(A) Schematic of the NOX2 enzymatic assay. O2- produced by NOX2 are converted into H2O2 by SOD. In the presence of H2O2, horseradish peroxidase (HRP) converts the nonfluorescent Amplex Red into resorufin, the fluorescence of which is measurable and proportional to the concentration of H2O2. (B) The activity of the NOX2-p22 complex in crude cell membrane measured using Amplex Red assay. The activity is determined by subtracting the background of the enzymatic assay buffer without crude cell membrane. Data are shown as means ± standard deviations; N=3 technical replicates. (C) The amounts of O2- produced by NOX2-p22-7D5-TP1170 complex in nanodisc in the presence of Trimera are plotted versus time. The represented data are shown. DPI, diphenyliodonium, NADPH oxidase inhibitor. (D) The rates of O2- production in C are summarized. Data are shown as means ± standard deviations; N=3 technical replicates. (E) Side view of the cryo-EM map of NOX2-p22-7D5-TP1170 complex. The approximate boundaries of the phospholipid bilayer are indicated as gray thick lines. p22 and NOX2 are colored in orange and light blue. 7D5 and TP1170 are colored in gray and light green. FAD, lipid, and glycosylation decoration are colored in pink, cyan, and gold, respectively. (F) A 90°-rotated side view compared to D. (G) The cut-open view of the cryo-EM map of NOX2-p22. Haem and FAD is colored in magenta and pink, respectively. (H) A 90°-rotated bottom view of E. (I) Topology of p22 and NOX2 subunits. Transmembrane helices are shown as cylinders, unmodeled disordered regions are shown as dashed lines. The phospholipid bilayer is shown as gray layers. Glycosylation sites were indicated by protrusions. DH, dehydrogenase domain of NOX2. (J) Structure of NOX2-p22 complex in cartoon representation. The colors are the same as in D.