GP91(PHOX) IS THE HEME-BINDING SUBUNIT OF THE SUPEROXIDE-GENERATING NADPH OXIDASE

The phagocyte NADPH oxidase flavocytochrome b(558) is a membrane-bound heterodimer comprised of a glycosylated subunit, gp91(phox), and a no nglycosylated subunit, p22(phox), It contains two nonidentical heme gr oups that mediate the final steps of electron transfer to molecular ox ygen (O-2), resulting in the generation of superoxide ion (O-2(-)). Ho wever, the location of the hemes within the flavocytochrome heterodime r remains controversial, In this study, we have used transgenic COS7 c ell lines expressing gp91(phox), p22(phox), or both polypeptides to ex amine the relative role of each flavocytochrome b(558) subunit in heme binding and O-2(-), formation, A similar membrane localization was ob served when gp91(phox) and p22(phox) were either expressed individuall y or coexpressed, as analyzed by confocal microscopy and immunoblottin g of subcellular fractions. Spectral analysis of membranes prepared fr om COS7 cell lines expressing either gp91(phox) or both gp91(phox) and p22phox showed a b-type cytochrome with spectral characteristics iden tical to those of human neutrophil flavocytochrome b(558). In contrast , no heme spectrum was detected in wild-type COS7 membranes or those c ontaining only p22(phox), Furthermore, redox titration studies suggest ed that two heme groups were contained in gp91(phox) expressed in COS7 membranes, with midpoint potentials of -264 and -233 mV that were ver y similar to those obtained for neutrophil flavocytochrome b(558) Thes e results provide strong support for the hypothesis that gp91(phox) is the sole heme binding subunit of flavocytochrome b(558) However, coex pression of gp91(phox) and p22(phox) in COS7 membranes was required to support O-2(-) production in combination with neutrophil cytosol, ind icating that the functional assembly of the active NADPH oxidase compl ex requires both subunits of flavocytochrome b(558).