Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta.
The conversion of cholesterol to pregnenolone by cytochrome P450scc is the rate-determining step in placental progesterone synthesis. The limiting component for placental cytochrome P450scc activity is the concentration of adrenodoxin reductase in the mitochondria, where it permits cytochrome P450scc to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce adrenodoxin as part of the electron transfer from NADPH to cytochrome P450scc. We therefore measured the proportion of adrenodoxin in the reduced form in intact mitochondria from the human placenta during active pregnenolone synthesis, using EPR. We found that the adrenodoxin pool was only 30% reduced, indicating that the adrenodoxin reductase concentration was insufficient to maintain the adrenodoxin in the fully reduced state. As both oxidized and reduced adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized adrenodoxin to act as a competitive inhibitor of pregnenolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted system comprising submitochondrial particles, purified adrenodoxin and adrenodoxin reductase. We found that oxidized adrenodoxin is an effective competitive inhibitor of placental cytochrome P450scc with a Ki value half that of the Km for reduced adrenodoxin. We conclude that the limiting concentration of adrenodoxin reductase present in placental mitochondria has a two-fold effect on cytochrome P450scc activity. It limits the amount of reduced adrenodoxin that is available to donate electrons to cytochrome P450scc and the oxidized adrenodoxin that remains, competitively inhibits the cytochrome.
Tuckey
R C
RC
Department of Biochemistry and Department of Chemistry, The University of Western Australia, Nedlands, Australia. rtuckey@cyllene.uwa.edu.au
McKinley
A J
AJ
Headlam
M J
MJ
D000325:Adrenodoxin / Q000378:metabolism*; D001667:Binding, Competitive; D002384:Catalysis; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D055590:Electromagnetic Phenomena; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism*; D011136:Polysorbates / Q000494:pharmacology; D011284:Pregnenolone / Q000096:biosynthesis; D013997:Time Factors
D003902:Detergents; D011136:Polysorbates; D000325:Adrenodoxin; D011284:Pregnenolone; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase; D010100:Oxygen