Flavocytochrome b558 is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O2 into the superoxide anion O2 in phagocytic cells. Flavocytochrome b558 is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox forphagocyte oxidase) (beta subunit) and a small protein p22phox (alpha subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b558 which becomes activated and generates O2-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O2- and the derived metabolites H2O2 and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b558. The p22phox subunit serves as a docking site for the cytoso lic phox proteins. This review provides an overview of current knowledge on the structural organization of the O2(-)-generating flavocytochrome b558, its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O2(-)-generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed.

Professional phagocytes (neutrophils, eosinophils, monocytes and macrophages) possess an enzymatic complex, the NADPH oxidase, which is able to catalyze the one-electron reduction of molecular oxygen to superoxide, 0;. The NADPH oxidase is dormant in non-activated phagocytes. It is suddenly activated upon exposure of phagocytes to the appropriate stimuli and thereby contributes to the microbicidal activity of these cells. Oxidase activation in phagocytes involves the assembly, in the plasma membrane, of membrane-bound and cytosolic components of the oxidase complex, which were disassembled in the resting state. One of the membrane-bound components in resting phagocytes has been identified as a low-potential b-type cytochrome, a heterodimer composed of two subunits of 22-kDa and 91-kDa. The link between NADPH and cytochrome b is probably a flavoprotein whose subcellular localization in resting phagocytes remains to be determined. Genetic defects in the cytochrome b subunits and in the cytosolic factors have been shown to be the molecular basis of chronic granulomatous disease, a group of inherited disorders in the host defense, characterized by severe, recurrent bacterial and fungal infections in which phagocytic cells fail to generate 0 , upon stimulation. The present review is focused on recent data concerning the signaling pathway which leads to oxidase activation, including specific receptors, the production of second messengers, the organization of the oxidase complex and the molecular defects responsible for granulomatous disease.

Two variants of the human N-formylpeptide chemoattractant receptor have been isolated from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with Bt2cAMP. Both recombinant receptors, fMLP-R26 and fMLP-R98, are 350 amino acids long (Mr 38,420); they differ from each other by two residue changes at positions 101 and 346 and by significant differences in the 5' and 3' untranslated regions. Both clones were able to transfer to COS-7 cells the capacity to specifically bind a new and highly efficient hydrophilic derivative of N-formyl-Met-Leu-Phe-Lys, referred to as fMLPK-Pep12. Photolabeling experiments revealed that the glycosylated form of the fMLP receptor in COS cells has a molecular weight (Mr 50,000-70,000) similar to that observed for the native receptor in differentiated HL-60 cells. Northern blot analysis revealed a major transcript of 1.6-1.7 kb and two minor hybridization signals of 2.3 and 3.1 kb, suggesting a related family of receptors. The complex hybridization pattern obtained with restricted genomic DNA was consistent with either two genes encoding fMLP receptor isoforms or a single gene with at least one intron in the coding sequence. Sequence comparison established that the fMLP receptor belongs to the G-protein-coupled receptor superfamily. The structural similarities observed with RDC1, a receptor isolated from a dog thyroid cDNA library, which shares weak homologies with other members of the family, suggests that the fMLP receptor is representative of a new subfamily.

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