13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing “Heart of Steel”

Lukoyanov, Dmitriy A.; Yang, Zhi-Yong; Pérez-González, Ana; Raugei, Simone; Dean, Dennis R.; Seefeldt, Lance C.; Hoffman, Brian M.

doi:10.1021/jacs.2c06149

Title: ¹³C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe₆ Core Is a Stabilizing “Heart of Steel”

Journal Article · Tue Sep 27 00:00:00 EDT 2022 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/jacs.2c06149· OSTI ID:1972334

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Northwestern Univ., Evanston, IL (United States)
Utah State Univ., Logan, UT (United States)
Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe–9S–C–Mo–homocitrate] contained within the MoFe protein α-subunit. The cofactor contains a CFe₆ core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted ¹³C labeling of the carbon enables electron-nuclear double resonance (ENDOR) spectroscopy to sensitively monitor the electronic properties of the Fe–C bonds and the spin-coupling scheme adopted by the FeMo-cofactor metal ions. Here this report compares ¹³CFe₆ ENDOR measurements for (i) the wild-type protein resting state (E₀; α-Val⁷⁰) to those of (ii) α-Ile⁷⁰, (iii) α-Ala⁷⁰-substituted proteins; (iv) crystallographically characterized CO-inhibited “hi-CO” state; (v) E₄(4H) Janus intermediate, activated for N₂ binding/reduction by accumulation of 4[^e–/H⁺]; (vi) E₄(2H)* state containing a doubly reduced FeMo-cofactor without Fe-bound substrates; and (vii) propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, both S = 1/2 and 3/2 exhibited near-zero ¹³C isotropic hyperfine coupling constants, $^Ca$ = [-1.3 ↔ +2.7] MHz. Density functional theory computations and natural bond orbital analysis of the Fe-C bonds show that this occurs because a (3 spin-up/3 spin-down) spin-exchange configuration of CFe₆ Fe-ion spins produces cancellation of large spin-transfers to carbon in each Fe–C bond. Previous X-ray diffraction and DFT both indicate that trigonal-prismatic geometry around carbon is maintained with high precision in all these states. The persistent structure and Fe–C bonding of the CFe₆ core indicate that it does not provide a functionally dynamic (hemilabile) “beating heart”-instead it acts as “a heart of steel”, stabilizing the structure of the FeMo-cofactor-active site during nitrogenase catalysis.

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Research Organization:: Northwestern Univ., Evanston, IL (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB)

Grant/Contract Number:: SC0019342; SC0010687; SC0010834; AC05-76RL01830

OSTI ID:: 1972334

Alternate ID(s):: OSTI ID: 1970140

Report Number(s):: PNNL-SA-174111

Journal Information:: Journal of the American Chemical Society, Vol. 144, Issue 40; ISSN 0002-7863

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (56)

Mechanism of Molybdenum Nitrogenase Burgess, Barbara K.; Lowe, David J. Chemical Reviews, Vol. 96, Issue 7 https://doi.org/10.1021/cr950055x	journal	January 1996
Duplication and extension of the Thorneley and Lowe kinetic model for Klebsiella pneumoniae nitrogenase catalysis using a mathematica software platform Wilson, P. E.; Nyborg, A. C.; Watt, G. D. Biophysical Chemistry, Vol. 91, Issue 3 https://doi.org/10.1016/s0301-4622(01)00182-x	journal	July 2001
A Confirmation of the Quench-Cryoannealing Relaxation Protocol for Identifying Reduction States of Freeze-Trapped Nitrogenase Intermediates Lukoyanov, Dmitriy; Yang, Zhi-Yong; Duval, Simon Inorganic Chemistry, Vol. 53, Issue 7 https://doi.org/10.1021/ic500013c	journal	March 2014
Hydride Conformers of the Nitrogenase FeMo-cofactor Two-Electron Reduced State E ₂ (2H), Assigned Using Cryogenic Intra Electron Paramagnetic Resonance Cavity Photolysis Lukoyanov, Dmitriy A.; Khadka, Nimesh; Yang, Zhi-Yong Inorganic Chemistry, Vol. 57, Issue 12 https://doi.org/10.1021/acs.inorgchem.8b00271	journal	March 2018
Reduction of Substrates by Nitrogenases Seefeldt, Lance C.; Yang, Zhi-Yong; Lukoyanov, Dmitriy A. Chemical Reviews, Vol. 120, Issue 12 https://doi.org/10.1021/acs.chemrev.9b00556	journal	March 2020
Evidence for Interstitial Carbon in Nitrogenase FeMo Cofactor Spatzal, T.; Aksoyoglu, M.; Zhang, L. Science, Vol. 334, Issue 6058 https://doi.org/10.1126/science.1214025	journal	November 2011
Cementite Bhadeshia, H. K. D. H. International Materials Reviews, Vol. 65, Issue 1 https://doi.org/10.1080/09506608.2018.1560984	journal	January 2019
The Interstitial Carbon of the Nitrogenase FeMo Cofactor is Far Better Stabilized than Previously Assumed Grunenberg, Jörg Angewandte Chemie International Edition, Vol. 56, Issue 25 https://doi.org/10.1002/anie.201701790	journal	May 2017
A Terminal N ₂ Complex of High-Spin Iron(I) in a Weak, Trigonal Ligand Field McSkimming, Alex; Harman, W. Hill Journal of the American Chemical Society, Vol. 137, Issue 28 https://doi.org/10.1021/jacs.5b06337	journal	July 2015
Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage Hoffman, Brian M.; Lukoyanov, Dmitriy; Yang, Zhi-Yong Chemical Reviews, Vol. 114, Issue 8 https://doi.org/10.1021/cr400641x	journal	January 2014
⁵⁷ Fe ENDOR Spectroscopy and ‘Electron Inventory’ Analysis of the Nitrogenase E ₄ Intermediate Suggest the Metal-Ion Core of FeMo-Cofactor Cycles Through Only One Redox Couple Doan, Peter E.; Telser, Joshua; Barney, Brett M. Journal of the American Chemical Society, Vol. 133, Issue 43 https://doi.org/10.1021/ja205304t	journal	November 2011
Electron Redistribution within the Nitrogenase Active Site FeMo-Cofactor During Reductive Elimination of H ₂ to Achieve N≡N Triple-Bond Activation Lukoyanov, Dmitriy A.; Yang, Zhi-Yong; Dean, Dennis R. Journal of the American Chemical Society, Vol. 142, Issue 52 https://doi.org/10.1021/jacs.0c07914	journal	December 2020
Exploring the Role of the Central Carbide of the Nitrogenase Active-Site FeMo-cofactor through Targeted ¹³C Labeling and ENDOR Spectroscopy Pérez-González, Ana; Yang, Zhi-Yong; Lukoyanov, Dmitriy A. Journal of the American Chemical Society, Vol. 143, Issue 24 https://doi.org/10.1021/jacs.1c04152	journal	June 2021
Testing if the Interstitial Atom, X , of the Nitrogenase Molybdenum−Iron Cofactor Is N or C: ENDOR, ESEEM, and DFT Studies of the S = ³ / ₂ Resting State in Multiple Environments Lukoyanov, Dmitriy; Pelmenschikov, Vladimir; Maeser, Nathan Inorganic Chemistry, Vol. 46, Issue 26 https://doi.org/10.1021/ic7018814	journal	December 2007
Iron-sulfur clusters in the molybdenum-iron protein component of nitrogenase. Electron paramagnetic resonance of the carbon monoxide inhibited state Davis, Lawrence C.; Henzl, Michael T.; Burris, R. H. Biochemistry, Vol. 18, Issue 22 https://doi.org/10.1021/bi00589a014	journal	October 1979
Structural Characterization of Two CO Molecules Bound to the Nitrogenase Active Site Buscagan, Trixia M.; Perez, Kathryn A.; Maggiolo, Ailiena O. Angewandte Chemie International Edition, Vol. 60, Issue 11 https://doi.org/10.1002/anie.202015751	journal	January 2021
Substrate Interaction at an Iron-Sulfur Face of the FeMo-cofactor during Nitrogenase Catalysis Barney, Brett M.; Igarashi, Robert Y.; Dos Santos, Patricia C. Journal of Biological Chemistry, Vol. 279, Issue 51 https://doi.org/10.1074/jbc.m410247200	journal	October 2004
Trapping H ^- Bound to the Nitrogenase FeMo-Cofactor Active Site during H ₂ Evolution: Characterization by ENDOR Spectroscopy Igarashi, Robert Y.; Laryukhin, Mikhail; Dos Santos, Patricia C. Journal of the American Chemical Society, Vol. 127, Issue 17 https://doi.org/10.1021/ja043596p	journal	May 2005
Connecting nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol and identification of the N2 binding state Lukoyanov, D.; Barney, B. M.; Dean, D. R. Proceedings of the National Academy of Sciences, Vol. 104, Issue 5 https://doi.org/10.1073/pnas.0610975104	journal	January 2007
High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E ₄ (4H) Hoeke, Veronika; Tociu, Laura; Case, David A. Journal of the American Chemical Society, Vol. 141, Issue 30 https://doi.org/10.1021/jacs.9b04474	journal	June 2019
Reversible Photoinduced Reductive Elimination of H ₂ from the Nitrogenase Dihydride State, the E ₄ (4H) Janus Intermediate Lukoyanov, Dmitriy; Khadka, Nimesh; Yang, Zhi-Yong Journal of the American Chemical Society, Vol. 138, Issue 4 https://doi.org/10.1021/jacs.5b11650	journal	January 2016
An Organometallic Intermediate during Alkyne Reduction by Nitrogenase Lee, Hong-In; Igarashi, Robert Y.; Laryukhin, Mikhail Journal of the American Chemical Society, Vol. 126, Issue 31 https://doi.org/10.1021/ja048714n	journal	August 2004
Localization of a Catalytic Intermediate Bound to the FeMo-cofactor of Nitrogenase Igarashi, Robert Y.; Dos Santos, Patricia C.; Niehaus, Walter G. Journal of Biological Chemistry, Vol. 279, Issue 33 https://doi.org/10.1074/jbc.m403194200	journal	June 2004
Localization of a Substrate Binding Site on the FeMo-Cofactor in Nitrogenase: Trapping Propargyl Alcohol with an α-70-Substituted MoFe Protein ^† Benton, Paul M. C.; Laryukhin, Mikhail; Mayer, Suzanne M. Biochemistry, Vol. 42, Issue 30 https://doi.org/10.1021/bi034595x	journal	August 2003
Intermolecular interactions from a natural bond orbital, donor-acceptor viewpoint Reed, Alan E.; Curtiss, Larry A.; Weinhold, Frank Chemical Reviews, Vol. 88, Issue 6 https://doi.org/10.1021/cr00088a005	journal	September 1988
What Is the Structure of the E ₄ Intermediate in Nitrogenase? Cao, Lili; Ryde, Ulf Journal of Chemical Theory and Computation, Vol. 16, Issue 3 https://doi.org/10.1021/acs.jctc.9b01254	journal	January 2020
The E ₂ state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H ₂ Evolution Thorhallsson, Albert Th.; Bjornsson, Ragnar Chemistry – A European Journal, Vol. 27, Issue 67 https://doi.org/10.1002/chem.202102730	journal	October 2021
Nitrogenase: A Draft Mechanism Hoffman, Brian M.; Lukoyanov, Dmitriy; Dean, Dennis R. Accounts of Chemical Research, Vol. 46, Issue 2 https://doi.org/10.1021/ar300267m	journal	December 2012
Photoinduced Reductive Elimination of H ₂ from the Nitrogenase Dihydride (Janus) State Involves a FeMo-cofactor-H ₂ Intermediate Lukoyanov, Dmitriy; Khadka, Nimesh; Dean, Dennis R. Inorganic Chemistry, Vol. 56, Issue 4 https://doi.org/10.1021/acs.inorgchem.6b02899	journal	February 2017
Catalytic and Biophysical Properties of a Nitrogenase Apo-MoFe Protein Produced by a n ifB -Deletion Mutant of Azotobacter v inelandii ^† Christiansen, Jason; Goodwin, Paul J.; Lanzilotta, William N. Biochemistry, Vol. 37, Issue 36 https://doi.org/10.1021/bi981165b	journal	September 1998
ENDOR of Metalloenzymes Hoffman, Brian M. Accounts of Chemical Research, Vol. 36, Issue 7 https://doi.org/10.1021/ar0202565	journal	July 2003
Simulating Suppression Effects in Pulsed ENDOR, and the ‘Hole in the Middle’ of Mims and Davies ENDOR Spectra Doan, Peter E.; Lees, Nicholas S.; Shanmugam, Muralidharan Applied Magnetic Resonance, Vol. 37, Issue 1-4 https://doi.org/10.1007/s00723-009-0083-6	journal	November 2009
Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N ₂ reduction Raugei, Simone; Seefeldt, Lance C.; Hoffman, Brian M. Proceedings of the National Academy of Sciences, Vol. 115, Issue 45 https://doi.org/10.1073/pnas.1810211115	journal	October 2018
Substrate Channel in Nitrogenase Revealed by a Molecular Dynamics Approach Smith, Dayle; Danyal, Karamatullah; Raugei, Simone Biochemistry, Vol. 53, Issue 14 https://doi.org/10.1021/bi401313j	journal	April 2014
Density-functional exchange-energy approximation with correct asymptotic behavior Becke, A. D. Physical Review A, Vol. 38, Issue 6 https://doi.org/10.1103/physreva.38.3098	journal	September 1988
Density-functional approximation for the correlation energy of the inhomogeneous electron gas Perdew, John P. Physical Review B, Vol. 33, Issue 12 https://doi.org/10.1103/physrevb.33.8822	journal	June 1986
Erratum: Density-functional approximation for the correlation energy of the inhomogeneous electron gas Perdew, John P. Physical Review B, Vol. 34, Issue 10 https://doi.org/10.1103/physrevb.34.7406	journal	November 1986
Density‐functional thermochemistry. III. The role of exact exchange Becke, Axel D. The Journal of Chemical Physics, Vol. 98, Issue 7, p. 5648-5652 https://doi.org/10.1063/1.464913	journal	April 1993
Ab Initio Calculation of Vibrational Absorption and Circular Dichroism Spectra Using Density Functional Force Fields Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F. The Journal of Physical Chemistry, Vol. 98, Issue 45, p. 11623-11627 https://doi.org/10.1021/j100096a001	journal	November 1994
Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy Weigend, Florian; Ahlrichs, Reinhart Physical Chemistry Chemical Physics, Vol. 7, Issue 18, p. 3297-3305 https://doi.org/10.1039/b508541a	journal	January 2005
Energy‐adjusted a b i n i t i o pseudopotentials for the rare earth elements Dolg, M.; Stoll, H.; Preuss, H. The Journal of Chemical Physics, Vol. 90, Issue 3 https://doi.org/10.1063/1.456066	journal	February 1989
A model for dinitrogen binding in the E ₄ state of nitrogenase Thorhallsson, Albert Th.; Benediktsson, Bardi; Bjornsson, Ragnar Chemical Science, Vol. 10, Issue 48 https://doi.org/10.1039/c9sc03610e	journal	January 2019
Extremely large differences in DFT energies for nitrogenase models Cao, Lili; Ryde, Ulf Physical Chemistry Chemical Physics, Vol. 21, Issue 5 https://doi.org/10.1039/c8cp06930a	journal	January 2019
Thermodynamically Favourable States in the Reaction of Nitrogenase without Dissociation of any Sulfide Ligand Jiang, Hao; Ryde, Ulf Chemistry – A European Journal, Vol. 28, Issue 14 https://doi.org/10.1002/chem.202103933	journal	February 2022
The ORCA program system: The ORCA program system Neese, Frank Wiley Interdisciplinary Reviews: Computational Molecular Science, Vol. 2, Issue 1 https://doi.org/10.1002/wcms.81	journal	June 2011
Iron-57 hyperfine coupling tensors of the FeMo cluster in Azotobacter vinelandii MoFe protein: determination by polycrystalline ENDOR spectroscopy True, Anne E.; Nelson, Mark J.; Venters, Ronald A. Journal of the American Chemical Society, Vol. 110, Issue 6 https://doi.org/10.1021/ja00214a045	journal	March 1988
Analysis of the Magnetic Properties of Nitrogenase FeMo Cofactor by Single-Crystal EPR Spectroscopy Spatzal, Thomas; Einsle, Oliver; Andrade, Susana L. A. Angewandte Chemie International Edition, Vol. 52, Issue 38 https://doi.org/10.1002/anie.201303000	journal	August 2013
Is Mo Involved in Hydride Binding by the Four-Electron Reduced (E ₄ ) Intermediate of the Nitrogenase MoFe Protein? Lukoyanov, Dmitriy; Yang, Zhi-Yong; Dean, Dennis R. Journal of the American Chemical Society, Vol. 132, Issue 8 https://doi.org/10.1021/ja910613m	journal	March 2010
Investigation of CO Binding and Release from Mo-Nitrogenase during Catalytic Turnover ^† Cameron, Linda M.; Hales, Brian J. Biochemistry, Vol. 37, Issue 26 https://doi.org/10.1021/bi972667c	journal	June 1998
Combining steady-state and dynamic methods for determining absolute signs of hyperfine interactions: Pulsed ENDOR Saturation and Recovery (PESTRE) Doan, Peter E. Journal of Magnetic Resonance, Vol. 208, Issue 1 https://doi.org/10.1016/j.jmr.2010.10.008	journal	January 2011
A bound reaction intermediate sheds light on the mechanism of nitrogenase Sippel, Daniel; Rohde, Michael; Netzer, Julia Science, Vol. 359, Issue 6383 https://doi.org/10.1126/science.aar2765	journal	March 2018
The Critical E ₄ State of Nitrogenase Catalysis Rohde, Michael; Sippel, Daniel; Trncik, Christian Biochemistry, Vol. 57, Issue 38 https://doi.org/10.1021/acs.biochem.8b00509	journal	July 2018
Ligand-Bound S = ¹ / ₂ FeMo-Cofactor of Nitrogenase: Hyperfine Interaction Analysis and Implication for the Central Ligand X Identity Pelmenschikov, Vladimir; Case, David A.; Noodleman, Louis Inorganic Chemistry, Vol. 47, Issue 14 https://doi.org/10.1021/ic7022743	journal	July 2008
Analysis of the 57Fe Hyperfine Coupling Constants and Spin States in Nitrogenase P-Clusters Mouesca, J. -M.; Noodleman, L.; Case, D. A. Inorganic Chemistry, Vol. 33, Issue 22 https://doi.org/10.1021/ic00100a004	journal	October 1994
Time-Resolved EPR Study of H₂ Reductive Elimination from the Photoexcited Nitrogenase Janus E₄(4H) Intermediate Lukoyanov, Dmitriy A.; Krzyaniak, Matthew D.; Dean, Dennis R. The Journal of Physical Chemistry B, Vol. 123, Issue 41 https://doi.org/10.1021/acs.jpcb.9b07776	journal	September 2019
Structural Enzymology of Nitrogenase Enzymes Einsle, Oliver; Rees, Douglas C. Chemical Reviews, Vol. 120, Issue 12 https://doi.org/10.1021/acs.chemrev.0c00067	journal	June 2020

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Title: 13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing “Heart of Steel”

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Title: ¹³C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe₆ Core Is a Stabilizing “Heart of Steel”