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.
Highlights
In the past few years, several proteins homologous to Nox2 were discovered to be superoxide/peroxide-producing NADPH oxidases (2, 3)
A53D, R54G, R54M, R54S, and A57E mutations led to an XϩCGD phenotype characterized by normal Nox2 expression (Fig. 3) but a drastic decrease (A53D, R54G, and R54S) or a total inhibition (R54M and A57E) of NADPH oxidase activity (Table 1)
The P56L mutant exhibited a decrease of Nox2 expression and proportional reduction in NADPH oxidase activity as typically observed in XϪCGD phenotypes (Fig. 3 and Table 1)
Summary
In the past few years, several proteins homologous to Nox were discovered to be superoxide/peroxide-producing NADPH oxidases (the Nox family of NADPH oxidases, Nox, Nox, Nox, and Nox5) (2, 3). The physiological functions of the Nox family members include host defense and cellular signaling, cell differentiation, and gene regulation They are widely involved in a range of pathological processes such as cardiovascular diseases and neurological disorders (4). According to the hydrophobicity pattern of the Nox amino acid sequences, the N-terminal half of the protein appears to be embedded in the plasma membrane and structured into six potential ␣-helices. This domain of the Nox proteins contains two nonidentical hemes coordinated by four histidine residues in the third and fifth transmembrane passages (17), three glycosylated asparagines in two external-loops (18), and the B(19) and D-intracellular-loops (see model Fig. 1A) (12, 20). The authors found that the R54S mutation affects the function of the heme moiety of cytochrome b558 and the electron transfer from the FAD moiety to heme
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