Probing the role of the carboxyl terminus of the gp91phox subunit of neutrophil flavocytochrome b558 using site-directed mutagenesis.
Site-directed mutagenesis was used to generate a series of substitutions and deletions in the carboxyl-terminal 11 residues of gp91phox, 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 p47phox 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 gp91phox is a member. Mutant proteins were expressed in human myeloid leukemia cells which lack expression of endogenous gp91phox due to targeted disruption of the X-linked gp91phox 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 gp91phox 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 gp91phox expression, suggesting that overall protein structure was perturbed. No stable gp91phox protein was detected upon deletion of the terminal 11 amino acids. Alanine substitution or deletion of the carboxyl-terminal Phe570 in gp91phox resulted in a 2-fold reduction in superoxide production. This contrasts with a approximately 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 gp91phox and other ferredoxin-NADP+ reductases.
Zhen
L
L
Herman B. Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, Indiana 46702, USA.
Yu
L
L
Dinauer
M C
MC
D000595:Amino Acid Sequence; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D006105:Granulomatous Disease, Chronic / Q000201:enzymology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D013481:Superoxides / Q000378:metabolism
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D013481:Superoxides; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases