Oxidoreductases generate hydrogen peroxide that drives iron-dependent lipid peroxidation during ferroptosis

Abstract

The inhibition of antioxidant systems of glutathione peroxidase 4 (GPX4) or ferroptosis suppressor protein 1 (FSP1) causes iron-dependent peroxidation of polyunsaturated phospholipids that leads to cell death, a process known as ferroptosis. The mechanisms underlying iron-dependent lipid peroxidation are under active debate. Here, we report that two endoplasmic reticulum-residing oxidoreductases, NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), are responsible for the iron-dependent peroxidation of polyunsaturated phospholipids and membrane disruption that executes ferroptosis. Genetic ablation of POR and CYB5R1 or mutations that eliminate PORs electron transfer activity blocked ferroptosis. In vitro enzymatic assays established that POR and CYB5R1 catalyze hydrogen peroxide production by transferring electrons from NADPH/NADH to oxygen, which is then used to carry out iron-dependent lipid peroxidation via a Fenton reaction. The lipid peroxidation reaction catalyzed by POR and CYB5R1 additively disrupts polyunsaturated phospholipid-containing liposomes. Finally, POR knockdown confers significant protective effects during concanavalin A-induced, ferroptosis-associated acute liver injury in vivo. Our study thus indicates that POR and CYB5R1 are the enzymes of the "oxidant" system that operates to contravene the antioxidant GPX4/FSP1 systems; the balance between these two systems determines cell commitment to ferroptosis.