Studies on P-450: VIII. Effects of Steroids on the Reaction with Oxygen and the Rate of Reduction of Partially Purified P-45) from Adrenocortical Mitochondria*

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Abstract
The effects of steroids on the reaction of P-450 with oxygen and on the rate of reduction of P-450 were studied using a reconstituted system for steroid 11β-hydroxylation. 1. Oxidation of NADPH by the reconstituted system was measured at various oxygen concentrations, obtained using a glucose oxidase-catalase system as a deoxygenating agent. The results showed that addition of deoxycorticosterone increased the maximal reaction velocity but did not affect the affinity of oxygen for the reconstituted system. The Km value was estimated to be approximately 9×10−6M. High concentrations of oxygen inhibited the oxidation of NADPH by the reconstituted system. 2. The initial rates of chemical and enzymatic reduction of P-450 were accelerated by deoxycorticosterone and decreased by pregnenolone. The reduction did not follow first order kinetics but was essentially biphasic with an initial, rapid phase and a later, slow phase. Greater reduction occurred during the rapid phase when deoxycorticosterone was present in the medium and when the concentration of residual oxygen was low. 3. The reduced forms of P-450 had different absorption spectra in the presence and absence of deoxycorticosterone, giving a “substrate difference spectrum of reduced P-450.” 4. Based on these observations, possible mechanisms of the oxidation-reduction cycle of P-450 during catalysis were discussed.

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