Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase. - Archive ouverte HAL Access content directly
Journal Articles Journal of Biological Chemistry Year : 2011

Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase.

(1) , (1) , (1) , (1) , (2) , (3) , (4)
1
2
3
4

Abstract

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.
Embargoed file
Embargoed file
Ne sera jamais visible
Loading...

Dates and versions

inserm-00763368 , version 1 (10-12-2012)

Identifiers

Cite

Antoine Picciocchi, Franck Debeurme, Sylvain Beaumel, Marie-Claire Dagher, Didier Grunwald, et al.. Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase.. Journal of Biological Chemistry, 2011, 286 (32), pp.28357-69. ⟨10.1074/jbc.M111.220418⟩. ⟨inserm-00763368⟩
792 View
2 Download

Altmetric

Share

Gmail Facebook Twitter LinkedIn More