Probing the role of the carboxyl terminus of the gp91(phox) subunit of neutrophil flavocytochrome b558 using site-directed mutagenesis

Ling Zhen, Lixin Yu, Mary C. Dinauer

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

Site-directed mutagenesis was used to generate a series of substitutions and deletions in the carboxyl-terminal 11 residues of gp91(phox), the 91-kDa subunit of the phagocyte NADPH oxidase flavocytochrome b558. This region encompasses 559RGVHFIF565, implicated as a contact point for the cytosolic oxidase subunit p47(phox) during oxidase activation, and a carboxyl-terminal phenylalanine (Phe570), which corresponds in position to a highly conserved aromatic residue that interacts with the flavin group in the ferredoxin-NADP+ reductase flavoenzyme family, of which gp91(phox) is a member. Mutant proteins were expressed in human myeloid leukemia cells which lack expression of endogenous gp91(phox) due to targeted disruption of the X- linked gp91(phox) gene. Although specific residues within 559RGVHFIF565 had previously been identified by alanine scanning as essential for peptide inhibition of oxidase activity in a cell-free assay, comparable substitutions in the gp91(phox) polypeptide had either no or only a modest effect on oxidase activity in whole cells. Replacement of nonpolar with polar or charged residues had greater effects on oxidase activity, but were also associated with decreased gp91(phox) expression, suggesting that overall protein structure was perturbed. No stable gp91(phox) protein was detected upon deletion of the terminal 11 amino acids. Alanine substitution or deletion of the carboxyl-terminal Phe570 in gp91(phox) resulted in a 2- fold reduction in superoxide production. This contrasts with a ≃300-800- fold reduction reported for comparable mutations in pea ferredoxin-NADP+ reductase, which suggests that structural or functional differences exist between the carboxyl terminus of gp91(phox) and other ferredoxin-NADP+ reductases.

Original languageEnglish
Pages (from-to)6575-6581
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number11
DOIs
StatePublished - Mar 13 1998
Externally publishedYes

Fingerprint Dive into the research topics of 'Probing the role of the carboxyl terminus of the gp91(phox) subunit of neutrophil flavocytochrome b<sub>558</sub> using site-directed mutagenesis'. Together they form a unique fingerprint.

  • Cite this