Mechanisms of lipid peroxidation dependent upon cytochrome P-450 LM2.

A mechanism of lipid peroxidation dependent on the oxidase activity of cytochrome P-450 LM2 in reconstituted membrane vesicles has been investigated. The rate of lipid peroxidation, determined as the formation of thiobarbituric-acid-reactive substances, was inhibited by CO. It increased concomitantly to the production of O-2 and H2O2, when cytochrome P-450 LM2 was incorporated into vesicles containing NADPH-cytochrome-P-450 reductase, until a 1:1 molar ratio between the enzymes was reached. Also the formation of lipid hydroperoxides was dependent on the presence of cytochrome P-450 LM2 in the membranes. This lipid peroxidation was not inhibited by hydroxyl radical scavengers and not specifically inhibited by scavengers of singlet oxygen. By contrast, superoxide dismutase was a very potent scavenger of the lipid peroxidation. A half-maximal effect at 3 ng/ml enzyme was registered, whereas a 100-fold higher concentration was necessary in order to inhibit O-2 formation as detected by succinylated cytochrome c or pyrogallol. The reason for this difference might be inherent in different types of kinetics in the interaction of O-2 with different scavengers or might possibly indicate that SOD scavenges another type of reactive oxygen, different from O-2, generated by cytochrome P-450 LM2. Iron chelators inhibited the P-450-dependent lipid peroxidation, whereas iron chelate interacted with NADPH-cytochrome-P-450 reductase in the membranes giving rise to reductase-dependent lipid peroxidation. Neither superoxide dismutase nor EDTA at high concentrations, inhibited CCl4-initiated lipid peroxidation, indicating the point of action of these compounds at the initiation step in the cytochrome-P-450-LM2-dependent lipid peroxidation. Superoxide generated by pyrogallol, in three times the amount produced by P-450 LM2, could not bring about lipid peroxidation. It is suggested that the cytochrome-P-450-dependent lipid peroxidation mechanism might be of importance for intracellular oxidative damage under certain conditions.