Deletion mutagenesis of p22^phox subunit of flavocytochrome b₅₅₈: Identification of regions critical for gp91^phox maturation and NADPH oxidase activity

The heterodimeric flavocytochrome b₅₅₈, comprised of the two integral membrane proteins p22^phox and gp91^phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22^phox required for maturation of gp91^phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22^phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91^phox and two other NADPH oxidase subunits, p47^phox, and p67^phox. The results demonstrate that p22^phox- dependent maturation of gp91^phox carbohydrate, cell surface expression of gp91^phox, and the enzymatic function of flavocytochrome b₅₅₈ are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22^phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22^phox (amino acids 142-195), maturation and cell surface expression of gp91^phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47 ^phox. Antibody binding studies indicate that the extreme N terminus of p22^phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22 ^phox extend into the cytoplasm, anchored by two membrane-embedded regions.