13C ENDOR spectroscopy of lipoxygenase-substrate complexes reveals the structural basis for C-H activation by tunneling

Masaki Horitani, Adam R. Offenbacher, Cody A. Marcus Carr, Tao Yu, Veronika Hoeke, George E. Cutsail, Sharon Hammes-Schiffer, Judith P. Klinman, Brian M. Hoffman

Research output: Contribution to journalArticle

Abstract

In enzymatic C-H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor-acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn2+ as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. 13C ENDOR then reveals the locations of 13C10 and reactive 13C11 of linoleic acid relative to the metal; 1H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDORderived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of ∼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.

Original languageEnglish (US)
Pages (from-to)1984-1997
Number of pages14
JournalJournal of the American Chemical Society
Volume139
Issue number5
DOIs
StatePublished - Feb 8 2017
Externally publishedYes

Fingerprint

Lipoxygenase
Electron Spin Resonance Spectroscopy
Soybeans
Spectrum Analysis
Hydrogen
soybean
Ground state
Substrates
Catalytic Domain
hydrogen
substrate
Enzymes
Chemical activation
Sampling
Heroin Dependence
probe
enzyme
sampling
Cape Verde
Beta-Globulins

ASJC Scopus subject areas

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

Cite this

Horitani, M., Offenbacher, A. R., Marcus Carr, C. A., Yu, T., Hoeke, V., Cutsail, G. E., ... Hoffman, B. M. (2017). 13C ENDOR spectroscopy of lipoxygenase-substrate complexes reveals the structural basis for C-H activation by tunneling. Journal of the American Chemical Society, 139(5), 1984-1997. DOI: 10.1021/jacs.6b11856

13C ENDOR spectroscopy of lipoxygenase-substrate complexes reveals the structural basis for C-H activation by tunneling. / Horitani, Masaki; Offenbacher, Adam R.; Marcus Carr, Cody A.; Yu, Tao; Hoeke, Veronika; Cutsail, George E.; Hammes-Schiffer, Sharon; Klinman, Judith P.; Hoffman, Brian M.

In: Journal of the American Chemical Society, Vol. 139, No. 5, 08.02.2017, p. 1984-1997.

Research output: Contribution to journalArticle

Horitani, M, Offenbacher, AR, Marcus Carr, CA, Yu, T, Hoeke, V, Cutsail, GE, Hammes-Schiffer, S, Klinman, JP & Hoffman, BM 2017, '13C ENDOR spectroscopy of lipoxygenase-substrate complexes reveals the structural basis for C-H activation by tunneling' Journal of the American Chemical Society, vol 139, no. 5, pp. 1984-1997. DOI: 10.1021/jacs.6b11856
Horitani M, Offenbacher AR, Marcus Carr CA, Yu T, Hoeke V, Cutsail GE et al. 13C ENDOR spectroscopy of lipoxygenase-substrate complexes reveals the structural basis for C-H activation by tunneling. Journal of the American Chemical Society. 2017 Feb 8;139(5):1984-1997. Available from, DOI: 10.1021/jacs.6b11856

Horitani, Masaki; Offenbacher, Adam R.; Marcus Carr, Cody A.; Yu, Tao; Hoeke, Veronika; Cutsail, George E.; Hammes-Schiffer, Sharon; Klinman, Judith P.; Hoffman, Brian M. / 13C ENDOR spectroscopy of lipoxygenase-substrate complexes reveals the structural basis for C-H activation by tunneling.

In: Journal of the American Chemical Society, Vol. 139, No. 5, 08.02.2017, p. 1984-1997.

Research output: Contribution to journalArticle

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abstract = "In enzymatic C-H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor-acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn2+ as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. 13C ENDOR then reveals the locations of 13C10 and reactive 13C11 of linoleic acid relative to the metal; 1H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDORderived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of ∼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.",
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