Cytochrome P-450-dependent oxidase activity and hydroxyl radical production in micellar and membranous types of reconstituted systems.
In view of conflicting results in the literature regarding the contribution of cytochrome P-450 to hydrogen peroxide production and formation of hydroxyl radicals in the microsomal electron transport chain, experiments were undertaken to evaluate this problem using reconstituted micellar and membranous systems containing NADPH-cytochrome P-450 reductase and cytochrome P-450 LM2 purified from rabbit liver. It was found that P-450 LM2 increased the rate of NADPH consumption in the vesicular system, reconstituted with microsomal phospholipids, much more than in the micellar system, based on dilauroylphosphatidylcholine (DLPC) under otherwise similar conditions. At small amounts of Fe(III)-EDTA (1-5 microM), the enhanced oxidase activity was manifested in a much higher dependency on P-450 LM2 for the production of hydroxyl radicals, as determined by the oxidation of dimethylsulphoxide (Me2SO) or 2-keto-4-thiomethylbutyric acid (KMBA), in the vesicular than in the micellar system. In the presence of high amounts of Fe(III)-EDTA (10-50 microM), the relative increase due to P-450 LM2 was less pronounced in both types of reconstituted systems, although the increase in absolute terms was about the same as at small Fe(III)-EDTA concentrations. The data indicate that in the presence of no or small amounts of chelated iron in negatively-charged membranous systems, most of the hydrogen peroxide and superoxide anions necessary for generation of hydroxyl radicals, are produced by cytochrome P-450 LM2. This appears to be due to a higher affinity between the reductase and P-450 LM2 in this system. In reconstituted micellar systems or in the presence of high amounts of chelated iron, "uncoupling" at the level of the reductase appears to take place, with a resulting production of hydroxyl radicals and other forms of reactive oxygen species.