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13C ENDOR Spectroscopy of Lipoxygenase–Substrate Complexes Reveals the Structural Basis for C–H Activation by Tunneling
journal contribution
posted on 2017-01-25, 14:04 authored by Masaki Horitani, Adam R. Offenbacher, Cody A. Marcus Carr, Tao Yu, Veronika Hoeke, George E. Cutsail, Sharon Hammes-Schiffer, Judith P. Klinman, Brian M. HoffmanIn 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 ENDOR-derived 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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ENDOR-derived restraintshydrogen DADsoybean lipoxygenasesolvated SLO modelreactive tunneling geometriesnonadiabatic tunnelingsite arrangement13 C ENDOR Spectroscopy1 H ENDORnonadiabatic quantum treatmentsX-ray diffractionTunneling-impaired SLO variants showStructural Basisground state substrate-bound structureENDOR probessite architecturenonadiabatic tunneling systemproton-coupled electron transferhydrogen wave function overlapsite ground statehydrogen tunnelingbarrier widthtunneling distancesite reactivityground statevan der Waals distancesreactive 13 C 1113 C 10van der Waals contactlinoleic acidSLO Fe ion13 C ENDORdynamics simulations
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