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-1 alpha (HIF-1 alpha) pathway, which is controlled by factor inhibiting HIF-1 alpha (FIH). FIB is a non-heme alpha-ketoglutarate (alpha KG)-dependent dioxygenase that inhibits HIF-1 alpha by hydroxylating the C-terminal transactivation domain (CTAD) of HIF-1 alpha at HIF-Asn(803). A tight coupling between CTAD binding and O-2 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 O-2 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 aKG 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 aKG [(D2O)k(cat)/K-M(CTAD) = 0.40 +/- 0.07, and (D2O)k(cat)/K-M(alpha KG) = 0.32 +/- 0.08], providing direct evidence of aquo release during steady-state turnover. Furthermore, the SIE at saturating concentrations of CTAD and aKG was inverse ((D2O)k(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 O-2 homeostasis in humans and points toward a strategy for tightly coupling O-2 activation with CTAD hydroxylation that relies on substrate triggering.