Mutagenesis of p22phoxHistidine 94

Karla J. Biberstine-Kinkade, Lixin Yu, Natalie Stull, Brendan LeRoy, Shelley Bennett, Andrew Cross, Mary C. Dinauer
2002 Journal of Biological Chemistry  
The NADPH oxidase is a multicomponent enzyme that transfers electrons from NADPH to O 2 to generate superoxide (O 2 . ), the precursor of microbicidal oxygen species that play an important role in host defense. Flavocytochrome b 558 , 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
more » ... hydrophobic N terminus of gp91 phox , but the role of p22 phox in heme binding is unclear. We compared biochemical and functional features of wild type flavocytochrome b 558 with those in cells co-expressing gp91 phox with p22 phox harboring amino acid substitutions at histidine 94, the only invariant histidine residue within the p22 phox subunit. Substitution with leucine, tyrosine, or methionine did not affect heterodimer formation or flavocytochrome b 558 function. The heme spectrum in purified preparations of flavocytochrome b 558 containing the p22 phox derivative was unaffected. In contrast, substitution of histidine 94 with arginine appeared to disrupt the intrinsic stability of p22 phox and, secondarily, the stability of mature gp91 phox and abrogated O 2 . production. These findings demonstrate that His 94 p22 phox is not required for heme binding or function of flavocytochrome b 558 in the NADPH oxidase. 1 The abbreviations used are: CGD, chronic granulomatous disease; mAb, monoclonal antibody; INT, iodonitrotetrazolium; CHO, Chinese hamster ovary; RT, reverse transcription; HPRT, hypoxanthine phosphoribosyltransferase.
doi:10.1074/jbc.m203993200 pmid:12042318 fatcat:ci4v2dvoonfuxjnssxaigjoomy