Gp91 phox is the heme binding subunit of the superoxide-generating NADPH oxidase

Abstract

The phagocyte NADPH oxidase flavocytochrome b₅₅₈ is a membrane-bound heterodimer comprised of a glycosylated subunit, gp91^phox, and a nonglycosylated subunit, p22^phox. It contains two nonidentical heme groups that mediate the final steps of electron transfer to molecular oxygen (O₂), resulting in the generation of superoxide ion (O₂⁻). However, the location of the hemes within the flavocytochrome heterodimer remains controversial. In this study, we have used transgenic COS7 cell lines expressing gp91^phox, p22^phox, or both polypeptides to examine the relative role of each flavocytochrome b₅₅₈ subunit in heme binding and O₂⁻ formation. A similar membrane localization was observed when gp91^phox and p22^phox were either expressed individually or coexpressed, as analyzed by confocal microscopy and immunoblotting of subcellular fractions. Spectral analysis of membranes prepared from COS7 cell lines expressing either gp91^phox or both gp91^phox and p22^phox showed a b-type cytochrome with spectral characteristics identical to those of human neutrophil flavocytochrome b₅₅₈. In contrast, no heme spectrum was detected in wild-type COS7 membranes or those containing only p22^phox. Furthermore, redox titration studies suggested that two heme groups were contained in gp91^phox expressed in COS7 membranes, with midpoint potentials of −264 and −233 mV that were very similar to those obtained for neutrophil flavocytochrome b₅₅₈. These results provide strong support for the hypothesis that gp91^phox is the sole heme binding subunit of flavocytochrome b₅₅₈. However, coexpression of gp91^phox and p22^phox in COS7 membranes was required to support O₂⁻ production in combination with neutrophil cytosol, indicating that the functional assembly of the active NADPH oxidase complex requires both subunits of flavocytochrome b₅₅₈.