Mutagenesis of p22(phox) histidine 94. A histidine in this position is not required for flavocytochrome b558 function.

The NADPH oxidase is a multicomponent enzyme that transfers electrons from NADPH to O2 to generate superoxide (O2*-), the precursor of microbicidal oxygen species that play an important role in host defense. Flavocytochrome b558, a heterodimeric oxidoreductase comprised of gp91(phox) and p22(phox) subunits, contains two nonidentical, bis-histidine-ligated heme groups imbedded within the membrane. Four histidine residues that appear to serve as noncovalent axial heme ligands reside within the hydrophobic N terminus of gp91(phox), but the role of p22(phox) in heme binding is unclear.

Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91(phox).

The phagocyte NADPH-dependent oxidase generates superoxide (O(2)) by reducing molecular oxygen through flavocytochrome b(558) (flavocytochrome b), a heterodimeric oxidoreductase composed of gp91(phox) and p22(phox) subunits. Although each flavocytochrome b molecule contains two heme groups, their precise distribution within the heterodimer is unknown. Among functionally and/or structurally related oxidoreductases, histidines at codons 101, 111, 115, 119, 209, 210, and 222 of gp91(phox) are conserved and potential candidates to ligate heme.

Mutagenesis of an arginine- and lysine-rich domain in the gp91(phox) subunit of the phagocyte NADPH-oxidase flavocytochrome b558.

Site-directed mutagenesis was used to generate a series of mutants harboring point or multiple substitutions within the hydrophilic, polybasic domain of gp91(phox) encompassed by residues 86-102, which was previously identified as a site of interaction with p47(phox) during phagocyte NADPH oxidase assembly. Recombinant wild-type or mutant gp91(phox) was expressed in a human myeloid leukemia cell line in which the endogenous gp91(phox) gene was disrupted by gene targeting. NADPH oxidase activity was measured in a cytochrome c reduction assay following granulocytic differentiation of cells that expressed recombinant gp91(phox).

Biosynthesis of flavocytochrome b558 . gp91(phox) is synthesized as a 65-kDa precursor (p65) in the endoplasmic reticulum.

The redox center of the phagocyte NADPH oxidase is flavocytochrome b558, a transmembrane protein with two subunits, gp91(phox) and p22(phox). In this study we investigated the identity, subcellular localization, and maturation of a putative 65-kDa gp91(phox) precursor (p65). Expressing the gp91(phox) cDNA in an in vitro transcription and translation system, we found that synthesis of p65 required endoplasmic reticulum (ER) microsomes.