Báo cáo Y học: Exploration of the diaphorase activity of neutrophil NADPH oxidase Critical assessment of the interaction of iodonitrotetrazolium with the oxidase redox components doc

Exploration of the diaphorase activity of neutrophil NADPH oxidase Critical assessment of the interaction of iodonitrotetrazolium with the oxidase redox components Alexandra Poinas 1 , Jacques Gaillard 2 , Pierre Vignais 1 and Jacques Doussiere 1 1 Laboratoire de Biochimie et Biophysique des Syste ` mes Inte ´ gre ´ s, UMR 5092 CEA-CNRS, De ´ partement de Biologie Mole ´ culaire et Structurale Grenoble, France; 2 De ´ partement de Recherche Fondamentale sur la Matie ´ re Condense ´ e SCIB-SCPM, CEA-Grenoble, France In the O 2 – generating flavocyto chrome b, t he membrane- bound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O 2 in the fol- lowing sequence: NADPH fi FAD fi heme b fi O 2 . Although p-iodonitrotetrazolium (INT) has frequently been used as a probe of the d iaphorase a ctivity o f t he neutrophil flavocytochrome b , the propensity of its radical to interact reversibly w ith O 2 led us to question its specificity. This study was undertaken to reexamin e the interaction of INT with the redox components of t he neutrophil flavocytochrome b . Two series of inhibitors were used, n amely the flavin analog 5-deaza FAD and the heme inhibitors bipyridyl and ben- zylimidazole. The following re sults indicate that INT reacts preferentially with the hemes rather than with the FAD redox center of flavocytochrome b and is not therefore a specific probe of the diaphorase activity of flavocyto- chrome b . First, i n a naerobiosis, reduced he me b in activa- ted m embranes was reoxidized by INT as efficiently a s by O 2 even in the presence of concentrations of 5-deaza FAD which fully inhibited t he NADPH oxidase activity. S econd, the t itration curve of dithionite-reduced heme b in neutro- phil membranes obtained by oxidation with increasing amounts of INT was strictly superimposable on that of dithionite-reduced hemin. Third, INT competitively inhib- ited the O 2 uptake by the activated NADPH oxidase in a cell-free system. Finally, the heme inhibitor bipyridyl com- petitively inhibited the reduction of INT in anaerobiosis, a nd the o xygen uptake in a erobiosis. Keywords: diaphorase; INT reductase; NADPH oxidase; neutrophils; fl avocytochrome b. Upon activation, the n eutrophil NADPH oxidase c omplex generates the superoxide anion O 2 – from which are derived microbicidal oxygen species, such as hydrogen peroxide and hypochloride. The active NADPH oxidase complex consists of a membrane-bound flavocytochrome b made of two subunits, gp91phox and p22phox (phox for phagocyte oxidase), and water-soluble proteins of cytosolic origin (p67phox, p47phox, p40phox and Rac 1/2) [1]. A defect in any o f the genes encoding gp91ph ox, p22phox, p47phox or p67phox results in chronic granulomatous disease (CGD) [2]. Physiological activation of NADPH oxidase can be mimicked byusing a cell-free system with flavocytochrome b, p47phox, p67phox, Rac, GTP and arachidonic acid as basic components. The large subunit of flavocytochrome b, gp91phox, contains all of t he redox components necessary for electron transfer from NADPH to O 2 , namely FAD and two hemes [3–6]. Like the yeast FRE1 reductase, the b cytochrome of the mitochondrial bc 1 complex and cyto- chrome b 6 of the b 6 f complex in chloroplasts, gp91phox contains mu ltiple hydrophobic domains, consistent with transmembrane a helices, and two pairs of histidine residues in these h ydrophobic domains, separated by 1 3 intervening amino acids (quoted from [7]). Based on these considera- tions, it has been postulated that the two hemes located in the N-terminal domain of gp91phox are coordinated by two pairs of histidine residues within two distinct a helices [7]. One of them (heme 1) is close to the c ytosolic face of the membrane, the other (heme 2 ) is o n the opposite s ide of t he membrane. The C-terminal region of gp91phox, which consists of predominantly hydrophilic amino a cid residues, is extramembranous a nd exposed t o the cytoso l. It co ntains binding sites for NADPH a nd FAD. The FAD binding site is thought to be in the close neighborhood of heme 1. The topographical a ssignment of the redox centers of gp91phox in this model indicates that electrons are transported from NADPH to O 2 across the membrane via a chain of redox components in the following sequence: NADPH fi FAD fi heme 1 fi heme 2 fi O 2 . Consistent with the presence of two distinct domains in gp91phox are reports showing that gp91phox may act as a diaphorase in the presence of appropriate electron acceptors such as dichlo- rophenol indophenol [8] or p-iodonitrotetrazolium violet (INT) [9–11]. Not only the oxidase a ctivity, but also the diaphorase activity required activation for full elicitation [8,9]. From these s tudies emerged the idea th at th e electron flux along the redox components of flavocytochrome b is Correspondence to J. Doussiere DBMS/BBSI, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France. Fax: +33 4 76 88 51 85, Tel.: +33 4 76 8 8 34 76, E-mail: jdoussiere@cea.fr Abbreviations: INT, P-iodonitrotetrazolium; NBT, nitroblue tetrazolium; 5-deaza FAD, 5 deazaflavin adenine dinucleotide; SOD, superoxide dismutase; CGD, chronic granulomatous disease; phox, phagocyte oxidase. (Received 2 4 September 20 01, revised 30 November 2001, a ccepted 3 January 2002) Eur. J. Biochem. 269, 1243–1252 (2002) Ó FEBS 2002 regulated both at t he level of the NA DPH-FAD portion of the electron transfer chain and at the leve l of he me b [12,13]. This idea was supported by the finding that neutrophils of a CGD (91X + ) patient with a point mutation (Arg54Ser) in the g p91phox subunit of flavocyto chrome b , a lthough unable to reduce O 2 into O 2 – despite the presence of heme b in gp91p hox, retained the capacity to reduce INT [14]. It was also shown th at electron tran sfer from NADPH to INT and from NADPH to O 2 could be activated independently of each other, depending on the presence of p67phox and p47phox [10,12]. From these data it appeared that measu- rement of INT r eductase could b e t aken as an index of the diaphorase activity o f flavocytochrome b. However, a recent paper [15] called a ttention to the possibility of the nonen- zymatic univalent reduction of te trazolium salts, particularly INT by O 2 – with concomitant production of the tetrazolium radical. In addition, t he INT r adical in an a erated medium can reduce O 2 to O 2 – [16,17]. These observations suggested that under certain circumstanc es INT did n ot probe the diaphorase activity of the NADPH oxidase. The present paper describes experiments in which INT and O 2 were compared for their ability to acce pt electrons from activated flavocytochrome b , u sing neutrophil membranes pretreated with 5-deaza FAD, an FAD analog inefficien t in electron transfer in flavocytochrome b , and with benzylimidazole and bipyridyl as heme inhibitors. The results show that INT is able to directly o xidize reduced heme b. EXPERIMENTAL PROCEDURES Materials NADPH, ATP, GTPcS were f rom Boehringer; horse heart cytochrome c type III, arachidonic a cid, dimethanesulfox- ide, diisopropyl fluorophosphate, benzylimidazole and hemin were f rom Sigma. I NT was from Amresco. 5 -Deaza FAD was a gift from V. Massey, Medical School Ann Harbor Michigan (USA). Biological preparations Neutrophil membranes and cytosol were prepared from bovine neutrophils in saline p hosphate buffer (NaCl/P i ) composed of 2.7 m M KCl, 136.7 m M NaCl, 1 .5 m M KH 2 PO 4 and 8.1 m M Na 2 HPO 4 , pH 7 .4 supplemented with 1 m M diisopropyl fluorophosphate and 1 m M EDTA [13]. Protein concentration was assayed with the BCA reagent u sing BSA as standard. P urified flavocytochrome b in detergent was obtained as reported p reviously [18]. Preparation of INT radical INT (60 mg) was solubilized in 1 mL of a mixture of dimethyl sulfoxide/H 2 O (2 : 1, v /v). The oxid iz ed INT was reduced by a few grains of sodium dithionite. The pale yellow s olution became rapidly orange, w hich is typical of the INT radical. This was immediately followed by four sequential extractions of INT by 2 mL chloroform. After each extraction, the mixture was centrifuged at 1000 g for 2 m in. The chloroform solutions co ntaining INT were collected and poole d. After evaporation under a flow of nitrogen, t he dry residue (57 mg) was taken up in 2 m L dimethyl sulfoxide and kept at )20 °C under a rgon. Assay of oxidase and INT diaphorase activities NADPH oxidase activity was a ssayed in a cell-free system [13], either by m easurement of the rate of p roduction of O 2 – or the rate of O 2 uptake. INT d iaphorase activity was assayed by the rate of reduction of INT into fo rmazan in the presence of superoxide dismutase ( SOD) or under anaero- biosis. In all cases, the assay of oxidase activity was preceded by an activation step at room temperature. Briefly, mem- branes obtained from resting neutrophils were mixed with 2m M MgSO 4 and an optim al amount of arachidonic acid. After 5 min, cytosol from resting cells (an amount corres- ponding to 10 · that of membrane protein), 20 l M GTPcS, 500 l M ATP and 2 m M MgSO 4 were added, and incubation was continued for another 5 min. In the case of O 2 – measurement, 10–20 lg aliquots of m embrane protein were used in a final volume of 20–50 lLofNaCl/P i . Following activation, the suspension was transferred to a photometric cuvette containing 200 l M NADPH and either 100 l M cytochrome c or 100 l M INTin2mLNaCl/P i .Cyto- chrome c reduction was r ecorded at 550 nm ( e ¼ 21.1 m M )1 Æcm )1 ) [19], and INT reduction at 500 nm (e ¼ 11 m M )1 Æ1cm )1 ) [20]. After 2–3 m in, 50 lgofSOD was added to quench O 2 – . In all preparations, cytochrome c reduction was inhibited to > 95% b y t he addition of SOD, indicating that O 2 – was the main product of reduction of O 2 . In contrast to the reduction of cytochrome c which is monoelectronic, reduction of INT to formazan requires two electrons. For normalization of t he data, the activitie s were calculated as lmol e – transferredÆmin )1 Æmg membrane pro- tein )1 When r eduction of INT was conducted under anaerobiosis, t he cuvette c ontaining the m edium was sealed with gas-tight rubber stoppers, into which two needles were inserted. One of the needles was u sed f or flushing nitrogen, the other for gas evacuation. When the oxidase activity was assayed by the rate of O 2 uptake, the suspension of activated particles was transferred to an oxygraphic cuvette containing 1.5 mL NaCl/P i supplemented with 250 l M NADPH and, when indicated, INT or heme inhibitors at different concentrations. The quantity of neutrophil membranes used in the oxygraphic assays was 10 · that used in the photometric assay. For measurement of the K m of activated oxidas e f or O 2 ,theO 2 concentration of the medium was d ecreased to 30–40% of the initial value by controlled N 2 bubbling prior to NADPH addition. Below 40–50 l M , the oxygraphic traces curved inward. The rates of o xygen u ptake were deduced from the slopes of the tangents to the oxygraphic traces, and the contact points of t he tangents with th e c urves were used t o determine the O 2 concentrations at which O 2 uptake proceeds [18]. All experiments were repeated two or three times, and t he reported results are representative o f at least two experiments. Optical spectra Absorption spectra of clear solutions were recorded at room temperature with an Uvikon 930 spectrophotometer. In the case of turbid suspensions, a double beam PerkinElmer 5 57 spectrophotometer was used. R eduction was a chieved with a few grains of sodium dithionite. The amount of heme b in neutrophil membranes was determined from difference spectra (dithionite-reduced vs. o xidized). The molar extinc- 1244 A. Poinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002 tion coefficients (De)were106m M )1 Æcm )1 at 425 nm (Soret Peak) and 21.6 m M )1 Æcm )1 at 558 nm [21]. The amount of heme b in neutrophil membranes varies from 0.35 to 0.65 nmolÆmg protein )1 depending on preparations. In experiments where reduced heme b was reoxidized by sequential additions of INT (cf. Fig. 8), the difference spectra relative to the Soret band were recorded and t he extent of reoxidation was assessed by the decrease of the peak of absorbancy by reference to a base-line (see Fig. 8). EPR spectra EPR spectra were recorded with a X -band Bruker EMX spectrometer equipped with an Oxford Instruments ESR- 900 continuous flow helium cryostat. RESULTS Effect of NADPH oxidase activation and oxygen on the rates of INT reductase and O 2 – production The experiment illustrated in Fig. 1 shows the effects of arachidonic acid and O 2 on the rates of electron transfer from NADPH to cytochrome c and INT. Arachidonic acid, an efficient amphiphile commonly u sed to elic it the production of O 2 – in a cell-free system of NADPH o xidase activation, was added at increasing concentrations to neutrophil membranes which were further supplemented with neutrophil cytosol, GTPcS and ATP. After completion of activation, the rates of cytochrome c reduction and INT reduction were measured either in a n a erated medium or in aN 2 saturated m edium. The data were expressed in terms of lmol e – transferred min )1 Æmg membrane protein )1 ,cor- recting for the fact t hat c ytochrome c is reduced by one electron and INT by two electrons. In t he aerated medium (Fig. 1 A), the cytochrome c reductase activity, referred as oxidase activity, and the INT reductase activity both peak ed at a concentration of 1.2–1.3 lmol arachidonic acidÆmg membrane protein )1 , the rates of electron transfer being 1.00 lmol and 0.78 lmol e – min )1 Æmg membrane pro- tein )1 , respectively. Both activities differed in their sensitiv- ity to SOD. The INT reductase was i nhibited % 50% by SOD, whereas reduction of cytochrome c was inhibited by > 95%, indicating that reduction of cytochrome c was essentially due to the superoxide anion O 2 – generated by the NADPH oxidase. In the oxygen-free medium (Fig. 1B) INT was reduced, but not cytochrome c . Thus, in contrast to INT, cyto- chrome c does not capture e lectrons from a redox center of flavocytochrome b . The rate of reduction of INT was even higher in anaerobiosis than in aerobiosis (0.94 lmol vs. 0.78 lmol e – transferred min )1 Æmg membrane protein )1 ), and nearly the same as the rate of O 2 – production in aerobiosis. This result m eans that the electron t ransfer s tep from NADPH to the redox center from w hich electrons are captured by INT controls the rate of the overall electron transfer from NADPH to O 2 . The SOD-sensitive reduction of INT by the activated NADPH oxidase in an aerated medium previously des- cribed and ascribed to the reduction of INT by O 2 – generated by the oxidase activity of flavocytochrome b [9,11] deserves some comments. An alternative explanation is that INT radicals generated b y direct c apture of electrons from reduced flavocytochrome b interact with O 2 to gen- erate oxidized INT (INT ox )andO 2 – [15,17] according to reaction 1: INT • +O 2 «INT ox +O 2 – . Eliminating O 2 – with SOD displaces the reaction to the right, with formation of INT ox . In this mechanism, the superoxide O 2 – is no longer considered as the product of reduction of O 2 at the heme level of fl avocytochrome b, but rather as the product of reduction of O 2 by INT, and the SOD-dependent inhibition of INT reduction appears to b e an indirect effect. On the other hand, the INT radical may generate by dismutation the fully reduced INT red according to reaction 2: INT • +INT • +H + fi INT red +INT ox . At low concentrations of INT, reaction 1 predominates, whereas a t high c oncentrations of INT reaction 2 (which is second order with respect to the INT concentration) is favored. Thus, the balance of I NT depends not only on the presence of O 2 , but also on the concentration of I NT. This may explain why the SOD-dependent sensitivity of INT reduction fluctuates depending on experimental conditions. For example, in a recent report, the extent o f inhibition of INT reduction by SOD was limited to 10% [9] compared with 50% in the present paper (Fig. 1). Characterization of the INT radical The reduction of tetrazolium salts into formazan, which involves the overall transfer of two electrons per molecule, proceeds by stepwise addition of individual electrons [22]. Fig. 1. Reduction of O 2 and INT by neutrophil membranes activated in a cell-free system. Effect o f increasing concentrations of arachidonic acid. Neutrophil membranes (2 0 lg protein) w ere i ncubated at room temperature with increasing concentrations of arachidonic acid, up to 3 lmolÆmg protein )1 ,and5m M MgSO 4 . After 5 min, cytosol (200 lg protein) was added, together w ith 0.5 m M ATP and 10 l M GTPcS. The final volume was adjusted to 50 lL with NaCl/P i and incuba tion was continued for a further 5 min The who le sample was transferred to a photometric cuvette in 2 mL N aCl/P i supplemented with 200 l M NADPH and either 100 l M cytochrome c (d) or 100 l M INT ( j), depending on the m easurement of the oxidase a ctivity (O 2 – produc- tion) or the INT reductase activity. The assays were carried out in aerobiosis (A) a nd in anaerobiosis (B) as described in Experim ental procedures. Production of O 2 – as a r educing agen t was quenched by addition of 50 lg of superoxide dismutase to the medium of the photometric cuvette containing eit her cytochrome c ( s)orINT(h) (A). Ó FEBS 2002 INT reductase activity of neutrophil oxidase (Eur. J. Biochem. 269) 1245 This process involves the formation of a tetrazolinyl free radical which was found by EPR spectroscopy to be relatively stable at 25 °C in hydrophobic media, e ven i n the presence of O 2 . The tetrazolinyl radical c an also accumulate by oxidation of formazan or by disproportionation of a mixture of formazan and the tetrazolium salt. The optical spectra illustrated in Fig. 2 A were recorded before and after addition of a small amount of sodium dithionite to a solution of INT in a mixture of dimethyl formamide and water 1 : 1, v/v). The recorded spectrum of oxidized INT above 4 00 nm was flat (Fig. 2A, trace a ). Following addition of sodium dithionite, an orange color rapidly developed, with a maximal optical absorbance at 449 nm (Fig. 2 A, trace b). In a few minutes, the color changed to red (Fig. 2 A, trace c), corresponding to a new spectrum with two maxima at 500 nm and 550 nm, which was character- istic o f t he monoformazan, i.e. the fully reduced product o f INT [15,17]. In t he following aeration of the medium, the absorbance of the spectral bands decreased, but the shape of the spectrum r emained t he same (Fig. 2A, trace d), which means that the fully reduced INT became reoxidized. I t was concluded that the transient absorbance at 449 nm corres- ponded to a partially reduced state of INT, most likely to the INT radical. The rate of transition from the oxidized state to the fully reduced state upon addition of sodium dithionite depended on the medium. When the solvent was water, the red- colored formazan accumulated in a few seconds. I n contrast, in a mixture o f d imethyl formamide and water of 2 : 1 ( v/v), t he INT radical was stable for more than 10 min (Fig. 2B), the stability of t he INT radical increasing with the increase in dimethyl formamide concentration. Detergents such as Triton X-100 or SDS at a final concentration o f 1% (w/v) also stabilize the INT radical (data not shown). This observation corroborates data showing that t he nitroblue tetrazolium (NBT) radical obtained by reduction of oxidized NBT by silver amalgam was stabilized in dimethoxyethane [23]. The stabilizing effect of SDS was not encountered with arachidonic acid which we routinely used as an activator of the NADPH o xidase. The first derivative EPR spectrum at 293 K of INT reduced by sodium dithionite in dimethyl formamide shows a radical structure centered at g ¼ 2.00 (Fig. 2C, upper trace). The spectrum was characterized by a 10-line pattern, nine of which resemble those o f t he EPR spectrum of the 2,3,5 triphenyltetrazolium chloride radical [23]. The 10-line pattern of the EPR spectrum of the I NT radical could b e simulated by assuming a structure containing four equivalent nitrogen a toms and a supernumerary atom with a spin of 1/2, namely a proton (Fig. 2C, bottom t race), with isotrop ic hyperfine splitting constants o f 0.5 mT between nitrogen atoms and 0.75 mT for the sup ernumer- ary proton. The specific chemical p roperties of INT and more particularly the stability of its radical in hydrophobic media may e xplain differences in the efficiency of elec tron capture by INT depending on experimental conditions of the assay of NADPH oxidase, for example the nature of the detergent used in the cell-free assay or the membrane concentration. Compared effects of the two heme inhibitors, benzylimidazole and bipyridyl on the optical and EPR spectra of hemin and flavocytochrome b In a preliminary experiment, we found that benzylimidazole and bipyridyl inhibited not only the production of O 2 – assayed by the SOD-sensitive reduction of cytochrome c , but also the r eduction of INT w ith the same efficiency. H alf inhibition was obtained with 2–3 m M bipyridyl and 5–7 m M benzylimidazole. Inhibition was largely reversed by d ilution, indicating that it was not due to denaturation of flavocyt- ochrome b . Complementary experiments u sing optical and EPR s pectra were carried out to assess the specificity o f the effects of bipyridyl and benzylimidazole on the heme(s) of flavocytochrome b . Hemin was chosen as a model to test by spectral modifications the ability of benzylimidazole and bipyridyl to react with heme iron. Because hemin solutions in detergent are not turbid, absolute spectra of oxidized and reduced hemin in the absence and presence of inhibitors were recorded directly (Fig. 3A, trace a, control, and trace b, presence of benzylimidazole). The difference spectra of dithionite-reduced hemin plus benzylimidazole and dithi- onite-reduced hemin plus bipyridyl minus reduced hemin exemplify typical changes in the s pectra consisting of the appearance of well defined peaks at 428 n m, 530 nm and 560 n m in the case of benzylimidazole (Fig. 3A, trace c) and at 437 nm in t hat o f b ipyrid yl (Fig. 3A, trace d). When t he same heme inhibitors were added t o neutrophil membranes Fig. 2. Evidence for accumulation of a stable INT radical during reduction of INT by sodium dithionite. The INT radical was prepared as described in Exp erimental procedures. ( A) Optical spectra show ing the progressive red uction of INT by so dium dithionite, in a m ixture of dimethyl formamide and H 2 O ( 1 : 1, v/v), from a fully oxidized state (a) to a fully reduced state (c) (after 5 min) via a semireduced state corresponding to the INT radical (b) (after 2 min). The spectrum taken 15 min after aeration (d) has a shape similar to that of the fully reduced INT, but its size was significantly decreased. ( B) Spectra o f the purified INT radical in a mixt ure of dimethyl formamide a nd H 2 O (2 : 1, v/v), before (solid line) and after addition of sodium dithionite (dotted line). (C) Upper trace: first derivative EPR spectrum of the INT radical recorded at r oom temperature. Microwave power 2 mW; modulation frequency 100 kHz, modulation amplitude 0.5 mT, microwave fre- quency 9.660 GHz. Bottom trace: simulation of the EPR spectrum shown in the upp er trace was obtained by a ssuming f our equivalen t nitrogen atoms coupled at 0.5 mT and one proton coupled at 0.75 mT. 1246 A. Poinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002 in the absence of arachidonic acid, the heme spectrum of flavocytochrome b was not modified (Fig. 3B, t race a). Neither was it in the presence of arachidonic acid a lone in the a bsence of inhibitors (Fig. 3B, trace b). However, when the heme inhibitors were added to the neutrophil mem- branes in the presence of arachidonic acid used at a concentration that elicited maximal oxidase activity, signi- ficant spectral modifications were recorded. These modifi- cations consisted in a decrease of the Soret peak accompanied by a slight blue shift and in a decrease of the a peak (Fig. 3B, trace c, benzylimidazole, and t race d, bipyridyl). Moreover, addition of benzylimidazole and bipyridyl resulted in opposite modifications of the sizes of the a and c peaks of heme b. Benzylimidazole d ecreased the a/c peak ratio f rom 4.5 to 3.4 whereas bipyridyl increased it from 4.5 to 6.1, suggesting different types of constraint applied to the hemes by the two inhibitors. Binding of benzylimidazole to t he heme iron of hemin and to the heme iron of purified flavocytochrome b was assessed by EPR spectroscopy (Fig. 4). Addition of benzy- limidazole to h e min resulted in the decre ase of the high spin signal at g ¼ 6.0 (Fig. 4, trace d), characteristic of the pentacoordinated form of the iron atom of the heme, and i n the concomitant emergence of a low spin signal with components at g 1 ¼ 2.97 and g 2 ¼ 2.25 (trace e). Purified flavocytochrome b in detergent displayed a mixture of penta- and h exacoordinated forms of heme b (Fig. 4, trace a). The high spin sig nal at g ¼ 6.0 s imilar to t hat of h emin accounted for the pentacoordinated form of t he heme iron. The hexacoordinated form was represented by two low spin g 1 signals at g ¼ 3.28 and g ¼ 2.85. The g 2 components are probably associated a t g ¼ 2.20. The s ignal at g ¼ 4.3 w as due to adventitious ferric specie s [24]. Thus, even in the absence of a rachidonic a cid, a f raction o f purified flavocyt- ochrome b is pentacoordinated and capable of reacting with O 2 or with heme ligands. The high spin fraction was significantly increased b y addition of 100 l M arachidonic acid, whereas the low spin sign als were totally erase d (data not shown) in accordance with previous results [18]. The high spin signal of purified flavocytochrome b (see control trace a ) w as decreased by addition of 25 m M benzylimidaz- ole (trace b), and nearly abolished at 50 m M benzylimidaz- ole (trace c), a concentration which also fully inhibited the NADPH oxidase activity. Concomitantly with the disap- pearance of the high spin signal a t g ¼ 6.0, a low spin signal with components g 1 and g 2 at g ¼ 2.97 and g ¼ 2.2 5 emerged at positions similar to t hose observed in the case of the hemin/benzylimidazo le c omplex (trace e), probably d ue to the binding of benzylimidazole as an axial ligand to the heme iron in hemin or in flavocytochrome b. The two low spin signals g 1 at g ¼ 3.28 and g ¼ 2.85 initially present in purified flavocytochrome b were not altered upon addition of 50 m M benzylimidazole. This behavior is reminiscent o f the absence of effect of benzylimidazole on the optical spectrum of resting neutrophil membranes in the absence of arachidonic acid. Thus, the fraction of purified flavocyto- chrome b characterized by low spin signals contains a hexacoordinated heme i ron unable to r eact with benzylim- idazole; this fraction, calculated by integration, represents roughly half of the total amount of flavocytochrome b. Fig. 4. Effect of benzylimidazole on the EPR s pectra of isolated flavo- cytochrome b and hemin. Traces a–c are EPR sp ectra of purified fl avo- cytochrome b (45 l M )insolutionin20m M P i , 20% glycerol, 0 .5 M NaCl and 0.1% Triton X-100, pH 7.4. Trace a corresponds to control flavocytochrome b,tracebtoflavocytochromebtreated with 2 5 m M benzylimidazole and trace c to flavocytochrome b treated with 50 m M benzylimidazole. Traces d and e correspond to hemin (1 m M )inDMF untreated and treated with 50 m M benzylimidazole, respectively. (A) and (B) show the high spin and low spin regions of the EPR spectra, respectively. Fig. 3. Effects of benzylimidazole and bipyridyl on optical spectra of hemin and membrane bound fl avocytochrome b . (A ) Traces a1 and a2: absolute spectra of oxidized and dithionite-reduced hemin (10 l M ), respectively. Traces b1 and b2: absolute spectra of oxidized and dithionite-reduced hemin in the presence of 5 m M imidazole. Trace c: difference spectrum of reduced hemin plus 5 m M benzylimidazole against reduced hemin (10 l M ). Trace d: Difference spectrum of reduced hemin p lus 10 m M bipyridyl a gainst reduced hemin (20 l M ). (B) Traces a –d: difference spectra (dithionite-reduced minus oxidized) at room temperature of neutrophil membranes in NaCl/P i (1 m g protein ÆmL )1 equivalent to 0.65 nmol heme b). Trace a, control; trace b, membranes supplemented by arachidonic acid (1.3 lmolÆmg protein )1 ); trace c, reduced membranes plus arachidonic acid treated for 5 min with 40 m M bipyridyl; trace d, reduced membranes plus arachidonic acid treated for 5 min with 25 m M benzylimidazole. Ó FEBS 2002 INT reductase activity of neutrophil oxidase (Eur. J. Biochem. 269) 1247 Dependence of NADPH oxidase inhibition by 5-deaza FAD on the activation state of flavocytochrome b The e ffect of 5-deaza F AD, a flavin a nalog and an obligate two-electron donor [25–27] was tested on the NADPH oxidase activity and the INT reductase activity of flavocyto- chrome b in the c ell-free system (Fig. 5). Its inhibitory effect depended on t he step at which it was added t o the medium. When 5 -deaza FAD was added to the activated cell-free system, the NADPH oxidase and the INT reductase were hardly inhibited. On the other hand, when 5-deaza FAD was preincubated with n eutrophil membranes together with arachidonic acid 5 min prior to addition of the other components of t he activation system, namely cytosol, GTPcS and ATP, both the elicited NADPH oxidase and INT reductase activities were inhibited efficiently (K i ¼ 25 l M ), and the extent of inhibition was the same for the two activities. The inhibition caused by 5-deaza FAD was prevented by addition of a 10 · excess of FAD (data not shown). Together these results suggest that arachidonic a cid induces by itself some structural modifica- tions in flavocytochrome b which result in the release of the bound FAD and its replacement by 5-deaza FAD. When the oxidase is fully activated, these modifications do n ot occur any more. It is noteworthy that, in contrast to the flavin analogs, the heme inhibitors benzylimidazole and bipyridyl were e qually effective w hen added either t o the activated cell-free system or to the membranes before NADPH oxidase activation. Effect of 5-deaza FAD and heme inhibitors on the reduced state of flavocytochrome b in activated neutrophil membranes In the experiment o f Fig. 6 conducted in anaerobiosis, 5-deaza FAD was preincubated with neutrophil membranes and arachidonic acid 5 min before the addition of cytosol, GTPcS and ATP, a condition required for the optimal inhibitory effect of the flavin analog on the o xidase activity. In the c ontrol assay ( Fig. 6, trace a), addition of NADPH resulted in an abrupt rise of heme b r eduction, followed by a plateau which corresponded to 40–50% of the full reduction obtained with sodium dithionite. After the reduction plateau had been reached, 10 nmol O 2 (dissolved in buffer) were added, which resulted in an abrupt, but limited, reoxidation of h eme b, rapidly counteracted by the electron flux issued from NADPH. Afte r a new redox equilibrium had been a ttained, two o ther redox cycles were initiated b y addition of 5 and 10 nmol of INT. Reoxidation of reduced heme b with 10 nmol INT was twice that with 5 nmol INT, indicating proportionality over this range of INT concen- tration. Moreover, 5 nmol INT ( a mediator which is reduced by a pair of electro ns) a re able to oxidize the s ame amount of reduced heme b a s 10 nmol O 2 , w hich speaks in favor of the idea that reduced heme b is the source of electrons for both INT and O 2 . If the major source of electrons for INT we re reduced FAD, assuming a back flow of electrons from reduced heme b t o FAD, the above stoichiometry would be different. The experiment was completed by addition of sodium dithionite in limiting amounts just sufficient to reduce % 95% of the heme components of flavocytochrome b. Addition of 10 nmol O 2 (dissolved in buffer) followed by 10 nmol INT resulted in oxidation cycles of heme b similar to those obtained in the presence of NADPH alone as reducing agent. Trace b (Fig. 6) r efers to the effect of 5-deaza FAD used at a concentration that inhibited % 80% of the NADPH- oxidase activity. The rate and the extent of the NADPH- dependent reduction of heme b were both largely decreased. This explains w hy the redox cycles initiated by addition of O 2 and I NT were smaller in size, compared t o the control (trace a); yet the dithionite-redu ced heme b was reoxidized by O 2 and INT to virtually the same rate and extent as in the control, despite the block imposed by 5-deaza FAD at the level of the fl avin redox center of flavocytochrome b.Witha higher concentration of 5-deaza FAD, which inhibited > 95% of the N ADPH-dependent reduction of h eme b, INT and O 2 were still able to reoxidize the d ithionite reduced heme b (trace c, Fig. 6). The two later traces (d and e, Fig. 6) refer to the action of the heme inhibitors, benzylimidazole and bipyridyl. Both inhibitors strongly interfered with the rate and extent of reoxidation of the heme b reduced in the presence of NADPH. Following addition of sodium dithi- onite, the cycles of reoxidation of heme b initiated by O 2 and Fig. 5. Dose-dependent inhibition of the NADPH oxidase a nd INT reductase activities of neutrophil membranes by the flavin analog 5-deaza FAD. Oxidase activation was performed as described in Experimental procedures and in the legend of Fig. 1. 5-deaza F AD was added with arachidonic ac id and M gSO 4 to t he membrane s uspension (20 lg protein). After 5 min at room temperature, oxidase activation was elicited by addition of cytosol and GTPcS. After an additional 5 min incubation, the o x idase activity was measured as O 2 – production (d) and the INT reductase activity by th e rate o f r eduction o f INT (j). A parallel experiment was carried out in which 5-deaza FAD was incubated for 5 min with the m embrane suspension after the cell-free activation of the NADPH oxidase; O 2 – production (s), INT r eductase activity (h). 1248 A. Poinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002 INT were a lso significantly diminished, compared to t he control (trace a). In summary, using the fl avin analog 5-deaza FAD and the heme inhibitors, bipyridyl and benzylimidazole, to limit or to block the flux of electrons along the redox centers of flavocyto chrome b, it is possible to demonstrate that INT is able to capture electrons from the heme c omponents of fl avocytochrome b. Kinetics of inhibition of the oxidase activity by INT and bipyridyl As INT appeared to capture electrons from the hemes of flavocytochrome b , we asked whether INT could compete with O 2 . Activated neutrophil membranes in a cell-free system in the presence of cytosol, GTPcS and arachidonic acid were placed in an oxygraphic cuvette co ntaining the NaCl/P i medium whose O 2 concentration had been pre vi- ously lowered to % 80 l M by controlled bubbling of N 2 . Then, O 2 uptake was initiated by addition of a saturating concentration of NADPH (250 l M ). Below 80 l M O 2 ,the oxygraphic traces curved inwards. In the portion of the traces correspond ing t o O 2 concentrations ranging between 80 l M and 20 l M ,theratesofO 2 uptake were deduced from the slopes o f tangents at different concentrations of O 2 . The assay was repeated with INT added to the medium at increasing concentrations. In the absence of INT, the reciprocal plots corresponded to a straight line from w hich a K m value of 25 l M for O 2 could b e deduced (Fig. 7A). At increasing concentrations of INT, the plots corresponded also to straight lines that intersected the 1/v axis at a common intercept, wh ich was the same as that of the control c urve, and the apparent K m for o xygen increased in proportion to the increase in INT concentration. These features are typical of a competitive inhibition. The K i found for INT was % 30 l M , a value which is of the same order as the K m found for O 2 [13]. Assuming that capture of electrons from reduced heme b by INT was responsible for the competitive effect of INT o n the oxidase activity of flavocytochrome b , it w as inferred that a heme b inhibitor should competitively inhibit in anaerobiosis the reduction of INT by membranes of neutrophils activated in a cell-free system. This w as in fact the case with bipyridyl as shown in Fig. 7 B. A similar competitive inhibition of O 2 uptake b y bipyridyl was found when activated neutrophil membranes were incubated with NADPH in an aerated medium (Fig. 7C). In both cases (Fig. 7 B,C) the calculated K i for bipyridyl were the same, namely 2 m M ±0.2m M . Twice higher values for K i were obtained with benzylimidazole (data not shown). INT-dependent reoxidation of dithionite-reduced heme b in flavocytochrome b and dithionite-reduced hemin In this experiment, we followed the stepwise oxidation of reduced flavocytochrome b and r educed hemin by sequen- tial additions of small amounts of INT. Neutrophil membranes pretreated by arachidonic acid were placed in photometric cuvettes under a flow of nitrogen. I t is known that pretreatment of m embranes by arachidonic a cid modifies the spin state of heme b [18], but is not sufficient per se to elicit the oxidase activity of flabocytochrome b. A parallel spectrophotometric assay was carried out with hemin. Hemin and flavocytochrome b were reduced to % 95% by the addition of a limited amount of sodium dithionite. To r eoxidize hemin and the h eme c omponent of flavocytochrome b , INT was a dded b y small increments to the anaerobic cuvettes. Absorbance was recorded using a double wavelength spectrophotometer, between 380 nm and 480 nm for hemin and between 400 nm and 465 nm for flavocytochrome b, corresponding to the Soret peak of the t wo pigments. Reoxidation of hemin or the heme of Fig. 6. Effect of 5-deaza FAD, bipyridyl and b enzylimidazole on the O 2 and INT-dependent reoxidation of re duced h eme b in a ctivated neutro- phil membranes. Activated membranes (2 mg protein in 1.7 mL, equivalent to 0 .70 n mol heme b) in the cell-free system (arachidonic acid, c ytosol and GTPcS) were placed in a photo metric c uvette. The medium (1.7 mL) was made anaerobic as described in Experimental procedures. The following c ompounds were injected i nto the medium in minimal volumes in the f ollowing sequence: NADPH, 1 lmol; O 2 , 10 nm ol (dissolved in b uffer); INT, 5 and 10 nmol; sodium dithionite, 1 lmol. Trace a, control membranes; trace b, membranes preincu- batedfor10minwith40l M 5-deaza F AD and arachidonic ac id fol- lowedbyadditionofcytosolandGTPcS; trace c, same conditions as in trace b except that 5-deaza FAD was used at 80 l M ;traced,same conditions as i n trace a with 10 m M benzylimidazole (BI) a dded t o the cuvette after reduction of heme b by NADPH; trace e, same conditions as in trace a with 10 m M bipyridyl (Bipy) added to the c uvette after reduction of heme b by NADPH. After full reduction of heme b with sodium dithio nite, f urther cycles of oxidation were i nitiated by addi- tion of O 2 and INT (respectively 10 and 5 nmol). Ó FEBS 2002 INT reductase activity of neutrophil oxidase (Eur. J. Biochem. 269) 1249 flavocytochrome b was assessed by the decrease of the absorbance (Fig. 8, insert). U p to 80% heme reoxidation, a linear relationship between the amount of added INT and the absorbance d ecrease was observed (Fig. 8). A bove 80% heme reoxidation, the curves departed from linearity. The two dose–response curves for hemin and flavocytochrome b were virtually superimposable. By extrapolation of the linear portions of the curves to t he abscissa, it could b e calculated that 0.45–0.48 mol INT w as nee ded to reoxidize 1 mol hemin or 1 mol heme of flavocytochrome b , which is consistent with the stoichiometry of 2 e – captured per INT molecule in both cases. Thus, like reduced hemin, the reduced heme of flavocytochrome b is able to donate electrons to INT. A b ack transfer of electrons from reduced heme b to F AD should not be significant because, as noted above, the oxidase activity of neutrophil membranes treated with arachidonic acid is dormant in the absence of cytosolic factors [18] and, consequently, electron transfer b etween the redox centers of fl avocytochrome b is maintained at a very low level. In summary, the value of the stoichiometric ratio of reduced INT to oxidized heme suggests that under our experimental conditions elec trons are e ssentially transferred from the heme components of flavocytochrome b to INT. DISCUSSION The postulate that the flavocytochrome b component of the neutrophil NADPH oxidase has the potential to display a diaphorase activity s tems from two different observations. First, artificial disruption of the electron transfer chain of flavocytochrome b by detergents allows capture of electrons upstream of heme b [8]. Second, a mutated flavocyto- chrome b from a CGD (91X + ) patient, that was unable to reduce O 2 into O 2 – , was still able to carry electrons from NADPH to INT. This latter observation led to the belief that INT is a suitable electron acceptor from the reduced FAD component of flavocytochrome b and thereby a suitable probe of the diaphorase activity of flavocyto- chrome b [9]. There were, however, in the mean time, reports that pointed to the r ather complex behavior of INT [15,17]. We therefore decided to determine b y classical techniques, using spec ific inhibitors of the electron flux in flavocyto- chrome b , which redox components interacted w ith INT. The flavin analog, 5-deaza FAD was used to i nhibit the flux of electrons at the FAD level. On the other hand, two efficient heme inhibitors, bipyridyl and benzylimidazole, were selected after validation by optical and EPR spectro- metric tests. The experiments described here led us to conclude that INT is able to capture electrons from the heme b components of the neutrophil flavocytochrome: (a) in activated membranes maintained in anaerob iosis, heme b reduced by NADPH was reoxidized by INT as efficiently as by O 2 (Fig. 6 ). Reoxidation of h eme b was insensitive to 5-deaza FAD, which contrasted with the inhibitory effect of this compound on the reduction of heme b by NADPH (Fig. 6). This result, which agrees with the well recognized function of 5-deaza FAD as a flavin inactive analog, rules out the possibility of a back reaction from heme to flavin and then from flavin t o INT; (b) INT was able to reoxidize hemin reduced by sodium dithionite (Fig. 7 ). As FAD was absent in this experiment, electrons were directly transferred from hemin to INT. The comparative titrations by INT of dithionite-reduced hemin and d ithionite-reduced heme b o f flavocytochrome b ended with the same stoichiometry of roughly 0.5 mol INT reduced by 1 mol hemin or by 1 mol heme b in flavocytochrome b ; (c) INT was able to compete with O 2 in an aerobic medium, and also with the heme inhibitor, bipyridyl, in anaerobiosis (Fig. 7). These results led us to conclude that the heme c omponents of flavocytochrome b interact directly with INT. Fig. 7. Kinetics o f inhibition o f the elicited NADPH oxidase a ctivity in a cell-free s ystem. (A) and (C), aliq uots of n eutrophil membranes (150 lg protein in A) activated in the cell-free system were placed in the oxygraphic cuvette containing 1.5 mL NaCl/P i supplemented with different fixed concentrations of INT i n A ( d,zero;s,30l M ;j,60l M ;h,90l M ;_,120l M ) and bipyridyl in C (j,zero;d,3m M ;h,7m M ;s,15m M ). The initial concent ration of O 2 (230 l M ) was decreased b y about two-third s by controlled bubbling of nitrogen prior to th e assay of O 2 uptake initiated by addition of 250 l M NADPH. The r ate of O 2 uptake was c alculated f rom the s lopes of the tangents to the ox ygraphic t races. (B) Activated membranes ( 40 lg protein per a ssay) were placed in a closed photometric cuvette containing 1 m L of a medium previously made an aerobic by N 2 bubbling. The medium consisted of NaCl/P i supplemented with 250 l M NADPH and different concentrations of INT ranging from 20 to 200 l M . The rate of INT r eductio n re corded at 500 nm was calculated using a molar e xtinct ion c oe fficient o f 11 m M )1 Æcm )1 . Bipyridyl was used at t he following concentrations: j,zero;d,3m M ;h,7m M ;s,15m M . 1250 A. Poinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Theoritically, INT might accept electrons from reduced FAD in activated flavocytochrome b inasmuch as the F AD binding site is believed to be located in the relatively hydrophilic C-terminal half of the g p91phox subunit of the flavocytochrome. Experimental data show that this is not the case, which raises the question of the capacity of I NT to probe specifically the diaphorase activity of neutrophil NADPH oxidase. It is however, not excluded t hat under specific conditions, for example t he presence of a d etergent or of a mutation such as Arg54Ser [14], the structural arrangement of the FAD binding domain of flavocyto- chrome b is modified, resulting in a loss of interaction of FAD with heme 1 and in a facilitated access o f INT to FAD. Along this line, one may recall that a peculiar behavior of FAD i n flavocytochrome b was recognized in the p ast [ 28], a nd explained in terms of a kinetic barrier between flavin and heme b. A peculiar structural arrange- ment of the peptide chain in t he FAD region of flavocyto- chrome b , possibly related to the oligomerization of the protein, might be responsible for its very unusual properties. Finally, we would like to point out that some of the unusual properties of INT might be due to the stabilization of its radical in hydrophobic media, for example the lipid phase of membranes. ACKNOWLEDGEMENTS We thank V. Massey for the gift of 5-deaza FAD, J. Willison for careful reading of the manuscript a nd J. Bourne t-Cauci for excellent secretarial assistance. This work was supported by funds fro m the Centre National de la Recherche Scientifique, t he Commissariat a ` l’Energie At om ique, the Universite ´ Joseph Fourier–Grenoble I, and the A ssociation p our la Recherche sur le Cancer (9996). REFERENCES 1. Babior, B.M. (1999) NADPH oxidase: an update. Blood 93, 1464– 1476. 2. Thrasher, A.J., Keep, N.H., Wientjes, F. & Segal, A.W. (1994) Chronic granu lomatous disease. Biochim. Biophys. Acta 1227, 1–24. 3. Segal, A.W., West, I., Wien tjes, F., Nugent, J.H., Chavan, A.J., Haley, B., Garcia, R.C., Rosen, H. & Scrace, G. (1992) Cyto- chrome b-245 is a fl avocytochrome containing FAD and the NADPH-binding s ite o f t he microbicidal oxidase of p hagocytes. Biochem. J. 284, 781–788. 4. Doussie ` re, J., Brandolin, G., Derrien, V. & Vignais, P.V. (1993 ) Critical assessment of the presence of an NADPH binding site on neutrophil cytochrome b558 by photoaffinity and immunochemical labeling. Biochemistry 32, 888 0–8887. 5. Doussie ` re, J ., Buzenet, G . & Vignais , P.V. (19 95) Photoaffinity labeling and photoinactivation of the O À 2 generating oxidase of neutrophils by an azido derivative o f FAD. Biochemistry 34, 1760–1770. 6. YuL., Quinn, M.T., Cross, A.R. & Dinauer, M.C. (1998) Gp91 (phox) is the heme binding subunit o f t he sup eroxide-generating NADPH o xidase. Proc. Natl Acad. Sci. USA 95, 7993–7998. 7. Finegold, A.A., Shatwell, K.P., Segal, A.W., K lausner, R.D. & Dancis, A. ( 1996) Intr amembrane b is-heme m otif for transmem- brane electron transport conserved in a yeast iron reductase and the human NADPH oxidase. J. Biol. Chem. 271 , 31021– 31024. 8. Doussie ` re, J. & Vignais, P.V. (1992 ) Diphenylene iodonium as an inhibitor of t he NADPH o xid ase complex of b ovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodo- nium in a c ell-free system of oxidase activation. Eur. J. Biochem. 208, 6 1–71. 9. Cross, A.R., Yarchover, J.L. & Curnutte, J.T. ( 1994) The super- oxide-generating system of hu man neutrophils possesses a novel diaphorase activity. Evidence for d istinct regulation of e lectron flow within NADPH oxidase b y p67-phox and p47-phox. J. Biol. Chem. 269, 21448–21454. 10. Cross, A.R. & Curnutte, J .T. (1995) The cytosolic ac tivating fac- tors p47phox and p67phox have distinct roles in the regulation of electron flow i n NADPH oxidase. J. Biol. C hem. 270, 6543–6548. 11. Li, J. & Guillory, R.J. (1997) Purified leukocyte cytochrome b558 incorporated into lip osomes catalyzes a cytosolic factor dependent diaphorase a ctivity. Biochemistry 36, 5 529–5537. Fig. 8. Effects of INT on reoxidation o f dithionite-reduced hemin and dithionite-reduced flavocytochrome b . Neutrophil membranes in NaCl/P i (2 mg proteinÆmL )1 ;0.60nmolhemebÆmg protein )1 ,total volume 1.7 m L) were preincubated for 10 m in at room temperature with arachidonic acid, 1.5 lmolÆmg protein )1 (j). A solution of freshly prepared hemin was used at the concentration of 10 l M in NaCl/P i supplemented with 1% Triton X-100 (s). Neutro phil membranes or hemin were placed in a photometric cuvette. The medium had previously been made anaerobic by bubb ling N 2 ,and maintained anaerobic during the optical assays as described in Experimental procedures. After a f ew minutes of an aerobiosis, small amounts of sodium dithionite solution were added to attain a level of reduction of heme b and hemin of 90–95%. Sequential additions of small aliquots (0.2 lL) of a solution of INT previously made anaerobic by N 2 bubbling w ere injected t o the me dium until heme b and hemin were fully reoxidized. After each a ddition the difference spectra (reduced minus oxidized) were recorded. As shown in the i nsert in the case of flavocytoch rome b, the loss of absorbance was measured between t he peak of absorb an ce of the f ully reduced s pectrum and a base-line drawn between the isosbestic point a t 4 16 nm and 450 nm. The results were norma lized in terms of p ercent of reduc ed heme and plotted against the calculated ratio of added INT to oxidized heme (mol/mol). A s imilar procedure was u sed in the case of hemin. Ó FEBS 2002 INT reductase activity of neutrophil oxidase (Eur. J. Biochem. 269) 1251 12. Cross, A.R., Erickson, R.W. & Curnutte, J.T. (1999) Simulta- neous presence of p47 (phox) and flavocyt ochrome b-245 are required for th e activatio n of N ADPH oxidase by anionic amphiphiles. Evidence for a n intermediate state of oxidase activation. J. Biol. Chem. 274, 1 5519–15525. 13. Doussie ` re, J., Bouzidi, F., P oinas, A., G aillard, J. & Vignais, P.V. (1999) Kinetic study of the a ctivation of t he neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and p henylarsine oxide. Biochemistry 38, 1639 4–16406. 14. Cross, A.R., H eyworth, P.G., Rae, J. & Curnutte, J.T. (1995) A variant X-linked chronic granulomatous disease patient (X91+) with p artially functional cytochrome b. J . Biol. Chem. 270, 819 4– 8200. [published e rratum in J. Biol. Chem. (19 95) 270, 1 7056]. 15. Liochev, S.I., Batinic-Haberle, I. & Fridovich, I. (1995) The effect of detergen ts on the reduction of tetrazolium salts. Arch. B iochem. Biophys. 324 , 48–52. 16. Auclair, C., Torres, M. & Hakim, J. (1978) Superoxide anion involvement in NBT re duction catalyzed by NADPH-cytochrome P-450 reductase: a pitfall. FEBS Lett. 89 , 26–28. 17. Auclair, C. & Voisin, E. (1986) Nitroblue tetrazolium reduction. In CRC Handbook of Methods for Oxygen Radical Research (Greenwald, R.A., ed.), pp. 123–132. CRC Press, Boca Raton, FL, USA. 18. Doussie ` re, J., Gaillard, J. & Vignais, P.V. (1996) Electron transfer across the O À 2 generating flavocytochrome b of neutrophils. Evidence fo r a transition from a low-spin state to a high-spin state of the h eme iron component. Biochemistry 35, 1340 0–13410. 19. Greenwood, C. & Palmer, G. (1965) Evidence for the e xistence of two functionally distinct forms cytochrome c monomer at alkaline pH. J. Biol. Chem. 240, 3660 –3663. 20. Pearse, A.G.E. (1961) Histochemistry: Theoretical and Applied, 2nd edn. L ittle, Brown and Co, Bosto n, MA, USA. 21. Cross, A.R., Higson, F.K., Jones, O.T.G., Harper, A.M. & Segal, A.W. (1982) T he enzymatic reduction and kinetics of cytochrome b -245 of neutrophils. Bioc hem. J. 20 4, 479–485. 22. Maender, O.W. & Russel, G.A. (1966) The formation of ra dical intermediates in formazan-tetrazolium salt systems. J. Org. Chem. 31, 442–446. 23. Deguchi, Y. & Takagi, Y. (1967) ESR spectrum of 2,3,5-triphenyl tetrazolium. Tetrahedron L ett. 33, 3179–3180. 24. Gadsby, P.M. & Thompson, A .J. (1986) Lo w t emperature EPR and near-infrared MCD studies of highly anisotropic low-spin ferrihaem species. FEBS L ett. 197, 2 53–257. 25. Light, D.R., Walsh, C., O’Callaghan, A.M., Goetzl, E.J. & Tauber, A.I. (1981) Characteristics of the cofactor requirement f or the superoxide-generating NADPH oxidase of human polymorphonuclear leukocytes. Biochemistry 20 , 1468–1476. 26. Spencer, R ., Fisher, J. & Walsh, C. (1976) Preparation, char- acterization, and chemical properties of the flavin coenzyme analogues 5-deazariboflavin, 5-deaza-riboflavi n 5¢-phosphate, a nd 5-deazariboflavin 5¢-diphosphate, 5¢-5 ¢ adenosine ester. B i oche m- istry 15, 1043–1053. 27. Fisher, J ., Spence r, R. & Walsh, C. (1976) Enzyme-catalyzed redox reactions with the flavin analogues 5-deazariboflavin, 5-deazariboflavin 5¢-phosphate and 5-deazariboflavin 5¢-diphos- phate, 5¢-5¢-adenosine ester. Bioc hemistry 15, 105 4–1064. 28. Koskhin, V., Lotan, O. & Pick, E. (1997) Electron transfer in the superoxide-generating NADPH oxidase complex reconstituted in vitro. Biochim. Biophys. Acta 1319, 139 –146. 1252 A. Poinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Exploration of the diaphorase activity of neutrophil NADPH oxidase Critical assessment of the interaction of iodonitrotetrazolium with the oxidase redox components Alexandra. reduction of INT by the activated NADPH oxidase in an aerated medium previously des- cribed and ascribed to the reduction of INT by O 2 – generated by the oxidase activity of flavocytochrome b [9,11]. L dimethyl sulfoxide and kept at )20 °C under a rgon. Assay of oxidase and INT diaphorase activities NADPH oxidase activity was a ssayed in a cell-free system [13], either by m easurement of the