Effect of spin state on reduction of cytochrome P-450 ( P-450 c21) from bovine adrenocortical microsomes

Abstract

The effect of spin state on cytochrome P-450 reduction was studied with a reconstituted system consisting of P-450 c21 and NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) purified from bovine adrenocortical microsomes. The absolute high spin contents of substrate-free, progesterone-bound and 17α-hydroxyprogesterone-bound P-450 c21 were estimated from the analysis of thermally induced difference spectra to be 25, 78 and 94% at 25°C, respectively, in 50 mM potassium phosphate buffer (pH 7.2) containing 20% glycerol, 0.1 mM EDTA and 0.5% Emulgen 913. The effect of the high spin content on P-450 c21 reduction by NADPH in the reconstituted system was analyzed by a steady-state method and by a stopped-flow method at 25°C. The steady-state results showed that the rate of P-450 c21 reduction was not affected by the high spin content of substrate-bound P-450 c21 but was very slow without a steroid substrate. Biphasic reduction of P450 c21 containing two first-order processes was observed in the stopped-flow experiment in the presence of either of the steroid substrates, but the reduction was very slow without the substrate. There were no significant differences in the rate and the amount of the fast phase of reduction between 17α-hydroxyprogesterone-bound and progesterone-bound P-450 c21. Both kinetic studies indicate that the spin state does not control the electron transfer from NADPH to P-450 c21 via NADPH-cytochrome P-450 reductase but the presence of substrate is essential for the reduction of P-450 c21.