The Soybean Lipoxygenase–Substrate Complex: Correlation between the Properties of Tunneling-Ready States and ENDOR-Detected Structures of Ground States

Hydrogen tunneling in enzymatic C–H activation requires a dynamical sampling among ground-state enzyme–substrate (E–S) conformations, which transiently generates a tunneling-ready state (TRS). The TRS is characterized by a hydrogen donor–acceptor distance (DAD) of 2.7 A, ∼0.5 A shorter than the dominant DAD of optimized ground states. Recently, a high-resolution, 13C electron–nuclear double-resonance (ENDOR) approach was developed to characterize the ground-state structure of the complex of the linoleic acid (LA) substrate with soybean lipoxygenase (SLO). The resulting enzyme–substrate model revealed two ground-state conformers with different distances between the target C11 of LA and the catalytically active cofactor [Fe(III)–OH]: the active conformer “a”, with a van der Waals DAD of 3.1 A between C11 and metal-bound hydroxide, and an inactive conformer “b”, with a distance that is almost 1 A longer. Herein, the structure of the E–S complex is examined for a series of six variants in which subtle structu...