©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Variant X-linked Chronic Granulomatous Disease Patient (X91) with Partially Functional Cytochrome b(*)

(Received for publication, December 6, 1994)

Andrew R. Cross (§) Paul G. Heyworth Julie Rae (¶) John T. Curnutte (¶)

From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Genetic analysis of a patient with the variant cytochrome b-positive form of chronic granulomatous disease revealed a missense mutation resulting in a Arg Ser substitution in the gp91 subunit of cytochrome b. As a consequence, although no O(2) is made, NADPH oxidase-associated FAD accepts electrons from NADPH in the cell-free activation system and becomes reduced. The reduced flavin exhibits normal levels of iodonitrotetrazolium violet diaphorase activity, and the patient's neutrophils exhibit high levels of intracellular oxidant production and show a low level of NBT staining in the NBT slide test. Thus, this mutation appears to render the heme center of NADPH oxidase present but nonfunctional, while leaving the flavin center fully functional.


INTRODUCTION

In response to a wide range of stimuli, neutrophils and other microbicidal phagocytes undergo a respiratory burst in which molecular oxygen is converted to superoxide (O(2)) (^1)and other reactive species(1) . The enzyme responsible for this activity, NADPH oxidase, is localized to the plasma membrane of the cells, which also forms the phagolysosomal membrane. The function of this oxidase system in phagocytes is to participate in the destruction of bacteria, fungi, parasites, virus-infected cells, and tumors by directing the reactive oxygen species against the target cells. In order to respond to a wide range of dissimilar stimuli and to minimize damage to normal tissues, the neutrophil has evolved a complex signal transduction mechanism to regulate NADPH oxidase. This signaling pathway is not yet fully understood. In part, the oxidase is regulated by separation of component parts in different cellular localizations. Thus, assembly of the oxidase at the plasma membrane, promoting electron transfer between the redox components of the oxidase, is one of the important steps in the activation process. An essential component of this oxidase system is a unique low potential b-type cytochrome, cytochrome b (also known as cytochrome b or cytochrome b)(2) . The cytochrome is a heterodimeric protein consisting of a 22-kDa alpha- subunit, termed p22(phagocyte oxidase), and a heavily glycosylated 91-kDa beta- subunit termed gp91. Both a flavin and a heme redox center are probably contained within the cytochrome heterodimer (3, 4) . Electrons supplied by the donor, NADPH, are believed to be transferred to oxygen as shown below.

We have recently provided evidence for the direct transfer of electrons from the reduced flavin center to the dye acceptor INT and have suggested that autoxidation of the reduced flavin results in low levels of intracellular hydrogen peroxide production in some forms of CGD(5) . In this inherited disorder, there is a failure of the phagocytic cells to generate O(2). Affected individuals suffer from severe recurrent infections due to the inability of the phagocytes to effectively kill infecting micro-organisms. Since the realization that defects in NADPH oxidase are the cause of CGD, it has proved possible to identify four essential polypeptide components of this system, defects in any one of which can lead to the clinical phenotype of CGD. Individuals with genetic lesions in either gp91 (type X91° CGD) or p22 (type A22° CGD) usually have phagocytes that do not express detectable levels of either cytochrome polypeptide, suggesting that the individual subunits are unstable in the absence of their partners(6, 7) . In a very few cases, missense mutations have resulted in normal levels of a spectrally normal, but nonfunctional cytochrome b (type X91 CGD) or a decreased abundance of a fully or partly functional cytochrome b (X91 CGD)(8) .

In addition to the membrane-bound redox centers of NADPH oxidase, there are at least three other components required for the fully active enzyme that are present in the cytosolic fraction of disrupted neutrophils. Defects in the genes coding for two of these proteins, p47 and p67, result in two of the recognized forms of CGD(8) . The third cytosolic factor is the low molecular weight GTP-binding protein, Rac. Initially, Rac1 was isolated from guinea pig macrophages (9) and Rac2 from human neutrophils(10) , based on their ability to substantially augment cell-free O(2) production. Subsequent studies using wholly or partially recombinant systems have shown that Rac (either form) is absolutely required for NADPH oxidase activity.

Here we describe a new patient (patient A) with neutrophils that express normal levels of a cytochrome that is non functional in terms of O(2) production, but retains normal dye-reductase activity. By comparison with another patient (patient B) previously described by Dinauer et al. (patient RC in (11) ) whose neutrophils express a cytochrome b in which there is a Pro His substitution in gp91, we can infer that patient A has a mutation affecting electron transfer processes subsequent to flavin reduction, whereas patient B has a mutation affecting the initial electron transfer processes preventing enzyme flavin reduction.


EXPERIMENTAL PROCEDURES

Preparation of Neutrophils and Their Subcellular Fractions

For experiments with intact neutrophils (including translocation studies) blood was collected by venipuncture and the neutrophils purified by dextran sedimentation, hypotonic lysis of erythrocytes, and centrifugation through Ficoll-Hypaque(12) . For the preparation of subcellular fractions neutrophils were obtained from normal subjects and CGD patients by leukapheresis (13) and purified as described above with the omission of the dextran sedimentation step. Neutrophils were treated with 2.5 mM diisopropyl fluorophosphate for 10 min at 4 °C, disrupted in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl(2), 1 mM ATP, 1.25 mM EGTA, 10 mM PIPES, pH 7.3) (14) by N(2) cavitation, and fractionated on discontinuous Percoll gradients. These methods, described in detail elsewhere(15, 16) , produce cytosol and plasma membrane fractions whose final concentrations were adjusted to 9 times 10^7 and 1.25 times 10^9 cell equivalents (cell eq)/ml, respectively. Fractions were stored at -80 °C for up to 1 year without loss of activity.

Translocation of Cytosolic Oxidase Components

Experiments to study the translocation of p47 and p67 from the soluble to the particulate neutrophil fractions were performed exactly as described by Heyworth et al.(17) , using PMA (100 ng/ml) as the stimulus.

Gel Electrophoresis and Protein Immunoblotting

Neutrophil fractions were subjected to SDS-polyacrylamide gel electrophoresis on homogeneous 10 or 13% acrylamide gels (see figure legends) with prestained Rainbow protein markers (Amersham Corp.) as molecular mass standards. Separated proteins were electrophoretically transferred to PVDF membrane (Bio-Rad) in a Transblot cell (Bio-Rad). Nonspecific binding sites were blocked with 3% bovine serum albumin, 10% goat serum in HEPES-buffered saline and the blots probed with antibodies to the appropriate subunits of NADPH oxidase. Antisera to the Phox proteins were raised in rabbits using synthetic peptides (coupled to keyhole limpet hemocyanin) corresponding to amino acid residues 340-355 of p47 (RPGPQSPGSPLEEERQ), 437-450 of p67 (DEPKESEKADANNQ), 153-170 of p22 (SNPPPRPPAEARKKPSEE), and 537-556 of gp91 (CGPEALAETLSKQSISNSES). The antisera to p47 and p22 were used without further purification at dilutions of 1:2000 and 1:1000 respectively. Antibodies to p67 and gp91 were affinity-purified on Affi-Gel columns (Bio-Rad) to which the cognate peptides had been coupled. A goat anti-rabbit IgG antibody-alkaline phosphatase conjugate (1:1000 dilution) (Bio-Rad), together with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium substrates (Bio-Rad) were used to detect bound primary antibody. The protein content of neutrophil membrane fractions was estimated using the BCA microassay (Pierce) with bovine serum albumin as a standard.

Functional Assays

Whole cell O(2) production was determined spectrophotometrically by the superoxide dismutase-inhibitable reduction of cytochrome c(15) . NBT slide tests were performed as described previously(18) . Intracellular production of oxidants was assayed using a flow cytometric assay of dichlorofluorescein fluorescence, using the method described by Bass (19) with minor modifications. Briefly, neutrophils were preincubated at a cell density of 10^6/ml for 15 min at 37 °C in phosphate-buffered saline (138 mM NaCl, 2.7 mM KCl, 8.1 mM Na(2)HPO(4), 1.47 mM KH(2)PO(4)) supplemented with glucose (5 mM) and gelatin (0.1%) containing 5 µM DCFH-DA. Tubes were mixed every 5 min. Stock solutions (5 mM) of DHFH-DA (Eastman Kodak Co.) in EtOH were prepared fresh daily and kept in the dark at room temperature. After preincubation, catalase was added (275 units/ml), and the samples were divided into two 1-ml aliquots. PMA (100 ng/ml in Me(2)SO) was added to one tube and Me(2)SO to the other. After 10- and 20-min incubation at room temperature, samples were read on a FACScan (Becton Dickinson, San Jose, CA) equipped with a 15-milliwatt argon laser and FACScan Research Software. 10,000 events were collected; cellular aggregates and debris were excluded by gating on the neutrophil population. Mean channel fluorescence was calculated for each sample using a linear scale, the unstimulated (+Me(2)SO) control values were subtracted from the stimulated (+PMA) fluorescence.

Biochemical Assays

INT reductase activities of NADPH oxidase activated in the cell-free system were assayed spectrophotometrically in 96-well microtiter plates using a kinetic plate reader as described previously(5) . O(2) assays were performed on membranes prepared from PMA-activated neutrophils and on membranes and cytosols prepared from unstimulated neutrophils using the cell-free activation system in microtiter plates by determination of the superoxide dismutase-inhibitable rate of cytochrome c reduction(20) . These assays were also used to determine the Michaelis parameters for NADPH oxidase.

Genetic Analysis

Molecular genetic analyses were undertaken as described previously using genomic DNA extracted from 3 ml of whole blood(21) . Details of these methods will be published fully elsewhere. (^2)

Spectroscopy

Reduced minus oxidized difference spectra were recorded as described previously using 2% (w/v) Triton X-100 extracts of whole neutrophils (15) or 2% (w/v) OG extracts of neutrophil plasma membranes that had been washed with 1 M NaCl to remove myeloperoxidase(22) . CO difference spectra were recorded by reduction of a detergent extract of membranes with a few grains of sodium dithionite, storing the spectrum in the memory of the spectrophotometer, then gently bubbling the contents of the cuvette with CO for 60 s (one to two bubbles/s through a fine pipette attached to the gas line) and re-recording the spectrum. The subtraction of the original trace results in the (reduced + CO) minus (reduced) spectrum. Reoxidation of reduced cytochrome b by oxygen was assessed by reducing an aliquot of detergent-solubilized membranes with a slight excess of crystalline sodium dithionite (0.5 mg), recording the absorbance spectrum, and reoxidizing the reduced flavin and cytochrome b by gentle end over end mixing of the contents of the cuvette to introduce atmospheric oxygen; after 30-s agitation the spectrum was re-recorded to determine changes in the degree of cytochrome b reduction. NADPH-dependent reduction of FAD and cytochrome b in 2% OG extracts of neutrophil plasma membranes after cell-free activation of NADPH oxidase activity was determined under conditions of reduced oxygen tension using the galactose and galactose oxidase system described previously(23) .


RESULTS

Patient A had been diagnosed with CGD at 7 years of age following a history of recurrent infections and an abnormal NBT slide test. The family history was highly suggestive of X-linked inheritance. On evaluation at Scripps Clinic, no O(2) production could be detected from the patient's neutrophils using either PMA or formylmethionylleucylphenylalanine as the stimulus, confirming the clinical diagnosis of CGD.

NBT Slide Test

NBT slide tests performed on normal subjects show 98-100% of PMN strongly staining with purple NBT formazan after stimulation of the cells with PMA(8) . Typical CGD patients (both X-linked (X91° and X91, including patient B) and autosomal recessive forms (A22°, A47°, and A67°)) show no detectable NBT staining. In contrast, neutrophils and monocytes from patient A displayed a uniform population (98-100%) of weakly staining cells (not shown). This staining pattern is similar to that observed in a very few other variant X-linked CGD patients (X91 CGD), with diminished levels (3-25%) of a functional cytochrome b. (^3)Unlike patient A, these X91 CGD patients have neutrophils that are capable of diminished levels of O(2) production that broadly correlate with their degree of cytochrome expression.

DCFH-DA Fluorescence

Measurements of intracellular oxidant production using the fluorescent probes DCFH-DA and rhodamine 123 have shown that in marked contrast to patients with mutations in gp91 or p22 in which cytochrome b is missing (X91° or A22° CGD) and who have essentially no production of intracellular oxidants, neutrophils from patients with p47 deficiency have levels of intracellular oxidant production intermediate between cytochrome b-deficient patients and normal controls. (^4)The reactive species responsible for the oxidation of the DCFH is probably H(2)O(2). It should be emphasized that with neither cytochrome-deficient nor p47-deficient CGD neutrophils can any extracellular O(2) or H(2)O(2) be detected. Analysis of intracellular oxidant production in the neutrophils of patient A gave a high intermediate fluorescence value of 85 after 10 min, and 231 after 20 min, PMA stimulation, compared with the normal control of 246 and 495 at 10 and 20 min, respectively (Table 1). These values are higher than that observed in p47 deficient CGD patients (35 and 129 at 10 and 20 min, respectively). In contrast, the PMN of patient B gave a low mean fluorescence values of 10 at 10 min and 55 at 20 min, similar to the values of 7 at 10 min and 16 at 20 min observed in cytochrome-deficient X91° CGD patients (Table 1).



Localization of the Defect in Patient A to the PMN Membrane

In order to define more closely the type of CGD, cell-free O(2) assays were performed in microtiter plates using combinations of membranes and cytosol from patient A and normal controls. By these means the defect in patient A was localized to the membranes, since the neutrophil cytosol from the patient A could support O(2) production in normal membranes, whereas the neutrophil membranes from patient A were incapable of O(2) generation under any conditions (see below).

Effects of Increasing NADPH Concentration

The mutation in patient B forms part of the region postulated to be the NADPH-pyrophosphate binding loop(3) . We hypothesized that this mutation might increase the K(m) of the oxidase for NADPH to such an extent that no activity would be seen in whole PMN (where the NADPH concentration is 34 µM(24) ) or under the conditions normally employed in the cell-free assay (160 µM NADPH). However, no superoxide dismutase-inhibitable cytochrome c reductase activity could be obtained at concentrations of NADPH up to 3 mM using membranes from either patient A or B. Above this concentration the rate of superoxide dismutase-insensitive activity made estimations of O(2)-dependent activity unreliable.

Cytochrome bSpectroscopy

Localization of the defect in patient A to the neutrophil membrane suggested a genetic lesion in one of the genes encoding cytochrome b (either p22 or gp91). Most commonly such mutations result in the absence of cytochrome, but reduced minus oxidized difference spectra of Triton X-100 extracts of neutrophils isolated on three different occasions from patient A, disclosed the presence of normal quantities of cytochrome b (51.8, 68.6, and 62.7 pmol/10^7 cells, compared with 59.8, 67.3, and 63.7 pmol/10^7 cells for normal controls processed in parallel). On first inspection, the absorbance maxima of the patient's cytochrome appeared grossly normal, but it was noted that the Soret band of the reduced minus oxidized spectrum was shifted slightly toward the red. In order to examine the cytochrome spectrum in more detail, 2% OG extracts were made of neutrophil plasma membrane fractions, and the absolute oxidized, absolute reduced (Fig. 1B) and reduced minus oxidized (Fig. 1A) difference spectra determined. The Soret band of the oxidized cytochrome b of patient A is shifted 1 nm hypsochromically from 414.6 to 413.6 nm. The reduced spectrum is shifted bathochromatically, from 426 to 426.5 nm. As a result, the Soret peak and trough are shifted in the reduced minus oxidized difference spectrum from 427 to 427.5 nm (peak) and from 412 to 411.6 nm (trough). No differences in absorption maxima were observed for the alpha (558 nm) or beta(529 nm) bands. The ratio of cytochrome b Soret/ absorbance was not significantly altered in the patient. In contrast, patient B (with a Pro His mutation in gp91) had no apparent abnormalities in any of the cytochrome b visible absorption spectra (Refs. 11, 25, and 26 and this work). Cytochrome b has been shown to bind CO with low affinity(27) . As a further probe of the heme environment of the two mutant cytochromes b, the ability of each cytochrome to form a complex with CO was compared with that of normal cytochrome b. In all three cases, CO was bound weakly with 30-40% of the cytochrome in each sample forming a CO complex (not shown). As a further test of cytochrome function, the ability of the reduced cytochrome to become reoxidized by atmospheric oxygen was approximated as described under ``Experimental Procedures.'' In all cases, cytochrome b was fully reoxidized within 30 s of mixing in air.


Figure 1: A, reduced minus oxidized difference spectra of normal and X91 CGD membranes. OG extracts were made of neutrophil plasma membranes, and reduced minus oxidized difference spectra were recorded as described under ``Experimental Procedures.'' Solid line, solubilized membranes from a normal donor; dashed line, solubilized membranes from patient A. Each spectrum contains approximately 290 nM cytochrome b and 180 nM FAD. The dashed spectrum has been offset for clarity. B, absolute absorption spectra of normal and patient A X91 CGD membranes. Absolute oxidized (solid and short dashed traces) and absolute dithionite reduced spectra (dotted and long dashed traces) were recorded of normal (solid and dotted traces) and X91 CGD neutrophil membrane extracts from patient A (dashed traces) as described under ``Experimental Procedures.'' The cytochrome b concentration was 290 nM.



Immunocytochemistry

Immunochemical methods confirmed the presence of cytochrome b in the neutrophils of CGD patient A and patient B (with the Pro His mutation in gp91). As shown in Fig. 2, neutrophil plasma membrane fractions from both patients contained both the p22 and gp91 subunits of the cytochrome. Although the amount of cytochrome present in the patients neutrophils and plasma membranes was normal as determined by visible spectroscopy of the heme (see above and Table 1), and the same preparations of membranes were used for both spectroscopic determination of cytochrome b and immunostaining, the apparent intensity of the immunostaining of the gp91 bands in both patients A and B was unexpectedly decreased. The peptide used to raise the gp91 antibody used in these experiments did not correspond with the regions of either mutation (see below and ``Experimental Procedures'').


Figure 2: Protein immunoblots showing presence of flavocytochrome b subunits in neutrophil membranes from CGD patients A and B. Neutrophil plasma membranes isolated from: normal control (lane 1), flavocytochrome b-deficient CGD patients (lane 2) (p22-deficient in A and gp91-deficient in B), CGD patient B (lane 3), CGD patient A (lane 4) (all 10 µg of protein/well) were subjected to SDS-polyacrylamide gel electrophoresis on 13% (A) and 10% (B) acrylamide gels. After electrophoretic transfer of the proteins to PVDF, the blots were probed with antipeptide antibodies to either p22 (1:1000 dilution of antiserum) (A) or gp91 (affinity-purified antibody) (B). The location and molecular mass (kDa times 10) of the marker proteins are indicated to the left of each blot. The glycoprotein gp91 runs as a broad band as indicated.



Genetic Analysis

Based on the localization of the defect in patient A to the membrane, the shifts in the Soret absorbance band of the cytochrome b spectrum, and the X-linked inheritance of the disease, we considered the underlying mutation in patient A was most likely to reside in the gene for gp91. Consequently, genomic DNA from the patient was sequenced revealing a single base substitution of a cytosine for guanine 173 in exon 2 of gp91 (AGG ACG). The predicted effect of this mutation is the nonconservative replacement of arginine 54 with a serine residue. This is unlikely to be a polymorphism as it has not been observed in more than 200 normal and CGD patients alleles evaluated at Scripps. No other abnormalities in the sequence were detected. The patients mother and two sisters were found to be heterozygous for the mutation, in keeping with their NBT test results as reported to us, other family members; including another sister, had normal gp91 sequences and NBT tests.

Translocation of p47 and p67 during Activation

We have previously shown that the absence of cytochrome b, in both the X91° and A22° forms of CGD, prevents the association of both p47 and p67 with the plasma membrane fraction of neutrophils(17) . In the case of patient A we were interested in determining if the Arg Ser mutation, which leads to normal levels of nonfunctional cytochrome b, disrupted the assembly of cytosolic components at the membrane. As shown in Fig. 3, translocation of p47 and p67 was normal in patient A, with both proteins appearing in the particulate (pellet) fraction upon stimulation of the normal and CGD neutrophils with PMA. Thus the mutation that renders the cytochrome inactive does not appear to disrupt the interaction with other oxidase components.


Figure 3: Difference absorption spectra showing the reduction of cytochrome b and flavin in detergent-solubilized membranes after the cell-free activation of NADPH oxidase. Membrane extracts, cell-free activation of NADPH oxidase, and spectrophotometry were performed as described under ``Experimental Procedures.'' in the presence of galactose and galactose oxidase. a, absorption spectrum obtained from mixing normal cytosol and normal membrane extract. b, absorption spectrum obtained from mixing X91 CGD membrane extract from patient A with normal cytosol.c, absorption spectrum obtained from mixing X91 CGD membrane extract from patient A with p47-deficient cytosol. d, dithionite-reduced minus oxidized difference absorption spectrum of 14 nM FAD.



Dye Reductase (Diaphorase) Activity

We have recently described a dye (INT) reductase (diaphorase) activity associated with NADPH oxidase (5) and have provided evidence that electrons are transferred to the dye at the level of the FAD redox center(5, 23) . INT diaphorase activity was absent from the membranes of patients with X91° or A22° forms of CGD (in which there is an absence of cytochrome b heme and a marked decrease or absence of flavin). We have examined the ability of the two X91 CGD patients neutrophil membranes to support INT reductase activity in the cell-free activation system (Table 1). The X91 patients A and B (who have normal levels of both cytochrome b and flavin) differ with regard to diaphorase activity, patient A has completely normal activity, whereas patient B has close to zero activity, typical of that of cytochrome-deficient membranes (Table 2). The cytosol from both patients was capable of supporting normal levels of dye-reductase activity in normal membranes, demonstrating the defect was cytochrome-associated. This suggests that the mutation in each of these patients affects different points in the pathway of electron flow, despite the fact that they affect the same (gp91) subunit of cytochrome b. We have recently provided evidence that electrons are transferred to INT from the FAD center of NADPH oxidase(5) . Thus, it appears that in patient B, electrons from NADPH cannot be transferred to the flavin, whereas in patient A, the flavin center can become reduced and electrons can be transferred to INT, but electrons cannot flow to oxygen. This could arise either because cytochrome b does not become reduced or because the reduced cytochrome does not react with oxygen. In order to resolve this question we examined the ability of the heme and flavin redox centers of the patients' cytochrome b to undergo reduction in the cell-free activation system in the presence of NADPH, using the methods we have developed previously(23) . In accord with the results of the INT diaphorase experiment, in patient A the NADPH oxidase-associated flavin underwent reduction in the cell-free activation system in the presence of NADPH (Fig. 4). In contrast, there was no observable reduction of flavin in the case of patient B (not shown). In neither patient was there any evidence for the reduction of cytochrome b, unlike that observed in extracts of normal membranes (Fig. 4, trace a). We have recently provided evidence that p67 and p47 have individual roles in controlling electron flow in NADPH oxidase. We showed that the presence of p67 is sufficient, in the absence of p47, to activate NADPH-oxidase to a state were electrons reduce the flavin center of the oxidase, but p47 was required for electron transfer to continue to the heme center. Consistent with that proposal, we found that p47-deficient cytosol was capable of inducing electron flow into the flavin redox center of NADPH oxidase in extracts of the neutrophil membranes of patient A (Fig. 4, trace c).




Figure 4: Translocation of p47 and p67 upon stimulation of neutrophils from a normal control and an X91 CGD (patient A). Neutrophils from a normal control and CGD patient A were incubated for 5 min with either PMA (+) or an equal volume of Me(2)SO carrier(-), disrupted by sonication, and fractionated as described under ``Experimental Procedures.'' Cytosol (1.5 times 10^6 cell eq) and particulate (pellet) (4.5 times 10^6 cell eq) fractions were subjected to SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels, the proteins transferred to PVDF membrane, and the blots probed with antibodies to p47 and p67. The location and molecular mass (kDa times 10) of the marker proteins are indicated on the left of the figure, and p47 and p67 are identified on the right.




DISCUSSION

The results reported above identify mutations in gp91 that give insight into the functions of particular regions of the protein. The Arg Ser mutation in patient A is in a region of gp91 that is predicted to be on the cytoplasmic side of the membrane, close to a transmembranous domain. The result of this mutation appears to mainly effect the function of the heme moiety of cytochrome b rather than the NADPH-binding or flavin-binding regions since: (a) there is a subtle shift in the optical absorbance properties of the heme; (b) the heme fails to become reduced on activation but the flavin is reduced normally; (c) the flavin is functional with regard to INT diaphorase activity; and (d) there is a high level of DCFH-DA fluorescence (possibly by autoxidation of reduced flavin generating intracellular H(2)O(2) as we have recently proposed).^4 The ability of the FAD to donate electrons to external acceptors is probably the cause of the slight positive staining observed in the NBT slide test of patient A. We (and others) have previously speculated that at least part of the NBT reduction seen in the NBT slide test, although NADPH-oxidase dependent, is O(2)-independent, since NBT reduction is only partly inhibited by saturating concentrations of superoxide dismutase or under anaerobic conditions(28, 29, 30) . The finding that the PMNs of patient A display a degree of NBT reduction in the absence of O(2) production supports this proposal. In contrast to patient A, patient B has a mutation that appears to principally effect the flavin and/or NADPH-binding domains, since (a) the physicochemical properties of the heme appear normal with regard to spectrum, redox potential, and CO binding ( (26) and this work); (b) the flavin does not become reduced on activation of NADPH oxidase; (c) no diaphorase activity is seen with INT (or NBT staining in the slide test); and (d) the intracellular oxidant production measured by DCFH-DA fluorescence is low. These findings are consistent with the proposal that the mutation in this patient is within a NADPH-pyrophosphate binding loop that was identified by comparison of the sequence of gp91 with the sequences of enzymes of the ferredoxin NADP reductase class(3, 4, 31) .

The experiments described above support the proposal that NADPH-derived electrons do not reduce the flavin center of patient B, and electrons cannot pass from the flavin to cytochrome b in patient A. The correlation of INT reductase activity with the ability of NADPH oxidase-associated FAD to become reduced in neutrophil membrane extracts from patient A is strong confirmatory evidence that the diaphorase activity we have previously identified is due to electron transfer from the reduced FAD to INT. Furthermore, the ability of p47-deficient cytosol to permit electrons to reduce the flavin in this patient provides further support for our proposal that p47 and p67 have separate roles in the control of electron flow in NADPH oxidase.

Recently, another X91 CGD patient was reported with a mutation resulting in the substitution of a glycine residue for Asp toward the C terminus of gp91(32) . The functional defect in this patient apparently arises from the failure of the cytosolic factors to translocate to the membrane during the activation process. The C terminus of gp91 has previously been implicated as being essential for translocation of the cytosolic factors p67 and p47 and activation of NADPH oxidase(17, 33, 34) . Translocation of these factors appeared to be completely normal in patients A and B. Thus the study of three naturally occurring mutations in gp91 has led to the identification of three separate functional domains within cytochrome b.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI24838 (to J. T. C.), General Clinical Research Center Grant RR00833, and by the Stein Endowment Fund. 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: Dept. of Molecular & Experimental Medicine, CAL-1, The Scripps Research Institute, 10666 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-3654; Fax: 619-554-6988.

Present address: Immunology Dept., Genentech Inc., South San Francisco, CA 94080.

(^1)
The abbreviations used are: O(2), superoxide anion radical; CGD, chronic granulomatous disease; INT, p-iodonitrotetrazolium violet; NBT, nitro blue tetrazolium; OG, octyl-beta-D-glucopyranoside; DCFH-DA, 2`,7`-dichlorofluorescein diacetate (2`,7`-dichlorodihydrofluorescein diacetate); PVDF, polyvinylidene difluoride; PMN, polymorphonuclear neutrophil; PMA, phorbol 12-myristate 13-acetate.

(^2)
J. Rae, P. J. Hopkins, and J. T. Curnutte, manuscript in preparation.

(^3)
P. J. Hopkins and J. T. Curnutte, unpublished data.

(^4)
Rae, J., and Curnutte, J. T., manuscript in preparation.


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