Abstract In this work, the results of photoacoustic calorimetry (PAC) studies involving CO photodissociation from horseradish peroxidase (HRP) and soybean peroxidase (SBP) are discussed. Both proteins contain Fe-protoporphyrin IX active sites and relatively open distal heme pockets (i.e., direct solvent access). In addition, it has been shown previously that SBP binds a Tris molecule in the distal pocket near the heme group potentially regulating ligand binding to the heme iron. Results of PAC studies indicate a fast (< ∼ 50 ns) relaxation for both HRP and SBP subsequent to CO photolysis in both phosphate and Tris buffers and with varying concentrations of Tris. However, the molar volume/enthalpy changes associated with CO release are distinct between the two proteins. In the case of HRP, CO photolysis results in an enthalpy change of ∼ 2 kcal mol − 1 and volume change of ∼ − 12 mL mol − 1 attributed to solvation/structural changes regardless of buffer conditions. In contrast, SBP exhibits buffer and ionic strength dependent enthalpy changes ranging from ∼ − 23 kcal mol − 1 in 50 mM phosphate buffer to ∼ 6 kcal mol − 1 in Tris buffer with volume changes similar to those observed in HRP. The results are consistent with a model in which photodissociation of CO from ferrous HRP or SBP leads to CO migration from the distal heme pocket to the bulk solvent with a corresponding input of a water molecule all occurring in < ∼ 50 ns. The differences in enthalpies are attributed to variations in hydrogen bond formation between the incoming water molecule(s) and the protein matrix in both HRP and SBP.