Substrate-induced changes in visible absorption and electron spin resonance properties of adrenal cortex mitochondrial P450.

The effect of steroid substrate addition on the visible absorption and electron spin resonance (ESR) properties of cytochrome P450 was studied in bovine adrenal cortex submitochondrial particles. The interaction of ll-deoxycortisol or deoxycorticosterone produced peaks at 385, 500, and 645 nm and troughs at 420, 535, and 570 run. 2OarHydroxycholesterol produced opposite spectral effects. llDeoxycortisol or deoxycorticosterone decreased and 20ahydroxycholesterol increased the g = 2.42, 2.26, and 1.91 low spin signals of P450. The spectral dissociation constants for the visible absorption changes, ESR changes, and Km values for these substrates were similar. A g = 7.9 ESR signal was found in these submitochondrial particles. The height of this signal was increased by deoxycorticosterone and decreased by 20cr-hydroxycholesterol interaction. The g = 7.9 signal was not altered by 9 mu cyanide or 100 mM fluoride but was abolished by deoxycholate conversion of P450 to P420 and by dithionite reduction. The substrateinduced changes in the visible absorption and ESR properties of P450 are consistent with a deoxycorticosteroneor lldeoxycortisol-induced low to high spin transition and a 20ac-hydroxycholesterol high to low spin transition of the Fe3+ in P450. The disappearance of the g = 7.9 signal with deoxycholate treatment and the inverse relationship between the steroid-induced changes in the g = 7.9 and the low spin signals suggest that the g = 7.9 signal is a high spin P450. Since all steroid substrates of P450, with the exception of 20ac-hydroxycholesterol, produce low to high spin spectral changes, this electronic effect may be important in the enzyme reaction mechanism. Cholesterol was found to inhibit competitively the 20ar-hydroxycholesterol-induced difference spectrum, indicating that 2Oarand 22R-hydroxylations occur at the same enzyme site. The exceptional electronic effects of 20a-hydroxycholesterol may be explained by its role as an enzyme-bound intermediate of cholesterol oxidation or by its ability to displace cholesterol from cholesterol oxidase.