The cytosolic component p47(phox) is not a sine qua non participant in the activation of NADPH oxidase but is required for optimal superoxide production.

The superoxide (O2)-generating NADPH oxidase of phagocytes is a multicomponent complex consisting of a membrane-associated flavocytochrome (cytochrome b559), bearing the NADPH binding site and two redox centers (FAD and heme) and three cytosolic activating components: p47phox, p67phox, and the small GTPase Rac (1 or 2). The canonical view is that the induction of O2 generation involves the stimulus-dependent assembly of all three cytosolic components with cytochrome b559, a process mimicked in vitro by a cell-free system activated by anionic amphiphiles. We studied the requirement for individual cytosolic components in the activation of NADPH oxidase in a cell-free system consisting of purified and relipidated cytochrome b559, recombinant p47phox, p67phox, and Rac1, and the amphiphile, lithium dodecyl sulfate. We found that pronounced activation of NADPH oxidase can be achieved by exposing cytochrome b559 to p67phox and Rac1, in the total absence of p47phox (turnover = 60 mol O2/s/mol cytochrome b559). However, maximal activation (turnover = 153 mol O2/s/mol cytochrome b559) could only be obtained in the presence of p47phox. O2 production, in the absence of p47phox, was dependent on: high molar ratios of p67phox and Rac1 to cytochrome b559, Rac1 being in the GTP-bound form, cytochrome b559 being saturated with FAD, and an optimal concentration of amphiphile. Single cytosolic components or combinations of two cytosolic components, other than p67phox and Rac1, were incapable of activation. We conclude that p67phox and Rac1 are the only cytosolic components directly involved in the induction of electron transport in cytochrome b559. p47phox appears to facilitate or stabilize the interaction of p67phox and, possibly, Rac1 with cytochrome b559, and is required for optimal generation of O2 under physiological conditions.

The production of oxygen radicals, in response to triggering of cell-surface receptors, is the paramount microbicidal mechanism of phagocytic cells. The primary reaction in this process is the generation of superoxide (O 2 . ), 1 derived by the one-electron reduction of molecular oxygen, catalyzed by a membranebound heterodimeric flavocytochrome (cytochrome b 559 ), utilizing NADPH as the source of electrons (reviewed in Refs. [1][2][3]. Electron flow from NADPH to oxygen is activated consequent to the interaction of cytochrome b 559 with three cytosolic proteins, p47 phox , p67 phox , and the small GTPase Rac (1 or 2), leading to the formation of a multimolecular structure, known as the NADPH oxidase complex (reviewed in Ref. 4). The elicitation of an oxidative burst in intact phagocytes is accompanied by the translocation to the plasma membrane of about 10% of p47 phox and p67 phox (5). Translocation of p47 phox to the membrane is linked to the phosphorylation of the component at multiple sites (6). Also, movement of p47 phox and p67 phox to the membrane requires the presence of cytochrome b 559 (7). It is of interest that membrane localization of p67 phox is dependent on p47 phox (7), suggesting that at least one of the functions of p47 phox is to escort p67 phox to the membrane. Whether activation or an oxidative burst is associated with translocation of Rac to the membrane is still controversial (8,9). A number of studies dealt with the interaction of cytosolic components with the cytoskeleton (10,11). Of special relevance to the subject of the present communication is the finding that, in preparations obtained from both resting and stimulated leukocytes, p47 phox is detected in the soluble fraction, whereas p67 phox is associated with the cytoskeleton, which is also the site of O 2 . production (11). The activation of NADPH oxidase can be mimicked in vitro by a cell-free system consisting, in its most basic form, of membranes and the cytosolic fraction of resting phagocytes exposed to a critical concentration of some anionic amphiphiles (12)(13)(14)(15)(16). Complete reconstitution of a functioning NADPH oxidase can be achieved in cell-free systems consisting exclusively of highly purified or recombinant components (17)(18)(19). The most commonly utilized version of this is the semi-recombinant system, consisting of purified relipidated and reflavinated cytochrome b 559 combined with recombinant p47 phox , p67 phox , and Rac (18). It is commonly accepted that the cell-free system contains all the basic components necessary for NADPH-dependent O 2 . generation, but is lacking at least one cytosolic component, probably active in the intact cell (p40 phox ) (20), and all signal transduction and regulatory elements. Experimental evidence has, so far, supported the canonical view that NADPH oxidase activation in the cell-free system is an "all or none" phenomenon, in the sense that all three cytosolic components, in rigorous molar proportions, are required for the activation of electron flow across the redox centers of cytochrome b 559 . In spite of recent advances in our understanding of the nature of protein-protein interactions among components, leading to the assembly of the NADPH oxidase complex (reviewed in Ref. 4), the molecular basis of the ensuing activation of electron transport in cytochrome b 559 remains obscure. Three pieces of evidence suggest that not all cytosolic components are directly involved in the "activation" of cytochrome b 559 or that individual components might have distinctive roles in this process. First, O 2 . production could be induced in solubilized membranes (21) or purified and relipidated cytochrome b 559 (22,23) by phosphatidic acid, in the absence of cytosolic components. Second, the finding (11) that p47 phox was absent from the cytoskeletal fraction of activated neutrophils, which contained the bulk of O 2 . forming activity, was difficult to reconcile with the concept that p47 phox is more than a transient constituent of the NADPH oxidase complex. Finally, Cross et al. (24) demonstrated that the activated NADPH oxidase possessed a diaphorase activity, which was dependent on p67 phox but not on p47 phox . They went on to show that the two cytosolic components have unequal roles in the regulation of electron flow in cytochrome b 559 ; p67 phox stimulated the flow from NADPH to FAD, whereas p47 phox was required for electron transport from FAD to heme and oxygen (25). The subject of this paper is the finding that purified relipidated and reflavinated cytochrome b 559 generates high amounts of O 2 . when combined in vitro with p67 phox , Rac1, and an anionic amphiphile, in the total absence of p47 phox .

EXPERIMENTAL PROCEDURES
Purification of Cytochrome b 559 -Cytochrome b 559 was purified from guinea pig peritoneal macrophage membranes, solubilized by n-octyl-␤-D-glucopyranoside (26), by a recent modification (27) of the original procedures (22,28), and had a specific content of 4 -6 nmol of heme/mg of protein. The material was concentrated by ultrafiltration to a concentration of 2-3 M and relipidated with 200 g/ml L-␣-phosphatidylcholine (type IV-S, Sigma) by dialysis against detergent-free buffer (27). The material was found free of possible contamination by cytosolic components by immunoblotting with polyclonal antibodies raised against purified recombinant p47 phox and p67 phox (29) (kind gifts of Dr. Thomas L. Leto) and with a polyclonal antibody to Rac1 (sc-217, Santa Cruz Biotechnology).
Preparation of Recombinant Cytosolic NADPH Oxidase Components-p47 phox and p67 phox were prepared in Sf9 cells, infected with baculoviruses carrying cDNAs for human cytosolic components (kind gifts of Dr. Thomas L. Leto), by a modification of the procedure described by Leto et al. (29). The differences from the original procedure were: the Sf9 cells were grown as suspension cultures in serum-free medium (SF-900 II SFM, Life Technologies); the cells were disrupted by sonication; purification of p67 phox on Q-Sepharose was done at pH 7.5, and purification of p47 phox , by cation exchange chromatography, was performed on SP-Sepharose (HighLoad 16/10, Pharmacia Biotech Inc.). The purified recombinant proteins were analyzed by SDS-polyacrylamide gel electrophoresis; p67 phox was 90% pure, whereas p47 phox was 99% pure. Both p67 phox and p47 phox were recognized, in an enzymelinked immunosorbent assay (30), by antibodies to p67 phox and p47 phox (29), respectively, and no evidence was found, by this methodology, for cross-contamination between the two recombinant proteins. Recombinant Rac1 was isolated from Escherichia coli transformed with Rac1 cDNA subcloned into the bacterial expression vector pGEx2T (a kind gift of Dr. Thomas L. Leto), as described by Kwong et al. (31). The preparation was found to be 99% pure and free of contamination by p47 phox and p67 phox , as tested by enzyme-linked immunosorbent assay. Nucleotide exchange on Rac1 to guanosine 5Ј-3-O-(thio)triphosphate (GTP␥S) was performed as described previously (32).

RESULTS AND DISCUSSION
We found that O 2 . was generated by a cell-free system consisting of purified and relipidated cytochrome b 559 , p67 phox , and Rac1 in the GTP␥S-bound form, activated by the anionic amphiphile, LiDS. The remarkable feature of this form of NADPH oxidase activation is that it occurs in the total absence of the cytosolic component, p47 phox . Fig. 1 illustrates a characteristic experiment, in which O 2 . production by the complete cell-free system, consisting of cytochrome b 559 combined with all three cytosolic components (trace 1), was compared with that proceeding in the absence of p47 phox (trace 2), Rac1 (trace 3), p67 phox (trace 4), or both p47 phox and Rac1 (trace 5). It is evident that the combination of cytosolic components p67 phox and Rac1-GTP␥S is both necessary and sufficient for the activation of electron transport in cytochrome b 559 from NADPH to oxygen, leading to the production of O 2 . , although the presence of p47 phox was required for maximal O 2 . production. The process was dependent on: the presence of NADPH (a K m for NADPH of 40 M was found), saturation of cytochrome b 559 with FAD (a 3-fold molar excess of FAD was present in the activation mixture), and an optimal concentration of LiDS. Heat denaturation of either p67 phox or Rac1 (95°C, 3 min) resulted in a total loss of NADPH oxidase activating ability. Recombinant Rac1 not subjected to exchange to GTP␥S was also inactive. The simultaneous presence of p67 phox and Rac1 during coincubation with cytochrome b 559 , in the presence of LiDS, was an absolute  Table I, turnover rates reaching 40% of those obtained in the complete cell-free system, were achieved by activation of cytochrome b 559 with the combination of p67 phox and Rac1, whereas single cytosolic components or all other combinations of two cytosolic components elicited turnover rates of less than 1.9% of those measured in the complete system. Fig. 2 illustrates the dose-response dependence of O 2 .
production, in the p47 phox -deficient cell-free system, on the concentration of p67 phox (A) and Rac1 (B) in the presence of a constant amount of cytochrome b 559 and a saturating amount of the second cytosolic component. It is apparent that maximal O 2 . production is obtained at molar excesses over cytochrome b 559 of 40:1, for p67 phox and 15:1 for Rac1. The lipid composition of the cytochrome b 559 liposomes utilized in the present experiments was identical to that described in our original reports on the methodology of cytochrome b 559 relipidation (26,28) and to that of liposomes described in other reports on the semirecombinant cell-free system, in which an absolute requirement for three cytosolic components was demonstrated (17,19).
These results are best explained by a novel interpretation of the mechanism of NADPH oxidase assembly. We propose that the induction of electron flow in cytochrome b 559 is mediated by the cooperative action of p67 phox and Rac1, in the GTP-bound state, and that p47 phox is not directly involved in this process. This proposal is compatible with a number of earlier findings. These are: (a) p67 phox is found in the cytosol of phagocytes as a complex with p47 phox (33,34), and it was suggested that they may translocate en bloc to the plasma membrane (34); (b) translocation of p67 phox to the cell membrane is absolutely dependent on the presence of p47 phox (7); (c) protein-protein interactions between p67 phox and p47 phox have been shown to be mediated by bidirectional affinities of SH3 domains on one component for proline-rich domains on the other component (35)(36)(37)(38)(39)(40), and, recently, also by what appears to be a chargerelated mechanism (41). Whereas some of these interactions are constitutive, others are enhanced by stimuli eliciting O 2 . generation (40); (d) stimulus-dependent translocation of p67 phox to the membrane in intact cells requires the interaction of its NH 2 -terminal domain (residues 1-246) with the two SH3 domains of p47 phox (40). It is of interest that the same aminoterminal domain of p67 phox , which lacks both SH3 domains, is sufficient for cell-free NADPH oxidase activation (36); (e) whereas there is an abundance of data on the molecular basis of the interaction between p47 phox and cytochrome b 559 , involving SH3 (  . production by a semirecombinant cell-free system in the presence and absence of p47 phox Cell-free activation of NADPH oxidase was performed as described under "Experimental Procedures." The concentration of cytochrome b 559 varied from 0.05 to 0.08 M. Recombinant components were added in the molar ratios to cytochrome b 559 indicated in the footnotes. Turnover rates represent means Ϯ S.E., derived from the number of experiments indicated in parentheses. Composition of cell-free system Turnover report by Uhlinger et al. (46), proposing, on the basis of kinetic analysis of NADPH oxidase activation in a semirecombinant cell-free system, that p67 phox interacts directly with cytochrome b 559 ; (f) there is evidence (47,48) for constitutive binding of Rac1 to the amino-terminal region of p67 phox , a domain also involved in stimulus-dependent interaction with the SH3 domains of p47 phox , and it was recently shown that p67 phox is required for the stimulus-elicited translocation of Rac1 to the membrane (49); (g) finally, indirect evidence was presented for an interaction between Rac2 and cytochrome b 559 by the finding of reduced membrane localization of Rac2 in cells of chronic granulomatous disease (CGD) patients lacking cytochrome b 559 (50). We conclude that, in a cell-free setting, a marked level of NADPH oxidase activation can be achieved in the absence of p47 phox . It is likely that the unassisted interaction of p67 phox (and Rac1?) with cytochrome b 559 is either of low affinity or possesses a high dissociation rate. p47 phox , by virtue of its ability to bind to both cytochrome b 559 and p67 phox , by molecular affinities enhanced by in vivo or in vitro activating mechanisms (37,40), serves as a bridge leading to the establishment of a stable bond. It is also possible that binding of p47 phox to p67 phox and cytochrome b 559 is not simultaneous but sequential, as recently proposed in another context (41).
Assembly of the NADPH oxidase complex in the absence of p47 phox is clearly nonphysiological, as demonstrated by the impairment of O 2 . production by phagocytes of patients with the p47 phox -deficient form of CGD, and there is little doubt that, in the intact cell, p47 phox is required for optimal O 2 . production. It is, however, significant, that CGD patients, lacking p47 phox , have a milder form of disease and higher intracellular oxidant production than those lacking cytochrome b 559 (cited in Ref. 25). The most likely reason for our observation not having been made earlier is that its occurrence is dependent on p67 phox and Rac1 being present in marked molar excesses in relation to cytochrome b 559 . This requirement is overcome, under physiological conditions, by the "bridging" action of p47 phox .
Note Added in Proof-While this manuscript was being prepared for printing we became aware of the report by Freeman and Lambeth (51), describing the activation of NADPH oxidase in vitro independently of p47 phox .