The Rate-Limiting Step of O(2) Activation in the α-Ketoglutarate Oxygenase Factor Inhibiting Hypoxia Inducible Factor

[Image: see text] Factor inhibiting HIF (FIH) is a cellular O(2)-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 O(2) activation step ( J. A. Hangasky, E. Saban, and M. J. Knapp (2013) Biochemistry52, 1594−160223351038). Here we characterize the rate-limiting step for O(2) activation by FIH using a suite of mechanistic probes on the second order rate constant k(cat)/K(M(O(2))). Steady-state kinetics showed that the rate constant for O(2) activation was slow (k(cat)/K(M(O(2)))(app) = 3500 M(–1) s(–1)) compared with other non-heme iron oxygenases, and solvent viscosity assays further excluded diffusional encounter with O(2) from being rate limiting on k(cat)/K(M(O(2))). Competitive oxygen-18 kinetic isotope effect measurements ((18)k(cat)/K(M(O(2))) = 1.0114(5)) indicated that the transition state for O(2) 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 O(2)(—) 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 O(2) activation among the broad family of αKG oxygenases.