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

-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.