Dynamics And Energetics Associated With Ligand Photodissociation From Co Bound Chloroperoxidase

Abstract

Chloroperoxidase (CPO) is the most versatile heme peroxidase that can function as peroxidase, catalase, halogenase/dehalogenase and monooxigenase. The active site of CPO strongly resembles that of cytochrome P450 since the heme iron in both proteins is coordinated with a Cys axial ligand. Two hydrophobic channels that connect the heme binding cavity with the surrounding are likely to provide ligand access into the active site. At alkaline pH the protein undergoes a conformational transition resulting in the population of six coordinated heme iron with the histidine residue in the position of the sixth ligand. Here we report the results of the photoacoustic study of ligand dissociation from CPO. The ligand photodissociation and migration out of the protein matrix occurs within 50 ns and no additional kinetics was determined (detected?) between 50 ns and 5 μs. At acidic pH ligand dissociation is associated with a positive volume change (ΦΔV = 10.9 ± 0.9 mL mol−1) and enthalpy change (ΦΔH = 24 ± 5 kcal mol−1), strongly indicating that the ligand release from the protein matrix does not result in significant changes in protein conformation and CO is likely to migrate through one of the hydrophobic channels. On the other hand, the thermodynamic parameters associated with CO photorelease at pH 10 are significantly smaller than those determined at acidic pH (ΦΔV = 3.2 ± 1.1 mL mol−1 and ΦΔH = -12.1 ± 5.3 kcal mol−1) due to changes in heme coordination upon CO dissociation.