title: Mutagenesis of p22(phox) histidine 94. A histidine in this position is not required for flavocytochrome b558 function.
abstract: 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. We compared biochemical and functional features of wild type flavocytochrome b558 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 b558 function. The heme spectrum in purified preparations of flavocytochrome b558 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 O2*- production. These findings demonstrate that His94 p22(phox) is not required for heme binding or function of flavocytochrome b558 in the NADPH oxidase.
authors.affiliation: Department of Pediatrics (Hematology/Oncology), Herman B. Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Indiana 46202-5225, USA.
authors.forename: Karla J
authors.initials: KJ
authors.lastname: Biberstine-Kinkade
authors.forename: Lixin
authors.initials: L
authors.lastname: Yu
authors.forename: Natalie
authors.initials: N
authors.lastname: Stull
authors.forename: Brendan
authors.initials: B
authors.lastname: LeRoy
authors.forename: Shelley
authors.initials: S
authors.lastname: Bennett
authors.forename: Andrew
authors.initials: A
authors.lastname: Cross
authors.forename: Mary C
authors.initials: MC
authors.lastname: Dinauer
mesh_terms: D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D003573:Cytochrome b Group / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D003573:Cytochrome b Group; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D006639:Histidine; C036888:cytochrome b558; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase