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Title: Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme

Abstract

Here, we have proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70Val→Ile substituted MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN=NH) reduction level bound to the catalytic FeMo-cofactor. In the present work, EPR/ENDOR and photophysical measurements show that a state freeze-trapped during N2 reduction by wild type (WT) MoFe protein is the same Janus intermediate, thereby establishing the α-70Val→Ile intermediate as a reliable guide to mechanism, and enabling new experimental tests of the re/oa mechanism with WT enzyme. These allow us to show that the re/oa mechanism accounts for the longstanding Key Constraints on mechanism. Monitoring the S = ½ FeMo-co EPR signal of Janus in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby explaining earlier observations and verifying the re/oa mechanism. Relaxation of E4(2N2H) tomore » the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium.« less

Authors:
 [1];  [2];  [2];  [3];  [2];  [1]
  1. Northwestern Univ., Evanston, IL (United States)
  2. Utah State Univ., Logan, UT (United States)
  3. Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)
Publication Date:
Research Org.:
Northwestern Univ., Evanston, IL (United States). Depts. of Chemistry and Molecular Biosciences
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1466796
Grant/Contract Number:  
SC0010687
Resource Type:
Accepted Manuscript
Journal Name:
Journal of the American Chemical Society
Additional Journal Information:
Journal Volume: 138; Journal Issue: 33; Journal ID: ISSN 0002-7863
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Lukoyanov, Dmitriy, Khadka, Nimesh, Yang, Zhi -Yong, Dean, Dennis R., Seefeldt, Lance C., and Hoffman, Brian M. Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme. United States: N. p., 2016. Web. doi:10.1021/jacs.6b06362.
Lukoyanov, Dmitriy, Khadka, Nimesh, Yang, Zhi -Yong, Dean, Dennis R., Seefeldt, Lance C., & Hoffman, Brian M. Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme. United States. https://doi.org/10.1021/jacs.6b06362
Lukoyanov, Dmitriy, Khadka, Nimesh, Yang, Zhi -Yong, Dean, Dennis R., Seefeldt, Lance C., and Hoffman, Brian M. Tue . "Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme". United States. https://doi.org/10.1021/jacs.6b06362. https://www.osti.gov/servlets/purl/1466796.
@article{osti_1466796,
title = {Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme},
author = {Lukoyanov, Dmitriy and Khadka, Nimesh and Yang, Zhi -Yong and Dean, Dennis R. and Seefeldt, Lance C. and Hoffman, Brian M.},
abstractNote = {Here, we have proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70Val→Ile substituted MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN=NH) reduction level bound to the catalytic FeMo-cofactor. In the present work, EPR/ENDOR and photophysical measurements show that a state freeze-trapped during N2 reduction by wild type (WT) MoFe protein is the same Janus intermediate, thereby establishing the α-70Val→Ile intermediate as a reliable guide to mechanism, and enabling new experimental tests of the re/oa mechanism with WT enzyme. These allow us to show that the re/oa mechanism accounts for the longstanding Key Constraints on mechanism. Monitoring the S = ½ FeMo-co EPR signal of Janus in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby explaining earlier observations and verifying the re/oa mechanism. Relaxation of E4(2N2H) to the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium.},
doi = {10.1021/jacs.6b06362},
journal = {Journal of the American Chemical Society},
number = 33,
volume = 138,
place = {United States},
year = {2016},
month = {8}
}

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Works referenced in this record:

Mechanism of Molybdenum Nitrogenase
journal, January 1996

  • Burgess, Barbara K.; Lowe, David J.
  • Chemical Reviews, Vol. 96, Issue 7
  • DOI: 10.1021/cr950055x

A nitrogen pressure of 50 atmospheres does not prevent evolution of hydrogen by nitrogenase
journal, June 1984


Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage
journal, January 2014

  • Hoffman, Brian M.; Lukoyanov, Dmitriy; Yang, Zhi-Yong
  • Chemical Reviews, Vol. 114, Issue 8
  • DOI: 10.1021/cr400641x

Nitrogenase: A Draft Mechanism
journal, December 2012

  • Hoffman, Brian M.; Lukoyanov, Dmitriy; Dean, Dennis R.
  • Accounts of Chemical Research, Vol. 46, Issue 2
  • DOI: 10.1021/ar300267m

Trapping H - Bound to the Nitrogenase FeMo-Cofactor Active Site during H 2 Evolution:  Characterization by ENDOR Spectroscopy
journal, May 2005

  • Igarashi, Robert Y.; Laryukhin, Mikhail; Dos Santos, Patricia C.
  • Journal of the American Chemical Society, Vol. 127, Issue 17
  • DOI: 10.1021/ja043596p

Is Mo Involved in Hydride Binding by the Four-Electron Reduced (E 4 ) Intermediate of the Nitrogenase MoFe Protein?
journal, March 2010

  • Lukoyanov, Dmitriy; Yang, Zhi-Yong; Dean, Dennis R.
  • Journal of the American Chemical Society, Vol. 132, Issue 8
  • DOI: 10.1021/ja910613m

Connecting nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol and identification of the N2 binding state
journal, January 2007

  • Lukoyanov, D.; Barney, B. M.; Dean, D. R.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 5
  • DOI: 10.1073/pnas.0610975104

On reversible H2 loss upon N2 binding to FeMo-cofactor of nitrogenase
journal, September 2013

  • Yang, Z. -Y.; Khadka, N.; Lukoyanov, D.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 41
  • DOI: 10.1073/pnas.1315852110

Identification of a Key Catalytic Intermediate Demonstrates That Nitrogenase Is Activated by the Reversible Exchange of N 2 for H 2
journal, March 2015

  • Lukoyanov, Dmitriy; Yang, Zhi-Yong; Khadka, Nimesh
  • Journal of the American Chemical Society, Vol. 137, Issue 10
  • DOI: 10.1021/jacs.5b00103

Catalytic and Biophysical Properties of a Nitrogenase Apo-MoFe Protein Produced by a n ifB -Deletion Mutant of Azotobacter v inelandii
journal, September 1998

  • Christiansen, Jason; Goodwin, Paul J.; Lanzilotta, William N.
  • Biochemistry, Vol. 37, Issue 36
  • DOI: 10.1021/bi981165b

Reversible Photoinduced Reductive Elimination of H 2 from the Nitrogenase Dihydride State, the E 4 (4H) Janus Intermediate
journal, January 2016

  • Lukoyanov, Dmitriy; Khadka, Nimesh; Yang, Zhi-Yong
  • Journal of the American Chemical Society, Vol. 138, Issue 4
  • DOI: 10.1021/jacs.5b11650

Metal dihydride complexes: Photochemical mechanisms for reductive elimination
journal, November 1998


Ultrafast reductive elimination of hydrogen from a metal carbonyl dihydride complex; a study by time-resolved IR and visible spectroscopy
journal, January 1997

  • Colombo, Mirco; George, Michael W.; Moore, John N.
  • Journal of the Chemical Society, Dalton Transactions, Issue 17
  • DOI: 10.1039/a704484d

Transient and matrix photochemistry of Fe(dmpe)2H2 (dmpe = Me2PCH2CH2Me2): dynamics of C-H and H-H activation
journal, September 1993

  • Whittlesey, Michael K.; Mawby, Roger J.; Osman, Robert
  • Journal of the American Chemical Society, Vol. 115, Issue 19
  • DOI: 10.1021/ja00072a016

The hydride route to the preparation of dinitrogen complexes
journal, January 2010

  • Ballmann, Joachim; Munhá, Rui F.; Fryzuk, Michael D.
  • Chemical Communications, Vol. 46, Issue 7
  • DOI: 10.1039/b922853e

The photoreversible oxidative-addition, reductive-elimination reactions iron + molecular hydrogen .dblharw. iron hydride (FeH2) in low-temperature matrixes
journal, February 1984

  • Ozin, Geoffrey A.; McCaffrey, John G.
  • The Journal of Physical Chemistry, Vol. 88, Issue 4
  • DOI: 10.1021/j150648a002

New routes to low-coordinate iron hydride complexes: The binuclear oxidative addition of H2
journal, August 2009


Studies of Low-Coordinate Iron Dinitrogen Complexes
journal, January 2006

  • Smith, Jeremy M.; Sadique, Azwana R.; Cundari, Thomas R.
  • Journal of the American Chemical Society, Vol. 128, Issue 3
  • DOI: 10.1021/ja052707x

The Reactivity Patterns of Low-Coordinate Iron−Hydride Complexes
journal, May 2008

  • Yu, Ying; Sadique, Azwana R.; Smith, Jeremy M.
  • Journal of the American Chemical Society, Vol. 130, Issue 20
  • DOI: 10.1021/ja710669w

Stretched exponential relaxation in molecular and electronic glasses
journal, September 1996


Mathematical functions for the analysis of luminescence decays with underlying distributions 1. Kohlrausch decay function (stretched exponential)
journal, August 2005


Selective Photochemistry at Stereogenic Metal and Ligand Centers of cis -[Ru(diphosphine) 2 (H) 2 ]: Preparative, NMR, Solid State, and Laser Flash Studies
journal, February 2012

  • Câmpian, Marius V.; Perutz, Robin N.; Procacci, Barbara
  • Journal of the American Chemical Society, Vol. 134, Issue 7
  • DOI: 10.1021/ja210568t

A Confirmation of the Quench-Cryoannealing Relaxation Protocol for Identifying Reduction States of Freeze-Trapped Nitrogenase Intermediates
journal, March 2014

  • Lukoyanov, Dmitriy; Yang, Zhi-Yong; Duval, Simon
  • Inorganic Chemistry, Vol. 53, Issue 7
  • DOI: 10.1021/ic500013c

Works referencing / citing this record:

Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N 2 reduction
journal, October 2018

  • Raugei, Simone; Seefeldt, Lance C.; Hoffman, Brian M.
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 45
  • DOI: 10.1073/pnas.1810211115

X-ray Magnetic Circular Dichroism Spectroscopy Applied to Nitrogenase and Related Models: Experimental Evidence for a Spin-Coupled Molybdenum(III) Center
journal, June 2019

  • Kowalska, Joanna K.; Henthorn, Justin T.; Van Stappen, Casey
  • Angewandte Chemie International Edition, Vol. 58, Issue 28
  • DOI: 10.1002/anie.201901899

N 2 -to-NH 3 Conversion by a triphos-Iron Catalyst and Enhanced Turnover under Photolysis
journal, May 2017

  • Buscagan, Trixia M.; Oyala, Paul H.; Peters, Jonas C.
  • Angewandte Chemie International Edition, Vol. 56, Issue 24
  • DOI: 10.1002/anie.201703244

Nitrogen Reduction to Ammonia on a Biomimetic Mononuclear Iron Centre: Insights into the Nitrogenase Enzyme
journal, December 2017

  • Kaczmarek, Monika A.; Malhotra, Abheek; Balan, G. Alex
  • Chemistry - A European Journal, Vol. 24, Issue 20
  • DOI: 10.1002/chem.201704688

A model for dinitrogen binding in the E 4 state of nitrogenase
journal, January 2019

  • Thorhallsson, Albert Th.; Benediktsson, Bardi; Bjornsson, Ragnar
  • Chemical Science, Vol. 10, Issue 48
  • DOI: 10.1039/c9sc03610e

Nitrogen Fixation Catalyzed by Transition Metal Complexes: Recent Developments: Nitrogen Fixation Catalyzed by Transition Metal Complexes: Recent Developments
journal, February 2018

  • Stucke, Nadja; Flöser, Benedikt M.; Weyrich, Thomas
  • European Journal of Inorganic Chemistry, Vol. 2018, Issue 12
  • DOI: 10.1002/ejic.201701326

High-Frequency Fe-H Vibrations in a Bridging Hydride Complex Characterized by NRVS and DFT
journal, June 2018

  • Pelmenschikov, Vladimir; Gee, Leland B.; Wang, Hongxin
  • Angewandte Chemie, Vol. 130, Issue 30
  • DOI: 10.1002/ange.201804601

Easily reduced bis-pincer ( NS 2 ) 2 molybdenum( iv ) to ( NHS 2 ) 2 Mo( ii ) by alcohols vs . redox-inert ( NS 2 )( NHS 2 )iron( iii ) complexes
journal, January 2018

  • Robles-Marín, Elvis; Mondragón, Alexander; Martínez-Alanis, Paulina R.
  • Dalton Transactions, Vol. 47, Issue 32
  • DOI: 10.1039/c8dt01562g

Is there computational support for an unprotonated carbon in the E 4 state of nitrogenase?
journal, December 2017

  • Siegbahn, Per E. M.
  • Journal of Computational Chemistry, Vol. 39, Issue 12
  • DOI: 10.1002/jcc.25145

Electrophile-promoted Fe-to-N 2 hydride migration in highly reduced Fe(N 2 )(H) complexes
journal, January 2018

  • Deegan, Meaghan M.; Peters, Jonas C.
  • Chemical Science, Vol. 9, Issue 29
  • DOI: 10.1039/c8sc02380h

X‐ray Magnetic Circular Dichroism Spectroscopy Applied to Nitrogenase and Related Models: Experimental Evidence for a Spin‐Coupled Molybdenum(III) Center
journal, June 2019

  • Kowalska, Joanna K.; Henthorn, Justin T.; Van Stappen, Casey
  • Angewandte Chemie, Vol. 131, Issue 28
  • DOI: 10.1002/ange.201901899

Synthesis and Diverse Transformations of a Dinitrogen Dititanium Hydride Complex Bearing Rigid Acridane‐Based PNP‐Pincer Ligands
journal, March 2020

  • Mo, Zhenbo; Shima, Takanori; Hou, Zhaomin
  • Angewandte Chemie International Edition, Vol. 59, Issue 22
  • DOI: 10.1002/anie.201916171

Computational Investigations of the Chemical Mechanism of the Enzyme Nitrogenase
journal, January 2020


Reactivity of hydride bridges in a high-spin [Fe 3 (μ-H) 3 ] 3+ cluster: reversible H 2 /CO exchange and Fe–H/B–F bond metathesis
journal, January 2017

  • Anderton, Kevin J.; Knight, Brian J.; Rheingold, Arnold L.
  • Chemical Science, Vol. 8, Issue 5
  • DOI: 10.1039/c6sc05583d

Synthesis and Diverse Transformations of a Dinitrogen Dititanium Hydride Complex Bearing Rigid Acridane‐Based PNP‐Pincer Ligands
journal, March 2020


High-Frequency Fe-H Vibrations in a Bridging Hydride Complex Characterized by NRVS and DFT
journal, June 2018

  • Pelmenschikov, Vladimir; Gee, Leland B.; Wang, Hongxin
  • Angewandte Chemie International Edition, Vol. 57, Issue 30
  • DOI: 10.1002/anie.201804601

N 2 -to-NH 3 Conversion by a triphos-Iron Catalyst and Enhanced Turnover under Photolysis
journal, May 2017

  • Buscagan, Trixia M.; Oyala, Paul H.; Peters, Jonas C.
  • Angewandte Chemie, Vol. 129, Issue 24
  • DOI: 10.1002/ange.201703244