Regulation of the Neutrophil Respiratory Burst Oxidase

Superoxide generation by the neutrophil respiratory burst oxidase (NADPH oxidase) can be reconstituted in a cell-free system using flavocytochrome b 558 and the cytosolic proteins p47phox, p67phox, and Rac. p47phox functions as an adaptor protein; it increases the affinity of p67phox and Rac in the NADPH oxidase complex, but is not essential when high concentrations of these proteins are used (Freeman, J. L., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 22578–22582), implying that p67phoxand/or Rac directly regulates enzyme activity. Herein, we describe an activation domain in p67phox that is essential for NADPH oxidase activity. A series of C-terminal truncation mutants of p67phox showed that residues 211 to the C terminus (residue 526) are not needed for cell-free activity. However, shorter truncations were inactive, pointing to an activation domain within the region spanning residues 199–210. p67phox mutated at single amino acid residues within this region showed diminished activity, and p67phox V204A was completely inactive. The effects of mutations on activity were independent of p47phox, and mutations did not affect the binding of p67phox to Rac. In the presence of wild-type p67phox, the V204A mutant was a potent inhibitor of superoxide generation, and inhibition was partially reversed by high concentrations of p67phox, but not by p47phox or Rac. The V204A mutant competed with native p67phox for translocation to neutrophil plasma membrane, indicating that p67phox V204A assembles to form an inactive complex. The data imply a direct activation of flavocytochrome b 558 by an activation domain in p67phox. Superoxide generation by the neutrophil respiratory burst oxidase (NADPH oxidase) can be reconstituted in a cell-free system using flavocytochrome b 558 and the cytosolic proteins p47phox, p67phox, and Rac. p47phox functions as an adaptor protein; it increases the affinity of p67phox and Rac in the NADPH oxidase complex, but is not essential when high concentrations of these proteins are used (Freeman, J. L., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 22578–22582), implying that p67phoxand/or Rac directly regulates enzyme activity. Herein, we describe an activation domain in p67phox that is essential for NADPH oxidase activity. A series of C-terminal truncation mutants of p67phox showed that residues 211 to the C terminus (residue 526) are not needed for cell-free activity. However, shorter truncations were inactive, pointing to an activation domain within the region spanning residues 199–210. p67phox mutated at single amino acid residues within this region showed diminished activity, and p67phox V204A was completely inactive. The effects of mutations on activity were independent of p47phox, and mutations did not affect the binding of p67phox to Rac. In the presence of wild-type p67phox, the V204A mutant was a potent inhibitor of superoxide generation, and inhibition was partially reversed by high concentrations of p67phox, but not by p47phox or Rac. The V204A mutant competed with native p67phox for translocation to neutrophil plasma membrane, indicating that p67phox V204A assembles to form an inactive complex. The data imply a direct activation of flavocytochrome b 558 by an activation domain in p67phox. During the respiratory burst, neutrophils generate superoxide and secondarily generate reactive oxygen species (H2O2, O·̄2, and HOCl) that together participate in killing invading microorganisms (1Badwey J.A. Karnovsky M.L. Annu. Rev. Biochem. 1980; 49: 695-726Crossref PubMed Scopus (843) Google Scholar, 2Clark R.A. J. Infect. Dis. 1990; 161: 1140-1147Crossref PubMed Scopus (179) Google Scholar, 3Segal A.W. Abo A. Trends Biochem. Sci. 1993; 18: 43-47Abstract Full Text PDF PubMed Scopus (570) Google Scholar, 4Chanock S.J. el Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar). Superoxide generation is catalyzed by a multicomponent enzyme, the respiratory burst oxidase (NADPH oxidase). The enzyme consists of a plasma membrane-associated flavocytochrome b 558composed of two subunits, gp91phox (where phox isphagocytic oxidase) and p22phox. The former contains all of the electron-carrying groups (one FAD and two hemes) needed to transfer electrons from NADPH to molecular oxygen as well as a candidate NADPH-binding site (5Nisimoto Y. Otsuka-Murakami H. Lambeth D.J. J. Biol. Chem. 1995; 270: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 6Quinn M.T. Mullen M.L. Jesaitis A.J. J. Biol. Chem. 1992; 267: 7303-7309Abstract Full Text PDF PubMed Google Scholar, 7Segal A.W. West I. Wientjes F. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrace G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (291) Google Scholar, 8Sumimoto H. Sakamoto N. Nozaki M. Sakaki Y. Takeshige K. Minakami S. Biochem. Biophys. Res. Commun. 1992; 186: 1368-1375Crossref PubMed Scopus (111) Google Scholar). In addition, three cytosolic factors, p47phox, p67phox, and Rac, are needed for optimal activity. In resting cells, p47phox and p67phox reside in the cytosol in a 240-kDa complex with a third component, p40phox (9Wientjes F.B. Hsuan J.J. Totty N.F. Segal A.W. Biochem. J. 1993; 296: 557-561Crossref PubMed Scopus (262) Google Scholar, 10Someya A. Nagaoka I. Yamashita T. FEBS Lett. 1993; 330: 215-218Crossref PubMed Scopus (72) Google Scholar, 11Fuchs A. Dagher M.-C. Vignais P.V. J. Biol. Chem. 1995; 270: 5695-5697Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Upon cell activation, p47phox and p67phox associate with the membrane, where they interact with flavocytochrome b 558(12Clark R.A. Volpp B.D. Leidal K.G. Nauseef W.M. J. Clin. Invest. 1990; 85: 714-721Crossref PubMed Scopus (330) Google Scholar, 13Dinauer M.C. Pierce E.A. Bruns G.A.P. Curnutte J.T. Orkin S.H. J. Clin. Invest. 1990; 86: 1729-1737Crossref PubMed Scopus (267) Google Scholar, 14Heyworth P.G. Curnutte J.T. Nauseef W.M. Volpp B.D. Pearson D.W. Rosen H. Clark R.A. J. Clin. 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Assembly and activation can be reconstituted in a cell-free system consisting of the protein components, phospholipid, GTPγS, 1The abbreviations used are: GTPγS, guanosine 5′-O-(3-thiotriphosphate); Gpp(NH)p, guanosine 5′-(β,γ-imido)triphosphate; PCR, polymerase chain reaction; mant, methylanthraniloyl; PIPES, 1,4-piperazinediethanesulfonic acid. and an anionic amphiphile such as arachidonate (19Bromberg Y. Pick E. J. Biol. Chem. 1985; 260: 13539-13545Abstract Full Text PDF PubMed Google Scholar, 20Curnutte J.T. J. Clin. Invest. 1985; 75: 1740-1743Crossref PubMed Scopus (199) Google Scholar, 21Ishida K. Takeshige K. Takasugi S. Minakami S. FEBS Lett. 1989; 243: 169-172Crossref PubMed Scopus (12) Google Scholar, 22Ligeti E. Tardif M. Vignais P.V. Biochemistry. 1989; 28: 7116-7123Crossref PubMed Scopus (34) Google Scholar, 23Uhlinger D.J. Burnham D.N. Lambeth J.D. J. Biol. Chem. 1991; 266: 20990-20997Abstract Full Text PDF PubMed Google Scholar, 24Abo A. Boyhan A. West I. Thrasher A.J. 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A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar, 31Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar, 32Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). p47phox contains tandem SH3 (Srchomology 3) domains, and one or more of these bind to a proline-rich region in the C terminus of p22phox. p67phox also contains two SH3 domains in the C-terminal half of the molecule, and one or both of these mediate binding to proline-rich regions in p47phox in the assembled oxidase. p47phoxand p67phox bind cooperatively in the active complex (33Uhlinger D.J. Taylor K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 22095-22098Abstract Full Text PDF PubMed Google Scholar), and p47phox is needed for the stable assembly of p67phox(27Leto T. Adams A. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar, 33Uhlinger D.J. Taylor K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 22095-22098Abstract Full Text PDF PubMed Google Scholar, 34Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar). Rac interacts with the membrane through its isoprenylated C terminus (35Heyworth P.G. Knaus U.G. Xu X. Uhlinger D.J. Conroy L. Bokoch G.M. Curnutte J.T. Mol. Biol. Cell. 1993; 4: 261-269Crossref PubMed Scopus (112) Google Scholar), which in Rac1 contains a polybasic region (36Didsbury J. Weber R.F. Bokoch G.M. Evans T. Snyderman R. J. Biol. Chem. 1989; 264: 16378-16382Abstract Full Text PDF PubMed Google Scholar). Membrane binding is important for stimulation of high rates of superoxide generation in a cell-free system (37Kreck M.L. Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 4161-4168Abstract Full Text PDF PubMed Google Scholar). In addition, Rac contains an effector region (residues 26–45) that interacts in a GTP-dependent manner with a binding site in the N terminus of p67phox (38Diekmann D. Abo A. Johnson C. Segal A. Hall A. Science. 1994; 265: 531-533Crossref PubMed Scopus (349) Google Scholar, 39Nisimoto Y. Freeman J.L.R. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Another region (the “insert region,” residues 124–135) is important for activation of superoxide generation by Rac (40Freeman J.L. Abo A. Lambeth J.D. J. Biol. Chem. 1996; 271: 19794-19801Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar); this region has been proposed to interact directly with the flavocytochrome (39Nisimoto Y. Freeman J.L.R. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Thus, a complex set of interactions among multiple proteins governs assembly of the active NADPH oxidase. Although knowledge about the protein interactions within the NADPH oxidase complex is becoming increasingly detailed, specific roles for individual cytosolic components have remained obscure. According to one hypothesis, p67phox regulates electron transfer in flavocytochrome b 558 from NADPH to FAD, whereas p47phox controls electron flow from FAD through the heme groups to oxygen (41Cross A.R. Yarchover J.L. Curnutte J.T. J. Biol. Chem. 1994; 269: 21448-21454Abstract Full Text PDF PubMed Google Scholar, 42Cross A.R. Curnutte J.T. J. Biol. Chem. 1995; 270: Full Text PDF PubMed Scopus Google Scholar). However, or rates of superoxide generation can be reconstituted in the of p47phox using high concentrations of p67phox and Rac J.L. Lambeth J.D. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, O. Pick E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). p47phox as an adaptor protein to the binding of the other two components by J.L. Lambeth J.D. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, p47phox not to in electron and p67phox or Rac is directly in the Herein, an activation domain in p67phox is and a is proposed in which binding of p67phox to both p47phox and Rac this domain in such a as to interact with and FAD, GTPγS, Gpp(NH)p, and were from in was from and was from was from and were from was from was from were and by the neutrophils were from from as S.J. PubMed Google Scholar). were as D.N. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). of b 558 from plasma membrane was as (5Nisimoto Y. Otsuka-Murakami H. Lambeth D.J. J. Biol. Chem. 1995; 270: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). p47phox and wild-type p67phox were in and to Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1993; 268: 8624-8631Abstract Full Text PDF PubMed Google Scholar, 33Uhlinger D.J. Taylor K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 22095-22098Abstract Full Text PDF PubMed Google Scholar). Rac was in as a protein and using by (37Kreck M.L. Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 4161-4168Abstract Full Text PDF PubMed Google Scholar). A series of and mutated of p67phox were in with by as (38Diekmann D. Abo A. Johnson C. Segal A. Hall A. Science. 1994; 265: 531-533Crossref PubMed Scopus (349) Google Scholar), and to concentrations were to Biochem. PubMed Scopus Google Scholar). A series of p67phox were by using p67phox in as the all the was to to the of the p67phox and to a site in by an the for was to to the p67phox to the region to be and to the and an site in the as was used with the as that the the wild-type a in the terminus of were the of the and for of the The were to that mutations were and to the Rac1 was with for to form the complex as (39Nisimoto Y. Freeman J.L.R. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The complex was with or p67phox, and the at was at were to a single site binding as R.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), for and Superoxide generation was by superoxide of as D.N. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google using a Rac was with of for at in the of (37Kreck M.L. Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 4161-4168Abstract Full Text PDF PubMed Google Scholar). the cell-free of plasma membrane protein, p47phox, Rac, GTPγS, and arachidonate in a of of were to and for at of NADPH and A and PIPES, was to the was by the at using an of J.D. Burnham D.N. Tyagi S.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the activity of reconstituted 558 in the of p47phox, the mutant p67phox, Rac with GTPγS, b and The was with arachidonate as D. Yeung C.L. Katkin J.P. J. Biol. Chem. 1993; 268: 14256-14260Abstract Full Text PDF PubMed Google Scholar). The was at for by the of NADPH and were by a of the of Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1993; 268: 8624-8631Abstract Full Text PDF PubMed Google Scholar). The of plasma membrane protein, p47phox, of p67phox Rac, GTPγS, and arachidonate in of A. The was at for and a of and in The were for at in a at in a the components was spanning the and the plasma membrane D.J. Inge K.L. M.L. Tyagi S.R. Neckelmann N. Lambeth J.D. Biochem. Biophys. Res. Commun. 1992; 186: PubMed Scopus (34) Google was from were with acid using as a The of was to with and of and of acid were The was on for and for at The was with and in of and the was to with The were on a and to a D.J. Inge K.L. M.L. Tyagi S.R. Neckelmann N. Lambeth J.D. Biochem. Biophys. Res. Commun. 1992; 186: PubMed Scopus (34) Google was using p67phox to p67phox was used for native p67phox, whereas to p67phox was used for V204A of by the was to the we showed that an form of both SH3 domains partially for p67phox to the respiratory burst whereas superoxide generation J.L. Lambeth J.D. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). on these a series of were and A and to the p67phox that cell-free superoxide In a of superoxide generation to of the with p67phox, as J.L. Lambeth J.D. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). shorter of the protein, and superoxide generation at the as wild-type p67phox Thus, the region from residues to is inhibitory to the of p67phox. of the proline-rich region (residues in which has been in binding to p47phox and translocation of p67phox to the membrane (27Leto T. Adams A. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar, 30Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar), did not the of p67phox to superoxide generation of the protein shorter amino acid residues to of superoxide Thus, p67phox contains an activation domain within the region spanning amino acid residues 199–210. mutant of were and mutant proteins were on residues within amino were mutated to whereas the two residues were to proteins were for to NADPH oxidase activity in the cell-free The was using the cell-free which p47phox, and plasma membrane the of and in a which p47phox and b 558 In both mutations within the region from residues to in a in the of p67phox to superoxide In p67phox V204A showed or to superoxide generation, and the showed of the wild-type activity. was independent of p47phox, indicating that the effects of these are not a of binding of p67phox to p67phox was to a site within the region by residues (38Diekmann D. Abo A. Johnson C. Segal A. Hall A. Science. 1994; 265: 531-533Crossref PubMed Scopus (349) Google Scholar). the presence of the activation domain was Rac binding to p67phox, we the binding (39Nisimoto Y. Freeman J.L.R. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). a of that to Rac1 and as a an in (39Nisimoto Y. Freeman J.L.R. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The concentrations of p67phox, and were as in and the in was three of p67phox Rac with about the p67phox and with of and Thus, the of the activation domain did not affect the of p67phox to bind to Rac. Superoxide generation was in the cell-free system wild-type in the of p67phox V204A to or in inhibition of superoxide Thus, in an in p67phox V204A functions as a for superoxide the of the of p47phox, wild-type p67phox, or Rac1 was in A and the of p47phox or Rac1 did not the inhibitory of p67phox p67phox V204A contains a a for inhibition is that the mutant protein with Rac in in this were the Rac the Thus, p67phox V204A be by this p67phox V204A not to be by In an of wild-type p67phox partially reversed the inhibitory of p67phox The for these data is that wild-type p67phox and with one in the NADPH oxidase complex and that the assembled complex p67phox V204A is inactive. not that p67phox V204A activity assembly the system wild-type by for with p67phox V204A at the of the superoxide when the system p67phox V204A was with wild-type p67phox was to the that it has a high affinity for the NADPH oxidase complex, p67phox on and the complex used a to that p47phox and p67phox are in the plasma membrane cell-free activation, that this is the presence of and that binding with a among p47phox, p67phox, and 558 D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1993; 268: 8624-8631Abstract Full Text PDF PubMed Google Scholar). this to directly p67phox V204A with wild-type p67phox for binding in the active NADPH oxidase complex. p67phox V204A and p67phox were from the plasma membrane by and by in concentrations of p67phox V204A in of wild-type p67phox in the plasma membrane which of p67phox V204A in the plasma membrane The of wild-type p67phox in the plasma membrane at concentrations of p67phox V204A was to the in this in the of not indicating that of the was to on with we that of wild-type to the plasma membrane In the presence of the of p67phox this is to whereas of p67phox V204A is with the Thus, the of wild-type mutant p67phox with the membrane is indicating that p67phox V204A with wild-type p67phox for binding in the assembled NADPH oxidase complex. A of the of protein at a membrane such as the plasma these an effector enzyme proteins functions are are for the of these protein and two or more protein components together within a small or on a the of as is the for the of the of complex to the plasma membrane by binding to on the The protein an for which is in the plasma of the effector enzyme activity by with other The of with the protein is of as an of this of and not and but also protein, membrane, and other proteins K. S. B. S. K. T. T. K. K. Y. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). The respiratory burst oxidase an of activation, and the to individual roles for of the cytosolic Herein, we have an activation domain in p67phox that we is directly in NADPH oxidase The domain was to amino acid residues on a series of mutants of p67phox. of p67phox the amino to the respiratory burst whereas shorter this were The activation domain was using a series of mutations within amino Although of of the residues within this activity, the at was important its in a of The by which and residues is not can be region is not in binding interactions with the other cytosolic of mutations in this region were the or not p47phox was with p47phox as an for the of the activation the binding of p67phox to Rac was the or not the activation domain of p67phox was Thus, it is that this region of p67phox directly interacts with and regulates the electron transfer from NADPH to oxygen within flavocytochrome b The specific electron transfer that are by the activation domain are not is in The a for the roles of the cytosolic in the respiratory burst oxidase. In to that directly with the proline-rich region in the small of b 558 the have that p47phox functions as a adaptor protein, the of which is to the binding of the other two cytosolic components J.L. Lambeth J.D. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). the binding on of p67phox by and that of Rac by Thus, p47phox a binding site for it with the of (the in to the plasma membrane in the of p47phox P.G. Bohl B.P. Bokoch G.M. Curnutte J.T. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google and with (16Quinn M.T. Evans T. Loetterle L.R. Jesaitis A.J. Bokoch G.M. J. Biol. Chem. 1993; 268: 20983-20987Abstract Full Text PDF PubMed Google Scholar, Malech Leto T.L. Biochemistry. 1994; PubMed Scopus Google Scholar). to the plasma membrane is in by and from the inhibitor protein RhoGDI in the have that its with the plasma membrane is important for activity (37Kreck M.L. Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 4161-4168Abstract Full Text PDF PubMed Google and that the effector region (residues 26–45) in Rac as a binding site for p67phox (39Nisimoto Y. Freeman J.L.R. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Rac contains an the that is important for assembly J.L.R. M.L. Uhlinger D.J. Lambeth J.D. Biochemistry. 1994; PubMed Scopus Google and that was proposed to interact directly with the (39Nisimoto Y. Freeman J.L.R. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Thus, both Rac and p47phox are the of and and both binding for p67phox. that these regions bind and p67phox in such a as to the activation domain in p67phox with a region on the The of this domain with 558 electron flow from NADPH to molecular a is in that it roles to of the cytosolic and a by which effector are by complex with multiple Hall for the and