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Title: Reductive Elimination of H 2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E 4(4H) Janus Intermediate in Wild-Type Enzyme

Abstract

Here, we have proposed a reductive elimination/oxidative addition ( re/oa) mechanism for reduction of N 2 to 2NH 3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70 Val→Ile substituted MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E 4(4H)). The mechanism postulates that obligatory re of the hydrides as H 2 drives reduction of N 2 to a state (denoted E 4(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 N 2 reduction by wild type (WT) MoFe protein is the same Janus intermediate, thereby establishing the α-70 Val→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 N 2 reduction under mixed-isotope condition, H 2O buffer/D 2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover,more » thereby explaining earlier observations and verifying the re/oa mechanism. Relaxation of E 4(2N2H) to the WT resting-state is shown to occur via oa of H 2 and release of N 2 to form Janus, followed by sequential release of two H 2, 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) (SC-22)
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. doi: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. doi: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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