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 [^D₂O<i>k</i>_cat/<i>K</i>_M(CTAD) = 0.40 ± 0.07, and ^D₂O<i>k</i>_cat/<i>K</i>_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 (^D₂O<i>k</i>_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

Substrate Positioning by Gln²³⁹ Stimulates Turnover in Factor Inhibiting HIF, an αKG-Dependent Hydroxylase

Nonheme Fe­(II)/αKG-dependent oxygenases catalyze diverse reactions, typically inserting an O atom from O₂ into a C–H bond. Although the key to their catalytic cycle is the fact that binding and positioning of primary substrate precede O₂ activation, the means by which substrate binding stimulates turnover is not well understood. Factor Inhibiting HIF (FIH) is a Fe­(II)/αKG-dependent oxygenase that acts as a cellular oxygen sensor in humans by hydroxylating the target residue Asn⁸⁰³, found in the C-terminal transactivation domain (CTAD) of hypoxia inducible factor-1. FIH-Gln²³⁹ makes two hydrogen bonds with CTAD-Asn⁸⁰³, positioning this target residue over the Fe­(II). We hypothesized the positioning of the side chain of CTAD-Asn⁸⁰³ by FIH-Gln²³⁹ was critical for stimulating O₂ activation and subsequent substrate hydroxylation. The steady-state characterization of five FIH-Gln²³⁹ variants (Ala, Asn, Glu, His, and Leu) tested the role of hydrogen bonding potential and sterics near the target residue. Each variant exhibited a 20–1200-fold decrease in <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_M(CTAD), but no change in <i>K</i>_M(CTAD), indicating that the step after CTAD binding was affected by point mutation. Uncoupled O₂ activation was prominent in these variants, as shown by large coupling ratios (<i>C</i> = [succinate]/[CTAD-OH] = 3–5) for each of the FIH-Gln²³⁹ → X variants. The coupling ratios decreased in D₂O, indicating an isotope-sensitive inactivation for variants, not observed in the wild type. The data presented indicate that the proper positioning of CTAD-Asn⁸⁰³ by FIH-Gln²³⁹ is necessary to suppress uncoupled turnover and to support substrate hydroxylation, suggesting substrate positioning may be crucial for directing O₂ reactivity within the broader class of αKG hydroxylases

The Rate-Limiting Step of O₂ Activation in the α‑Ketoglutarate Oxygenase Factor Inhibiting Hypoxia Inducible Factor

Factor inhibiting HIF (FIH) is a cellular O₂-sensing enzyme, which hydroxylates the hypoxia inducible factor-1α. Previously reported inverse solvent kinetic isotope effects indicated that FIH limits its overall turnover through an O₂ activation step (Hangasky, J. A., Saban, E., and Knapp, M. J. (2013) Biochemistry 52, 1594−1602). Here we characterize the rate-limiting step for O₂ activation by FIH using a suite of mechanistic probes on the second order rate constant <i>k</i>_cat/<i>K</i>_M(O₂). Steady-state kinetics showed that the rate constant for O₂ activation was slow (<i>k</i>_cat/<i>K</i>_M(O₂)^app = 3500 M^–1 s^–1) compared with other non-heme iron oxygenases, and solvent viscosity assays further excluded diffusional encounter with O₂ from being rate limiting on <i>k</i>_cat/<i>K</i>_M(O₂). Competitive oxygen-18 kinetic isotope effect measurements (¹⁸<i>k</i>_cat/<i>K</i>_M(O₂) = 1.0114(5)) indicated that the transition state for O₂ activation resembled a cyclic peroxohemiketal, which precedes the formation of the ferryl intermediate observed in related enzymes. We interpret this data to indicate that FIH limits its overall activity at the point of the nucleophilic attack of Fe-bound O₂^ on the C-2 carbon of αKG. Overall, these results show that FIH follows the consensus mechanism for αKG oxygenases, suggesting that FIH may be an ideal enzyme to directly access steps involved in O₂ activation among the broad family of αKG oxygenases