Electron Leakage from the Mitochondrial NADPH-Adrenodoxin Reductase-Adrenodoxin-P450scc (Cholesterol Side Chain Cleavage) System

In electron (e−) transfer systems some e− may "leak," reducing O2 to a superoxide radical. This study examined the sites and kinetics of e− leakage from the mitochondrial P450scc system. Adrenodoxin reductase alone oxidized NADPH, reducing O2 to a superoxide radical at a very low rate. However, the reductase-adrenodoxin system reduced O2 at a rapid steady-state rate with Michaelis- Menten dependence on [adrenodoxin](Vmax = 3.5 μM e−/min). After depletion of NADPH, reduced adrenodoxin was oxidized (autooxidation) with pseudo first order kinetics and the rate of e− transfer decreased 10-fold. Ca2+ (<1 mM) stimulated e− leakage in both phases. The reductase-adrenodoxin-P450scc system exhibited the highest rate of leakage (Vmax = 7.8 μM e−/min). At low [adrenodoxin] the majority of e− leaked through P450scc and not through adrenodoxin. In the presence of the substrate, cholesterol, leakage drastically decreased to <0.5 μM e−/min. These results indicate that the mitochondrial P450 systems can leak e−, producing O2 derived free radicals. Reduction of leakage during P450scc conversion of cholesterol to pregnenolone provides a clue to understanding physiological mechanisms that control e− leakage. These may include coregulation of NADPH and cholesterol availability to the P450scc system and a system of antioxidants for quenching the oxygen radicals.