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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A heterodimer of prenylated Rac1 and Rho GDP dissociation inhibitor was purified and found to be competent in NADPH oxidase activation. Small angle neutron scattering experiments confirmed a 1:1 stoichiometry. The crystal structure of the Rac1-RhoGDI complex was determined at 2.7 A resolution. In this complex in which Rac1 is bound to GDP, the switch I region of Rac1 is in the GDP conformation whereas the switch II region resembles that of a GTP-bound GTPase. Two types of interaction between RhoGTPases and RhoGDI were investigated. The lipid-protein interaction between the geranylgeranyl moiety of Rac1 and RhoGDI resulted in numerous structural changes in the core of RhoGDI. The interactions between Rac1 and RhoGDI occur through hydrogen bonds which involve a number of residues of Rac1, namely, Tyr64(Rac), Arg66(Rac), His103(Rac), and His104(Rac), conserved within the Rho family and localized in the switch II region or in its close neighborhood. Moreover, in the switch II region of Rac1, hydrophobic interactions involving Leu67(Rac) and Leu70(Rac) contribute to the stability of the Rac1-RhoGDI complex. Inhibition of the GDP-GTP exchange in Rac1 upon binding to RhoGDI partly results from interaction of Thr35(Rac) with Asp45(GDI). In the Rac1-RhoGDI complex, the accessibility of the effector loops of Rac1 probably accounts for the ability of the Rac1-RhoGDI complex to activate the NADPH oxidase.

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Diphenylene iodonium (Ph21), a lipophilic reagent, is an efficient inhibitor of the production of 0; by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[y-thio]triphosphate, MgC12 and arachidonic acid, or in membranes isolated from neutrophils activated by 4P-phorbol 1Zmyristate 13-acetate, addition of a reducing agent, e. g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph21. The membrane fraction was found to contain the PhJ-sensitive component(s). In the presence of a concentration of PhzI sufficient to fully inhibit 0; production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (C121nd) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The C12-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph21 (100 nmol/mg membrane protein), the C1,Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph21 sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph21 (< 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph21 is the heme binding component of cytochrome b558. Higher concentrations of Ph21 were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [1251]Ph21. However, the radiolabellng of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of 0; production by Ph2T. The 0;-generating form of xanthine oxidase was also inhibited by Ph21. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph21 had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of 0, production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by C1,Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.The neutrophil NADPH oxidase is an 0, -generating enzyme complex, which is dormant in circulating neutrophils and becomes abruptly active when the neutrophils are stimulated by particulate or soluble specific ligands. The activated oxidase complex is located in the plasma membrane of neutrophils. It is thought to consist of two membrane-bound proteins with a redox function, namely an NADPH flavoCor...

A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formylpeptide receptor is 34%.