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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

Journal Article · · Journal of the American Chemical Society
DOI:https://doi.org/10.1021/jacs.6b06362· OSTI ID:1466796
 [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)
+ Show Author Affiliations

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.

Research Organization:
Northwestern Univ., Evanston, IL (United States). Depts. of Chemistry and Molecular Biosciences
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
Grant/Contract Number:
SC0010687
OSTI ID:
1466796
Journal Information:
Journal of the American Chemical Society, Vol. 138, Issue 33; ISSN 0002-7863
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 107 works
Citation information provided by
Web of Science

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Cited By (16)

Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N 2 reduction journal October 2018
X-ray Magnetic Circular Dichroism Spectroscopy Applied to Nitrogenase and Related Models: Experimental Evidence for a Spin-Coupled Molybdenum(III) Center journal June 2019
N 2 -to-NH 3 Conversion by a triphos-Iron Catalyst and Enhanced Turnover under Photolysis journal May 2017
Nitrogen Reduction to Ammonia on a Biomimetic Mononuclear Iron Centre: Insights into the Nitrogenase Enzyme journal December 2017
A model for dinitrogen binding in the E 4 state of nitrogenase journal January 2019
Nitrogen Fixation Catalyzed by Transition Metal Complexes: Recent Developments: Nitrogen Fixation Catalyzed by Transition Metal Complexes: Recent Developments journal February 2018
Survey of the Geometric and Electronic Structures of the Key Hydrogenated Forms of FeMo-co, the Active Site of the Enzyme Nitrogenase: Principles of the Mechanistically Significant Coordination Chemistry journal January 2019
High-Frequency Fe-H Vibrations in a Bridging Hydride Complex Characterized by NRVS and DFT journal June 2018
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
Is there computational support for an unprotonated carbon in the E 4 state of nitrogenase? journal December 2017
X‐ray Magnetic Circular Dichroism Spectroscopy Applied to Nitrogenase and Related Models: Experimental Evidence for a Spin‐Coupled Molybdenum(III) Center journal June 2019
Synthesis and Diverse Transformations of a Dinitrogen Dititanium Hydride Complex Bearing Rigid Acridane‐Based PNP‐Pincer Ligands journal March 2020
Computational Investigations of the Chemical Mechanism of the Enzyme Nitrogenase journal January 2020
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
N 2 -to-NH 3 Conversion by a triphos-Iron Catalyst and Enhanced Turnover under Photolysis journal May 2017

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