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.
- Key Words: cytochrome b
- NADH-cytochrome breductase
- ferric complexes, lipid peroxidation