©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reconstitution of Flavin-depleted Neutrophil Flavocytochrome b with 8-Mercapto-FAD and Characterization of the Flavin-reconstituted Enzyme (*)

Yukio Nisimoto (1)(§), Hidetsugu Otsuka-Murakami (1), David J. Lambeth (2)

From the (1)Department of Biochemistry, Aichi Medical University, Nagakute, Aichi 480-11, Japan and the (2)Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cytochrome b isolated from human neutrophils was inactive and contained no detectable FAD. However, high NADPH oxidase activity was seen upon reconstitution of the cytochrome with either native FAD or 8-mercapto-FAD in the presence of phospholipids (phosphatidylcholine/phosphatidylethanolamine/phosphatidylinositol/sphingomyelin/cholesterol, 4:2:1:3:3 (w/w)). Their cell-free superoxide-generating activities were 40.5 and 35.5 mol/s/mol of heme, respectively, which corresponded to 70 and 61% of the original activity of the plasma membranes. Both flavins co-eluted with heme and protein on gel exclusion chromatography. The respective specific flavin content was 6.45 and 7.93 nmol/mg of protein and corresponded to a flavin:heme molar ratio of 0.41 and 0.51 consistent with a 2:1 ratio of heme to flavin. Mixing of 8-mercapto-FAD with flavin-depleted cytochrome b caused a red-shift of the flavin absorption maximum from 520 nm to around 560 nm, as has been seen when a variety of other apoflavoprotein dehydrogenases bind this analog. The 8-mercapto-FAD reconstituted into the cytochrome reacted readily with either iodoacetamide (k = 38.8 Mmin) or iodoacetic acid (k = 12.1 Mmin) to give a fluorescence spectrum characteristic of a 8-mercaptoflavin derivative, 8-SCHCONH FAD or 8-SCHCOOH FAD. These results indicate that position 8 of FAD bound to the protein is freely accessible to solvent. These studies support the idea that cytochrome b is a flavocytochrome.


INTRODUCTION

Upon ingestion of microbes, polymorphonuclear leukocytes (e.g. neutrophils) undergo a marked increase in oxygen consumption, a process referred to as the respiratory burst(1) . Respiration is accompanied by oxidation of NADPH and generation of superoxide anion, a precursor of other antimicrobicidal oxidants. Activation of the burst is thought to require the assembly of cytosolic regulatory proteins with the plasma membrane-associated cytochrome, cytochrome b. The membrane-bound cytochrome b consists of - and -subunits, with respective molecular masses of 22 and 91 kDa(2, 3, 4, 5, 6) . The cytosolic components include the p47, p67, and the low molecular weight GTP-binding protein, Rac 1 or Rac 2(7, 8, 9, 10, 11) . The cytosolic factors are presumed to regulate electron flow within the complex, but their mechanism of action is unknown.

It has long been assumed that the electron flow from NADPH through cytochrome and finally to oxygen must utilize a flavin moiety. This was based not only on chemical precedent, but also on early studies that showed that FAD could partially stabilize oxidase activity in detergent extracts from membranes of activated neutrophils(12) . In addition, it was found that during turnover, a flavin-like semiquinone ESR signal was seen in close proximity to the heme iron(13) . Until recently it was assumed that the NADPH binding/FAD-containing moiety was distinct from the cytochrome. Purified preparations of cytochrome b typically contain heme but have little or no FAD(14, 15, 16, 17) . A variety of candidate flavoproteins have been proposed based on purification approaches(12, 16, 17, 18, 19, 20, 21) and chemical modification with chemically reactive NADPH analogues(22, 23, 24) . Supporting the idea of a separate FAD-component, NADPH-cytochrome c reductase purified from neutrophil membranes (25) was able to form superoxide anion when combined with purified cytochrome b in the presence of phospholipids. However, the rate of Ogeneration was low, undermining this interpretation. In addition, none of the other candidate flavoproteins have held up as good candidates for an exogenous oxidase-linked component.

Recent evidence has suggested that the NADPH-interacting, FAD-binding moiety is cytochrome b itself. The large subunit contains a region of homology with the NADPH-binding region of several flavoenzymes including cytochrome P-450 reductase and ferredoxin-NADP reductase(26, 27, 28) . Homology was also detected in the region of the FAD-isoalloxazine-binding site approximately 90 amino acids upstream of the NADPH-ribose-interacting site of the large subunit(27) , but the homology was rather weak. Although the flavin is completely lost from the protein as the purification proceeds, it is possible to restore activity to either native or recombinant preparations of cytochrome b by incubating in the presence of phospholipids and FAD(26, 27, 28, 29) . Following such an incubation, FAD fluorescence comigrates with oxidase activity and heme absorption in gel exclusion chromatography(26) . Thus, the cytochrome b has been proposed to bind FAD and to contain all obligate electron transporting groups of the oxidase(30, 31) .

Several chemically modified forms of flavins including 8-mercaptoflavin have been developed in recent years and have proven useful as active site probes of flavoproteins(32, 33) . While binding of FAD to apoflavoproteins typically produces small changes in the visible spectrum of the native flavin, binding generates a large red shift in the spectrum of 8-mercaptoflavin. In addition, this analog can be used to investigate the solvent accessibility of the protein-bound flavin (32, 33). This approach exploits the chemical reactivity of the 8-SH group on the flavin and the large fluorescence change that occurs upon modification. In the present study, we have used 8-mercapto-FAD to probe the flavin binding site of cytochrome b as well as the solvent accessibility of the bound flavin.


EXPERIMENTAL PROCEDURES

Materials

Cytochrome c (type VI), polyacrylamide gel electrophoresis standards, NADPH, n-octyl glucoside, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and N-p-tosyl-L-lysine chloromethyl ketone were from Sigma. GTPS was purchased from Boehringer Mannheim. HESSOL (6% hetastarch in 0.9% NaCl) was from Green Cross Co., and lymphocyte separation medium (LSM, 6.2% Ficoll plus 9.4% sodium diatrizoate) was obtained from American Critical Care Division of American Hospital Supply (McGaw Park, IL) and Biotics (Kensington, MD), respectively. Superoxide dismutase and dithiothreitol were from Wako Pure Chemical Co., and prestained molecular weight standards for SDS-PAGE were obtained from Bio-Rad. Heparin-Sepharose CL-6B, DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, and -aminooctyl-agarose were purchased from Pharmacia Biotech Inc. L--Phosphatidylcholine (bovine brain), L--phosphatidylethanolamine (bovine brain), L--phosphatidylserine (bovine brain), L--phosphatidylinositol (bovine brain), and sphingomyelin (bovine erythrocyte) were purchased from Sigma. All other reagents were of the highest grade available commercially.

Isolation of Human Neutrophils

Human neutrophils were obtained from peripheral blood of normal healthy donors after obtaining informed consent. Erythrocytes were sedimented with HESSOL, and the mononuclear cells were removed from the resulting supernatant by centrifugation through lymphocyte separation medium(34) . The resulting cells were greater than 95% neutrophilic granulocytes. Neutrophils were resuspended in buffer A (6 mM PIPES, pH 7.35, containing 60 mM KCl, 18 mM NaCl, 2.3 mM MgCl, 1 µM diisopropyl fluorophosphate, 1 mM PMSF, 1 µg/ml TLCK, and 6% (w/w) sucrose).

Plasma Membrane Preparation and Solubilization

Membranes were prepared as described previously(25) . Cells (6 10) in 20 ml of ice-cold buffer A were disrupted by nitrogen cavitation after being pressurized at 500 p.s.i. for 25 min at 3 °C(35) . The cavitate was centrifuged (800 g, 5 min) to remove nuclei and unbroken cells. The supernatant was loaded onto discontinuous sucrose gradient consisting of 50 and 30% sucrose in buffer A and centrifuged at 150,000 g for 1 h. Plasma membrane and specific granule fractions were collected, and 5 volumes of relaxation buffer (10 mM PIPES, pH 7.35, containing 100 mM KCl, 3 mM NaCl, and 3.5 mM MgCl) were added. The membrane suspension was centrifuged at 250,000 g for 1.5 h, and the pellets were resuspended in 5 ml of buffer B (0.1 M Tris acetate buffer, pH 7.4, containing 0.1 M KCl, 20% glycerol, 1 mM dithiothreitol, 1 mM EGTA, 1 mM PMSF, 1 µg/ml TLCK, 40 mMn-octyl glucoside, and 0.5% sodium cholate). The material was centrifuged at 150,000 g for 1 h, and the supernatant was used for purification of cytochrome b.

Purification of Cytochrome bfrom Solubilized Membranes

Detergent-solubilized cytochrome b was purified by the method of Segal et al. (27) with some modifications. The solubilized membrane fraction was passed through a mixed bed of DEAE-Sepharose, CM-Sepharose, and -aminooctyl-agarose (1.6 ml bed volume of each), followed by chromatography on heparin-Sepharose (2.36 ml bed volume). The columns were equilibrated and washed well with buffer B, and cytochrome b was adsorbed to the heparin-Sepharose. The latter was washed stepwise with 50 ml of buffer B containing 0.1 M NaCl and then with 25 ml of buffer B containing 0.5 M NaCl, which eluted the cytochrome. These cytochrome b-containing fractions were pooled, concentrated, and applied to a desalting column (Excellulose GF-5, Pierce) equilibrated with buffer C (50 mM Tris acetate buffer, pH 7.4, containing 20% glycerol, 2 mM MgCl, 1 mM dithiothreitol, 1 mM EGTA, 1 mM PMSF, and 1 µg/ml TLCK) to remove NaCl. Samples with specific heme contents greater than 14.3 nmol heme/mg protein were pooled and applied to a HiTrap Q column (Pharmacia) and the eluting heme-containing fractions were collected. The concentration of cytochrome b was determined by reduced-minus-oxidized absorbance difference spectroscopy or by directly measuring the oxidized absorbance spectrum (350 nm to 600 nm) on a Hitachi 156 dual beam scanning spectrophotometer, assuming a reduced-minus-oxidized Soret band (427-440 nm) extinction coefficient of 161 mMcm or an oxidized Soret band (414 nm) extinction coefficient of 131 mMcm(36) . The pooled fractions had a heme content of 16.2 nmol/mg protein. Flavin was analyzed according to Nisimoto et al.(37) , using reverse phase HPLC with a fluorescence detector. FMN and FAD obtained from Sigma were purified by HPLC and used as the authentic standards. Protein was quantified by the method of Lowry et al.(38) , using bovine serum albumin as the standard.

Western Blot Analysis

Proteins separated by SDS-PAGE (10% gel) were transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA)(39) . The membrane was incubated at 25 °C for 2 h in 4% skim milk in 20 mM phosphate buffer, pH 7.3, containing 0.14 M NaCl and 2.7 mM KCl. Antibodies used were those to two synthetic peptides corresponding to the COOH termini of residues 175-195 of the small cytochrome subunit and 558-570 of the large cytochrome subunit, prepared as described in the previous paper(40) . After washing, the membrane was reacted with the antibodies (3.0 µg/ml) and then with horseradish peroxidase-linked second antibody (IgG, 1:5000 dilution) raised in goat. Antibodies were diluted with 20 mM phosphate-buffered saline, pH 7.3, containing 0.1% Tween 20, and each incubation was carried out for 1 h at 25 °C. The Immobilon-P membrane was washed extensively 3 times with 20 mM phosphate-buffered saline, pH 7.3, containing 0.1% Tween 20 (20 min each). The Western blots were stained for 15 min with 3,3`-diaminobenzidine in 20 mM phosphate-buffered saline, pH 7.3, containing 0.03% HO.

Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis was carried out according to the method by Rudolph and Krueger(41) . Following electrophoresis for 2 h, the gels were stained with two-dimensional-silver stain II reagent (Daiichi Pure Chemical Co. Ltd.).

Assay of the Superoxide-generating Activity

Cell-free NADPH oxidase activity was assayed by measuring superoxide dismutase-inhibitable ferricytochrome c reduction. The assay was carried out in the absence and presence of exogeneous native or 8 mercapto-FAD in the final assay medium. The cell-free system consisted of cytochrome b (without or with added FAD or FAD analog) or plasma membrane, cytosol, phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)), 10 µM GTPS, and 0.25 mM arachidonate in 0.2 ml of medium A (10 mM PIPES, pH 7.45, containing 4 mM MgCl, 3 mM NaCl, 0.1 M KCl, 1 mM EGTA, and 0.5 mM PMSF). This mixture was preincubated for 10 min at 25 °C. At the end of the preincubation period, an aliquot (95 µl) of the preincubation mixture was employed for the assay of superoxide generation. Superoxide dismutase (80 µg) was added to the reference cuvette. The reaction was initiated by addition of 0.2 mM NADPH (final) to 1 ml of assay medium containing 0.1 mM cytochrome c and 0.5 mg of phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)). When plasma membrane (8.5 pmol as heme) was used, phospholipids were omitted from the assay mixture. An extinction coefficient of 18.5 mMcm at 550 nm was used to calculate the quantity of cytochrome c reduced (42).

Purification of FAD Synthetase from Brevibacterium ammoniagenes

Culture of B. ammoniagenes and enzyme purification were performed as in starting with 160 g of frozen cell paste(43) .

Preparation of 8-Mercapto-FAD

The starting material, 8-chlororiboflavin(44) , was generously provided by Dr. Dale E. Edmondson, Emory University. This was converted to 8-chloro-FAD using partially purified FAD synthetase from B. ammoniagenes. 8-Chlororiboflavin (76.8 µM) was incubated in a final volume of 8.5 ml of 50 mM Tris-HCl buffer, pH 8.0, containing 5 mM ATP and 20 mM MgCl, and the reaction was started by the addition of 2 ml of partially purified FAD synthetase. After reaction at 36 °C for 12 h in the dark, an aliquot (15 µl) was removed and applied to thin-layer chromatography (Eastman Kodak Co., Chromagram, 13255 cellulose). The 8-chloro-FAD and unreacted 8-chlororiboflavin were identified on the thin-layer chromatography cellulose sheet by fluorescence detector. The total reaction mixture (10.5 ml) was then loaded onto a Bio-Gel P-2 column (5 60 cm). The first yellow band eluted was identified as 8-chloro-FAD by thin-layer chromatography using native FAD (R value, 0.07) as a standard. The thin-layer chromatography was developed using 1-butanol/acetic acid/water (4:2:2). The appropriate 8-mercapto-FAD was prepared just before use by reaction of 8-chloro-FAD, buffered at pH 8.0, with 5 mM NaS. Excess unreacted sodium sulfide was removed by P-2 column (2 65 cm) chromatography. The course of the reaction was monitored at 520 nm and is accompanied by a color change from yellow to bright red. The resulting 8-mercapto-FAD was reasonably stable at pH 7.5 and was used immediately for the spectral and kinetic studies.

Reconstitution of Flavin-depleted Cytochrome b with Either Native FAD or 8-Mercapto-FAD

Purified FAD-depleted cytochrome b (194 pmol heme/12 µg of protein) was incubated in 0.3 ml of 0.05 M Tris acetate buffer, pH 7.45, containing 0.05 M KCl, 10% glycerol, 1 mM dithiothreitol, 1 mM EGTA, 1 mM PMSF, 1 µg/ml TLCK (Buffer I) and 0.64 mg of phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)) at 3 °C for 3 h and then it was mixed with a 7.4-fold excess of either FAD or 8-mercapto-FAD. After keeping the mixture at 3 °C for 12 h, it was passed through a Sephadex G-25 column (12 200 mm) equilibrated with the Buffer I to separate FAD-reconstituted cytochrome b from excess free flavin. Cytochrome fractions showing high flavin fluorescence were pooled, and FAD was quantified by fluorescence spectroscopy. The binding of 8-mercapto-FAD to cytochrome b is also evidenced and determined by the measurement of absorption spectrum using the extinction coefficient of 30 mMcm for the FAD analog(32) . Pooled fractions were used for the assay of superoxide production by complementation with cytosol in the cell-free activation system.

Reaction of 8-Mercapto-FAD-reconstituted Cytochrome bwith Either Monoiodoacetamide or Monoiodoacetate

8-SH FAD-reconstituted cytochrome b was used to investigate the chemical reactivity of position 8 of the flavin. The reconstituted cytochrome b contained 15.5 nmol of protoheme and 7.93 nmol of 8-SH FAD/mg of protein. If it is assumed that the heme and flavin-binding sites are 1 mol each/mol of protein ( plus -subunits), the reflavination is 51% effective. If two or more hemes per FAD is assumed, as has been proposed(26, 45) , then reflavination is complete. Reaction with either iodoacetamide or iodoacetic acid was initiated by adding an aliquot of the reagent (0.1 M stock solution) to 0.2 ml of the 8-SH FAD-reconstituted cytochrome. In a control experiment, the thiol reagent was reacted with free 8-mercapto-FAD. The resulting fluorescence increase at 519 nm was monitored as the generation of 8-SCHCONH FAD or 8-SCHCOOH FAD when excited at 473 nm. Since it appears that the reactions show a single second-order dependence of the observed rate (k) on reagent concentration, a one-step reaction between reagent and flavinated cytochrome b is considered. The reaction is expressed as follows.

On-line formulae not verified for accuracy

REACTION 1

In this reaction, the appearance of product (8-SCHCONHFAD-cytochrome b) could be measured, and the second-order rate constant (k) was determined. Likewise, the cytochrome b-bound FAD analog could react directly with iodoacetic acid, which has a negatively charged group, and simple second-order kinetics were also found.


RESULTS

Effect of FAD and 8-Thio-FAD on NADPH Oxidase Cell-free Activity

Upon analysis by silver-stained SDS-PAGE (Fig. 1A, lane2) the purified cytochrome b showed a major band at 22 kDa and a diffuse major band at 90-95 kDa. The latter represents the glycosylated large subunit, which is known to behave in this manner. Two minor bands were also present at around 75 and 24 kDa, respectively. The two main protein bands were identified as the small and large subunits of cytochrome b by Western blot analysis (Fig. 1B, lane2). The antibody raised to the synthetic peptide corresponding to the COOH terminus of the small subunit detected the 22 kDa band of cytochrome b, whereas that raised to the synthetic COOH-terminal peptide of the large subunit gave diffuse staining around 92-kDa region and also appeared to stain the minor 75 kDa band, suggesting that the latter is a degradation (proteolysis or partial loss of carbohydrates) product of the large subunit. The reduced minus oxidized difference absorption spectrum of the purified preparation had absorption maxima at 427, 530, and 558 nm, which are characteristic of cytochrome b (data not shown). The final preparation eluted from an ion-exchange column (HiTrap Q) contained 16.2 nmol heme/mg of protein, but less than 0.01 nmol FAD/mg of protein.


Figure 1: SDS-PAGE pattern and immunoblot analysis of purified cytochrome b. The purified cytochrome b (1.2 µg) was loaded onto SDS-PAGE, followed by silver staining to examine the purity (laneA-2). Major protein peaks corresponded to apparent molecular masses of 22 kDa (I) and 92 kDa (II). After transfer to an Immobilon-P membrane, the membrane was incubated with a mixture of rabbit polyclonal antibodies (3.0 µg/ml) against two synthetic carboxyl-terminal peptides corresponding to the human cytochrome b small and large subunits as described under ``Experimental Procedures'' (laneB-2). LaneA-1is silver staining of detergent-solubilized plasma membrane (10.7 µg). LaneM shows prestained SDS-PAGE standards (phosphorylase b, 106 kDa; bovine serum albumin, 80 kDa; ovalbumin, 50 kDa; carbonic anhydrase, 32.5 kDa; soybean trypsin inhibitor, 27.5 kDa; lysozyme, 14.5 kDa).



The superoxide-generating activity of the purified cytochrome b was initially measured in a cell-free assay system consisting of cytosol, arachidonate, and GTPS with or without FAD in the assay mixture. In the presence of 5 µM FAD, the purified FAD-depleted cytochrome b showed low superoxide-generating activity (1.34 mol O/s/mol of cytochrome b)(40) , but superoxide production was negligible in the absence of FAD (0.05 mol O/s/mol of cytochrome). These results confirm previous reports (26, 27) that added flavin is required for superoxide generation. Flavin-depleted cytochrome b was also incubated with FAD or 8-SH FAD in the presence of phosphatidylcholine, and an aliquot was used for the assay for superoxide generation. In the presence of phospholipids and either FAD or 8-thio-FAD added to the cell-free assay, somewhat higher superoxide generation was seen. The highest value (11.7 mol/s/mol of cytochrome b seen with heme) was only 20% of that seen when native plasma membranes were used as the source of the intact cytochrome. 8-Mercapto-FAD exhibited almost the same ability to reconstitute superoxide-generating activity using this protocol. The low activity is incomplete probably due to in the reflavination of cytochrome b under these conditions or possibly due to dissociation of flavin upon dilution of the flavocytochrome in the assay mixture.

However, high activity was observed when the reconstitution was carried out in the presence of PC/PE/PI/SM/cholesterol (4:2:1:3:3 (w/w)). Koshkin and Pick (45, 46) recently demonstrated that purified cytochrome b exhibits varying levels of O-producing activity depending upon the nature of phospholipids serving for lipidation and flavination. Our present studies confirm that binding of flavin to cytochrome b is affected by the composition of phospholipids. Flavin-depleted cytochrome b was incubated with phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)) and either native FAD or 8-mercapto-FAD as detailed under ``Experimental Procedures,'' and then subjected to gel filtration to separate flavin-bound cytochrome from free flavin (Fig. 2, A and B, respectively). An initial peak of flavin fluorescence coeluted with the purified cytochrome b, which migrated identically in the absence of added flavin (Fig. 2C). In the absence of the cytochrome, but with all other components present (Fig. 2D), the early eluting peak of flavin fluorescence was not detected. The weak fluorescence seen in the first peak of Figs. 2, C and D, was not due to flavin but to scattering due to the presence of phospholipids in the fractions. The major fractions with high flavin fluorescence and heme absorbance were pooled to examine the ability of either native FAD or 8-mercapto-FAD to reconstitute NADPH oxidase activity, and the results are summarized in . The FAD-reconstituted cytochrome b exhibited O-producing activity amounting to 70% of the original activity of the plasma membranes on a per heme basis. Likewise, 8-mercapto-FAD-reconstituted cytochrome b indicated a greatly increased NADPH oxidase activity, which corresponded to about 61% of the original activity of the plasma membranes. These results suggest that the flavin-reconstituted cytochrome b has a high affinity for 8-mercapto-FAD as well as native FAD, and stabilized binding of flavin to the protein leads to high catalytic activity. The difference in the redox potentials of native FAD (-219 mV) (47) and 8-SH FAD (-290 mV) (48) does not appear to change significantly the oxidase activity of the flavin-reconstituted cytochrome b.


Figure 2: Reconstitution of cytochrome b with either native FAD or 8-mercapto-FAD. FAD-depleted cytochrome b (194 pmol as heme) was incubated (3 h, 3 °C) with 0.64 mg of phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)) in total of 0.3 ml of 0.05 M Tris acetate buffer, pH 7.45, containing 0.05 M KCl, 10% glycerol, 1 mM EGTA, 1 mM PMSF, and 1 µg/ml TLCK (Buffer I). This sample was then mixed with 0.2 ml of either 7.1 µM native FAD (panelA) or 7.0 µM 8-SH FAD (panelB) or with 0.2 ml of Buffer I instead of flavin (panelC). As a control, 0.64 mg of phospholipids in 0.3 ml of Buffer I was mixed with 0.2 ml of 7.0 µM 8-SH FAD in the absence of cytochrome (panelD). After keeping the mixture at 3 °C for 12 h, it was applied to a Sephadex G-25 column (12 200 mm) equilibrated with Buffer I to separate bound from free flavin. Fractions (0.85 ml) were collected, and each fraction was used for the measurements of absorbances at 280 and 414 nm. For emission measurements of native FAD, an aliquot (0.20 ml) of each fraction was mixed with 0.1% SDS and heated for 3 min in a boiling water bath. The supernatant (6,000 g, 15 min) was used for the assay of flavin content. The excitation and emission wavelengths were 450 and 525 nm, respectively. For determination of 8-SH FAD, the supernatant was reacted with 10 mM iodoacetamide for 30 min at pH 7.8. The fluorescence at 519 nm was measured when excited at 473 nm (bandwidth of 3 nm). The peak fractions (fraction number 9, 10, and 11) indicated by the horizontalbars in panelsA and B were individually pooled, and aliquots were subjected to the assay for the cell-free NADPH oxidase activity and spectral studies.



Spectrophotometric Changes of 8-Mercapto-FAD Induced by Binding to Cytochrome b

When 8-mercapto-FAD is mixed and incubated with the flavin-depleted cytochrome b at pH 7.45, there was a large red-shift in the visible absorption spectrum (Fig. 3), with the peak shifting from about 520 nm to about 560 nm. No detectable absorbance or fluorescence change was seen with native FAD binding to the cytochrome. A stable spectrum was reached within 3 h at 3 °C, and no further change occurred over this period. A major peak at 560 nm with a weak residual shoulder at about 520 nm was seen using a 1.7-fold excess of flavin-depleted cytochrome b (heme) to the 8-mercapto-FAD. Although difficult to quantify precisely, these data indicate that most of the 8-mercapto-FAD was bound to protein under these conditions.


Figure 3: Effect of flavin-depleted cytochrome b on the absorption spectrum of 8-mercapto-FAD. The 8-SH FAD (0.21 µM in 1 ml) was titrated with 0.5, 0.9, and 1.1 ml of 0.32 µM cytochrome b in Buffer I containing 0.64 mg/ml phospholipids, and spectra D, E, and F were recorded, respectively. The molar ratios of the heme to 8-SH FAD were 0.76, 1.37, and 1.68, respectively. Curves B and C, respectively, show the absorption of the cytochrome (0.32 µM as heme) and of 8-SH FAD (0.21 µM) measured in the presence of phospholipids (0.64 mg/ml), and curve A is a base line containing the phospholipids alone in both sample and reference cuvettes. The titrated mixture was allowed to stand for 3 h at 3 °C and then each spectrum was scanned at the same temperature. Dry nitrogen gas was circulated through the cell compartments.



Consistent with the titration data, the 8-mercapto-FAD-reconstituted cytochrome b chromatographed on Sephadex G-25 column (Fig. 2B) also showed a broad peak with the absorption maximum around 560 nm, indicating that on binding to the protein, the 8-SH flavin is stabilized in its p-quinoid state (Fig. 4). Heme and 8-mercapto-FAD concentrations of the flavin-reconstituted cytochrome b were determined from its absorption spectrum. Their concentrations were 0.15 µM (heme) and 0.085 µM (8-SH FAD), respectively, indicating a molar ratio of FAD analog to protoheme of 0.57. The flavin of 8-mercapto-FAD-reconstituted cytochrome b was also quantified by fluorimetric assay, and its content (8-SH FAD/heme, 0.51) was almost the same as the value obtained by the spectrophotometric method.


Figure 4: Absorption spectrum of 8-mercapto-FAD-reconstituted cytochrome b. The cytochrome was incubated with a 7.4-fold molar excess of 8-thio-FAD, and the reconstituted cytochrome was resolved from free flavin by gel filtration, as in Fig. 2. The spectrum of the flavinated cytochrome b, which subtracted a base line containing the phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)) alone, was recorded from 350 to 650 nm at 3 °C.



Solvent Accessibility of the 8-SH Position

We examined the reactivity of iodoacetamide with 8-mercapto-FAD in order to explore the effect of the protein environment on the reaction kinetics. The 8-mercapto-FAD reacts readily with this small electrophilic reagent producing a large increase in fluorescence(33) . Fig. 5shows typical time courses of fluorescence increase at 519 nm for the reaction of both free and cytochrome b-bound 8-mercapto-FAD with excess iodoacetamide. Plotting the observed initial rate constant as a function of iodoacetamide concentration gave a straight line passing through the origin with a slope equal to the second-order rate constant, which was calculated to be 48.5 Mmin for free 8-SH FAD at pH 7.45 (Fig. 6), a value that is comparable with the rate reported previously(33) . The reaction of the thiol reagent with 8-mercapto-FAD bound to the cytochrome occurred with a second order rate constant of 38.8 Mmin, approximately 20% slower than that found for free 8-mercapto FAD. This relatively large rate constant suggested that position 8 of the protein-bound flavin was freely available to solvent. Second-order kinetics was also seen upon reacting iodoacetic acid with 8-mercapto-FAD bound to cytochrome b (k = 12.1 Mmin). The rate constant was close to 16.4 Mmin, which was calculated for the reaction of free 8-SH FAD with iodoacetic acid (data not shown). This water-soluble electrophile also appears to indicate that the position 8 of isoalloxazine ring is accessible to solvent.


Figure 5: Time course for the reaction of iodoacetamide with free 8-mercapto-FAD and 8-SH FAD-reconstituted cytochrome b. Free 8-SH FAD (0.075 µM, curve A), and 8-SH FAD-reconstituted cytochrome b prepared as in Fig. 2 (0.070 µM as flavin, curve B) were incubated in 0.2 ml of Buffer I containing 0.16 mg of phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)). The reaction was initiated by adding 9.2 mM CHICONH to the incubation mixture. Fluorescence was monitored using excitation and emission wavelengths of 473 and 519 nm, respectively. In a control reaction, 9.2 mM iodoacetamide was added to the reaction mixture without added 8-SH FAD (curve C).




Figure 6: Rate constants for the reaction of iodoacetamide with free 8-mercapto-FAD and 8-SH FAD-reconstituted cytochrome b. The apparent rate constant (K) obtained in experiments as in Fig. 5 was plotted as a function of CHICONH concentration, and the second-order rate constant was determined from the slope (see ``Experimental Procedures''). Opencircles show the reaction with free 8-SH FAD, and filledcircles are that with 8-SH FAD-reconstituted cytochrome b.




DISCUSSION

The present studies provide additional evidence that cytochrome b is a flavocytochrome. As purified, the cytochrome loses flavin so that in its purified form it lacks both measurable flavin and activity. Either native FAD or 8-substituted FAD analog can readily be bound to the cytochrome in such a way as to reconstitute activity. As was found by other groups(26, 27) , reconstitution of activity with FAD requires the presence of phospholiplids. Our data are thus consistent with the proposal that FAD binding requires phospholipids or a membrane environment and that detergent solubilization results in a cytochome conformation from which flavin is readily lost. In the presence of excess FAD or analog in the assay medium, the activity that can be maximally achieved was only about 20% of that seen with the cytochrome in its native plasma membrane environment (based on activity/heme). It may be either that flavin reconstitution is incomplete under these conditions or that the native and active conformation of the cytochrome has not been completely restored (or both), for example due to a nonoptimal phospholipid composition. In this regard it is worth noting earlier studies (45, 46, 49) in which phospholipid composition has significant effects on activity in detergent solubilized preparations. The most effective flavination was found using cytochrome b reconstituted with a mixture of PC, PE, PI, SM, and cholesterol (4/2/1/3/3 (w/w)). Under these conditions, the FAD:heme ratio was found to be 0.4-0.5 mol/mol, consistent with the previously proposed ratio of two hemes/flavin. Binding of 8-SH FAD to cytochrome b was demonstrated by co-elution of flavin and heme by gel exclusion chromatography, and also by the perturbation of the 8-thioflavin spectrum by the apoflavocytochrome. The high degree of reflavination of cytochrome b in the presence of the above lipids was accompanied by a corresponding increase in O producing activity, but this was slightly less than the activity seen in native plasma membranes suggesting that the plasma membrane provides a more optimal environment than does the defined lipid mixture.

Additional evidence for flavin is that binding to the cytochrome is provided by the pronounced spectral shift upon interaction of 8-SH FAD to the apoflavocytochrome. Spectral changes upon binding of native flavins are typically small, and we were unable to detect such changes for binding of FAD itself to the flavin-depleted cytochrome using either UV-visible absorption or fluorescence methods (data not shown). However, much larger red shifts of the spectrum have been seen upon binding of 8-SH flavins to a variety of apoflavoproteins(32) . These include flavoenzymes of the dehydrogenase-oxidase class such as glucose oxidase, D-amino acid oxidase, lactate oxidase, and old yellow enzyme(32) . Such spectral shifts were readily observed in the present studies and are indicative of flavin binding to a protein environment. These spectral shifts were larger than those previously seen (18) upon incubation of Triton X-100-solubilized neutrophil membranes with 8-SH FAD, possibly because of more complete saturation of the binding site with an excess of flavin-depleted cytochrome or because of complicating binding of the flavin to other species in the crude detergent extract.

The present studies also indicate that in the bound flavin, the 8-thio position of 8-SH FAD is readily accessible to a soluble electrophile. We observed only small decrease in the reaction rate of either iodoacetamide or iodoacetate with the flavin following binding to the flavin-depleted cytochrome. These data suggest that as in many other flavoproteins, the 7-8-flavin edge is on the surface of the cytochrome exposed to solvent, while other portions of the flavin are likely to be deeply buried. The relatively high fluorescence of the iodoacetamide and iodoacetate-derivatized flavins may prove to be a useful reporter group for investigating other aspects of oxidase assembly and function.

  
Table: Superoxide-generating activity of FAD-reconstituted cytochrome b in a cell-free assay system

An aliquot of either flavin-depleted cytochrome b (0.73 µg) or FAD-reconstituted cytochrome b (0.70 µg) was incubated with 0.22 mg of cytosol, 0.25 mg of phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)), 10 µM GTPS, and 0.25 mM arachidonate in 0.2 ml of medium A as described under ``Experimental Procedures.'' The assay was carried out in the absence of added flavin in the reaction medium. FAD-reconstituted cytochrome b was obtained from Sephadex G-25 column chromatography as described in Fig. 2. Flavin content of FAD-reconstituted cytochrome b was estimated by fluorimetric method.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI22809. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: O, superoxide; GTPS, guanosine 5`-(-thio)triphosphate; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; TLCK, N-tosyl-L-lysine chloromethyl ketone; PIPES, 1,4-piperazinediethanesulfonic acid; PC, L--phosphatidylcholine; PE, L--phosphatidylethanolamine; PI, L--phosphatidylinositol; SM, sphingomyelin; HPLC, high-performance liquid chromatography; P-450 reductase, microsomal NADPH-cytochrome P-450 reductase from rabbit liver.


ACKNOWLEDGEMENTS

We thank Dr. Dale E. Edmondson (Emory University, School of Medicine) for providing 8-chlororiboflavin and for helpful advice.


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