A microsomal membrane component associated with iron reduction in NADPH-supported lipid peroxidation

Area of Science:

• Biochemistry
• Cell Biology
• Enzymology

Background:

• Lipid peroxidation in rat liver microsomes is often supported by nicotinamide adenine dinucleotide phosphate (NADPH).
• The precise mechanism and specific factors involved in NADPH-supported iron reduction, which drives lipid peroxidation, remain incompletely understood.

Purpose of the Study:

• To investigate a factor responsible for reduced NADPH-supported lipid peroxidation.
• To determine if this factor is involved in iron reduction via cooperation with NADPH-cytochrome P450 reductase.

Main Methods:

• Anaerobic incubation of rat liver microsomes with NADPH and ferric pyrophosphate.
• Utilizing reconstituted systems with purified NADPH-cytochrome P450 reductase.
• Employing a cytochrome P450-free fraction from microsomes, subjected to cholate solubilization and laurate sepharose chromatography.
• Testing the effect of heat treatment and trypsin digestion on the identified fraction.

Main Results:

• NADPH-dependent reduction of ferric pyrophosphate in microsomes was independent of cytochrome P450 levels and unaffected by carbon monoxide (CO).
• A cytochrome P450-free fraction was essential for iron reduction in a reconstituted system, leading to lipid peroxidation.
• This fraction facilitated iron reduction using NADPH as a reducing equivalent via NADPH-cytochrome P450 reductase, and the activity was heat-labile and sensitive to trypsin.

Conclusions:

• A heat-labile component, likely a protein distinct from cytochrome P450, is associated with iron reduction in rat liver microsomes.
• This component plays a critical role in mediating NADPH-supported lipid peroxidation.
• The findings elucidate a novel mechanism in microsomal redox cycling and lipid peroxidation.