Characterization of a phagocyte cytochrome b558 91-kilodalton subunit functional domain: identification of peptide sequence and amino acids essential for activity.
The phagocyte NADPH oxidase is a multicomponent membrane-bound electron transport chain that catalyzes the reduction of O2 to superoxide. Cytochrome b558, the terminal electron donor to O2, is an integral membrane heterodimer containing 91- and 22-kDa subunits (gp91-phox and p22-phox, respectively). Synthetic peptides, whose amino acid sequences correspond to a gp91-phox carboxyl-terminal domain, inhibit superoxide production by blocking assembly of the oxidase from membrane and cytosol components. In this study, we examined the amino acid sequence requirements of a series of synthetic truncated gp91-phox peptides for inhibition of human neutrophil NADPH oxidase activation. RGVHFIF, corresponding to gp91-phox residues 559-565, was the minimum sequence capable of inhibiting superoxide generation. Contributions of individual amino acids to overall RGVHFIF inhibitory activity were determined by comparing the abilities of alanine-substituted RGVHFIF peptides to inhibit superoxide production. Substitution of alanine for arginine, valine, isoleucine, or either of the phenylalanines (but not glycine or histidine) within RGVHFIF resulted in loss of inhibitory activity. Synthetic gp91-phox carboxyl-terminal peptides are likely to be competitive inhibitors of the corresponding carboxyl-terminal domain of native gp91-phox by virtue of amino acid identity. We conclude that properties of arginine valine, isoleucine, and phenylalanine side chains within an RGVHFIF-containing domain of gp91-phox contribute significantly to cytochrome b558-mediated activation of the oxidase.
Kleinberg
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Baltimore Veterans Affairs Medical Center, Maryland.
Mital
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Rotrosen
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Malech
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HL
D000595:Amino Acid Sequence; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases; D010586:Phagocytes / Q000201:enzymology*; D013481:Superoxides / Q000378:metabolism
D003573:Cytochrome b Group; D013481:Superoxides; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases