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Title: Mechanism of Nitrogenase H 2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects

Abstract

Nitrogenase catalyzes the reduction of dinitrogen (N2) to ammonia (NH3) with obligatory reduction of protons (H+) to dihydrogen (H2) through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides at its active site FeMo-cofactor. The overall 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 MoFe protein without Fe protein and ATP hydrolysis, thereby eliminating the normal rate-limiting step. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, changes linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate limiting step. 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 protonmore » from S-H+ moves to the hydride in Fe-H-, predicting the number and magnitude of the observed H/D isotope effect. This study not only reveals the mechanism of H2 formation, but also illustrates a strategy for mechanistic study that can be applied to other enzymes and to biomimetic complexes.« less

Authors:
 [1]; ORCiD logo [2];  [1];  [3];  [4]; ORCiD logo [2];  [5]; ORCiD logo [3]; ORCiD logo [1]
  1. Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States
  2. Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States
  3. Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States
  4. Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, United States
  5. Pacific Northwest National Laboratory, Richland, Washington 99352, United States
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1406710
Report Number(s):
PNNL-SA-128474
Journal ID: ISSN 0002-7863; KC0304020
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of the American Chemical Society; Journal Volume: 139; Journal Issue: 38
Country of Publication:
United States
Language:
English
Subject:
KeyWords Plus:PHOTOINDUCED REDUCTIVE ELIMINATION; E-4(4H) JANUS INTERMEDIATE; RATE-LIMITING STEP; CATALYTIC CYCLE; FEMO-COFACTOR; MOFE PROTEIN; STATE; IDENTIFICATION; EXCHANGE; BINDING

Citation Formats

Khadka, Nimesh, Milton, Ross D., Shaw, Sudipta, Lukoyanov, Dmitriy, Dean, Dennis R., Minteer, Shelley D., Raugei, Simone, Hoffman, Brian M., and Seefeldt, Lance C. Mechanism of Nitrogenase H 2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects. United States: N. p., 2017. Web. doi: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 H 2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects. United States. doi:10.1021/jacs.7b07311.
Khadka, Nimesh, Milton, Ross D., Shaw, Sudipta, Lukoyanov, Dmitriy, Dean, Dennis R., Minteer, Shelley D., Raugei, Simone, Hoffman, Brian M., and Seefeldt, Lance C. Fri . "Mechanism of Nitrogenase H 2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects". United States. doi:10.1021/jacs.7b07311.
@article{osti_1406710,
title = {Mechanism of Nitrogenase H 2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects},
author = {Khadka, Nimesh and Milton, Ross D. and Shaw, Sudipta and Lukoyanov, Dmitriy and Dean, Dennis R. and Minteer, Shelley D. and Raugei, Simone and Hoffman, Brian M. and Seefeldt, Lance C.},
abstractNote = {Nitrogenase catalyzes the reduction of dinitrogen (N2) to ammonia (NH3) with obligatory reduction of protons (H+) to dihydrogen (H2) through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides at its active site FeMo-cofactor. The overall 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 MoFe protein without Fe protein and ATP hydrolysis, thereby eliminating the normal rate-limiting step. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, changes linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate limiting step. 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 proton from S-H+ moves to the hydride in Fe-H-, predicting the number and magnitude of the observed H/D isotope effect. This study not only reveals the mechanism of H2 formation, but also illustrates a strategy for mechanistic study that can be applied to other enzymes and to biomimetic complexes.},
doi = {10.1021/jacs.7b07311},
journal = {Journal of the American Chemical Society},
number = 38,
volume = 139,
place = {United States},
year = {Fri Sep 15 00:00:00 EDT 2017},
month = {Fri Sep 15 00:00:00 EDT 2017}
}