The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O2•−), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91phox and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47phox (aa 1-286), p67phox (aa 1-212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy. Highlights ### Competing Interest Statement The authors have declared no competing interest. * aa : amino acid AFM : Atomic Force Microscopy DPI : Diphenylene Iodonium FRET : Förster Resonance Energy Transfer FLIM : Fluorescence-Lifetime Imaging Microscopy FP : Fluorescent Protein HRP : Horseradish Peroxidase IR : Infra-Red NADPH : Nicotinamide Adenine Dinucleotide Phosphate reduced form NMDG : N-Methyl-D-glucamine PMA : Phorbol 12-Myristate 13-Acetate ROI : Region of Interest ROS : Reactive Oxygen Species

NADPH oxidases (NOX) are transmembrane proteins that professionally produce reactive oxygen species

NADPH oxidases (NOX) are transmembrane proteins that professionally produce reactive oxygen species