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 micro M 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 microM 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 microM 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.
Hanukoglu
I
I
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Rapoport
R
R
Weiner
L
L
Sklan
D
D
D000302:Adrenal Cortex / Q000201:enzymology; D000325:Adrenodoxin / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D004583:Electrons; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
D005609:Free Radicals; D013481:Superoxides; D000325:Adrenodoxin; D009249:NADP; D002784:Cholesterol; D006861:Hydrogen Peroxide; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase; D010100:Oxygen; D002118:Calcium