Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450sccin 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 com ponent for placental cytochrome P450scc activity is the concentration of ad renodoxin reductase in the mitochondria, where it permits cytochrome P450sc c to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce adrenodoxin as part of the electron transfer from NADPH to cytochrom e P450scc. We therefore measured the proportion of adrenodoxin in the reduc ed form in intact mitochondria from the human placenta during active pregne nolone synthesis, using EPR. We found that the adrenodoxin pool was only 30 % reduced, indicating that the adrenodoxin reductase concentration was insu fficient to maintain the adrenodoxin in the fully reduced state. As both ox idized and reduced adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized adrenodoxin to act as a competitive inhibitor of pregn enolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted syst em comprising submitochondrial particles, purified adrenodoxin and adrenodo xin reductase. We found that oxidized adrenodoxin is an effective competiti ve inhibitor of placental cytochrome P450scc with a K-i value half that of the K-m for reduced adrenodoxin. We conclude that the limiting concentratio n of adrenodoxin reductase present in placental mitochondria has a two-fold effect on cytochrome P450scc activity. It limits the amount of reduced adr enodoxin that is available to donate electrons to cytochrome P450scc and th e oxidized adrenodoxin that remains, competitively inhibits the cytochrome.