Metal Coordination Dynamics Governs Selective Epoxidation in Hyoscyamine 6β-Hydroxylase: Integrated Experimental and Computational Insights

Iron(II)- and 2-oxoglutarate-dependent (Fe(II)/2OG) enzymes catalyze a wide range of C–H bond activation and functionalization reactions and play essential roles in biosynthesis and metabolic regulation. Despite extensive mechanistic studies, the principles governing selectivity between canonical hydroxylation and alternative transformations remain incompletely understood. Here, we investigate the catalytic mechanism of hyoscyamine 6β-hydroxylase (H6H), a Fe(II)/2OG-dependent oxygenase that sequentially catalyzes the 6β-hydroxylation of hyoscyamine followed by 6,7-exo-epoxidation of 6β-hydroxyhyoscyamine to generate scopolamine. Combined molecular dynamics and QM/MM calculations reveal that an inline Fe(IV)-oxo intermediate initiates hydrogen atom abstraction from the substrate C7 position. The resulting Fe(III)–OH species subsequently deprotonates the substrate hydroxyl group in a process coupled to substrate coordination to the iron center and an in-line-to-off-line rearrangement of the Fe(III)–OH moiety. This coordination dynamics machinery is further supported by the observed chlorination reactivity on the same substrate. Importantly, this coordination switch favors epoxide formation over hydroxyl rebound, thereby directing the reaction toward selective epoxidation. Further computational analysis of the L290F variant demonstrates that steric constraints imposed by L290 are essential for suppressing hydroxylation, revealing a bidirectional regulatory mechanism governing epoxidation/hydroxylation selectivity. Whereas iron coordination dynamics promote epoxidation reactivity, precise substrate positioning and protein-derived steric effects suppress the competing hydroxylation pathway. These findings are consistent with available experimental observations and establish metal coordination dynamics as a key determinant of selective C–H functionalization in Fe(II)/2OG enzymes.