Inverse Solvent Isotope Effects Arising from Substrate Triggering in the Factor Inhibiting Hypoxia Inducible Factor

Oxygen homeostasis plays a critical role in angiogenesis, erythropoiesis, and cell metabolism. Oxygen homeostasis is set by the hypoxia inducible factor-1α (HIF-1α) pathway, which is controlled by factor inhibiting HIF-1α (FIH). FIH is a non-heme Fe­(II), α-ketoglutarate (αKG)-dependent dioxygenase that inhibits HIF-1α by hydroxylating the C-terminal transactivation domain (CTAD) of HIF-1α at HIF-Asn⁸⁰³. A tight coupling between CTAD binding and O₂ activation is essential for hypoxia sensing, making changes in the coordination geometry of Fe­(II) upon CTAD encounter a crucial feature of this enzyme. Although the consensus chemical mechanism for FIH proposes that CTAD binding triggers O₂ activation by causing the Fe­(II) cofactor to release an aquo ligand, experimental evidence of this has been absent. More broadly, this proposed coordination change at Fe­(II) has not been observed during steady-state turnover in any αKG oxygenase to date. In this work, solvent isotope effects (SIEs) were used as a direct mechanistic probe of substrate-triggered aquo release in FIH, as inverse SIEs (SIE < 1) are signatures for pre-equilibrium aquo release from metal ions. Our mechanistic studies of FIH have revealed inverse solvent isotope effects in the steady-state rate constants at limiting concentrations of CTAD or αKG [^D₂Ok_cat/K_M(CTAD) = 0.40 ± 0.07, and ^D₂Ok_cat/K_M(αKG) = 0.32 ± 0.08], providing direct evidence of aquo release during steady-state turnover. Furthermore, the SIE at saturating concentrations of CTAD and αKG was inverse (^D₂Ok_cat = 0.51 ± 0.07), indicating that aquo release occurs after CTAD binds. The inverse kinetic SIEs observed in the steady state for FIH can be explained by a strong Fe–OH₂ bond. The stable Fe–OH₂ bond plays an important part in FIH’s regulatory role over O₂ homeostasis in humans and points toward a strategy for tightly coupling O₂ activation with CTAD hydroxylation that relies on substrate triggering.