Abstract

Herein, we present photoacoustic calorimetry and transient absorption studies of the dynamics and energetics associated with dissociation of a ligand from Fe(2+) dehaloperoxidase (DHP) from Amphitrite ornata. Our data show that CO photodissociation is associated with an endothermic (DeltaH = 8 +/- 3 kcal mol(-1)) volume expansion (DeltaV = 9.4 +/- 0.6 mL mol(-1)) that occurs within 50 ns upon photodissociation. No additional thermodynamics were detected on slower time scales (up to 10 micros), suggesting that the dissociated ligand rapidly escapes from the heme-binding pocket into the surrounding solvent. Similar volume and enthalpy changes were observed for CO photodissociation in the presence of the substrate, 2,4-dichlorophenol or 4-bromophenol, indicating that either the substrate does not bind in the protein distal cavity at ambient temperature or its presence does not impact the thermodynamic profile associated with ligand dissociation. We attribute a fast ligand exchange between the protein active site and the surrounding solvent to the high flexibility of the distal histidine residue, His55, that provides a direct pathway between the heme-binding pocket and the protein exterior. The dynamics and energetics of conformational changes observed for dissociation of a ligand from DHP differ significantly from those measured previously for photodissociation of CO from the structural homologue myoglobin, suggesting that structural dynamics in DHP are fine-tuned to enhance the peroxidase function of this protein.