Cytochrome P450 is the Source of NADPH-Dependent Signal in Cell-Free Assays for Nox Activity

Measuring Nox-derived reactive oxygen species is a constant challenge in redox biology. All probes available display limitations regarding sensitivity, specificity or demand highly specialized technique. To overcome such limitations, numerous studies have used NADPH-stimulated assays in membrane fractions to address Nox activity. In our previous work we found an unchanged activity in a triple Nox knockout (Nox1-Nox2-Nox4-/-) compared to its wild type. Moreover, results in intact cells overexpressing Noxes could not be recapitulated in NADPH-stimulated membrane assays showing that such assays are unable to measure Nox activity. Here we further investigate and validate the real source of NADPH-depend signal in membranes. By combining native protein electrophoresis, NADPH-stimulated assays and mass spectrometry we found mitochondrial proteins and cytochrome P450 as possible candidates responsible for the signal. Cells lacking functional mitochondrial complexes (I, III, IV and IV) showed no difference in NADPH-stimulated membrane assays suggesting that mitochondrial oxidoreductases and dehydrogenases are unlikely to generate the signal. However, in microsomes overexpressing P450 proteins there was a remarkable signal upon NADPH stimulation in lucigenin assay but also with LO12 and DHE. Knockdown of P450 reductase by CRISP/Cas9 technology abolished the signal in NADPH-stimulated assays showing that cytochrome P450 system accounts for the majority of the signal which has been for so long addressed to NADPH oxidases.