Abstract
Abstract 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 11-deoxycortisol or deoxycorticosterone produced peaks at 385, 500, and 645 nm and troughs at 420, 535, and 570 nm. 20α-Hydroxycholesterol produced opposite spectral effects. 11-Deoxycortisol or deoxycorticosterone decreased and 20α-hydroxycholesterol 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 20α-hydroxycholesterol interaction. The g = 7.9 signal was not altered by 9 mm cyanide or 100 mm fluoride but was abolished by deoxycholate conversion of P450 to P420 and by dithionite reduction. The substrate-induced changes in the visible absorption and ESR properties of P450 are consistent with a deoxycorticosterone- or 11-deoxycortisol-induced low to high spin transition and a 20α-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 20α-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 20α-hydroxycholesterol-induced difference spectrum, indicating that 20α- and 22R-hydroxylations occur at the same enzyme site. The exceptional electronic effects of 20α-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.
Highlights
Submitochondrial particles were prepared from bovine adrenal cortex as described previously [14]
The S: fraction was the TPNH-cytochrome c reductase activity obtained after Sephadex G-100 gel filtration of the supernatant fraction from sonitally disrupted adrenal cortex mitochondria as described by
Hydroxylation of Substrates by Submitochondrial Particles-Previous experiments have indicated that adrenal cortex submitochondrial particles were capable of ll@hydroxylation
Summary
Preparation of Xubmitochondrkl Particles and Fraction 24’~. Submitochondrial particles were prepared from bovine adrenal cortex as described previously [14]. The S: fraction was the TPNH-cytochrome c reductase activity obtained after Sephadex G-100 gel filtration of the supernatant fraction from sonitally disrupted adrenal cortex mitochondria as described by. Submitochondrial particles and LYz were stored at -60” before use. Incubation Conditions for Determination of K, Values for lipand .%?R-Hydroxylation-Incubations were done at 23’ in Erlenmeyer flasks, which were shaken continuously in a Dubnoff shaker. Each flask contained submitochondrial particles (10.5 mg of protein per ml, 11.5 ELM P450, 3.4 pM P420), S’z having. TPNH-cytcchrome c reductase activity of 1.62 pmoles of cytochrome c reduced per min, 45 mu sodium phosphate buffer For llfi-hydroxylation, ll-deoxycortisol concentration was 2.5 to 22 PM, containing 0.125
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