Homoprotocatechuate 2,3-dioxygenase (HPCD) is a member of the extradiol dioxygenase family of non-heme iron enzymes. These enzymes catalyze the ring-cleavage step in the aromatic degradation pathway commonly found in soil bacteria. In this study, isothermal titration calorimetry (ITC) is used to measure the equilibrium constant (K = 1.1 ± 0.6 × 10(6)) and enthalpy change (ΔH = -17.0 ± 1.7 kcal/mol) associated with homoprotocatechuate binding to HPCD. The ITC data are consistent with the release of approximately 2.6 protons upon binding of the substrate to HPCD. These results raise new questions regarding the relationships between substrate, protein, and the oxygen activation mechanism for this class of non-heme metalloenzymes.