skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Exploring the Role of the Central Carbide of the Nitrogenase Active-Site FeMo-cofactor through Targeted 13C Labeling and ENDOR Spectroscopy

Journal Article · · Journal of the American Chemical Society
DOI:https://doi.org/10.1021/jacs.1c04152· OSTI ID:1837054
 [1]; ORCiD logo [2]; ORCiD logo [3]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [3]
  1. Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)
  2. Utah State Univ., Logan, UT (United States)
  3. Northwestern Univ., Evanston, IL (United States)
+ Show Author Affiliations

Mo-dependent nitrogenase is a major contributor to global biological N2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe7MoS9C-homocitrate) contains a carbide (C4–) centered within a trigonal prismatic CFe6 core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. Here, to explore this role, the carbide has been in effect selectively enriched with 13C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13C-carbide ENDOR of the S = 3/2 resting state (E0) is remarkable, with an extremely small isotropic hyperfine coupling constant, Ca = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the 13C carbide isotropic hyperfine-coupling constant essentially unchanged, Ca = –1.30 MHz. This indicates that both the E0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to Ca from three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropichyperfine coupling constant undergoes a symmetry change upon conversion of E0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe6 core structures of E0 and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or tretching/breaking of carbide–Fe bonds.

Research Organization:
Northwestern Univ., Evanston, IL (United States); Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Utah State Univ., Logan, UT (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES); USDA; National Science Foundation (NSF); National Institutes of Health (NIH)
Grant/Contract Number:
SC0021314; SC0010687; SC0010834; SC0019342; VA-160085; MCB-1908587; GM111097
OSTI ID:
1837054
Journal Information:
Journal of the American Chemical Society, Vol. 143, Issue 24; ISSN 0002-7863
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
English

References (39)

The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase journal January 2021
Current status of structure function relationships of vanadium nitrogenase journal February 2003
Catalytic and Biophysical Properties of a Nitrogenase Apo-MoFe Protein Produced by a n ifB -Deletion Mutant of Azotobacter v inelandii † journal September 1998
Ligand-Bound S = 1 / 2 FeMo-Cofactor of Nitrogenase: Hyperfine Interaction Analysis and Implication for the Central Ligand X Identity journal July 2008
Insight into the Iron–Molybdenum Cofactor of Nitrogenase from Synthetic Iron Complexes with Sulfur, Carbon, and Hydride Ligands journal June 2016
Cementite journal January 2019
Ligand spin densities in blue copper proteins by q-band proton and nitrogen-14 ENDOR spectroscopy journal February 1991
Reactivity, Mechanism, and Assembly of the Alternative Nitrogenases journal March 2020
Making hyperfine selection in Mims ENDOR independent of deadtime journal May 1997
Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes journal January 2012
Iron-sulfur clusters in the molybdenum-iron protein component of nitrogenase. Electron paramagnetic resonance of the carbon monoxide inhibited state journal October 1979
Low-coordinate iron complexes as synthetic models of nitrogenase journal April 2005
Distribution of Nitrogen‐Fixation Genes in Prokaryotes Containing Alternative Nitrogenases journal April 2020
Structure−Function Relationships of Alternative Nitrogenases journal January 1996
Iron-Only and Vanadium Nitrogenases: Fail-Safe Enzymes or Something More? journal September 2020
Analysis of the Magnetic Properties of Nitrogenase FeMo Cofactor by Single-Crystal EPR Spectroscopy journal August 2013
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 journal December 2007
Iron-57 hyperfine coupling tensors of the FeMo cluster in Azotobacter vinelandii MoFe protein: determination by polycrystalline ENDOR spectroscopy journal March 1988
Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation journal June 2018
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 journal November 2011
Characterization of an Intermediate in the Reduction of Acetylene by the Nitrogenase α-Gln 195 MoFe Protein by Q-band EPR and 13 C, 1 H ENDOR journal June 2000
Simulating Suppression Effects in Pulsed ENDOR, and the ‘Hole in the Middle’ of Mims and Davies ENDOR Spectra journal November 2009
Structural Enzymology of Nitrogenase Enzymes journal June 2020
Evidence for Interstitial Carbon in Nitrogenase FeMo Cofactor journal November 2011
Large-scale purification of high activity Azotobacter vinelandii nitrogenase journal July 1980
Nitrogenases—A Tale of Carbon Atom(s) journal May 2016
Q-Band Pulsed Electron Spin-Echo Spectrometer and Its Application to ENDOR and ESEEM journal March 1996
Structural Characterization of Two CO Molecules Bound to the Nitrogenase Active Site journal January 2021
Mechanism of Molybdenum Nitrogenase journal January 1996
The Spectroscopy of Nitrogenases journal April 2020
X-ray Emission Spectroscopy Evidences a Central Carbon in the Nitrogenase Iron-Molybdenum Cofactor journal November 2011
CO Binding to the FeMo Cofactor of CO-Inhibited Nitrogenase:  13 CO and 1 H Q-Band ENDOR Investigation journal October 1997
Interplay of hemilability and redox activity in models of hydrogenase active sites journal October 2017
High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E 4 (4H) journal June 2019
Combining steady-state and dynamic methods for determining absolute signs of hyperfine interactions: Pulsed ENDOR Saturation and Recovery (PESTRE) journal January 2011
The Interstitial Carbon of the Nitrogenase FeMo Cofactor is Far Better Stabilized than Previously Assumed journal May 2017
Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N 2 reduction journal October 2018
Reduction of Substrates by Nitrogenases journal March 2020
Time-Resolved Binding of Carbon Monoxide to Nitrogenase Monitored by Stopped-Flow Infrared Spectroscopy journal July 1997

Similar Records

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 · OSTI ID:1837054
+4 more
Exploring Electron/Proton Transfer and Conformational Changes in the Nitrogenase MoFe Protein and FeMo-cofactor Through Cryoreduction/EPR Measurements
Journal Article · Fri Jul 29 00:00:00 EDT 2016 · Israel Journal of Chemistry · OSTI ID:1837054
+5 more
Hydride Conformers of the Nitrogenase FeMo-cofactor Two-Electron Reduced State E 2 (2H), Assigned Using Cryogenic Intra Electron Paramagnetic Resonance Cavity Photolysis
Journal Article · Sat Mar 24 00:00:00 EDT 2018 · Inorganic Chemistry · OSTI ID:1837054
+3 more

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY