Functional analysis of two-amino acid substitutions in gp91<Emphasis Type="Italic">phox</Emphasis> in a patient with X-linked flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>558</Subscript>-positive chronic granulomatous disease by means of transgenic PLB-985 cells

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. The most common form is caused by mutations in the CYBB gene encoding gp91phox protein, the heavy chain of cytochrome b₅₅₈, which is the redox element of NADPH oxidase. In some rare cases, the mutated gp91phox is normally expressed but no NADPH oxidase can be detected. This type of CGD is called X91⁺ CGD. We have previously reported an X⁺ CGD case with a double-missense mutation in gp91phox. Transgenic PLB-985 cells have now been made to study the impact of each single mutation on oxidase activity and assembly to rule out a possible new polymorphism in the CYBB gene. The His303Asn/Pro304Arg gp91phox transgenic PLB-985 cells exactly mimic the phenotype of the neutrophils of the X⁺ CGD patient. The His303Asn mutation is sufficient to inhibit oxidase activity in intact cells and in a broken cell system, whereas in the Pro304Arg mutant, residual activity suggests that the Pro304Arg substitution is less devastating to oxidase activity than the His303Asn mutation. The study of NADPH oxidase assembly following the in vitro and in vivo translocation of cytosolic factors p47phox and p67phox has demonstrated that, in the double mutant and in the His303Asn mutant, NADPH oxidase assembly is abolished, although the translocation is only attenuated in Pro304Arg mutant cells. Thus, even though the His303Asn mutation has a more severe inhibitory effect on NADPH oxidase activity and assembly than the Pro304Arg mutation, neither mutation can be considered as a polymorphism.Clara Bionda and Xing Jun Li contributed equally to this work     

Drug-selected complete restoration of superoxide generation in Epstein-Barr virus-transformed B cells from p47 phox -deficient chronic granulomatous disease patients by using a bicistronic retrovirus vector encoding a human multi-drug resistance gene (MDR1) and the p47 phox gene

Chronic granulomatous disease (CGD) is a group of disorders characterized by the failure of phagocytes to produce superoxide. One-third of the cases of CGD in the USA and Europe results from defects in the gene encoding p47 ^phox , a cytoplasmic component of NADPH oxidase for superoxide generation. In this study, we constructed the bicistronic retrovirus vector Ha-MDR-IRES-p47, which carries cDNAs for a human multi-drug-resistance gene (MDR1) and p47 ^phox . The amphotropic retroviral producer cells were generated, and the supernatant of the producer cells was used to transduce Epstein-Barr virus-transformed B (EBV-B) cells, established from B cells of p47 ^phox -deficient CGD patients, as an in vitro model of gene therapy for p47 ^phox -deficient CGD. The transduced cells expressed both P-glycoprotein and p47 ^phox protein, and the expression levels were increased after appropriate vincristine selection. The levels of superoxide production in the vincristine-selected cells were increased to a level similar to normal EBV-B cells. This result suggests that it is possible to achieve 100% correction of the CGD defect in p47 ^phox -deficient EBV-B cells by using the bicistronic vector. This strategy could be employed not only in vitro, but also in vivo, in the gene therapy of a number of inherited diseases. Received: 8 June 1998 / Accepted: 5 August 1998     

Identification of allele-specific p22-phox mutations in a compound heterozygous patient with chronic granulomatous disease by mismatch PCR and restriction enzyme analysis

A rare subgroup (approx. 5%) of all chronic granulomatous disease (CGD) patients suffers from mutations in the gene encoding the small p22-phox subunit of the flavocytochrome b ₅₅₈ heterodimer, the terminal redox component of the phagocyte NADPH oxidase. A male CGD patient with neutrophil granulocytes devoid of any spectrometrically detectable cytochrome b ₅₅₈ owing to autosomally inherited p22-phox deficiency (phenotype, A22^–) is reported. The patient was identified as being compound heterozygous for two independent mutations of his p22-phox alleles. On the maternal allele a single base substitution (A186 to T) was found that predicts a nonconservative replacement of Glu 53 by Val. On his paternal p22-phox allele a G was found to be added to a G stretch between nucleotides G195 and G199 in the cDNA sequence. The resulting frame shift predicts an aberrant open reading frame, 16 amino acids longer than the normal p22-phox polypeptide. Genomic DNA was tested for the presence of the mutant allele by mismatch PCR (polymerase chain reaction). For this purpose, a single base mismatch was introduced at nucleotide position 189, leading to digestion of the normal allele by the restriction enzyme HinfI. The maternal allele was found to be present in 50% of the patient's DNA and in 50% of the DNA from his mother. The same mismatch PCR analysis with control DNA from 35 healthy individuals ruled out the possibility that the single base substitution (A186 to T) represents a common polymorphism. Inheritance of the second allelic mutation (G insertion) was verified by restriction enzyme analysis using BslI [CC(N)₇GG] to digest PCR-amplified genomic DNA at the mutation site. PCR in combination with restriction enzyme analysis proved to be a powerful tool for verification of point mutations in the compound heterozygous CGD patient analyzed and may be used for prenatal diagnosis in this family.Abbreviations phox phagocyte oxidase - CGD chronic granulomatous disease - PCR polymerase chain reaction     

Monoclonal antibody 7D5 recognizes the R147 epitope on the gp91phox,phagocyte flavocytochrome b558 large subunit

Human phagocyte flavocytochrome b₅₅₈ (Cyt b), the catalytic center of nicotinamide adenine dinucleotide phosphate oxidase, consists of a heavily glycosylated large subunit (gp91^phox; Nox2) and a small subunit (p22^phox). Cyt b is a membrane‐spanning complex enzyme. Chronic granulomatous disease (CGD) is predominantly caused by a mutation in the CYBB gene encoding gp91^phox on the X‐chromosome. Because the phagocytes of patients with CGD are not able to generate the superoxide anion, these patients are susceptible to severe infections that can be fatal. It has been suggested that the extracellular region of gp91^phox is necessary for and critical to forming the epitope of mAb 7D5 and that 7D5 provides a useful tool for rapid screening of X‐linked CGD by FACS. To further elucidate the mAb 7D5 epitope on human gp91^phox, chimeric DNA expressed human and mouse gp91^phox recombinant protein were constructed. The fusion proteins were immunostained for mAb 7D5 and analyzed by FACS and western blot analysis. The ¹⁴³ELGDRQNES¹⁵¹ region was found to reside at the extracellular surface on human gp91^phox and to be an important epitope for the interaction with mAb 7D5, as analyzed by FACS analysis. In particular, amino acid R147 is a unique epitope on the membrane‐associated Cyt b for mAb 7D5. In conclusion, it is proposed that FACS analysis using mAb 7D5 is a valuable tool for early diagnosis of CGD.

     

Expression of components of the superoxide generating NADPH oxidase by human leucocytes and other cells

Summary NADPH oxidase of phagocytic leucocytes contains a membrane cytochromeb with two subunits, gp91 ^phox and p22 ^phox , together with three cytosolic proteins, p47 ^phox , p67 ^phox and p2 ^rac . The presence of some of these components has been sought in non-phagocytes, using Western blot analysis for protein expression and PCR to amplify and detect mRNA. All components were detected in EBV-transformed B lymphocytes and peripheral blood B lymphocytes. Fibroblasts and human kidney mesangial cells contained mRNA for p67 ^phox , p47 ^phox , and p22 ^phox but not gp91 ^phox . Levels of expression varied with growth conditions, but it appears possible than an isozyme of cytochromeb which lacks gp9 ^phox is present in these cells. Proteins of p47 ^phox and p67 ^phox were expressed, in low concentrations, in these two cell types. Expression of mRNA for p47 ^phox and p67 ^phox was found to be widespread in many cell types.Abbreviations IL-1 interleukin 1 - PMA phorbol myristate acetate - CGD chronic granulomatous disease - EBV-BL Epstein-Barr virus transformed B-lymphocytes - PBBL peripheral blood B lymphocytes     

Characterization of six novel mutations in the CYBB gene leading to different sub-types of X-linked chronic granulomatous disease

Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and so cannot generate superoxide anions (O₂^–). The most common form is caused by mutations in CYBB encoding gp91 phox, the heavy chain of flavocytochrome b₅₅₈ (XCGD). We investigated 11 male patients and their families suspected of suffering from X-linked CGD. These XCGD patients were classified as having different variants (X91⁰, X91^– or X91⁺) according to their cytochrome b₅₅₈ expression and NADPH oxidase activity. Nine patients had X91⁰ CGD, one had X91^– CGD and one had X91⁺ CGD. Six mutations in CYBB were novel. Of the four new X91⁰ CGD cases, three were point mutations: G65A in exon 2, G387T in exon 5 and G970T in exon 9, leading to premature stop codons at positions Try18, Try125 and Glu320, respectively, in gp91 phox. One case of X91⁰ CGD originated from a new 1005G deletion detected in exon 9. Surprisingly, four nonsense mutations in exon 5 led to the generation of two mRNAs, one with a normal size containing the mutation and the other in which exon 5 had been spliced. A novel X91^– CGD case with low gp91 phox expression was diagnosed. It was caused by an 11-bp deletion in the linking region between exon 12 and intron 12, activating a new cryptic site. Finally, a new X91⁺ CGD case was detected, characterized by a missense mutation Leu505Arg in the potential NADPH-binding site of gp91 phox. No clear correlation between the severity of the clinical symptoms and the sub-type of XCGD could be established.     

Regulation of the NADPH-oxidase complex of phagocytic leukocytes. Recent insights from structural biology,molecular genetics,and microscopy

The NADPH-oxidase complex is a multisubunit enzyme complex that catalyzes the formation of superoxide (O₂^–) by phagocytic leukocytes. This paper reviews some of the major advances in understanding the assembly and regulation of this enzyme system that have occurred during the past decade. For example, novel domains/motifs have been identified in p47-phox (PX and super SH3 domains) and p67-phox (tetratricopeptide repeat motifs). X-ray crystallography and NMR spectroscopy have provided detailed structural data on these domains and how p47-phox and p67-phox interact with p22-phox and activated Rac, respectively. Site-directed mutagenesis and knockout experiments have identified the critical phosphorylation sites in p47-phox, revealed an activation domain in p67-phox, and demonstrated that a specific pathway exists for activating Rac to participate in oxidase assembly/activation. Cytochemistry and immunofluorescence microscopy have provided new insights into the assembly of the oxidase and reveal a level of complexity not previously appreciated.John A. Badwey has recently died     

p47phox Phox Homology Domain Regulates Plasma Membrane but Not Phagosome Neutrophil NADPH Oxidase Activation

The assembly of cytosolic subunits p47^phox, p67^phox, and p40^phox with flavocytochrome b₅₅₈ at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47^phox subunit plays a critical role in oxidase assembly. Recent studies showed that the p47^phox Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47^phox PX domain in neutrophils is unclear, we investigated its role using p47^phox knock-out (KO) mouse neutrophils to express human p47^phox and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47^phox proteins were expressed at levels similar to endogenous murine p47^phox, with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47^phox rescued p47^phox KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47^phox with Arg⁹⁰ substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47^phox Arg⁹⁰ mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47^phox PX domain for neutrophil NADPH oxidase activation.     

A Fluorescently Tagged C-Terminal Fragment of p47phox Detects NADPH Oxidase Dynamics during Phagocytosis

The assembly of cytosolic p47^phox and p67^phox with flavocytochrome b₅₅₈ at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47^phox and p67^phox are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67^phox translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67^phox to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47^phox PRR. This fragment was recruited in a flavocytochrome b₅₅₈-dependent, p67^phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67^phox SH3b domain and suggest that the p47^phox/p67^phox tail-to-tail interaction is disrupted after oxidase assembly such that the p67^phox-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.     

Cloning of putative subunits of the soybean plasma membrane NADPH oxidase involved in the oxidative burst by antibody expression screening

Summary Plant cells respond to a variety of external signals with the production of reactive-oxygen species. The enzyme system generating these reactive-oxygen species is believed to be an NADPH oxidase located in the plasma membrane and sharing similarities with the NADPH oxidase from mammalian macrophages. Antibodies directed against individual subunits (p22 ^phox , p47 ^phox , p67 ^phox ) of the human NADPH oxidase cross-react with soybean proteins of a similar size and subcellular location. An extensive expression screening of a soybean cDNA-library with the anti-human NADPH oxidase antibodies gave a single class of cDNA-clones for each antibody. However, the sequence analysis of these clones clearly demonstrates that the different antibodies recognise proteins which are unrelated to the expected oxidase subunits. The anti-p22 ^phox antibody recognised a microsomal protein with no significant homology to any known protein in the database. One anti-p47 ^phox antibody cross-reacted with the UDP-glucose dehydrogenase and another antibody bound to the chaperon peptidyl prolyl-cis-trans isomerase, both soluble cytosolic proteins. The anti-p67 ^phox antibody detected the soluble enzyme acetohydroxy acid reductoisomerase. Chromatography of soybean protein extracts on an ion-exchange column (MonoQ, FPLC) gave a perfect comigration of the enzyme activity with the antibody signal, thus confirming these unexpected results by independent biochemical experiments.Abbreviations AARI acetohydroxy acid reductoisomerase - DPI diphenylene iodonium - GST glutathione-S-transferase - phox NADPH oxidase of phagocytes - ROS reactive-oxygen species     

Conformational changes in truncated p47phox proteins monitored by fluorescent labeling

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to O^– ₂ at the expense of NADPH. During activation, the cytosolic oxidase components p47^phox and p67^phox, each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane. p47^phox and p67^phox associate with cytochrome b₅₅₈, a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid, as an activating agent. Activators of the oxidase in vitro cause exposure of the SH3 domains of p47^phox, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the fluorescence exhibited by the covalently labeled N,N-di-methyl-N(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD) was increased when N-terminal-truncated p47^phox-(SH3)₂-C was treated with anionic amphiphiles. This finding was similar to the results obtained with the full-length p47^phox. However, the fluorescence of C-terminal-truncated p47^phox-N-(SH3)₂ and that of both C-terminal and N-terminal truncated p47^phox-(SH3)₂ were not altered by the activators. These results indicate that the C-terminal region of p47^phox is a primary target of the conformational change during the activation of NADPH oxidase.     

Adenosine A(2A) receptor activation limits chronic granulomatous disease-induced hyperinflammation

Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase complex and is characterized by an increased susceptibility to infection. Other significant complications of CGD include autoimmunity and non-infectious hyperinflammatory disorders. We show that a gp91^phox deficiency leads to the development of phenotypically altered T lymphocytes in mice and that this abnormal, hyperactive phenotype can be modulated by activation of the adenosine A_2A receptor. T cells isolated from CGD mice produce significantly higher levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF-α, IL-4 and IL-13 than do WT cells after TCR-mediated activation; treatment with the selective adenosine A_2A receptor agonist, CGS21680, potently inhibits this response. Additionally, the over exuberant inflammatory response elicited by thioglycollate challenge in gp91^phox deficient mice is attenuated by CGS21680. These data suggest that treatment with A_2AR agonists may be an effective therapy by which to regulate the immune system hyperactivity that results from a gp91^phox deficiency.     

A structural model for the nucleotide binding domains of the flavocytochrome b-245 beta-chain.

NADPH is a system in phagocytic cells that generates O2- and hydrogen peroxide in the endocytic vacuole, both of which are important for killing of the engulfed microbe. Dysfunction of this oxidase results in the syndrome of chronic granulomatous disease, characterized by a profound predisposition to bacterial and fungal infections. A flavocytochrome b is the site of most of the mutations causing this syndrome. The FAD and NADPH binding sites have been located on the beta subunit of this molecule, the C-terminal half of which showed weak sequence similarity to other reductases, including the ferredoxin-NADP reductase (FNR) of known structure. This enabled us to build a model of the nucleotide binding domains of the flavocytochrome using this structure as a template. The model was built initially using a novel automatic modeling method based on distance-matrix projection and then refined using energy minimization with appropriate side-chain torsional constraints. The resulting model rationalized much of the observed sequence conservation and identified a large insertion as a potential regulatory domain. It confirms the inclusion of the neutrophil flavocytochrome b-245 (Cb-245) as a member of the FNR family of reductases and strongly supports its function as the proximal electron transporting component of the NADPH oxidase.     

The regulation of superoxide production by the NADPH oxidase of neutrophils and other mammalian cells

Superoxide is produced by a NADPH oxidase of phagocytic cells and contributes to their microbicidal activities. The oxidase is activated when receptors in the neutrophil plasma membrane bind to the target microbe. These receptors recognise antibodies and complement fragments which coat the target cell. The oxidase electron transport chain, located in the plasma membrane, comprises a low potential cytochrome b heterodimer (gp 91-phox and p22-phox) associated with FAD. It is non-functional until at least three proteins, p67-phox, p47-phox and p21rac (and possibly others), move from the cytosol to dock on the cytochrome b. The docking involves the interaction of SH₃ domains may become exposed follwoing phosphorylation of p47-phox by protein kinase C or, in model systems, by addition of arachidonic acid to reconstitution mixtures. Following the docking process the electron-transporting component is able to transfer electrons from NADPH to oxygen. This electrogenic event is charge-compensated by the opening of a prton channel. Components of the oxidase are expressed in non-phagocytes, where their function is uncretain but could be related to some signal function of superoxide.     

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47^phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47^phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47^phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47^phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22^phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47^phox−/− coronary microvascular cells. Compared with wild-type p47^phox cDNA transfected cells, the single mutation of S379A completely blocked p47^phox membrane translocation, binding to p22^phox and endothelial O₂^⨪ production in response to acute stimulation of PKC. p47^phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47^phox conformational changes and NADPH oxidase-dependent superoxide production by cells.     

Superoxide Anion Production and Expression of gp91phox and p47phox Are Increased in Glomeruli and Proximal Tubules of Cisplatin‐Treated Rats

The chemotherapeutic drug cisplatin has some side effects including nephrotoxicity that has been associated with reactive oxygen species production, particularly superoxide anion. The major source of superoxide anion is nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. However, the specific segment of the nephron in which superoxide anion is produced has not been identified. Rats were sacrificed 72 h after cisplatin injection (7.5 mg/kg), and kidneys were obtained to isolate glomeruli and proximal and distal tubules. Cisplatin induced superoxide anion production in glomeruli and proximal tubules but not in distal tubules. This enhanced superoxide anion production was prevented by diphenylene iodonium, an inhibitor of NADPH oxidase. Consistently, this effect was associated with the increased expression of gp91^phox and p47^phox, subunits of NADPH oxidase. The enhanced superoxide anion production in glomeruli and proximal tubules, associated with the increased expression of gp91^phox and p47^phox, is involved in the oxidative stress in cisplatin‐induced nephrotoxicity.     

Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47phox-deficient chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O₂^?) and other microbicidal oxidants due to mutations in one of the five components of the O₂^?-generating NADPH oxidase complex. The most common autosomal subtype of CGD is caused by mutations in NCF1, encoding the NADPH subunit p47^phox. Usually, these mutations are the result of unequal exchange of chromatid between NCF1 and one of its two pseudogenes. We have now investigated in detail the breakpoints within or between these (pseudo) NCF1 genes in 43 families with p47^phox-deficient CGD by means of multiplex ligase-dependent probe amplification (MLPA). In 24 families the patients totally lacked NCF1 sequences, indicating that in these families the cross-over points are located between NCF1 and its pseudogenes. Six other families were compound heterozygous for a total NCF1 deletion and another mutation in NCF1 on the other allele. In 8 families, the patients lacked NCF1 exons 1–4 but had retained NCF1 exons 6–10, indicating that a cross-over point is located within NCF1 between exons 4 and 6. Similarly, in 4 families a cross-over point was located within NCF1 between exons 2 and 4. Similar cross-overs, in heterozygous form, were observed in family members of the patients. Several patients were compound heterozygous for total and partial NCF1 deletions. Thus, at least three different cross-over points exist within the NCF1 gene cluster, indicating that autosomal p47^phox-deficient CGD is genetically heterogeneous but can be dissected in detail by MLPA.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.