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