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 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.

Altered specificity of 15-LOX-1 in the biosynthesis of 7S,14S-diHDHA implicates 15-LOX-2 in biosynthesis of resolvin D5

“…In order to obtain a better understanding of this binding event, DHA and 7S-HDHA were docked to a model of h15-LOX-1. In the case of DHA, both C12 and C15 are at a reasonable distance for hydrogen abstraction (65), whereas in case of 7S-HDHA, C12 is closer to the metal ion than C15, supporting the experimental results ( Table 9). The model explains this result by having the hydroxyl group at C7 in 7S-HDHA forming a hydrogen bond with the backbone carbonyl of I399, which subsequently positions C12 closer to the metal ion.…”

EPR Spectroscopic Studies of Lipoxygenases

“…ENDOR is atechnique that couldc ontribute understanding to the molecular origin of variations in ferric lipoxygenase spectra. [28] The specificity of redox reactions at lipoxygenase iron has been examined by EPR. The initial oxidation from ferrous to ferric consumes one equivalent of the enzymatic hydroperoxide product, but other long carbon chain secondary hydroperoxidesa re reactive too.…”

“…[11,43] andt he bend occurs close to the catalytic iron atom. Extensive quantum-chemical calculations have been directed to understanding [28,44,45] the isotope effect in SBL1 and, in particular,w hether the required donoracceptord istance (substrate C 11 to Oo fm etal coordinated water)f or hydrogen tunneling ( % 2.7 )c an be met. The relevant lipoxygenase crystal structures with as ubstrate, or substrate-like, fatty acid bound suggest donor-acceptor distances significantly longerthan this, 3.4 [9] and 3.7 [23] ,r espectively.…”

“…ENDOR measurements and quantum calculations addressing proton-coupled electron transferi nS BL1r evealed two donoracceptord istances in the frozen-solution structure of linoleic acid in complexw ith am anganese-substituted SBL1. [28] The linoleic acid carbon from whichh ydrogen is abstracted in forming 13-HPOD is C11. Both linoleic acid C10a nd,s eparately C11, were substituted with 13 Cf or the measurements.…”

“…Calculation of 50 ns MD trajectories, based on the ENDOR-derived distances, gave 0.2 %o ft he framesr eaching ad istance 2.9 ,s uggesting that the required distance for hydrogen transfer can be attained by conformational sampling. [28]…”