Role of Cytochrome b₅in NADH-Dependent Microsomal Reduction of Ferric Complexes, Lipid Peroxidation, and Hydrogen Peroxide Generation

The NADH-dependent microsomal electron transfer system consists of NADH-cytochrome b₅reductase and cytochrome b₅, which donates reducing equivalents to fatty acyl desaturase, cytochrome P450, and other reactions. A study was carried out to investigate the interaction of NADH with several ferric complexes and to evaluate the role of cytochrome b₅in these interactions. NADH-dependent microsomal lipid peroxidation was stimulated by ferric-ATP, ferric-histidine, and ferric-ammonium sulfate, but not by ferric-EDTA. Anti-cytochrome b₅IgG produced a concentration-dependent inhibition of lipid peroxidation catalyzed by all three ferric complexes. Addition of purified cytochrome b₅to the microsomes increased the rate of lipid peroxidation with all three ferric complexes. Lipid peroxidation in control and the cytochrome b₅-fortified microsomes was not sensitive to superoxide dismutase, catalase, or DMSO and was completely inhibited by trolox and propylgallate. Ferric-EDTA stimulated NADH-dependent microsomal production of H₂O₂and NADH consumption. Anti-cytochrome b₅IgG had only a small inhibitory effect on this stimulation by ferric-EDTA. NADH supported microsomal reduction of ferric complexes in the order ferric-ATP > ferric-histidine ≈ ferric-ammonium sulfate > ferric-EDTA. Anti-cytochrome b₅IgG inhibited, whereas added cytochrome b₅stimulated, the reduction of ferric-ATP, ferric-histidine, and ferric-ammonium sulfate, whereas reduction of ferric-EDTA was not affected by these additions. Ferric-ATP, at high concentrations, was more effective than ferric-histidine or ferric-ammonium sulfate in stimulating lipid peroxidation and in becoming reduced by NADH-dependent microsomal electron transport; anti-cytochrome b₅IgG was less inhibitory and added b₅was less stimulatory at 50 μMferric-ATP compared to 5 μMferric-ATP or 50 μMferric-histidine or 50 μMferric-ammonium sulfate. It is concluded that cytochrome b₅is required for reduction of low and high concentrations of ferric-histidine and ferric-ammonium sulfate and low concentrations of ferric-ATP and for the lipid peroxidation catalyzed by these ferric complexes. The reductase, not cytochrome b₅, is involved in interaction with ferric-EDTA. Higher concentrations of ferric-ATP can also interact with the reductase, as well as with cytochrome b₅.