Thermodynamic Studies of the Protein - Protein Interactions Cytochrome P - 450 and Cytochrome b 5

The interactions between purified rat hepatic m rosomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b6 have been studied in the presence of phospholipid using difference spectrophotometry. Cytochrome bs was shown to interact with cytochrome P-450 to form a tight 1:1 complex (Kd = 275 nM), in which the proportion of high spin cytochrome P-450 was increased from 7 to 30%. The presence of saturating cytochrome bs was shown to cause a decrease in the apparent Kdfor benzphetamine binding from 11 1 p~ to 40 PM. Likewise, the presence of benzphetamine was shown to cause a d crease in the apparent dissociation constant for cytochrome bs binding to cytochrome P-450 ( & = 90 nM). The above interactions were resolved into the basic equilibria inter-relating the various ligation states of the hemoprotein in an energetically closed eight-state free energy coupling model and the relative magnitudes of the microequilibria were analyzed to determine the degree of coupling of the interactions between cytochrome P-450 and both benzphetamine and cytochrome bs. Consequently, the spin state changes in cytochrome P-450 induced by benzphetamine and cytochrome bs binding were shown to arise because these ligands interact 7 and 4 times more tightly with high spin cytochrome P-450, respectively. Furthermore, the data revealed that these ligands interact at independent sites on cytochrome P-450. Thus the effects of cytochrome b5 upon benzphetamine binding and vice versa were rationalized simply in terms of an increase in the proportion of a high spin (high affinity) conformation of cytochrome P-450 brought about by preequilibration with the effector ligand, with the intrinsic binding affinities of the two ligands for the low or high spin states remaining relatively unaltered. The thermodynamic parameters associated with the interactions between cytochrome P-450 and cytochrome bs, determined from the temperature dependence of these interactions, revealed that these protein interactions are entropy driven and probably occur by a hydrophobic mechanism.