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)
摘要
Flavocytochrome b558 (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22phox 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 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 b558 for p67phox. 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.
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关键词
Electron Transfer,Genetic Diseases,Innate Immunity,NADPH Oxidase,Neutrophil,Nox,Chronic Granulomatous Disease,Nox2,Dehydrogenase,Rare X-CGD Variants
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