Electron leakage from the adrenal cortex mitochondrial P450scc and P450c11 systems: NADPH and steroid dependence

In the present study we examined the coupling of NADPH oxidation to substrate hydroxylation and the effects of steroids on this process in reconstituted P450scc and P450c11 systems. To determine the relative rates of substrate hydroxylation vs electron leakage we assayed both the steroid product and H₂O₂ in the same sample. For both P450 systems the rates of steroid product and superoxide formation increased as NADPH concentration was increased, However, P450c11 was found to be more leaky. The leakage from the P450scc system was not affected by pregnenolone, the product of cholesterol side chain cleavage. In contrast, corticosterone, the product of P450c11, increased the rate of futile NADPH oxidation by the P450c11 system. We also tested a series of steroids to analyze the stereospecificity of their effects. Relative to the control without steroid, both C-19 and C-21 steroids with 11α-hydroxy groups (11α-OH-testosterone and 11α-OH-cortisol) decreased leakage, and those with 11β-OH groups (11β-OH-testosterone and cortisol) stimulated both NADPH oxidation and electron leakage as measured by H₂O₂ formation. The results revealed a correlation between the effects previously observed in living cells and in our reconstituted systems. These findings provide further evidence that mitochondrial P450 systems indeed function as a significant source of oxygen radicals in steroidogenic cells.