Photothermal studies of CO photodissociation from peroxidases from horseradish and soybean

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.