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J Biol Chem, Vol. 273, Issue 11, 6575-6581, March 13, 1998

Probing the Role of the Carboxyl Terminus of the gp91^phox Subunit of Neutrophil Flavocytochrome b₅₅₈ using Site-directed Mutagenesis

Ling Zhen^§, Lixin Yu^§, and Mary C. Dinauer^§¶

From the  Herman B. Wells Center for Pediatric Research, Departments of ^§ Pediatrics (Hematology/Oncology) and ^¶ Medical and Molecular Genetics, James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, Indiana 46702

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.

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