Temperature Dependence of Cholesterol Binding to Cytochrome P-450,,, of the Rat Adrenal

A type I absorbance change is observed in suspensions of adrenal cortical mitochondria as the temperature is increased from O-22”. This “heat-generated” type I absorbance change is similar in magnitude to the pregnenolone-induced type II absorbance change of these mitochondria. Studies with inhibitors of cholesterol side chain cleavage indicate that the heat-generated type I absorbance change represents the specific interaction of cytochrome P-454,, with endogenous cholesterol in the mitochondria. This finding is confirmed by low temperature EPR spectroscopy on temperatureequilibrated, quick frozen adrenal mitochondrial samples. The EPR resonance at g = 8.2, which is that of the high spin cholesterol-bound cytochrome P-450,,,, is absent in the samples incubated at 0 and increases in magnitude with increasing temperature of incubation. Studies of the pH dependence of the heat-generated type I and pregnenolone-induced type II absorbance changes reveal that both are diminished by increasing pH over the range 6 to 8. Adrenocorticotropic hormone (ACTH) treatment of rats results in adrenal mitochondria which show a greatly increased heat-generated type I absorbance change. The latter correlates with an increased pregnenolone-induced type II absorbance change and increased EPR g = 8.2 signal. Prior treatment of animals with cycloheximide eliminated the ACTH-induced increase in the heat-generated type I absorbance change, the pregnenolone-induced type II absorbance change and the EPR g = 8.2 signal. We estimate that the hydrophobic bonding of cholesterol to cytochrome P-450,,, occurs with a MO’ of approximately + 15 kcallmol and a AS”’ of approximately +55 cal/mol deg. Our data support the concept of a labile protein which participates directly in this process.