%0 Journal Article %T Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase. %+ TheREx %+ ANTE-INSERM U836, équipe 4, Muscles et pathologies %+ Department of Microbiology %+ TheREx %A Picciocchi, Antoine %A Debeurme, Franck %A Beaumel, Sylvain %A Dagher, Marie-Claire %A Grunwald, Didier %A Jesaitis, Algirdas, J. %A Stasia, Marie-José %Z National Institutes of Health Grant N01-AI-30070 from the Immunodeficiency Network and the Primary Immunodeficiency Disease Consortium; CGD Research Trust Grant Award reference J4G/09/09, UK; grants from Joseph Fourier University Medical School and the Delegation for Clinical Research and Innovations, Grenoble, France %< avec comité de lecture %@ 0021-9258 %J Journal of Biological Chemistry %I American Society for Biochemistry and Molecular Biology %V 286 %N 32 %P 28357-69 %8 2011-08-12 %D 2011 %R 10.1074/jbc.M111.220418 %M 21659519 %Z Life Sciences [q-bio]/Cellular BiologyJournal articles %X Flavocytochrome b(558) (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22(phox) and gp91(phox). 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 α-helices coordinating two hemes. We studied the role of the second transmembrane α-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. %G English %2 https://inserm.hal.science/inserm-00763368/document %2 https://inserm.hal.science/inserm-00763368/file/Picciocchi_2011_Role_of_ME.pdf %L inserm-00763368 %U https://inserm.hal.science/inserm-00763368 %~ INSERM %~ CEA %~ UGA %~ IMAG %~ CNRS %~ UNIV-GRENOBLE1 %~ INPG %~ INRA %~ U836 %~ TIMC-IMAG %~ TIMC-IMAG-THEREX %~ AGREENIUM %~ CEA-DRF %~ BIG %~ IRIG %~ CEA-GRE %~ UNIV-LYON %~ INRAE