Gp91phox is the heme binding subunit of the superoxide-generating NADPH oxidase

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-07, Vol.95 (14), p.7993-7998
• Main Authors: Yu, Lixin, Quinn, Mark T, Croos, Andrew R, Dinauer, Mary C
• Format: Article
• Language: English
• Subjects: Biochemistry; Biological Sciences; Cellular biology; Genes; Immunology; Proteins
• ISSN: 0027-8424; 1091-6490

The phagocyte NADPH oxidase flavocytochrome b 558 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 (O 2 ), resulting in the generation of superoxide ion (O 2 − ). 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 b 558 subunit in heme binding and O 2 − 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 b 558 . 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 b 558 . These results provide strong support for the hypothesis that gp91 phox is the sole heme binding subunit of flavocytochrome b 558 . However, coexpression of gp91 phox and p22 phox in COS7 membranes was required to support O 2 − production in combination with neutrophil cytosol, indicating that the functional assembly of the active NADPH oxidase complex requires both subunits of flavocytochrome b 558 .