EPR spectroscopy reveals glycerol-dependent activation of cysteamine dioxygenase (ADO) enables bidentate substrate coordination

In mammals, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO) are the only known thiol dioxygenases (TDOs). These enzymes employ a mononuclear non-heme iron active site to catalyze the O 2 -dependent oxidation of L-cysteine (CYS) and cysteamine (CA), producing cysteine sulfinic acid (CSA) and hypotaurine (HT), respectively. Notably, ADO also exhibits N-terminal cysteinyl dioxygenase (NCO) activity, oxidizing protein N-terminal cysteine (N t -CYS) residues to initiate N-end rule degradation of G-protein regulators. Thus, ADO functions both as a small-molecule thiol dioxygenase and as an O 2 -dependent regulator of protein stability. Although TDO substrates typically bind the ferrous iron active site in a bidentate manner, the nature of substrate coordination at the ADO Fe-site is debated. Here, we show that molecular crowding by glycerol shifts the CA binding equilibrium toward bidentate coordination at the ADO Fe site, matching the coordination observed for the N t -CYS RGS5 polypeptide. Continuous-wave and pulsed EPR spectroscopy (ESEEM) show that glycerol occupies the outer coordination sphere of the Fe-site and stabilizes bidentate CA binding via a thiolate and neutral amine. Concomitant with this shift toward bidentate substrate coordination is a ∼7-fold increase in catalytic efficiency, suggesting that this coordination environment represents the catalytically relevant O 2 -activating enzyme–substrate complex. Supporting spectroscopic measurements (CD and Mössbauer) and computational DFT models are consistent with enzymatic activation being triggered by a switch in substrate-binding denticity rather than by changes in protein secondary structure. Together, these results identify glycerol as a conformational activator of ADO and resolve the ambiguous nature of substrate-binding denticity at the enzymatic Fe-site.