High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E4(4H)

Veronika Hoeke, Laura Tociu, David A. Case, Lance C. Seefeldt, Simone Raugei, Brian M. Hoffman

Research output: Contribution to journalArticle

Abstract

We have shown that the key state in N2 reduction to two NH3 molecules by the enzyme nitrogenase is E4(4H), the "Janus" intermediate, which has accumulated four [e-/H+] and is poised to undergo reductive elimination of H2 coupled to N2 binding and activation. Initial 1H and 95Mo ENDOR studies of freeze-trapped E4(4H) revealed that the catalytic multimetallic cluster (FeMo-co) binds two Fe-bridging hydrides, [Fe-H-Fe]. However, the analysis failed to provide a satisfactory picture of the relative spatial relationships of the two [Fe-H-Fe]. Our recent density functional theory (DFT) study yielded a lowest-energy form, denoted as E4(4H)(a), with two parallel Fe-H-Fe planes bridging pairs of "anchor" Fe on the Fe2,3,6,7 face of FeMo-co. However, the relative energies of structures E4(4H)(b), with one bridging and one terminal hydride, and E4(4H)(c), with one pair of anchor Fe supporting two bridging hydrides, were not beyond the uncertainties in the calculation. Moreover, a structure of V-dependent nitrogenase resulted in a proposed structure analogous to E4(4H)(c), and additional structures have been proposed in the DFT studies of others. To resolve the nature of hydride binding to the Janus intermediate, we performed exhaustive, high-resolution CW-stochastic 1H-ENDOR experiments using improved instrumentation, Mims 2H ENDOR, and a recently developed pulsed-ENDOR protocol ("PESTRE") to obtain absolute hyperfine interaction signs. These measurements are coupled to DFT structural models through an analytical point-dipole Hamiltonian for the hydride electron-nuclear dipolar coupling to its "anchoring" Fe ions, an approach that overcomes limitations inherent in both experimental interpretation and computational accuracy. The result is the freeze-trapped, lowest-energy Janus intermediate structure, E4(4H)(a).

Original languageEnglish
Pages (from-to)11984-11996
Number of pages13
JournalJournal of the American Chemical Society
Volume141
Issue number30
DOIs
Publication statusPublished - Jul 31 2019

Fingerprint

Nitrogenase
Electron Spin Resonance Spectroscopy
Hydrides
Spectrum Analysis
Spectroscopy
Density functional theory
Anchors
Structural Models
Hamiltonians
Uncertainty
Electrons
Ions
Enzymes
Chemical activation
Hydrogen
Molecules
Experiments

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E4(4H). / Hoeke, Veronika; Tociu, Laura; Case, David A.; Seefeldt, Lance C.; Raugei, Simone; Hoffman, Brian M.

In: Journal of the American Chemical Society, Vol. 141, No. 30, 31.07.2019, p. 11984-11996.

Research output: Contribution to journalArticle

Hoeke, Veronika ; Tociu, Laura ; Case, David A. ; Seefeldt, Lance C. ; Raugei, Simone ; Hoffman, Brian M. / High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E4(4H). In: Journal of the American Chemical Society. 2019 ; Vol. 141, No. 30. pp. 11984-11996.
@article{ab75e49e2d284f51aa1c66397c337a45,
title = "High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E4(4H)",
abstract = "We have shown that the key state in N2 reduction to two NH3 molecules by the enzyme nitrogenase is E4(4H), the {"}Janus{"} intermediate, which has accumulated four [e-/H+] and is poised to undergo reductive elimination of H2 coupled to N2 binding and activation. Initial 1H and 95Mo ENDOR studies of freeze-trapped E4(4H) revealed that the catalytic multimetallic cluster (FeMo-co) binds two Fe-bridging hydrides, [Fe-H-Fe]. However, the analysis failed to provide a satisfactory picture of the relative spatial relationships of the two [Fe-H-Fe]. Our recent density functional theory (DFT) study yielded a lowest-energy form, denoted as E4(4H)(a), with two parallel Fe-H-Fe planes bridging pairs of {"}anchor{"} Fe on the Fe2,3,6,7 face of FeMo-co. However, the relative energies of structures E4(4H)(b), with one bridging and one terminal hydride, and E4(4H)(c), with one pair of anchor Fe supporting two bridging hydrides, were not beyond the uncertainties in the calculation. Moreover, a structure of V-dependent nitrogenase resulted in a proposed structure analogous to E4(4H)(c), and additional structures have been proposed in the DFT studies of others. To resolve the nature of hydride binding to the Janus intermediate, we performed exhaustive, high-resolution CW-stochastic 1H-ENDOR experiments using improved instrumentation, Mims 2H ENDOR, and a recently developed pulsed-ENDOR protocol ({"}PESTRE{"}) to obtain absolute hyperfine interaction signs. These measurements are coupled to DFT structural models through an analytical point-dipole Hamiltonian for the hydride electron-nuclear dipolar coupling to its {"}anchoring{"} Fe ions, an approach that overcomes limitations inherent in both experimental interpretation and computational accuracy. The result is the freeze-trapped, lowest-energy Janus intermediate structure, E4(4H)(a).",
author = "Veronika Hoeke and Laura Tociu and Case, {David A.} and Seefeldt, {Lance C.} and Simone Raugei and Hoffman, {Brian M.}",
year = "2019",
month = "7",
day = "31",
doi = "10.1021/jacs.9b04474",
language = "English",
volume = "141",
pages = "11984--11996",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "30",

}

TY - JOUR

T1 - High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E4(4H)

AU - Hoeke, Veronika

AU - Tociu, Laura

AU - Case, David A.

AU - Seefeldt, Lance C.

AU - Raugei, Simone

AU - Hoffman, Brian M.

PY - 2019/7/31

Y1 - 2019/7/31

N2 - We have shown that the key state in N2 reduction to two NH3 molecules by the enzyme nitrogenase is E4(4H), the "Janus" intermediate, which has accumulated four [e-/H+] and is poised to undergo reductive elimination of H2 coupled to N2 binding and activation. Initial 1H and 95Mo ENDOR studies of freeze-trapped E4(4H) revealed that the catalytic multimetallic cluster (FeMo-co) binds two Fe-bridging hydrides, [Fe-H-Fe]. However, the analysis failed to provide a satisfactory picture of the relative spatial relationships of the two [Fe-H-Fe]. Our recent density functional theory (DFT) study yielded a lowest-energy form, denoted as E4(4H)(a), with two parallel Fe-H-Fe planes bridging pairs of "anchor" Fe on the Fe2,3,6,7 face of FeMo-co. However, the relative energies of structures E4(4H)(b), with one bridging and one terminal hydride, and E4(4H)(c), with one pair of anchor Fe supporting two bridging hydrides, were not beyond the uncertainties in the calculation. Moreover, a structure of V-dependent nitrogenase resulted in a proposed structure analogous to E4(4H)(c), and additional structures have been proposed in the DFT studies of others. To resolve the nature of hydride binding to the Janus intermediate, we performed exhaustive, high-resolution CW-stochastic 1H-ENDOR experiments using improved instrumentation, Mims 2H ENDOR, and a recently developed pulsed-ENDOR protocol ("PESTRE") to obtain absolute hyperfine interaction signs. These measurements are coupled to DFT structural models through an analytical point-dipole Hamiltonian for the hydride electron-nuclear dipolar coupling to its "anchoring" Fe ions, an approach that overcomes limitations inherent in both experimental interpretation and computational accuracy. The result is the freeze-trapped, lowest-energy Janus intermediate structure, E4(4H)(a).

AB - We have shown that the key state in N2 reduction to two NH3 molecules by the enzyme nitrogenase is E4(4H), the "Janus" intermediate, which has accumulated four [e-/H+] and is poised to undergo reductive elimination of H2 coupled to N2 binding and activation. Initial 1H and 95Mo ENDOR studies of freeze-trapped E4(4H) revealed that the catalytic multimetallic cluster (FeMo-co) binds two Fe-bridging hydrides, [Fe-H-Fe]. However, the analysis failed to provide a satisfactory picture of the relative spatial relationships of the two [Fe-H-Fe]. Our recent density functional theory (DFT) study yielded a lowest-energy form, denoted as E4(4H)(a), with two parallel Fe-H-Fe planes bridging pairs of "anchor" Fe on the Fe2,3,6,7 face of FeMo-co. However, the relative energies of structures E4(4H)(b), with one bridging and one terminal hydride, and E4(4H)(c), with one pair of anchor Fe supporting two bridging hydrides, were not beyond the uncertainties in the calculation. Moreover, a structure of V-dependent nitrogenase resulted in a proposed structure analogous to E4(4H)(c), and additional structures have been proposed in the DFT studies of others. To resolve the nature of hydride binding to the Janus intermediate, we performed exhaustive, high-resolution CW-stochastic 1H-ENDOR experiments using improved instrumentation, Mims 2H ENDOR, and a recently developed pulsed-ENDOR protocol ("PESTRE") to obtain absolute hyperfine interaction signs. These measurements are coupled to DFT structural models through an analytical point-dipole Hamiltonian for the hydride electron-nuclear dipolar coupling to its "anchoring" Fe ions, an approach that overcomes limitations inherent in both experimental interpretation and computational accuracy. The result is the freeze-trapped, lowest-energy Janus intermediate structure, E4(4H)(a).

UR - http://www.scopus.com/inward/record.url?scp=85070787882&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85070787882&partnerID=8YFLogxK

U2 - 10.1021/jacs.9b04474

DO - 10.1021/jacs.9b04474

M3 - Article

VL - 141

SP - 11984

EP - 11996

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 30

ER -