Carbon monoxide binding to myoglobin was characterized using the photothermal beam deflection method. The volume and enthalpy changes coupled to CO dissociation were found to be 9.3±0.8 mL mol-1 and 7.4±2.8 kcal mol-1, respectively. The corresponding values observed for CO rebinding have the same magnitude but opposite sign: ΔV= -8.6±0.9 mL mol-1 and ΔH= -5.8±5.9 kcal mol-1. Ligand rebinding occurs as a single conformational step with a rate constant of 5×10 M-1 s-1 and with activation enthalpy of 7.1±0.8 kcal mol-1 and activation entropy of -22.4±2.8 cal mol-1 K-1. Activation parameters for the ligand binding correspond to the activation parameters previously obtained using the transient absorption methods. Hence, at room temperature the CO binding to Mb can be described as a two-state model and the observed volume contraction occurs during CO-Fe bond formation. Comparing these results with CO dissociation reactions, for which two discrete intermediates were characterized, indicates differences in mechanism by which the protein modulates ligand association and dissociation.
