title: 13C ENDOR Spectroscopy of Lipoxygenase-Substrate Complexes Reveals the Structural Basis for C-H Activation by Tunneling.
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 of13C10 and reactive13C11 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.
authors.affiliation: Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Masaki
authors.identifier: 0000-0002-8881-0600
authors.initials: M
authors.lastname: Horitani
authors.forename: Adam R
authors.initials: AR
authors.lastname: Offenbacher
authors.forename: Cody A Marcus
authors.initials: CA
authors.lastname: Carr
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Tao
authors.initials: T
authors.lastname: Yu
authors.affiliation: Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Veronika
authors.initials: V
authors.lastname: Hoeke
authors.affiliation: Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: George E
authors.identifier: 0000-0002-7378-9474
authors.initials: GE
authors.lastname: Cutsail
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Sharon
authors.identifier: 0000-0002-3782-6995
authors.initials: S
authors.lastname: Hammes-Schiffer
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
authors.affiliation: Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Brian M
authors.identifier: 0000-0002-3100-0746
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D002247:Carbon Isotopes; D018360:Crystallography, X-Ray; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D013379:Substrate Specificity
chemical_list: D002247:Carbon Isotopes; D006859:Hydrogen; D008084:Lipoxygenase