzicka, Zhi-Yong Zhang, Lauren Pellows, Mark Willis, Katherine Brown, Lance Seefeldt, John Peters, Gordana Dukovic, Paul King

Research output: Contribution to journalArticlepeer-review

2 Scopus Citations


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.

Original languageAmerican English
Pages (from-to)21165-21169
Number of pages5
JournalJournal of the American Chemical Society
Issue number39
StatePublished - 2023

Bibliographical note

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society

NREL Publication Number

  • NREL/JA-2700-87497


  • ammonia
  • catalytic mechanism
  • CdS
  • electron paramagnetic resonance spectroscopy
  • nanocrystal
  • nitrogenase
  • photochemical


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