Mapping of functional domains in the p22^phox subunit of flavocytochrome b₅₅₉ participating in the assembly of the NADPH oxidase complex by "peptide walking"

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membranal heterodimeric flavocytochrome (cytochrome b₅₅₉), composed of gp91^phox and p22^phox subunits, and four cytosolic proteins, p47^phox, p67^phox, p40^phox, and the small GTPase Rac (1 or 2). All redox stations involved in electron transport from NADPH to oxygen are located in gp91^phox. NADPH oxidase activation is the consequence of assembly of cytochrome b₅₅₉ with cytosolic proteins, a process reproducible in a cell-free system, consisting of phagocyte membranes, and recombinant cytosolic components, activated by an anionic amphiphile. p22^phox is believed to act as a linker between the cytosolic components and gp91^phox. We applied "peptide walking" to mapping of domains in p22^phox participating in NADPH oxidase assembly. Ninety one synthetic overlapping pentadecapeptides, spanning the p22^phox sequence, were tested for the ability to inhibit NADPH oxidase activation in the cell-free system and to bind individual cytosolic NADPH oxidase components. We conclude the following. 1) The p22^phox subunit of cytochrome b₅₅₉ serves as an anchor for both p47^phox and p67^phox. 2) p47^phox binds not only to the proline-rich region, located at residues 151-160 in the cytosolic C terminus of p22^phox, but also to a domain (residues 51-63) located on a loop exposed to the cytosol. 3) p67^phox shares with p47^phox the ability to bind to the proline-rich region (residues 151-160) and also binds to two additional domains, in the cytosolic loop (residues 81-91) and at the start of the cytosolic tail (residues 111-115). 4) The binding affinity of p67^phox for p22^phox peptides is lower than that of p47^phox. 5) Binding of both p47^phox and p67^phox to proline-rich p22^phox peptides occurs in the absence of an anionic amphiphile. A revised membrane topology model of p22^phox is proposed, the core of which is the presence of a functionally important cytosolic loop (residues 51-91).