Role of Putative Second Transmembrane Region of Nox2 Protein in the Structural Stability and Electron Transfer of the Phagocytic NADPH Oxidase

Abstract

Flavocytochrome b 558 (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22 phox and gp91phox . The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/ NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane ␣-helices coordinating two hemes. We studied the role of the second transmembrane ␣-helix, which contains a "hot spot" for mutations found in rare X ؉ and X ؊ chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b 558 for p67 phox . By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production.

Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation

“…From 1.10 10 unstimulated WT-NOX2 or mutated NOX2 PLB-985 cells, we obtained 18 ± 2 μg of pure cytochromes b 558 as previously described [29] (Fig. 4A).…”

Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

“…31 Cells were stained with the 7D5 antibody (1:20) for 1 h at 4°C followed by incubation with an Alexa Fluor ® 488 goat-F(ab′) 2 fragment anti-mouse IgG1 (H+L) for 1 h at room temperature. Nuclei were stained with 1 µg/mL Hoechst 33258.…”

“…31,34 After incubation for 10 min at room temperature with arachidonic acid, NADPH oxidase activity was measured by adding 200 µM NADPH to initiate the superoxide production in the presence of 100 µM NBT. A negative control was performed in the absence of NADPH.…”

“…31 The reduced minus-oxidized difference spectra were recorded at room temperature with a spectrophotometer (BioTek Synergy HT, Winooski, VT, USA). Cytochrome b 558 integrity was validated by the presence of two absorption bands at 426 nm (Soret) and 558 nm (α band) and the valley at ∼410-411 nm.…”

“…31,33,34 The cytochrome b 558 heme concentration given by the 426-nm peak was used for the calculation of NADPH oxidase activity. In this reconstituted system, the NADPH oxidase activity of NOX2/p22 phox liposomes was 228.7 moles of superoxides O 2 •− /s/mol of cytochrome b 558 heme (±14.7; n=3), whereas in the negative liposomes the activity was only residual ( Figure 4A and B).…”