Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects

Nimesh Khadka, Ross D. Milton, Sudipta Shaw, Dmitriy Lukoyanov, Dennis R. Dean, Shelley D. Minteer, Simone Raugei, Brian M. Hoffman, Lance C. Seefeldt

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Nitrogenase catalyzes the reduction of dinitrogen (N2) to two ammonia (NH3) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H2. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H2. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to the MoFe protein allowing examination of the mechanism of H2 formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, was found to change linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate-limiting step of H2 formation. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H2 formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein, either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H2 formation where the S-H+ bond breaks and H+ attacks the Fe-hydride, and explains the observed H/D isotope effect. This study not only reveals the nitrogenase mechanism of H2 formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other oxidoreductase enzymes and to biomimetic complexes.

Original languageEnglish
Pages (from-to)13518-13524
Number of pages7
JournalJournal of the American Chemical Society
Volume139
Issue number38
DOIs
Publication statusPublished - Jan 1 2017

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Molybdoferredoxin
Nitrogenase
Electrocatalysis
Protonation
Hydrides
Isotopes
Metals
Hydrogen
Protons
Electrons
Proteins
Catalytic Domain
Equipment and Supplies
Biomimetics
Adenosinetriphosphate
Substrates
Ammonia
Peptides
Density functional theory
Amino acids

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Khadka, N., Milton, R. D., Shaw, S., Lukoyanov, D., Dean, D. R., Minteer, S. D., ... Seefeldt, L. C. (2017). Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects. Journal of the American Chemical Society, 139(38), 13518-13524. https://doi.org/10.1021/jacs.7b07311

Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects. / Khadka, Nimesh; Milton, Ross D.; Shaw, Sudipta; Lukoyanov, Dmitriy; Dean, Dennis R.; Minteer, Shelley D.; Raugei, Simone; Hoffman, Brian M.; Seefeldt, Lance C.

In: Journal of the American Chemical Society, Vol. 139, No. 38, 01.01.2017, p. 13518-13524.

Research output: Contribution to journalArticle

Khadka, N, Milton, RD, Shaw, S, Lukoyanov, D, Dean, DR, Minteer, SD, Raugei, S, Hoffman, BM & Seefeldt, LC 2017, 'Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects', Journal of the American Chemical Society, vol. 139, no. 38, pp. 13518-13524. https://doi.org/10.1021/jacs.7b07311
Khadka, Nimesh ; Milton, Ross D. ; Shaw, Sudipta ; Lukoyanov, Dmitriy ; Dean, Dennis R. ; Minteer, Shelley D. ; Raugei, Simone ; Hoffman, Brian M. ; Seefeldt, Lance C. / Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects. In: Journal of the American Chemical Society. 2017 ; Vol. 139, No. 38. pp. 13518-13524.
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abstract = "Nitrogenase catalyzes the reduction of dinitrogen (N2) to two ammonia (NH3) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H2. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H2. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to the MoFe protein allowing examination of the mechanism of H2 formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, was found to change linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate-limiting step of H2 formation. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H2 formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein, either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H2 formation where the S-H+ bond breaks and H+ attacks the Fe-hydride, and explains the observed H/D isotope effect. This study not only reveals the nitrogenase mechanism of H2 formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other oxidoreductase enzymes and to biomimetic complexes.",
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