Membrane Binding and Pro‐Ferroptotic Activity of NQO1

Peroxidation of polyunsaturated fatty acids in cellular membranes, when extensive and unrepaired, can lead to a form of eukaryotic cell death known as ferroptosis. A complex network of proteins and small molecules has evolved to modulate this peroxidation, thereby suppressing or enhancing ferroptosis. Within this network, the quinone reductase NQO1 has long been recognized for its ability to reduce and regenerate the membrane-resident antioxidant ubiquinone. Surprisingly, recent studies have also implicated NQO1 in pro-ferroptotic processes. Here, we present an experimental model designed to disentangle the opposing activities of NQO1 using a simple in vitro system composed of phospholipid liposomes. The biochemical setup enabled us to recapitulate the membrane association of the normally cytosolic NQO1 and to demonstrate that this association weakens in the presence of its cognate electron donor, NADH. The effect required the C-terminal tail of the enzyme and is likely linked to the higher disorder propensity of its last 50 amino acids. In the presence of NQO1, an increase in iron-driven liposome peroxidation was observed. Without quinone substrates in the system, these results support the idea that the NQO1 cofactor flavin adenine dinucleotide can reduce ferric (Fe3+) iron, thereby promoting reactive oxygen species generation and lipid peroxidation.