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J Biol Chem, Vol. 274, Issue 15, 10451-10457, April 9, 1999

Mutagenesis of an Arginine- and Lysine-rich Domain in the gp91^phox Subunit of the Phagocyte NADPH-oxidase Flavocytochrome b₅₅₈

Karla J. Biberstine-Kinkade, 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 School of Medicine, Indianapolis, Indiana 46202

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. Expression of a gp91^phox mutant in which amino acids 89-97 were replaced with nine alternate amino acids abolished NADPH oxidase activity. Expression of gp91^phox mutants R89T, D95A, D95R, R96A, R96E, or K102T did not significantly affect NADPH oxidase activity. However, mutations of individual or paired arginine residues at positions 91 and 92 had substantial effects on superoxide generation. The R91E/R92E mutation completely abolished both NADPH oxidase activity and membrane-translocation of the cytosolic oxidase proteins p47^phox, p67^phox, Rac1, and Rac2. The phorbol 12-myristate 13-acetate-induced rate of superoxide production was reduced by ~75% in cells expressing R91T/R92A, R91E, or R92E gp91^phox along with an increased lag time to the maximal rates of superoxide production relative to cells expressing wild-type gp91^phox. Taken together, these results demonstrate that Arg⁹¹ and Arg⁹² of gp91^phox are essential for flavocytochrome b₅₅₈ function in granulocytes and suggest that these residues participate in the interaction of gp91^phox with the cytosolic oxidase proteins.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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