Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O₂) concentrations. Here, we have reprogrammed cellular hypoxia (low O₂) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O₂ sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O₂ to PHD2's catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2's gas tunnel. Using kinetic stopped-flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5-fold enhancement in its association rate to the iron center of tunnel-engineered mutants. Our most effectively designed mutant displays 9-fold enhanced catalytic efficiency (k_cat/K_M=830±40 M^-1 s^-1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 (k_cat/K_M=90±9 M^-1 s^-1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK-293T mammalian cells reveal significant reduction of HIF-1α and downstream hypoxia response transcripts under hypoxic conditions of 1 % O₂. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.