THERMODYNAMIC ASPECTS OF THE CO-BINDING REACTION TO CYTOCHROME P-450(CAM) - RELEVANCE WITH THEIR BIOLOGICAL SIGNIFICANCE AND STRUCTURE

The CO-binding kinetics of cytochrome P-450(cam)(+) and P-450(cam)(-) have been measured in the millisecond time domain using a flash photol ysis method. We have determined the reaction coordinates for free ener gy, enthalpy and entropy from the temperature dependence of the overal l rate constants of the bimolecular forward (on) and backward (off) re actions. Comparing the thermodynamic profiles of P-450(cam) with that of myoglobin (Mb) reported so far, the enthalpy and the entropy coordi nates exhibit the following remarkable characteristics. The CO-binding equilibrium: The stability of the CO-complex is perfectly entropy-dri ven for P-450(cam) enthalpy-driven for Mb. This entropy-driven feature for P-450(cam) is enhanced by the dissociating d-camphor. The on and off activation processes: The on and off reactions for P-450(cam) are dominantly controlled by the enthalpy and entropy terms, respectively, while those for Mb are rather the reverse of the case of P-450(cam). The dissociation of d-camphor has a significant effect on the on react ion but no effect on the off reaction. Analyzing these thermodynamic f eatures on the basis of the physical chemistry in the solution reactio n, it was found that these characteristic profiles arise from the diff erence in the global structural change between the proteins. Namely, d uring the equilibrium process of the CO binding, this structural chang e is accompanied by a larger increase in the degree of freedom in P-45 0(cam) than in Mb. We discussed the correlations between the structura l changes and their biological significance.