Interaction of Ferric Complexes with NADH–Cytochrome b5 Reductase and Cytochrome b5: Lipid Peroxidation, H2O2Generation, and Ferric Reduction

NADH is reactive in interacting with iron and liver microsomes to catalyze the formation of reactive oxygen species. NADH-dependent microsomal electron transfer involves the enzymes NADH–cytochrome b5 reductase and cytochrome b5. Experiments were carried out to evaluate the ability of reconstituted systems containing purified reductase in the absence or presence of b5 to reduce several ferric complexes, to generate H2O2, and to catalyze lipid peroxidation. The reductase directly reduced ferric–EDTA; addition of b5 inhibited this reduction probably due to competition for the reductase. Cytochrome b5 was required for reduction of low (5 μM) and high (50 μM) concentrations of ferric–histidine and ferric–ammonium sulfate and low concentrations of ferric–ATP. The reductase could interact directly with high (50 μM) concentrations of ferric–ATP. Peroxidation of phospholipids extracted from liver microsomes by the reductase required b5. Molar ratios of b5 to reductase approximating those found in liver microsomes (e.g., 10) were effective in catalyzing lipid peroxidation and ferric reduction. The role of b5 in catalyzing lipid peroxidation appears to involve reduction of the ferric catalyst to help form an initiation complex and degradation of lipid hydroperoxides by the hemeprotein to catalyze propagation of the peroxidation cycle. In contrast to results with microsomes, lipid peroxidation by the complete reconstituted system was sensitive to superoxide dismutase; this sensitivity was decreased if the reconstituted system was dialyzed overnight to form vesicular preparations, indicating that accessibility of enzymes to sites of peroxidation was important. High rates of H2O2formation were observed in the presence of ferric–EDTA plus reductase; rates of H2O2formation with the other ferric complexes were low even in the presence of b5. These results indicate that the ability of NADH reductase and cytochrome b5 to interact with various ferric complexes depends on the nature of the chelating agent used to complex the iron and on the concentration of the iron.