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Originally published In Press as doi:10.1074/jbc.M104373200 on August 14, 2001

J. Biol. Chem., Vol. 276, Issue 42, 38852-38861, October 19, 2001
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Identification of a Spectrally Stable Proteolytic Fragment of Human Neutrophil Flavocytochrome b Composed of the NH2-terminal Regions of gp91phox and p22phox*

Thomas R. Foubert, Justin B. Bleazard, James B. Burritt, Jeannie M. Gripentrog, Danas Baniulis, Ross M. Taylor, and Algirdas J. JesaitisDagger

From the Department of Microbiology, Montana State University, Bozeman, Montana 59717-3520

Received for publication, May 14, 2001, and in revised form, July 30, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A heme-bearing polypeptide core of human neutrophil flavocytochrome b558 was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochrome b that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60-66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 °C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60-66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH2-terminal 320-363 amino acid residues of gp91phox and the NH2-terminal 169-171 amino acid residues of p22phox. These findings provide direct evidence that the primarily hydrophobic NH2-terminal regions of flavocytochrome b are responsible for heme ligation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The NADPH oxidase of human neutrophils is a multisubunit, membrane-associated complex that is crucial for host protection against invading pathogens (1-7). The redox center and the only membrane-spanning component of the oxidase is flavocytochrome b558 (also known as flavocytochrome b, cytochrome b558, cytochrome b559), a heterodimeric protein composed of an extensively glycosylated, 91-kDa large subunit (570 amino acid residues), gp91phox,1 and a 22-kDa non-glycosylated small subunit (192 amino acid residues), p22phox (8, 9). Intracellular binding sites for both FAD and NADPH have been identified on gp91phox (10-14). Flavocytochrome b functions as the terminal electron carrier prior to reduction of extracellular molecular oxygen to the antimicrobial precursor, superoxide anion (O&cjs1138;2) (2, 15-18). Flavocytochrome b has also been proposed to function as a voltage-gated proton transporter that maintains intracellular pH and membrane potential during the oxidative burst (19, 20).

The characteristic absorbance spectrum of flavocytochrome b is attributed to the presence of heme prosthetic groups that are non-covalently coordinated by histidine residues within the protein. Due to the tenuous nature of this ligation scheme, the heme spectrum is lost under conditions that separate the individual subunits (21), although heme remains associated with both subunits during electrophoresis at low temperature with lithium dodecyl sulfate-PAGE (22). Additionally, the detergent-solubilized protein is highly susceptible to proteolysis by the numerous endogenous phagocyte proteinases. These and other factors have prevented direct identification of the regions of the flavocytochrome b heterodimer that are responsible for heme coordination. There are, however, several lines of indirect evidence deriving from mutational (23, 24) and spectroscopic analyses (25, 26) that implicate regions of gp91phox with heme binding.

Inferences have also been made of a stacked heme orientation within the membrane bilayer, coordinated by two transmembrane helices of gp91phox based on similarities in primary sequence, spectral properties, and redox potentials between flavocytochrome b and select members of the ferredoxin-NADP+ reductase family, including FRE1 ferric reductase of Saccharomyces cerevisiae (27-30). Likewise, heme localization within gp91phox has been inferred based on similarities to heme-coordinating regions of cytochrome P450 of Pseudomonas putida (31) and the beta -subunit of cytochrome b559 of Synechocystis 6803 photosystem II (32). These arguments and the intrinsic hydrophobic nature of the heme molecule would suggest placement within the NH2-terminal putative membrane-spanning regions of flavocytochrome b2 (33-35), although there is no direct evidence to support this assumption.

The intent of this study was to isolate and identify proteinase-stable heme-ligating regions of flavocytochrome b. Partially purified flavocytochrome b was exposed to staphylococcal V8 proteinase (endoproteinase Glu-C). HPLC size exclusion chromatography was then used to isolate proteolytic fragments that retained the characteristic 414-nm heme absorbance spectrum. With this approach, we successfully isolated a spectrally native polypeptide core that was then characterized by amino acid sequencing and immunoblotting. The fragment was found to be a heterodimer composed of the NH2-terminal 336-363 amino acid residues of gp91phox and the NH2-terminal 169-171 amino acid residues of p22phox. The core fragment retained 74% of the native heme absorbance, suggesting that it contained all of the heme-ligating regions of flavocytochrome b. These findings suggest that the hemes are positioned intra- or juxtamembrane within the NH2-terminal predicted transmembrane-spanning regions of the protein.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- KCl, NaCl, EDTA, NaN3, silver nitrate, sodium carbonate, gelatin, Trizma (Tris base), EGTA, and syringe filters (Whatman, 25-mm diameter, polyethersulfone membrane, 0.2-µm pore size) were purchased from Fisher. GlcNAc, N,N'-diacetylglucosamine (chitobiose), heparin-Sepharose® 4B beads, N-formyl-Met-Leu-Phe, dihydrocytochalasin B, Na2ATP, chymostatin, wheat germ agglutinin, diisopropyl fluorophosphate, sodium dithionite, bovine serum albumin, Hanks' balanced salts, MgCl2, NaH2PO4, Trizma-HCl (Tris-HCl), hemin chloride (bovine), hydrogen peroxide 30% (w/w) solution, lithium dodecyl sulfate, pyridine (HPLC grade), glutathione, free acid, reduced form, thioglycolic acid (thioglycollate, mercaptoacetic acid), free acid, trifluoromethanesulfonic acid, iodoacetamide, 3,3',5,5'-tetramethylbenzidine (TMBZ) (free base), and proteinase inhibitor mixture (P8340) were from Sigma. Polyvinylidene difluoride membrane (0.2-µm pore size) was from Bio-Rad. Triton X-100 detergent was from EM Sciences Co. or Sigma. HEPES, Gammabind®, and CNBr-activated Sepharose 4B beads were purchased from Amersham Pharmacia Biotech. Ultrapure SDS was purchased from U. S. Biochemical Corp. N-Octyl-beta -D-glucopyranoside (OG, octyl glucoside), dithiothreitol (DTT, Cleland's reagent), and phenylmethylsulfonyl fluoride (PMSF), were from Calbiochem-Novabiochem. The BCA protein assay and BlueRanger® prestained molecular weight markers for SDS-PAGE were from Pierce. Other prestained molecular weight markers were supplied by Life Technologies, Inc. Endoproteinase Glu-C (proteinase V8 salt-free, lyophilized, sequencing grade) from Staphylococcus aureus V8 was from Roche Molecular Biochemicals. Nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate alkaline phosphatase developer kit for immunoblots was from Kirkegaard & Perry Laboratories (Gaithersburg, MD).

Buffers-- The following buffers were used in this work: heparin wash buffer: 50 mM NaH2PO4, 1 mM EGTA, 1 mM MgCl2, 0.1% (v/v) Triton X-100, 2 mM NaN3, pH 7.4, supplemented with 0.1 mM DTT, 10 µg/ml chymostatin, 0.2 mM PMSF (final concentrations) just prior to use; heparin elution buffer: either 75 mM or 2.0 M NaCl, 50 mM NaH2PO4, 2 mM NaN3, pH 7.4, 1 mM EGTA, 1 mM MgCl2, 0.1% (v/v) Triton X-100, supplemented with 0.1 mM DTT, 10 µg/ml chymostatin, 0.2 mM PMSF (final concentrations) immediately prior to use; HPLC column buffer: 150 mM NaCl, 50 mM NaH2PO4, 1 mM EGTA, 1 mM MgCl2, 0.1% (v/v) Triton X-100, 2 mM NaN3, 0.1 mM DTT, pH 7.4; TS buffer; 200 mM Tris base, 2% SDS, pH 8.0.

Partial Purification of Flavocytochrome b-- Isolation of human polymorphonuclear leukocytes from whole blood and purification of flavocytochrome b was carried out as described previously (9, 36) or with the following modifications. The wheat germ agglutinin affinity steps were eliminated to reduce the degree of sample handling, thus resulting in higher recoveries of flavocytochrome b without having an apparent effect on the overall purity. After loading, the heparin column containing the flavocytochrome b was washed with 10-20 bed volumes of heparin wash buffer and eluted using either a 50 mM to 2.0 M NaCl gradient or a 6-ml bolus of 1.0 M NaCl, both in heparin elution buffer. 1-ml fractions were collected, and the peak flavocytochrome b-containing fractions were pooled and concentrated to a final volume of ~1 ml using a 30-kDa nominal molecular weight cutoff centrifugal concentration device. As a final purification step prior to digestion, the retentate was subjected to an HPLC size exclusion chromatography step, as described below, and collected in 400-µl aliquots. Aliquots that were reserved for predigested controls had PMSF and chymostatin added to 1 mM and 10 µg/ml, respectively. Beginning with the solubilization step, all purification and digestion steps were accomplished in 1 day with all samples kept on wet ice prior to digestion. Flavocytochrome b heme content prior to heparin purification was quantitated using the reduced minus oxidized spectrum at 558 nm using Delta epsilon 558 = 29.3 (mM cm)-1 (37), blanked against control buffer, and reduced by addition of freshly mixed sodium dithionite in deionized water to a final concentration of 10 mM. After heparin purification, flavocytochrome b heme quantitation was carried out using epsilon 414 = 130.8 (mM cm)-1 (37) blanked against buffer. Absorbance values were determined using either a Hewlett-Packard HP 8452A diode array UV-visible spectrophotometer or a Molecular Dynamics Spectra-Max 250, environmentally controlled, 96-well microtiter plate, UV-visible spectrophotometer. When necessary, the samples were sonicated using either a Fisher 50 probe style Sonic Dismembrator, model XL2005, or a Fisher brand bath sonicator, model FS30.

HPLC Size Exclusion Chromatography of Flavocytochrome b-- All HPLC analyses were carried out using a Hitachi, LS-6200 HPLC with an F-1050 fluorescence detector connected in series with an L-7450A UV-visible Diode Array Detector. Size exclusion chromatography was performed using a Amersham Pharmacia Biotech Superdex 200 HR®, 10-30 column, maintained at 4 °C with a flow rate of 0.4 ml/min, equilibrated for a minimum of 1.5 h prior to sample injection. The column was calibrated using size exclusion chromatography standards (Bio-Rad catalog number 1511901) that included thyroglobulin, 670 kDa; gamma -globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa, and vitamin B12, 1.35 kDa. The standard curve was fitted using a non-linear regression algorithm (GraphPad Prism version 3.01 for Windows 95, GraphPad Software, San Diego, www.graphpad.com). Prior to HPLC size exclusion chromatography, all samples were bath sonicated for 1-2 s and passed through a 0.2-µm pore size syringe filter. HPLC elution fractions were collected from the column in 400-µl aliquots at 1-min intervals. Time point samplings for V8 digestions were removed from the digestion vessel and placed on wet ice with proteinase inhibitors prior to separation by HPLC size exclusion using the same procedures as for intact flavocytochrome b. All absorbance values were corrected for dilution and differences in HPLC injection volumes.

V8 Digestion of Flavocytochrome b-- Lyophilized V8 proteinase was reconstituted at 50-500 µg/ml in either distilled water or HPLC column buffer and added to the partially purified flavocytochrome b samples, while mixing, to a final ratio of 1:15 (w/w), proteinase to flavocytochrome b heme (assuming a flavocytochrome b heterodimer molecular mass of 110 kDa). The digestion was carried out at 37 °C in a continuously stirred vessel and terminated by placement on wet ice and addition of PMSF and chymostatin to final concentrations of 1 mM and 10 µg/ml, respectively. Digested samples were stored on ice at 4 °C until further analyses were conducted. Initial quantities of purified flavocytochrome b used for digestion ranged between 1.8 and 2.7 nmol of heme. The fractions retained from the HPLC runs had additional PMSF and chymostatin added to the same concentrations as above and were kept on ice until further analyses were conducted.

Modified TMBZ Heme Quantitation Assay-- Heme quantitation of HPLC fractions was carried out by modifying the procedure originally described (38, 39) to include addition of ethanol to the assay mixture to a nominal concentration of 50% (v/v). Assays were conducted in 96-well microtiter plates, and absorbance values were measured with a Molecular Dynamics Spectra-Max 250 UV-visible, environmentally controlled microtiter plate spectrophotometer. Reagents used in the assay were prepared immediately prior to use. The short incubation times combined with the large number of samples that were simultaneously tested required the use of a multitipped pipette to reduce timing errors in the mixing of the individual microtiter plate wells. Hemin standard solutions were prepared for initial quantitation as reduced, alkaline pyridine-solubilized hemochrome (37) by addition of 0.5 N NaOH to a final sample concentration of 0.075 N and 4.0 M pyridine to 2.1 M final concentration. The standards were then reduced by addition of sodium dithionite to a final concentration of 10 mM and quantitated using the reduced-oxidized molar extinction coefficient, Delta epsilon 556-540 = 20.7 (mM cm)-1 (37). TMBZ solution consisted of 10 mg of dry TMBZ mixed with 500 µl of glacial acetic acid, filtered through a 0.2 µm pore size polyethersulfone membrane filter. 15 µl of the TMBZ solution was mixed, per well, with 30 µl of hemochrome sample, 100 µl of absolute ethanol and incubated at ambient temperature for 2 min. At the end of the 2 min, 15-µl aliquots of 3% H2O2 were added per well and the mixtures incubated at ambient temperature for 5 min. The absorbance was then measured at 660 nm, and the hemin concentration of the samples was determined relative to the standards. All absorbance values were corrected for dilution and differences in HPLC injection volumes.

Immunoblot Analyses and Silver Staining-- Flavocytochrome b containing fractions were resolved by SDS-PAGE using 5-20% acrylamide gradient gels (40), and electrophoretic transfer of protein to nitrocellulose for immunoblotting was performed as described (41). Anti-gp91phox primary antibodies used were mouse mAbs NL10, CL5, NL7,3 and 54.1 (42, 43) and anti-peptide rabbit polyclonal antibodies KIS2 and KQS (44). Anti-p22phox primary antibodies used were mouse mAbs NS1, NS2, NS5, CS9,3 and 44.1 (42, 43), the rabbit polyclonal antibodies, anti-peptide EAR (45), and R3179 (9) produced by injecting rabbits with intact flavocytochrome b. Nitrocellulose transfers probed with primary antibodies were then probed with goat anti-rabbit, or goat anti-mouse, alkaline phosphatase-conjugated secondary antibodies and visualized using the Kirkegaard & Perry Laboratories nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate developer kit. Silver staining of polyacrylamide gels was carried out by overnight rocking in 50% methanol, 12% acetic acid in water, followed by three 20-min washes in 50% ethanol/water. Gels were then drained, completely submerged for 1 min in a solution containing 0.2 g/liter sodium thiosulfate, and washed 3 times for 30 s each with distilled water. After draining, 100 ml of a solution containing 0.2 g/liter silver nitrate, 1.0 ml/liter formaldehyde in water was added, and the gels were rocked for 20-30 min. The gels were then rinsed three times with water for 30 s each and developed by addition of 200 ml of 60 g/liter sodium carbonate, 1.0 ml/liter 37% (w/w) formaldehyde, and 30 ml/liter of the 0.2 g/liter sodium thiosulfate in distilled water and stopped by addition of 25% isopropyl alcohol and 10% acetic acid in water.

Preparation of Flavocytochrome b for Amino Acid Sequence Analysis-- NH2-terminal sequence analyses by Edman degradation were performed by Harvard Microchem (Cambridge, MA), and the samples were prepared as per their recommendations. To reduce the quantity of ubiquitous keratin in the SDS-PAGE reagents, twice recrystallized SDS (46) and distilled water that had been filtered through a 10-kDa nominal molecular weight cutoff filter were used. All other aqueous reagents were filtered through a 0.2-µm pore size membrane filter.

Inadvertent chemical modification of the NH2 termini of peptides to be sequenced was avoided by including the following modifications to the SDS-PAGE protocol (40). 5-20% gradient polyacrylamide resolving gels were allowed to polymerize overnight at ambient temperature. The stacking gel was poured, allowed to polymerize, and then pre-run without sample for 45 min at 4 mA constant current in the presence of Running buffer containing 5 µM reduced glutathione. The glutathione-containing buffer was removed; the samples were loaded, and the electrophoresis was conducted at 40 mA constant current using new Running buffer that contained 100 µM thioglycolic acid (47). Following SDS-PAGE, the proteins were transferred to polyvinylidene difluoride membrane, stained with Amido Black for visualization (46), excised from the membrane, and the strips washed 3× by gentle vortexing for 15 s each in filtered distilled water. The individual strips were then air-dried and placed in sealed plastic tubes for shipment. The diffuse band centered at ~90 kDa, and the consolidated bands at 22 kDa were excised for NH2-terminal sequence analyses of the nondigested flavocytochrome b. The single band at 17 kDa and the entire broad band from ~50 to 70 kDa were excised from the polyvinylidene difluoride membrane for NH2-terminal sequence analyses of the 1-h digest fragments.

Reduction and Alkylation-- Samples were reduced by addition of an equal volume of DTT solution (50 mM DTT in TS buffer) and heated 4-5 min at 90 °C. Alkylation was performed by adding 0.1 volume (sample + DTT mixture) of iodoacetamide stock (46 mg of iodoacetamide dissolved in 200 mM Tris base, pH 8.0), followed by incubation at 90 °C for 3-4 min with IgG used as a procedural control. Iodoacetamide was added without DTT treatment to sample controls. All samples were then added to sample loading buffer, separated by SDS-PAGE, and immunoblotted as described above.

Deglycosylation of Flavocytochrome b-- For chemical deglycosylation, both intact and digested polypeptides were precipitated by addition of 80% (v/v) trichloroacetic acid in distilled water to a final sample concentration of 15% (v/v). Samples were then cooled at -20 °C for 30 min, followed by centrifugation at 180,000 × g for 15 min. The supernatant fraction was removed, and the pellet was then washed twice in -20 °C acetone to remove the trichloroacetic acid and either allowed to air dry or purged under a stream of dry argon at room temperature. The pellet was then resuspended into 100 µl of neat trifluoromethanesulfonic acid (48, 49) by bath sonication in sealed tubes, purged with argon, and incubated on ice for 3 h in a ventilation hood. At the end of the incubation, the samples were cooled to less than -20 °C by immersion in a mixture of dry ice and ethanol. Neat pyridine, likewise cooled to less than -20 °C, was then slowly added to terminate the reaction. The volatile organic phase was removed under a stream of dry argon at room temperature, and the resulting gel was resuspended in distilled water and dialyzed against several changes of 10 mM phosphate-buffered saline at 4 °C. The protein was precipitated by addition of trichloroacetic acid to 15% (v/v), incubated at -20 °C for 15 min, and pelleted by centrifugation at 20,000 × g for 15 min at 4 °C. The resulting pellets were washed twice with -20 °C neat acetone and pelleted each time by centrifugation at 20,000 × g for 10 min at 4 °C. Samples were then reduced and alkylated as described above, mixed with loading buffer, and added directly to the lanes for separation by SDS-PAGE. Enzymatic deglycosylation of flavocytochrome b was carried out using peptide N-glycosidase F HPLC size exclusion chromatography fractions were denatured by addition of SDS to 0.5% in the presence of 10 mM DTT and 1 µl/ml proteinase inhibitor mixture and then heated to 100 °C for 10 min. Reagents supplied by the manufacturer were then added as per their instructions and incubated at 37 °C for 1 h with intermittent mixing.

Mass Determination of V8 Digest Fragments-- All predicted mass analyses based on primary sequence were done using either General Protein Mass Analysis for Windows, version 4.04, Lighthouse Data, or Statistical Analysis of Protein Sequences (SAPS) (50), available at www.isrec.isb-sib.ch/software/SAPS. Molecular mass determination by SDS-PAGE was extrapolated from pre-stained standards from separate suppliers consisting of either lysozyme, trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin, phosphorylase B, and myosin (heavy-chain), with respective molecular masses of 18, 28, 39, 60, 84, 120, and 215 kDa, (Life Technologies, Inc.), or lysozyme, beta -lactoglobulin, carbonic anhydrase, ovalbumin, bovine serum albumin, phosphorylase B, myosin (heavy-chain) with respective molecular masses of 15, 20, 30, 47, 73, 121, and 216 kDa (Pierce).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study was designed to isolate and identify proteinase-stable, heme-coordinating regions of human neutrophil flavocytochrome b558. Our approach used limited digestion of partially purified, detergent-solubilized flavocytochrome b with staphylococcal V8 proteinase. Following digestion, HPLC size exclusion chromatography was used to isolate heme-associated polypeptides. Heme and protein content were simultaneously measured by monitoring the absorbance spectrum from 230 to 600 nm with a UV-visible diode array detector (DAD) connected in series with a fluorometer (lambda ex = 280 nm, lambda em = 340 nm). Fig. 1 shows a three-dimensional chromatogram of partially purified flavocytochrome b (695 pmol of heme) during the final HPLC size exclusion purification step prior to digestion with V8 proteinase. Flavocytochrome b is readily identifiable as the prominent 414 nm absorbance peak eluting at 27 min, corresponding to an apparent mass of 300-330 kDa relative to soluble size exclusion chromatography protein standards. This apparently anomalous elution mass is independent of the detergent used4 (Triton X-100, dodecyl maltoside, and OG, with detergent micelle molecular masses of ~90, 24, and 18 kDa respectively) for either solubilization or HPLC column buffer. Since detergent-solubilized flavocytochrome b under similar conditions has been shown to have a 1:1 subunit stoichiometry (21, 51, 52), the apparent elution mass probably arises from a combination of the asymmetric geometry of the protein (21) and the roughly gram per gram ratio of bound detergent typical of transmembrane proteins (21, 53). Additionally, purification conditions similar to those used in this work have yielded flavocytochrome b-Rap1A complexes (36, 54). We thus infer that the apparent 300-330-kDa elution mass represents the unit size of monodisperse detergent-bound flavocytochrome b or possibly a flavocytochrome b-Rap1A complex. The absorbance peaks between 250 and 320 nm that coeluted with flavocytochrome b derive from protein and associated Triton X-100. 400-µl fractions were collected from the column at 1-min intervals from consecutive runs, and the peak flavocytochrome b-containing fractions that eluted between 25 and 29 min were pooled and digested.


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  		Fig. 1.   HPLC size exclusion purification of detergent-solubilized flavocytochrome b. Shown is a three-dimensional, UV-visible diode array spectrophotometer chromatogram of detergent-solubilized flavocytochrome b (1.6 nmol of heme) during the final HPLC size exclusion chromatography step prior to digestion with V8 proteinase, as described under "Experimental Procedures." The axes are as follows: x, elution time (min); y, absorbance wavelength (nm); and z, absolute absorbance (AU). Flavocytochrome b is the prominent peak eluting at ~27 min with an oxidized heme absorbance maximum (lambda max) at 414 nm. Coeluting absorbance maxima between 250 and 320 nm derive from protein and Triton X-100. The numbers and their corresponding colors in the upper left of the figure are a scale of absolute absorbances in absorbance units. This chromatogram is typical of results obtained from at least five separate experiments conducted on different days.

Evolution of Flavocytochrome b Heme Absorbance Spectrum during Proteolysis-- Digestion of flavocytochrome b for periods up to 4.5 h produced consolidated, heme-containing peaks during HPLC size exclusion chromatography, while longer digestion periods resulted in a gradual loss of recoverable heme activity. Absorbance measurements taken from three time points during a 4.5-h digestion of flavocytochrome b with V8 proteinase are shown in Fig. 2, illustrating the evolution of the oxidized flavocytochrome b Soret absorbance spectrum. Samples were removed at 30-min intervals from the digestion milieu, placed on wet ice, and supplemented with a proteinase/inhibitor mixture to inhibit further digestion. The absorbance spectrum of each time point was then recorded, and the absorbance maxima (lambda max) were plotted over time (Fig. 2B). After 60 min of digestion, a slight broadening of the Soret peak was observed, whereas lambda max remained at 414 nm with ~86% of the starting 414 nm absorbance retained (Fig. 2A). By 270 min of digestion, the Soret absorbance peak had broadened further, concurrent with a blue shift in lambda max from 414 to 408-410 nm (Fig. 2A) and an overall reduction in the Soret absorbance to 62% of the starting value (Fig. 2B).


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  		Fig. 2.   Evolution of flavocytochrome b heme oxidized Soret absorbance spectrum during proteolysis. Detergent-solubilized, partially purified flavocytochrome b (2.5 nmol of heme) was exposed to V8 proteinase as described under "Experimental Procedures" for a total of 4.5 h. Aliquots were removed from the digestion vessel and their absorbance spectra recorded. A, oxidized Soret heme absorbance spectra of intact (---), and following 1 (···) and 4.5 (- - -) h of digestion. Both intact and 1-h digested flavocytochrome b lambda max is at 414 nm, and ~86% of the heme absorbance is retained after 1 h of digestion. At 4.5 h, 62% of the initial heme absorbance is retained, and lambda max is shifted to 408-410 nm. B, triangles represent 414 nm absorbance values measured at 30-min intervals throughout the 4.5-h digestion. These results are typical of values obtained from at least five separate experiments conducted on different days.

Isolation of Heme-bearing Proteolytic Fragments of Flavocytochrome b after 1 h of Digestion-- Samples that were collected from the digestion milieu at 30-min intervals were again subjected to HPLC size exclusion chromatography to isolate possible heme-associated peptides (Fig. 3). Prior to digestion, flavocytochrome b eluted as a single 414 nm absorbance peak at 27 min corresponding to a molecular mass of 329 kDa relative to soluble globular protein standards (Fig. 3, top). The DAD spectrum (Fig. 1) revealed that the small absorbance peak centered at 32 min elution time was not from heme but was contributed by the large absorbance shoulder of micellar Triton X-100 from the injection bolus. Consistent with buffer blanks, the high level of fluorescence associated with this elution time was also contributed primarily by micellar Triton X-100. Silver-stained SDS-PAGE gels of this fraction showed only negligible levels of protein (not shown). The small, broad fluorescence peak centered at 39-40 min is a contaminant of unknown origin that was also present in buffer blanks.


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  		Fig. 4.   Heme distribution of intact and partially proteolyzed flavocytochrome b after separation by HPLC size exclusion chromatography. 400-µl fractions were collected every minute throughout the HPLC size exclusion runs shown in Fig. 3 and analyzed for heme content using a modified TMBZ assay as described under "Experimental Procedures." The heme content of each HPLC size exclusion fraction is shown as a percent of the total heme of the HPLC runs of intact (top) and following 1 (middle) and 4.5 (bottom) h of digestion.  denotes average ± SD. values obtained from two separate TMBZ assays corresponding to the indicated HPLC elution times. Top, intact flavocytochrome b. 84% of the total heme is contained within the fractions that eluted prior to 35 min, and 72% of the total heme is contained by the fractions that elute between 24 and 30 min. Middle, flavocytochrome b after 1 h of digestion. 85% of the total heme eluted before 35 min, and 70% of the total heme is contained within the 24-30-min elution fractions. Bottom, flavocytochrome b after 4.5 h of digestion. 73% of the remaining heme elutes before 35 min, mainly distributed between the three peaks eluting between 18-24, 24-29, and 29-34 min, representing 22, 23, and 28%, respectively.

Digestions for 1 h consistently produced a prominent heme-associated protein fragment that eluted as a single peak (Fig. 3, middle) with a lambda max at 414 nm. Integration of the 414 nm absorbance profiles of the intact and 1-h digested flavocytochrome b indicated a 95% recovery of the total heme absorbance after 1 h of digestion. Comparison of the integrated 414 nm absorbance values of the principal 25-30-min elution peaks for the intact and 1-h digested fractions indicated that 74% of the heme absorbance was retained by the digest fragment. The elution time of the peak fraction was retarded ~0.6-0.8 min relative to nondigested controls, corresponding to a reduced mass of roughly 30-34 kDa from the initial 329-kDa. Additionally, the 414 nm absorbance profile began to transform during the 1st h of digestion. An aggregated species was observed at an elution time of 19-25 min, corresponding to mass range spanning 1300 to 461 kDa respectively. An increase in the 414 nm absorbance was also observed at an elution time of 32 min, suggesting an accumulation of heme or heme-bearing protein fragments that either coeluted with or were partitioned into Triton X-100 micelles. The DAD absorbance spectra of both the aggregated and the smaller species, however, revealed a lambda max at 408-410 nm (not shown) suggesting an altered heme environment. The fluorescence intensity profile during the 1st h of digestion also exhibited a redistribution trend similar to the absorbance. The fluorescence associated with the prominent absorbance peak eluting at 25-30 min decreased slightly in intensity, concurrent with a slight increase in the aggregate fractions (elution time = 19-25 min) and a 20% increase in the fraction eluting at 32 min.

Isolation of Heme-bearing Proteolytic Fragments of Flavocytochrome b after 4.5 h of Digestion-- Continued digestion beyond 1 h resulted in a gradual division of the 414 nm absorbance profile into a trimodal distribution that reached a maximum accumulation at 4.5 h (Fig. 3, bottom). Integration of the 414 nm absorbance profiles from this time point indicated that ~63% of the total initial heme absorbance was recovered. The first peak to elute at 20.5 min corresponds to an aggregated species with a molecular mass of ~1000 kDa, and the second peak, centered at 27-28 min elution time, is the partially proteolyzed intermediate fragment, prominent after 1 h of digestion (see above). The third peak, coeluting with micellar Triton X-100 at 32 min, corresponds to a molecular mass of ~140 kDa. The DAD absorbance spectrum of the 32-min elution peak showed a large shoulder from Triton X-100, similar to that observed after 1 h of digestion but also revealed a distinct lambda max at 408-410 nm indicating the presence of heme in these fractions (not shown). The fluorescence associated with the 25-30-min elution fractions was also reduced, and a continued redistribution to the aggregated species eluting between 20 and 25 min was observed. The fluorescence intensity associated with the 32-min elution peak remained constant.

Heme Content of HPLC Fractions-- Throughout the 4.5-h digestion period, the majority of the heme absorbance appeared confined to the prominent peaks that eluted prior to 35 min. The heme distribution throughout the elution profile was confirmed using a modified 3,3',5,5'-tetramethylbenzidine (TMBZ) assay to allow direct heme quantitation of each HPLC fraction (Fig. 4). The assay also provided a 100-fold increase in heme detection sensitivity, which allowed heme quantitation of HPLC fractions that were otherwise hindered by low absorbance levels, changes in the heme absorbance spectrum, or absorbance interference contributed by Triton X-100. The TMBZ assay profiles (Fig. 4) indicated that 84% of the heme from intact flavocytochrome b (Fig. 4, top) eluted prior to 35 min, with 71% contained within the prominent peak fractions that eluted between 24 and 30 min. Following 1 h of digestion and HPLC size exclusion chromatography (Fig. 4, middle), the heme distribution remained relatively constant with 85% contained within the fractions that eluted prior to 35 min, and 70% contained within the prominent absorbance peak fractions that eluted between 24 and 30 min. By 4.5 h of digestion (Fig. 4, bottom), the heme distribution evolved to a primarily trimodal distribution with 73% eluting prior to 35 min. Minor heme-containing fractions were also evident, eluting at 37 and 47 min.


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  		Fig. 3.   HPLC size exclusion chromatograms of flavocytochrome b at three time points during the 4.5-h digestion. Detergent-solubilized, partially purified flavocytochrome b was digested with V8 proteinase for 4.5 h, and aliquots were removed every 30 min and subjected to HPLC size exclusion chromatography as described under "Experimental Procedures." Shown in each panel are the normalized 414 nm absorbance (---) and fluorescence (- - -) (lambda ex = 280 nm, lambda em = 340 nm) chromatograms from samples collected at the indicated time points. Top, intact flavocytochrome b elutes as a monodisperse peak centered at 27 min, with lambda max at 414 nm, corresponding to a mass of ~329 kDa relative to globular size exclusion chromatography standards. The fluorescence peak at eluting at 32 min is due to micellar Triton X-100 from the injection bolus and corresponds to a mass of ~135 kDa. Middle, flavocytochrome b at 1 h of digestion. After 1 h of digestion, all of the flavocytochrome b heme absorbance is recovered. The integrated 414 nm heme absorbance of the prominent peak is equal to 74% of the corresponding non-digested flavocytochrome b peak. The peak elution time is retarded by 0.6-0.8 min relative to non-digested flavocytochrome b, corresponding to an ~30-34-kDa reduction in mass. A small amount of the 414 nm absorbance is redistributed within an aggregate species that eluted between 19 and 25 min, corresponding to a mass range spanning 1300 to 461 kDa, respectively. A smaller heme-associated species began to accumulate in the 32-min elution fraction, coeluting with the micellar Triton X-100. A similar redistribution of the fluorescence intensity was observed up to 30 min of elution, and a 20% increase was observed at 32 min. Bottom, flavocytochrome b at 4.5 h of digestion. The 414 nm heme absorbance is distributed between three peaks with an overall loss of 37% relative to non-digested flavocytochrome b. The first peak eluting at ~20.5 min corresponds to an aggregated species with a mass of ~1000 kDa; the second peak at 27 min is the remaining partially proteolyzed 1-h digest fragment, and the third peak at 32 min corresponds to a mass of ~135 kDa. The fluorescence intensity profile again paralleled the redistribution of the 414 nm absorbance during the first 30 min of the elution profile, whereas the 32-min peak remained constant. The results shown here are typical of at least three separate experiments.

Identification of Heme-bearing Proteolytic Species by SDS-PAGE and Immunoblotting-- We next identified the polypeptide fragments of flavocytochrome b that coeluted with heme spectral activity by separation of the individual HPLC elution fractions on SDS-PAGE, followed by silver staining or immunoblotting with previously characterized (see "Experimental Procedures") flavocytochrome b-specific antibodies (Fig. 5 and Table I). The silver-stained gel (Fig. 5A, lane 2) and the immunoblot (Fig. 5B, lane 1) show non-digested gp91phox as a diffuse band, centered at ~90 kDa. Silver staining and immunoblotting of non-digested flavocytochrome b with alpha -p22phox antibodies show a consolidated band at 22 kDa (Fig. 5A, lane 2, Fig. 5B, lane 3 respectively), although the silver stain is less well represented due to its atypical staining characteristics (9). Following 1 h of digestion, the prominent feature on the silver-stained gel was a diffuse, asymmetrically stained band with a centroid mass between 60 and 66 kDa (Fig. 5A, lane 3), and immunoblots of the same fraction exhibited a similar but more homogeneous staining pattern for gp91phox (Fig. 5B, lane 2). The silver-stained gel also shows an accumulation of a 17-kDa band (Fig. 5A, lane 3) concurrent with an identical shift in the molecular mass of p22phox as indicated by immunoblotting with alpha -p22phox antibodies (Fig. 5B, lane 4). Thus, during the 1st h of proteolysis, the average molecular mass of gp91phox was reduced from 90 to 60-66 kDa, and the molecular mass of p22phox was reduced from 22 to ~17 kDa.


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  		Fig. 5.   Heme-associated proteolytic fragments of flavocytochrome b following HPLC size exclusion chromatography. 400-µl fractions were collected every min throughout the HPLC runs shown in Fig. 3. Fractions were separated by SDS-PAGE and either silver-stained or transferred to nitrocellulose and immunoblotted with either alpha -gp91phox (CL5), alpha -p22phox (NS5 or 44.1) mAbs as described under "Experimental Procedures." A, composite from silver-stained SDS-PAGE gels consisting of molecular mass markers (lane 1); intact (lane 2), 1-h digested, 27-28-min elution fraction (lane 3); or 4.5-h digested, 32-33-min elution fraction (lane 4). Arrows indicate intact (lane 2) or prominent digest fragments (lanes 3 and 4) of gp91phox and p22phox. B, an immunoblot composite of samples corresponding to those shown in A. Intact gp91phox is recognized by mAb CL5 as a diffuse band centered at ~90 kDa (lane 1). At 1 h of digestion, the large subunit mass was reduced to an ~60-kDa diffuse band (lane 2). Intact p22phox was recognized by mAbs 44.1 (not shown) and NS5 (lane 3) as a consolidated band at 22 kDa. After 1 h of digestion, p22phox is recognized by mAb NS5 as a band at 17 kDa (lane 4). At 4.5 h of digestion, none of the alpha -gp91phox immunoblots were positive for the 32-33-min elution fraction, but alpha -p22phox immunoblots were positive for bands at 17, 15, and <15 kDa (lane 5, arrows). The immunoblots shown are representative of multiple analyses that are summarized in Table I.

                              
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Table I
Immunoblot analyses of partially proteolysed flavocytochrome b
Detergent-solubilized, partially purified flavocytochrome b was digested with V8 proteinase for 1 h and subjected to HPLC size exclusion chromatography. The heme-bearing core polypeptide from the 27-28-min elution fraction shown in Fig. 5 was analyzed by immunoblot using either alpha -gp91phox or alpha -p22phox antibodies as described under "Experimental Procedures." The abbreviations used are: **, conformational epitope; -, epitope not detected by immunoblot; (M) and (P), monoclonal and polyclonal antibody. Molecular masses shown in bold were the predominantly staining species within the particular fraction. The indicated time point samplings are analogous to lanes 2 and 3 of Fig. 5A. Immunoblots are from three separate digestion experiments from different days, with each antibody tested twice against positive control lanes (not shown) consisting of aliquots of intact flavocytochrome b removed from the final size exclusion purification step prior to addition of V8 proteinase. Superscript numbers denote primary sequence designation with the NH2-terminal initiation methionine as number 1 of each respective subunit.

The immunoblots shown in Fig. 5B are representative of results obtained from multiple analyses that are summarized in Table I. The information derived from the immunoblot analyses indicated that the epitope regions 383PKIAVDGP390, 498EKDVITGRKQ507, and 548KQSISNSESGP558 of gp91phox and 183PQVNPI188 of p22phox were lost within the 1st h of digestion (Table I). The apparent mass reduction of gp91phox during the 1st h of digestion did not appear to be due to loss of carbohydrate as indicated by the consonant heterogeneity between the digested and non-digested samples (Fig. 5A, compare lanes 2 and 3, Fig. 5B, compare lanes 1 and 2). This observation, in combination with the results of the immunoblotting, suggests that the 60-66-kDa proteolytic fragment possesses all of the gp91phox glycosylation sites and provides additional evidence supporting a previous report that proposed Asn131, Asn148, and Asn239 as the sites of gp91phox glycosylation (55).

By 4.5 h of digestion, immunoblots of the 32-min elution peak fraction (Fig. 3, lower) using our panel of alpha -gp91phox antibodies were negative (Table I) even though multiple bands with molecular masses ranging from <15 to ~32 kDa were evident by silver staining (Fig. 5A, lane 4, arrows). However, immunoblots of the same elution fraction with alpha -p22phox antibodies were positive for multiple fragments ranging from 17 to <15 kDa (Fig. 5B, lane 5). The transition from the 1-h digest heme distribution to the trimodal distribution seen at 4.5 h of digestion (Fig. 3) suggested the possibility of a lower molecular mass component that would provide more precise information regarding the heme-ligating regions of flavocytochrome b. However, in repeated experiments, the peptide fragments associated with the 32-min elution peak varied in both composition and quantity. Additionally, the numerous peptides (Fig. 5A, lane 4) that coeluted with micellar Triton X-100 at 32 min (Fig. 3) introduced ambiguities that prohibited assignment of heme ligation to specific peptide components.

Identification of Heme-bearing Fragments after 1 h of Digestion-- We thus chose to focus our efforts on identification of the polypeptide components present in the 27-28-min fraction from the 1-h digestion using a combination of NH2-terminal sequence analysis, SDS-PAGE, and immunoblotting. Table II shows the results of the NH2-terminal sequence analyses, identifying the 1-h digest 60-66- and 17-kDa proteolytic fragments (Fig. 5A, lane 3 and Fig. 5B, lanes 2 and 4) as the intact NH2 termini of gp91phox and p22phox, respectively. Interestingly, the absence of the initiating methionine on both peptide sequences suggests that they are removed during the maturation of the protein. This result confirms our previous observations (9) but contrasts two previous reports of modification of the NH2 terminus of the small subunit (56, 57).

                              
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Table II
NH2-terminal sequences of V8 proteolysed flavocytochrome b digested for 1 h with V8 proteinase
Detergent-solubilized, partially purified flavocytochrome b was digested with V8 proteinase and subjected to HPLC size exclusion chromatography. Elution fractions that retained heme absorbance were pooled and prepared for NH2-terminal sequencing as described under "Experimental Procedures." The polypeptides transferred to PVDF membrane for sequencing were analogous to those shown in Fig. 5A, lane 3. The discrete band at 17 kDa and the entire broad bands from ~50 to 70 kDa were excised separately and analyzed by NH2-terminal sequence analysis. Superscript numbers denote primary sequence designation with the NH2-terminal initiation methionine as number 1 of each respective subunit, and dashes indicate non-matching residues. Of the 10 cycles requested for each analysis, 10 of 10 residues from the 60-66-kDa fragment matched the NH2-terminal end of gp91phox, and 8 of 10 residues from the 17 kDa molecular mass fragment matched the NH2-terminal end of p22phox.

It is interesting that the contaminating proteins present in the partially purified flavocytochrome b (Fig. 5A, lane 2) that was used for the subsequent digestion steps did not interfere with our ability to obtain NH2-terminal sequence data following proteolysis. After the 1st h of digestion, silver-stained SDS-PAGE gels indicate that the majority of the signal is consolidated to only two bands (Fig. 5A, lane 3, arrows). The NH2-terminal sequence data (Table II) was obtained by excising the entire diffuse band from ~50 to 70 kDa and the consolidated band at 17 kDa for analysis. Since the NH2-terminal sequencing process is exquisitely sensitive to background signal from contaminating protein, the ability to obtain unambiguous sequencing data suggests that the additional proteins present during the initial purification step were more susceptible to proteolysis than flavocytochrome b.

Mass Determination of gp91phox and p22phox Digestion Fragments-- Identification of the COOH-terminal proteinase cut sites of the 1-h digested, 17- and 60-66-kDa fragments was first investigated using SDS-PAGE analysis. To circumvent the anomalous migration behavior of glycosylated, integral membrane proteins on SDS-PAGE, accurate mass determination of the peptide components from the 27-28-min elution peak from the 1-h digestion (Fig. 5A, lane 3) required that we first deglycosylate and then reduce and alkylate the proteolytic fragments prior to SDS-PAGE analysis.

Enzymatic deglycosylation of intact gp91phox with peptide N-glycosidase F, followed by reduction and alkylation prior to separation by SDS-PAGE, resulted in the appearance of multiple discrete bands by immunoblot (Fig. 6A, lane 4). The smallest species (Fig. 6A, lane 4, arrow) corresponded to an apparent mass of 60-66 kDa, thus consistent with the predicted mass of the polypeptide core of gp91phox based on primary sequence (33, 35). Similarly, immunoblots of nondigested gp91phox that were deglycosylated using chemical methods (see "Experimental Procedures"), followed by reduction and alkylation, showed only a single consolidated band of the same mass (not shown). Although SDS-PAGE migration of the deglycosylated core polypeptide of gp91phox has been reported previously with apparent molecular masses ranging from 49 to 58 kDa (9, 58, 59), these reports did not include reduction and alkylation following deglycosylation, suggesting that inclusion of these steps promotes more complete unfolding of the protein during SDS-PAGE separation.


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  		Fig. 6.   Mass determination of deglycosylated fragments of flavocytochrome b. Intact flavocytochrome b and polypeptides isolated from the 1-h digest, 27-28-min HPLC elution fractions were deglycosylated, reduced, and alkylated, separated by SDS-PAGE, and immunoblotted using either alpha -p22phox or alpha -gp91phox primary antibodies as described under "Experimental Procedures." A, alpha -gp91phox immunoblots (mAb CL5) of flavocytochrome b samples showing prestained molecular mass markers (lane 1); intact, non-reduced and alkylated (lane 2); intact, reduced, and alkylated (lane 3); intact, deglycosylated, reduced, and alkylated (lane 4); and 1-h digested, deglycosylated, reduced, and alkylated (lane 5) flavocytochrome b. B, alpha -p22phox immunoblots of samples identical to A with primary mAb NS1, lanes 2 and 3, or mAb NS5, lanes 4 and 5. Arrows in both panels indicate lowest apparent molecular mass species following deglycosylation, reduction, and alkylation.

Reduction and alkylation of non-digested flavocytochrome b resulted in a slight increase in the apparent molecular mass of gp91phox (Fig. 6A, compare lanes 2 and 3) but had no effect on the electrophoretic migration of p22phox (Fig. 6B, compare lanes 2 and 3). However, simultaneous exposure of p22phox to the conditions used for enzymatic deglycosylation of gp91phox, followed by reduction and alkylation, resulted in a slight increase in apparent molecular mass (Fig. 6B, lane 4). Identical treatment of the 1-h digested polypeptide core had no effect on the electrophoretic migration of the 17-kDa proteolytic fragment of p22phox (Fig. 6B, lane 5). Thus, after 1-h of digestion, the remaining heme-bearing core polypeptide component of flavocytochrome b was composed of fragments of both gp91phox and p22phox, with respective molecular masses of 39 and 17 kDa. Mass predictions from the primary sequence of gp91phox, less the start methionine, suggest that the most likely COOH-terminal proteinase target sites that correspond to the apparent 39-kDa molecular mass are Glu320, Glu336, Glu347, Glu348, and Glu363, yielding masses of 36.7, 38.5, 39.9, 40.0, and 41.7 kDa, respectively.

Similarly, beginning at the NH2 terminus for p22 phox less the start methionine, the most likely cut site for the proteinase corresponding to the 17-kDa band would be at Glu162, yielding a predicted mass of 17.7 kDa. However, the positive immunoblot signal from both the polyclonal anti-peptide EAR, 162EARKKPSEEEAAA174, and the mAb CS9, 165KKPSE169, antibody (Table I) indicates that the 17-kDa fragment retains at least part of these epitopes. The next most likely proteolytic cleavage site lies within the glutamate repeats Glu169 to Glu171, yet cleavage within this site would yield predicted masses of 18.5-18.8 kDa, not the 17-kDa mass observed by SDS-PAGE. Since the mAb 44.1 epitope region, 183PQVNPI188 (Table I), is removed during the 1st h of digestion, and there are no other Glu residues COOH-terminal to Glu171, we conclude that the V8 cleavage site resides within the glutamate repeats Glu169 to Glu171. We remain uncertain as to the anomalous migration behavior of the p22phox fragment on SDS-PAGE. In conclusion, the combined results of the antibody, NH2-terminal sequence, and SDS-page analyses, identify the heme-bearing proteolytic polypeptide core as the NH2-terminal 320-363 amino acids of gp91phox and the NH2-terminal 169-171 amino acids of p22phox.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The principal conclusion of this study is that a flavocytochrome b heme-binding domain resides within a polypeptide core region consisting of the NH2-terminal 320-363 amino acids of gp91phox and the NH2-terminal 169-171 amino acids of p22phox. The fragment, isolated by exposure of partially purified flavocytochrome b to Staphylococcal V8 proteinase and size exclusion chromatography, was shown to retain a native heme absorbance spectrum.

Our initial digestion experiments were conducted for periods of up to 18 h to establish conditions rendering the smallest proteolytic fragments of flavocytochrome b that still retained native spectral activity and were separable by size exclusion chromatography. Digestion for 1 h reduced the mass of the flavocytochrome b by roughly 35% but produced a fragment that was spectrally stable for days at 4 °C in detergent-containing buffer. Longer digestion periods destabilized the heme environment as evidenced by blue-shifted Soret maxima and an inability to reproducibly recover smaller heme-associated fragments. Interestingly, free hemin introduced to identical HPLC size exclusion chromatography conditions, with either OG or Triton X-100-containing column buffers, coeluted with the detergent micelles (not shown) and displayed a lambda max of 396 nm, not the observed 408-410 nm. These observations suggest that the non-native heme absorbance spectrum associated with the more digested fractions may derive from heme that remains ligated to a peptide component but with a modified ligand environment. Although the intrinsic hydrophobic nature of the heme molecule could promote random interactions with the hydrophobic peptide regions of the digested flavocytochrome b, such an association would not serve to explain the lambda max at 408-410. The possibility of a spurious histidine-heme ligation is also unlikely given the relatively low association constants (5 × 103 M-1) observed for reconstituted polypeptide maquettes (60).

The data presented herein are consistent with previous findings implicating specific regions within the NH2-terminal half of flavocytochrome b with heme ligation. Examination of human mutations responsible for CGD revealed that the largest number of lesions occurred within the NH2-terminal half of gp91phox, including substitutions at His101 and His209 (24). Fujii et al. (25, 26) noticed a pyridine-induced shift in the ESR spectrum of flavocytochrome b toward that of cytochrome P450 of P. putida. X-ray crystallographic data from cytochrome P450 from P. putida (31) showed the hemes coordinated by a species-conserved motif, FXXGXXXCLG, a sequence similar to residues 78FXXGXXXC85 of gp91phox. The proximity of this heme-coordinating motif to His101 and His209 suggested that they were the most likely candidates for the heme ligands of the flavocytochrome b. Another His101 right-arrow Tyr mutation in gp91phox (23) resulted in a complete loss of the heme absorbance spectrum without affecting flavocytochrome b expression. In a separate CGD case, stemming from an Arg54 right-arrow Ser mutation in gp91phox, flavocytochrome b expression levels were unaffected, but changes in the dithionite-reduced Soret absorbance spectrum were observed (61), suggesting that the mutation had a direct effect on the heme environment. Interestingly, a heme-coordinating motif, 15TXXLAVHXXXV25, originally found in the beta -subunit of cytochrome b559 of Synechocystis 6803 photosystem II (32), is similar to the 232TXXXLAVHXXXV243 region of human neutrophil gp91phox. The stretch encompassing this region is predicted by hydropathy analysis to be relatively hydrophilic. This prediction is supported by the presence of the purported glycosylation site (55) immediately adjacent to His239 at Asn240. Also, the region is flanked by two previously characterized antibody epitope regions, 226RIVRG230 of mAb 7D5 (62) and 247KISEWGKIKE256 of polyclonal antibody KIS3 (Table I), both of which were shown to be accessible on the extracellular aspect of intact human neutrophils. Thus, if His239 is solvent-accessible, it would probably not be suitable for heme ligation based on thermodynamic considerations (18).

The possibility that p22phox alone coordinates a heme is unlikely due to spectroscopic data that implies a bis-histidyl ligation scheme (25, 26, 63, 64) and the presence of only a single invariant histidine, His94 (65-67). Since the gp91phox, p22phox subunit stoichiometry has been determined to be 1:1 (21, 51, 52), p22phox would be able to provide only a single histidine for heme ligation. Therefore, heme coordination by His94 of p22phox would necessitate that the second histidine ligand be provided by gp91phox (51, 68), thereby ligating the large and small subunits. The possibility that p22phox is involved with heme ligation is supported by our previous observations showing that heme remains associated with both p22phox and gp91phox following separation on lithium dodecyl sulfate-PAGE at 4 °C (69). We have also shown previously that the subunits of flavocytochrome b are separable only under denaturing conditions that result in loss of the heme absorbance spectrum (21), and most recently in collaborations with other colleagues that the processing and maturation of flavocytochrome b expressed in X-CGD promyelocytic leukemia cells requires heme incorporation as a prerequisite for heterodimer assembly. In the present work, the 1-h digest spectrally stable core polypeptide was heterodimeric. Additionally, the chromatographic distribution of a non-native heme absorbance spectrum correlated well with the presence of His94-containing fragments of p22phox. Thus, the correlation between the presence of heme and a heterodimeric structure suggests that both subunits contribute to the stability of the heme environment. The spectral stability of the 1-h digest fragment further suggests that the primary interfacial contact regions between gp91phox and p22phox remain intact within the polypeptide core.

Although transgenic expression of fully processed, spectrally similar gp91phox was demonstrated in the absence of p22phox in two non-promyelocytic cell lines, monkey kidney COS-7 cells and murine 3T3 fibroblasts5 (70), the reduced Soret band absorbance spectrum from COS-7 gp91phox alone was similar but not identical to either cells expressing both subunits or native human neutrophil flavocytochrome b. Moreover, in the absence of coexpressed COS-7 p22phox, COS-7 gp91phox was unable to produce superoxide (71). These observations, combined with our data, thus suggest that the presence of p22phox is probably necessary for stabilization of the native heme environment of flavocytochrome b.

Native flavocytochrome b contains 19 histidines, 6-8 of which are removed during the 1st hour of digestion depending on the precise COOH-terminal cleavage site of gp91phox. Since the 1-h digested polypeptide core retains 74% of the heme absorbance of native flavocytochrome b, the COOH-terminal domains of the protein are probably not involved with heme ligation. Of the 11-13 histidines that remain within the 1-h digested core polypeptide, the polymorphic His72 of p22phox (65-67) is ruled out and the extracellular His239 of gp91phox is probably an unlikely candidate for heme ligation. Therefore, the remaining 9-11 histidines, including His94 of p22phox, are the most likely candidates for this function. This number prohibits assignment of specific histidines for the ligation; however, their close proximity to the predicted transmembrane spanning regions3 of flavocytochrome b (33-35) suggests a transmembrane or juxtamembrane heme placement.

In conclusion, this study provides direct evidence that the regions of flavocytochrome b composed of the NH2-terminal 320-363 amino acids of gp91phox, and the NH2-terminal 169-171 amino acids of p22phox are responsible for heme coordination. Both regions encompass the predicted transmembrane spanning domains of each subunit and appear to retain the glycosylation sites for gp91phox as well as the sites of interaction between p22phox and gp91phox.

    ACKNOWLEDGEMENT

We thank Selisa C. Rausch for assistance in preparation of the graphics used for this manuscript.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant R01 AI 26711 (to A. J. J.) and American Heart Association SDG Award 30156N (to J. B. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Microbiology, Montana State University, 109 Lewis Hall, Bozeman, MT 59717-3520. Tel.: 406-994-4811; Fax: 406-994-4926; E-mail: umbaj@montana.edu.

Published, JBC Papers in Press, August 14, 2001, DOI 10.1074/jbc.M104373200

5 M. C. Dinauer, personal communication.

2 D. Baniulis, J. Burritt, and A. Jesaitis, unpublished observations.

3 J. Burritt and A. Jesaitis, unpublished observations.

4 T. Foubert and A. Jesaitis, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: phox, phagocyte oxidase; PAGE, polyacrylamide gel electrophoresis; CGD, chronic granulomatous disease; DAD, diode array detector; DTT, dithiothreitol; Glu-C, glutamic acid, carboxyl-terminal side; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; lambda max, absorbance maximum; mAb, monoclonal antibody; OG, octyl glucoside, octyl beta -glucopyranoside; TMBZ, 3,3',5,5'-tetramethylbenzidine; V8 proteinase, staphylococcal V8, endoproteinase Glu-C; PMSF, phenylmethylsulfonyl fluoride.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Nauseef, W. M., Volpp, B. D., McCormick, S., Leidal, K. G., and Clark, R. A. (1991) J. Biol. Chem. 266, 5911-5917[Abstract/Free Full Text]
2. DeLeo, F. R., and Quinn, M. T. (1996) J. Leukocyte Biol. 60, 677-691[Abstract]
3. Wientjes, F. B., and Segal, A. W. (1995) Semin. Cell Biol. 6, 357-365[Medline] [Order article via Infotrieve]
4. Leusen, J. H. W., Verhoeven, A. J., and Roos, D. (1996) J. Lab. Clin. Med. 128, 461-476[CrossRef][Medline] [Order article via Infotrieve]
5. Rotrosen, D., Yeung, C. L., and Katkins, J. P. (1993) J. Biol. Chem. 268, 14256-14260[Abstract/Free Full Text]
6. Babior, B. M. (1999) Blood 93, 1464-1476[Free Full Text]
7. Nauseef, W. M. (1999) Proc. Assoc. Am. Physicians 111, 373-382[Medline] [Order article via Infotrieve]
8. Segal, A. W. (1987) Nature 326, 88-91[CrossRef][Medline] [Order article via Infotrieve]
9. Parkos, C. A., Allen, R. A., Cochrane, C. G., and Jesaitis, A. J. (1987) J. Clin. Invest. 80, 732-742
10. Segal, A. W., West, I., Wientjes, F. B., Nugent, J. H. A., Chavan, A. J., Haley, B., Garcia, R. C., Rosen, H., and Scrace, G. (1992) Biochem. J. 284, 781-788
11. Yoshida, L. S., Chiba, T., and Kakinuma, K. (1992) Biochim. Biophys. Acta 1135, 245-252[Medline] [Order article via Infotrieve]
12. Rotrosen, D., Yeung, C. L., Leto, T. L., Malech, H. L., and Kwong, C. H. (1992) Science 256, 1459-1462[Abstract/Free Full Text]
13. Doussière, J., Brandolin, G., Derrien, V., and Vignais, P. V. (1993) Biochemistry 32, 8880-8887[CrossRef][Medline] [Order article via Infotrieve]
14. Doussière, J., Buzenet, G., and Vignais, P. V. (1995) Biochemistry 34, 1760-1770[CrossRef][Medline] [Order article via Infotrieve]
15. Isogai, Y., Iizuka, T., and Shiro, Y. (1995) J. Biol. Chem. 270, 7853-7857[Abstract/Free Full Text]
16. Segal, A. W. (1996) J. Clin. Invest. 83, 1785-1793[CrossRef]
17. Schrezel, J., Serrander, L., Banfi, B., Nübeta e, O., Fouyouzi, R., Lew, D. P., Demaurex, N., and Krause, K. H. (1998) Nature 392, 734-737[CrossRef][Medline] [Order article via Infotrieve]
18. Jesaitis, A. J. (1995) J. Immunol. 155, 3286-3288[Medline] [Order article via Infotrieve]
19. Henderson, L. M., and Meech, R. W. (1999) J. Gen. Physiol. 114, 771-786[Abstract/Free Full Text]
20. Henderson, L. M., Chappell, J. B., and Jones, O. T. (1987) Biochem. J. 246, 325-329[Medline] [Order article via Infotrieve]
21. Parkos, C. A., Allen, R. A., Cochrane, C. G., and Jesaitis, A. J. (1988) Biochim. Biophys. Acta 932, 71-83[Medline] [Order article via Infotrieve]
22. Quinn, M. T., Mullen, M. L., and Jesaitis, A. J. (1991) Clin. Res. 39, 353
23. Tsuda, M., Kaneda, M., Sakiyama, T., Inana, I., Owada, M., Kiryu, C., Shiraishi, T., and Kakinuma, K. (1998) Hum. Genet. 103, 377-381[CrossRef][Medline] [Order article via Infotrieve]
24. Bolscher, B. G., de Boer, M., de Klein, A., Weening, R. S., and Roos, D. (1991) Blood 77, 2482-2487[Abstract/Free Full Text]
25. Fujii, H., Yonetani, T., Miki, T., and Kakinuma, K. (1995) J. Biol. Chem. 270, 3193-3196[Abstract/Free Full Text]
26. Fujii, H., Finnegan, M. G., Miki, T., Crouse, B. R., Kakinuma, K., and Johnson, M. K. (1995) FEBS Lett. 377, 345-348[CrossRef][Medline] [Order article via Infotrieve]
27. Finegold, A. A., Shatwell, K. P., Segal, A. W., Klausner, R. D., and Dancis, A. (1996) J. Biol. Chem. 271, 31021-31024[Abstract/Free Full Text]
28. Taylor, W. R., Jones, D. T., and Segal, A. W. (1993) Protein Sci. 2, 1675-1685[Medline] [Order article via Infotrieve]
29. Shatwell, K. P., Dancis, A., Cross, A. R., Klausner, R. D., and Segal, A. W. (1996) J. Biol. Chem. 271, 14240-14244[Abstract/Free Full Text]
30. Dancis, A., Roman, D. G., Anderson, G. J., Hinnebusch, A. G., and Klausner, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3869-3873[Abstract/Free Full Text]
31. Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16130[Abstract/Free Full Text]
32. Pakrasi, H. B., De Ciechi, P., and Whitmarsh, J. (1991) EMBO J. 10, 1619-1627[Medline] [Order article via Infotrieve]
33. Royer-Pokora, B., Kunkel, L. M., Monaco, A. P., Goff, S. C., Newburger, P. E., Baehner, R. L., Cole, F. S., Curnutte, J. T., and Orkin, S. H. (1986) Nature 322, 32-37[CrossRef][Medline] [Order article via Infotrieve]
34. Dinauer, M. C., Orkin, S. H., Brown, R., Jesaitis, A. J., and Parkos, C. A. (1987) Nature 327, 717-721[CrossRef][Medline] [Order article via Infotrieve]
35. Teahan, C., Rowe, P., Parker, P., Totty, N., and Segal, A. W. (1987) Nature 327, 720-721[CrossRef][Medline] [Order article via Infotrieve]
36. Quinn, M. T., Parkos, C. A., and Jesaitis, A. J. (1995) Methods Enzymol. 255, 477-487
37. Lutter, R., Van Shaik, M. L. J., Van Zwieten, R., Wever, R., Roos, D., and Hamers, M. N. (1985) J. Biol. Chem. 260, 2237-2244[Abstract/Free Full Text]
38. Thomas, P. E., Ryan, D., and Levin, W. (1976) Anal. Biochem. 75, 168-176[CrossRef][Medline] [Order article via Infotrieve]
39. Holland, V. R., Saunders, B. C., Rose, F. L., and Walpole, A. L. (1974) Tetrahedron 30, 3299-3302[CrossRef]
40. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
41. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract/Free Full Text]
42. Burritt, J. B., Busse, S. C., Gizachew, B., Siemsen, D. W., Quinn, M. T., Bond, C. W., Dratz, E. A., and Jesaitis, A. J. (1998) J. Biol. Chem. 273, 24847-24852[Abstract/Free Full Text]
43. Burritt, J. B., Quinn, M. T., Jutila, M. A., Bond, C. W., and Jesaitis, A. J. (1995) J. Biol. Chem. 270, 16974-16980[Abstract/Free Full Text]
44. Jesaitis, A. J., Buescher, E. S., Harrison, D., Quinn, M. T., Parkos, C. A., Livesey, S., and Linner, J. (1990) J. Clin. Invest. 85, 821-835
45. Quinn, M. T., Parkos, C. A., Walker, L. E., Orkin, S. H., Dinauer, M. C., and Jesaitis, A. J. (1989) Nature 342, 198-200[CrossRef][Medline] [Order article via Infotrieve]
46. (1999) Current Protocols in Protein Science (Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., eds) p. 10.8.2, John Wiley & Sons Inc., New York
47. Hunkapiller, M. W., Lujan, E., Ostrander, F., and Hood, L. E. (1983) Methods Enzymol. 91, 227-236[Medline] [Order article via Infotrieve]
48. Sojar, H. T., and Bahl, O. P. (1987) Methods Enzymol. 138, 341-350[Medline] [Order article via Infotrieve]
49. Sojar, H. T., and Bahl, O. P. (1987) Arch. Biochem. Biophys. 259, 52-57[CrossRef][Medline] [Order article via Infotrieve]
50. Brendel, V., Bucher, P., Nourbakhsh, I. R., Blaisdell, B. E., and Karlin, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2002-2006[Abstract/Free Full Text]
51. Wallach, M., and Segal, A. W. (1996) Biochem. J. 320, 33-38
52. Huang, J., Hitt, N. D., and Kleinberg, M. E. (1995) Biochemistry 34, 16753-16757[CrossRef][Medline] [Order article via Infotrieve]
53. Boulanger, P., le Maire, M., Bonhivers, M., Dubois, S., Desmadril, M., and Letellier, L. (1996) Biochemistry 35, 14216-14224[CrossRef][Medline] [Order article via Infotrieve]
54. Bokoch, G. M., Quilliam, L. A., Bohl, B. P., Jesaitis, A. J., and Quinn, M. T. (1991) Science 254, 1794-1796[Abstract/Free Full Text]
55. Wallach, T. M., and Segal, A. W. (1997) Biochem. J. 321, 583-585
56. Yamaguchi, T., Hayakawa, T., Kaneda, M., Kakinuma, K., and Yoshikawa, A. (1989) J. Biol. Chem. 264, 112-118[Abstract/Free Full Text]
57. Pember, S. O., Heyl, B. L., Kinkade, J. M., Jr., and Lambeth, J. D. (1984) J. Biol. Chem. 259, 10590-10595[Abstract/Free Full Text]
58. Kleinberg, M. E., Rotrosen, D., and Malech, H. L. (1989) J. Immunol. 143, 4152-4157[Abstract]
59. Harper, A. M., Chaplin, M. F., and Segal, A. W. (1985) Biochem. J. 227, 783-788[Medline] [Order article via Infotrieve]
60. Gibney, B. R., and Dutton, P. L. (1999) Protein Sci. 8, 1888-1898[Medline] [Order article via Infotrieve]
61. Cross, A. R., Heyworth, P. G., Rae, J., and Curnutte, J. T. (1995) J. Biol. Chem. 270, 8194-8200[Abstract/Free Full Text]
62. Burritt, J. B., DeLeo, F. R., McDonald, C. L., Prigge, J. R., Dinauer, M. C., Nakamura, M., Nauseef, W. M., and Jesaitis, A. J. (2001) J. Biol. Chem. 276, 2053-2061[Abstract/Free Full Text]
63. Fujii, H., and Kakinuma, K. (1992) Biochim. Biophys. Acta 1136, 239-246[Medline] [Order article via Infotrieve]
64. Miki, T., Fujii, H., and Kakinuma, K. (1992) J. Biol. Chem. 267, 19673-19675[Abstract/Free Full Text]
65. Parkos, C. A., Dinauer, M. C., Walker, L. E., Allen, R. A., Jesaitis, A. J., and Orkin, S. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3319-3323[Abstract/Free Full Text]
66. Dinauer, M. C., Pierce, E. A., Bruns, G. A., Curnutte, J. T., and Orkin, S. H. (1990) J. Clin. Invest. 86, 1729-1737
67. Sumimoto, H., Nozaki, M., Sasaki, H., Takeshige, K., Sakaki, Y., and Minakami, S. (1989) Biochem. Biophys. Res. Commun. 165, 902-906[CrossRef][Medline] [Order article via Infotrieve]
68. Ueno, I., Fujii, S., Ohya-Nishiguchi, H., Iizuka, T., and Kanegasaki, S. (1991) FEBS Lett. 281, 130-132[CrossRef][Medline] [Order article via Infotrieve]
69. Quinn, M. T., Mullen, M. L., and Jesaitis, A. J. (1992) J. Biol. Chem. 267, 7303-7309[Abstract/Free Full Text]
70. Yu, L., Zhen, L., and Dinauer, M. C. (1997) J. Biol. Chem. 272, 27288-27294[Abstract/Free Full Text]
71. Yu, L., Quinn, M. T., Cross, A. R., and Dinauer, M. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7993-7998[Abstract/Free Full Text]

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