Kinetic Studies of the Hydrogen Atom Transfer in a Hypoxia-Sensing Enzyme, FIH-1: KIE and O₂ Reactivity

Cellular hypoxia plays a crucial role in tissue development and adaptation to pO₂. Central to cellular oxygen sensing is factor-inhibiting HIF-1α (FIH), an α-ketoglutarate (αKG)/non-heme iron(II)-dependent dioxygenase that hydroxylates a specific asparagine residue of hypoxia inducible factor-1α (HIF-1α). The high K_M(O and rate-limiting decarboxylation step upon O₂ activation are key features of the enzyme that classify it as an oxygen sensor and set it apart from other αKG/Fe(II)-dependent dioxygenases. Although the chemical intermediates following decarboxylation are presumed to follow the consensus mechanism of other αKG/Fe(II)-dependent dioxygenases, experiments have not previously demonstrated these canonical steps in FIH. In this work, a deuterated peptide substrate was used as a mechanistic probe for the canonical hydrogen atom transfer (HAT). Our data show a large kinetic isotope effect (KIE) in steady-state kinetics (^Dk_cat = 10 ± 1), revealing that the HAT occurs and is partially rate limiting on k_cat. Kinetic studies showed that the deuterated peptide led FIH to uncouple O₂ activation and provided the opportunity to spectroscopically observe the ferryl intermediate. This enzyme uncoupling was used as an internal competition with respect to the fate of the ferryl intermediate, demonstrating a large observed KIE on the uncoupling (^Dk₅ = 1.147 ± 0.005) and an intrinsic KIE on the HAT step (^Dk > 15). The close energy barrier between αKG decarboxylation and HAT distinguishes FIH as an O₂-sensing enzyme and is crucial for ensuring substrate specificity in the regulation of cellular O₂ homeostasis.