Kinetics of O2 and CO Binding to adrenal cytochrome P-450scc. Effect of cholesterol, intermediates, and phosphatidylcholine vesicles.

The kinetics of O2 and CO binding to purified adrenal cytochrome P-450scc has been measured at 25 degrees C by stopped flow spectrophotometry. Measurements were made on the cytochrome in both aqueous buffer and phosphatidylcholine vesicles; little difference in the kinetic constants between the two was observed. 22R-Hydroxycholesterol and 20 alpha,22R-dihydroxycholesterol, intermediates in the cytochrome P-450scc-catalyzed conversion of cholesterol to pregnenolone, markedly influenced rates and equilibria of O2 and CO binding. Binding of the intermediates greatly decreased the association rate of both O2 (80-160-fold) and CO (30-80-fold) relative to cholesterol-bound cytochrome. They had opposite effects on O2 and CO dissociation rates, causing a 5-fold increase in the CO dissociation rate relative to cholesterol and a 150-900-fold decrease in the O2 dissociation rate. Kd values for CO, determined by titration of the reduced cytochrome with CO, increased from 0.3 microM for substrate-depleted cytochrome to 0.7-1.0 microM, 100 microM, and 213-244 microM, respectively, for cholesterol, 22R-hydroxycholesterol, and 20 alpha,22R-dihydroxycholesterol. Conversely, Kd values for O2, determined from the ratio of dissociation and association rate constants, decreased from 23 microM for cholesterol-bound cytochrome to 12 microM and 5 microM for 22R-hydroxycholesterol and 20 alpha,22R-hydroxycholesterol, respectively, indicating a thermodynamic stabilization of the Fe2+ . O2 complex by these intermediates.