Mechanistic Insights into Dioxygen Transport Routes in the PHD2 Oxygenase from Long-Time Scale Simulations

Understanding how dioxygen accesses buried catalytic centers in metalloenzymes is critical for elucidating enzymatic kinetics and guiding strategies to modulate catalytic activity. Here, we report over 20 μs of classical molecular dynamics simulations of the PHD2 oxygenase, a metalloenzyme regulating hypoxia signaling via HIF-1α hydroxylation. Our extended simulations reveal multiple dynamic dioxygen transport routes from solvent-exposed regions through the cupin fold to the metal active site, capturing transient interconverting channels and kinetic heterogeneity inaccessible to prior short-time scale studies. Dioxygen transport occurs on widely differing time scales, from rapid exchange (∼250 ps) to long residence times within internal hydrophobic cavities lasting hundreds of nanoseconds. These internal cavities act as dynamic reservoirs, modulating dioxygen availability and potentially contributing to the high K_m and slow oxidative turnover by PHD2. Analysis of cavity-lining residues identifies hydrophobic positions that may be targeted to tune catalytic rates. Collectively, our results refine the mechanistic model of dioxygen access in PHD2 and demonstrate how high-resolution simulations can uncover functionally relevant kinetic landscapes, providing principles applicable to the design and regulation of molecular catalysts.