From the
Cytochrome b
Upon ingestion of microbes, polymorphonuclear leukocytes (e.g. neutrophils) undergo a marked increase in oxygen
consumption, a process referred to as the respiratory burst(1) .
Respiration is accompanied by oxidation of NADPH and generation of
superoxide anion, a precursor of other antimicrobicidal oxidants.
Activation of the burst is thought to require the assembly of cytosolic
regulatory proteins with the plasma membrane-associated cytochrome,
cytochrome b
It has long been
assumed that the electron flow from NADPH through cytochrome and
finally to oxygen must utilize a flavin moiety. This was based not only
on chemical precedent, but also on early studies that showed that FAD
could partially stabilize oxidase activity in detergent extracts from
membranes of activated neutrophils(12) . In addition, it was
found that during turnover, a flavin-like semiquinone ESR signal was
seen in close proximity to the heme iron(13) . Until recently it
was assumed that the NADPH binding/FAD-containing moiety was distinct
from the cytochrome. Purified preparations of cytochrome b
Recent evidence has suggested
that the NADPH-interacting, FAD-binding moiety is cytochrome b
Several chemically modified forms
of flavins including 8-mercaptoflavin have been developed in recent
years and have proven useful as active site probes of
flavoproteins(32, 33) . While binding of FAD to
apoflavoproteins typically produces small changes in the visible
spectrum of the native flavin, binding generates a large red shift in
the spectrum of 8-mercaptoflavin. In addition, this analog can be used
to investigate the solvent accessibility of the protein-bound flavin
(32, 33). This approach exploits the chemical reactivity of the 8-SH
group on the flavin and the large fluorescence change that occurs upon
modification. In the present study, we have used 8-mercapto-FAD to
probe the flavin binding site of cytochrome b
On-line formulae not verified for accuracy REACTION 1
In this reaction, the appearance of product
(8-SCH
However, high activity was observed when the
reconstitution was carried out in the presence of
PC/PE/PI/SM/cholesterol (4:2:1:3:3 (w/w)). Koshkin and Pick (45, 46) recently demonstrated that purified cytochrome b
The present studies provide additional evidence that
cytochrome b
Additional evidence for flavin
is that binding to the cytochrome is provided by the pronounced
spectral shift upon interaction of 8-SH FAD to the apoflavocytochrome.
Spectral changes upon binding of native flavins are typically small,
and we were unable to detect such changes for binding of FAD itself to
the flavin-depleted cytochrome using either UV-visible absorption or
fluorescence methods (data not shown). However, much larger red shifts
of the spectrum have been seen upon binding of 8-SH flavins to a
variety of apoflavoproteins(32) . These include flavoenzymes of
the dehydrogenase-oxidase class such as glucose oxidase, D-amino acid oxidase, lactate oxidase, and old yellow
enzyme(32) . Such spectral shifts were readily observed in the
present studies and are indicative of flavin binding to a protein
environment. These spectral shifts were larger than those previously
seen (18) upon incubation of Triton X-100-solubilized neutrophil
membranes with 8-SH FAD, possibly because of more complete saturation
of the binding site with an excess of flavin-depleted cytochrome or
because of complicating binding of the flavin to other species in the
crude detergent extract.
The present studies also indicate that in
the bound flavin, the 8-thio position of 8-SH FAD is readily accessible
to a soluble electrophile. We observed only small decrease in the
reaction rate of either iodoacetamide or iodoacetate with the flavin
following binding to the flavin-depleted cytochrome. These data suggest
that as in many other flavoproteins, the 7-8-flavin edge is on
the surface of the cytochrome exposed to solvent, while other portions
of the flavin are likely to be deeply buried. The relatively high
fluorescence of the iodoacetamide and iodoacetate-derivatized flavins
may prove to be a useful reporter group for investigating other aspects
of oxidase assembly and function.
An aliquot of either flavin-depleted cytochrome b
We thank Dr. Dale E. Edmondson (Emory University,
School of Medicine) for providing 8-chlororiboflavin and for helpful
advice.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
isolated from human
neutrophils was inactive and contained no detectable FAD. However, high
NADPH oxidase activity was seen upon reconstitution of the cytochrome
with either native FAD or 8-mercapto-FAD in the presence of
phospholipids
(phosphatidylcholine/phosphatidylethanolamine/phosphatidylinositol/sphingomyelin/cholesterol,
4:2:1:3:3 (w/w)). Their cell-free superoxide-generating activities were
40.5 and 35.5 mol/s/mol of heme, respectively, which corresponded to 70
and 61% of the original activity of the plasma membranes. Both flavins
co-eluted with heme and protein on gel exclusion chromatography. The
respective specific flavin content was 6.45 and 7.93 nmol/mg of protein
and corresponded to a flavin:heme molar ratio of 0.41 and 0.51
consistent with a 2:1 ratio of heme to flavin. Mixing of 8-mercapto-FAD
with flavin-depleted cytochrome b
caused a
red-shift of the flavin absorption maximum from 520 nm to around 560
nm, as has been seen when a variety of other apoflavoprotein
dehydrogenases bind this analog. The 8-mercapto-FAD reconstituted into
the cytochrome reacted readily with either iodoacetamide (k = 38.8 M
min
) or
iodoacetic acid (k = 12.1 M
min
) to give a
fluorescence spectrum characteristic of a 8-mercaptoflavin derivative,
8-SCH
CONH
FAD or 8-SCH
COOH FAD.
These results indicate that position 8 of FAD bound to the protein is
freely accessible to solvent. These studies support the idea that
cytochrome b
is a flavocytochrome.
. The membrane-bound cytochrome b
consists of
- and
-subunits, with
respective molecular masses of 22 and 91
kDa(2, 3, 4, 5, 6) . The
cytosolic components include the p47
,
p67
, and the low molecular weight GTP-binding protein,
Rac 1 or Rac
2(7, 8, 9, 10, 11) . The
cytosolic factors are presumed to regulate electron flow within the
complex, but their mechanism of action is unknown.
typically contain heme but have little or no
FAD(14, 15, 16, 17) . A variety of
candidate flavoproteins have been proposed based on purification
approaches(12, 16, 17, 18, 19, 20, 21) and
chemical modification with chemically reactive NADPH
analogues(22, 23, 24) . Supporting the idea of a
separate FAD-component, NADPH-cytochrome c reductase purified
from neutrophil membranes (25) was able to form superoxide anion
when combined with purified cytochrome b
in the
presence of phospholipids. However, the rate of
O
generation was
low, undermining this interpretation. In addition, none of the other
candidate flavoproteins have held up as good candidates for an
exogenous oxidase-linked component.
itself. The large subunit contains a region of
homology with the NADPH-binding region of several flavoenzymes
including cytochrome P-450 reductase and ferredoxin-NADP
reductase(26, 27, 28) . Homology was also
detected in the region of the FAD-isoalloxazine-binding site
approximately 90 amino acids upstream of the NADPH-ribose-interacting
site of the large subunit(27) , but the homology was rather
weak. Although the flavin is completely lost from the protein as the
purification proceeds, it is possible to restore activity to either
native or recombinant preparations of cytochrome b
by incubating in the presence of phospholipids and
FAD(26, 27, 28, 29) . Following such an
incubation, FAD fluorescence comigrates with oxidase activity and heme
absorption in gel exclusion chromatography(26) . Thus, the
cytochrome b
has been proposed to bind FAD and
to contain all obligate electron transporting groups of the
oxidase(30, 31) .
as
well as the solvent accessibility of the bound flavin.
Materials
Cytochrome c (type VI),
polyacrylamide gel electrophoresis standards, NADPH, n-octyl
glucoside, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride
and N-p-tosyl-L-lysine
chloromethyl ketone were from Sigma. GTP
S was purchased from
Boehringer Mannheim. HESSOL (6% hetastarch in 0.9% NaCl) was from Green
Cross Co., and lymphocyte separation medium (LSM, 6.2% Ficoll plus 9.4%
sodium diatrizoate) was obtained from American Critical Care Division
of American Hospital Supply (McGaw Park, IL) and Biotics (Kensington,
MD), respectively. Superoxide dismutase and dithiothreitol were from
Wako Pure Chemical Co., and prestained molecular weight standards for
SDS-PAGE were obtained from Bio-Rad. Heparin-Sepharose CL-6B,
DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, and
-aminooctyl-agarose
were purchased from Pharmacia Biotech Inc. L-
-Phosphatidylcholine (bovine brain), L-
-phosphatidylethanolamine (bovine brain), L-
-phosphatidylserine (bovine brain), L-
-phosphatidylinositol (bovine brain), and sphingomyelin
(bovine erythrocyte) were purchased from Sigma. All other reagents were
of the highest grade available commercially.
Isolation of Human Neutrophils
Human neutrophils
were obtained from peripheral blood of normal healthy donors after
obtaining informed consent. Erythrocytes were sedimented with HESSOL,
and the mononuclear cells were removed from the resulting supernatant
by centrifugation through lymphocyte separation medium(34) . The
resulting cells were greater than 95% neutrophilic granulocytes.
Neutrophils were resuspended in buffer A (6 mM PIPES, pH 7.35,
containing 60 mM KCl, 18 mM NaCl, 2.3 mM
MgCl, 1 µM diisopropyl fluorophosphate, 1
mM PMSF, 1 µg/ml TLCK, and 6% (w/w) sucrose).
Plasma Membrane Preparation and
Solubilization
Membranes were prepared as described
previously(25) . Cells (6 10
) in 20 ml of
ice-cold buffer A were disrupted by nitrogen cavitation after being
pressurized at 500 p.s.i. for 25 min at 3 °C(35) . The
cavitate was centrifuged (800
g, 5 min) to remove
nuclei and unbroken cells. The supernatant was loaded onto
discontinuous sucrose gradient consisting of 50 and 30% sucrose in
buffer A and centrifuged at 150,000
g for 1 h. Plasma
membrane and specific granule fractions were collected, and 5 volumes
of relaxation buffer (10 mM PIPES, pH 7.35, containing 100
mM KCl, 3 mM NaCl, and 3.5 mM MgCl
) were added. The membrane suspension was
centrifuged at 250,000
g for 1.5 h, and the pellets
were resuspended in 5 ml of buffer B (0.1 M Tris acetate
buffer, pH 7.4, containing 0.1 M KCl, 20% glycerol, 1
mM dithiothreitol, 1 mM EGTA, 1 mM PMSF, 1
µg/ml TLCK, 40 mMn-octyl glucoside, and 0.5%
sodium cholate). The material was centrifuged at 150,000
g for 1 h, and the supernatant was used for purification of
cytochrome b
.
Purification of Cytochrome b
Detergent-solubilized
cytochrome bfrom Solubilized Membranes
was purified by the method of Segal et al. (27) with some modifications. The solubilized
membrane fraction was passed through a mixed bed of DEAE-Sepharose,
CM-Sepharose, and
-aminooctyl-agarose (1.6 ml bed volume of
each), followed by chromatography on heparin-Sepharose (2.36 ml bed
volume). The columns were equilibrated and washed well with buffer B,
and cytochrome b
was adsorbed to the
heparin-Sepharose. The latter was washed stepwise with 50 ml of buffer
B containing 0.1 M NaCl and then with 25 ml of buffer B
containing 0.5 M NaCl, which eluted the cytochrome. These
cytochrome b-containing fractions were pooled, concentrated,
and applied to a desalting column (Excellulose GF-5, Pierce)
equilibrated with buffer C (50 mM Tris acetate buffer, pH 7.4,
containing 20% glycerol, 2 mM MgCl
, 1 mM
dithiothreitol, 1 mM EGTA, 1 mM PMSF, and 1 µg/ml
TLCK) to remove NaCl. Samples with specific heme contents greater than
14.3 nmol heme/mg protein were pooled and applied to a HiTrap Q column
(Pharmacia) and the eluting heme-containing fractions were collected.
The concentration of cytochrome b
was determined
by reduced-minus-oxidized absorbance difference spectroscopy or by
directly measuring the oxidized absorbance spectrum (350 nm to 600 nm)
on a Hitachi 156 dual beam scanning spectrophotometer, assuming a
reduced-minus-oxidized Soret band (427-440 nm) extinction
coefficient of 161 mM
cm
or an oxidized Soret band (414 nm) extinction coefficient of 131
mM
cm
(36) .
The pooled fractions had a heme content of 16.2 nmol/mg protein. Flavin
was analyzed according to Nisimoto et
al.(37) , using reverse phase HPLC with a
fluorescence detector. FMN and FAD obtained from Sigma were purified by
HPLC and used as the authentic standards. Protein was quantified by the
method of Lowry et al.(38) , using bovine
serum albumin as the standard.
Western Blot Analysis
Proteins separated by
SDS-PAGE (10% gel) were transferred to an Immobilon-P membrane
(Millipore Corp., Bedford, MA)(39) . The membrane was incubated
at 25 °C for 2 h in 4% skim milk in 20 mM phosphate
buffer, pH 7.3, containing 0.14 M NaCl and 2.7 mM KCl. Antibodies used were those to two synthetic peptides
corresponding to the COOH termini of residues 175-195 of the
small cytochrome subunit and 558-570 of the large cytochrome
subunit, prepared as described in the previous paper(40) . After
washing, the membrane was reacted with the antibodies (3.0 µg/ml)
and then with horseradish peroxidase-linked second antibody (IgG,
1:5000 dilution) raised in goat. Antibodies were diluted with 20 mM phosphate-buffered saline, pH 7.3, containing 0.1% Tween 20, and
each incubation was carried out for 1 h at 25 °C. The Immobilon-P
membrane was washed extensively 3 times with 20 mM phosphate-buffered saline, pH 7.3, containing 0.1% Tween 20 (20
min each). The Western blots were stained for 15 min with
3,3`-diaminobenzidine in 20 mM phosphate-buffered saline, pH
7.3, containing 0.03% HO
.
Polyacrylamide Gel
Electrophoresis
SDS-polyacrylamide gel electrophoresis was
carried out according to the method by Rudolph and Krueger(41) .
Following electrophoresis for 2 h, the gels were stained with
two-dimensional-silver stain II reagent (Daiichi Pure Chemical Co.
Ltd.).
Assay of the Superoxide-generating
Activity
Cell-free NADPH oxidase activity was assayed by
measuring superoxide dismutase-inhibitable ferricytochrome c reduction. The assay was carried out in the absence and presence
of exogeneous native or 8 mercapto-FAD in the final assay medium. The
cell-free system consisted of cytochrome b (without or with added FAD or FAD analog) or plasma membrane,
cytosol, phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)), 10
µM GTP
S, and 0.25 mM arachidonate in 0.2 ml
of medium A (10 mM PIPES, pH 7.45, containing 4 mM MgCl
, 3 mM NaCl, 0.1 M KCl, 1 mM EGTA, and 0.5 mM PMSF). This mixture was preincubated for
10 min at 25 °C. At the end of the preincubation period, an aliquot
(95 µl) of the preincubation mixture was employed for the assay of
superoxide generation. Superoxide dismutase (80 µg) was added to
the reference cuvette. The reaction was initiated by addition of 0.2
mM NADPH (final) to 1 ml of assay medium containing 0.1
mM cytochrome c and 0.5 mg of phospholipids
(PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)). When plasma membrane (8.5
pmol as heme) was used, phospholipids were omitted from the assay
mixture. An extinction coefficient of 18.5
mM
cm
at 550 nm was
used to calculate the quantity of cytochrome c reduced (42).
Purification of FAD Synthetase from Brevibacterium
ammoniagenes
Culture of B. ammoniagenes and enzyme
purification were performed as in starting with 160 g of frozen cell
paste(43) .
Preparation of 8-Mercapto-FAD
The starting
material, 8-chlororiboflavin(44) , was generously provided by
Dr. Dale E. Edmondson, Emory University. This was converted to
8-chloro-FAD using partially purified FAD synthetase from B.
ammoniagenes. 8-Chlororiboflavin (76.8 µM) was
incubated in a final volume of 8.5 ml of 50 mM Tris-HCl
buffer, pH 8.0, containing 5 mM ATP and 20 mM MgCl, and the reaction was started by the addition of
2 ml of partially purified FAD synthetase. After reaction at 36 °C
for 12 h in the dark, an aliquot (15 µl) was removed and applied to
thin-layer chromatography (Eastman Kodak Co., Chromagram, 13255
cellulose). The 8-chloro-FAD and unreacted 8-chlororiboflavin were
identified on the thin-layer chromatography cellulose sheet by
fluorescence detector. The total reaction mixture (10.5 ml) was then
loaded onto a Bio-Gel P-2 column (5
60 cm). The first yellow
band eluted was identified as 8-chloro-FAD by thin-layer chromatography
using native FAD (R
value, 0.07) as a
standard. The thin-layer chromatography was developed using
1-butanol/acetic acid/water (4:2:2). The appropriate 8-mercapto-FAD was
prepared just before use by reaction of 8-chloro-FAD, buffered at pH
8.0, with 5 mM Na
S. Excess unreacted sodium
sulfide was removed by P-2 column (2
65 cm) chromatography. The
course of the reaction was monitored at 520 nm and is accompanied by a
color change from yellow to bright red. The resulting 8-mercapto-FAD
was reasonably stable at pH 7.5 and was used immediately for the
spectral and kinetic studies.
Reconstitution of Flavin-depleted Cytochrome b
Purified
FAD-depleted cytochrome b with Either Native FAD or 8-Mercapto-FAD
(194 pmol heme/12
µg of protein) was incubated in 0.3 ml of 0.05 M Tris
acetate buffer, pH 7.45, containing 0.05 M KCl, 10% glycerol,
1 mM dithiothreitol, 1 mM EGTA, 1 mM PMSF, 1
µg/ml TLCK (Buffer I) and 0.64 mg of phospholipids
(PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)) at 3 °C for 3 h and then
it was mixed with a 7.4-fold excess of either FAD or 8-mercapto-FAD.
After keeping the mixture at 3 °C for 12 h, it was passed through a
Sephadex G-25 column (12
200 mm) equilibrated with the Buffer I
to separate FAD-reconstituted cytochrome b
from
excess free flavin. Cytochrome fractions showing high flavin
fluorescence were pooled, and FAD was quantified by fluorescence
spectroscopy. The binding of 8-mercapto-FAD to cytochrome b
is also evidenced and determined by the
measurement of absorption spectrum using the extinction coefficient of
30 mM
cm
for the
FAD analog(32) . Pooled fractions were used for the assay of
superoxide production by complementation with cytosol in the cell-free
activation system.
Reaction of 8-Mercapto-FAD-reconstituted Cytochrome
b
8-SH FAD-reconstituted cytochrome bwith Either Monoiodoacetamide or
Monoiodoacetate
was used to investigate the chemical
reactivity of position 8 of the flavin. The reconstituted cytochrome b
contained 15.5 nmol of protoheme and 7.93 nmol
of 8-SH FAD/mg of protein. If it is assumed that the heme and
flavin-binding sites are 1 mol each/mol of protein (
plus
-subunits), the reflavination is 51% effective. If two or more
hemes per FAD is assumed, as has been proposed(26, 45) ,
then reflavination is complete. Reaction with either iodoacetamide or
iodoacetic acid was initiated by adding an aliquot of the reagent (0.1 M stock solution) to 0.2 ml of the 8-SH FAD-reconstituted
cytochrome. In a control experiment, the thiol reagent was reacted with
free 8-mercapto-FAD. The resulting fluorescence increase at 519 nm was
monitored as the generation of 8-SCH
CONH
FAD or
8-SCH
COOH FAD when excited at 473 nm. Since it appears that
the reactions show a single second-order dependence of the observed
rate (k
) on reagent concentration, a one-step
reaction between reagent and flavinated cytochrome b
is considered. The reaction is expressed as follows.
CONH
FAD-cytochrome b
) could be measured, and the second-order rate
constant (k) was determined. Likewise, the cytochrome b
-bound FAD analog could react directly with
iodoacetic acid, which has a negatively charged group, and simple
second-order kinetics were also found.
Effect of FAD and 8-Thio-FAD on NADPH Oxidase Cell-free
Activity
Upon analysis by silver-stained SDS-PAGE (Fig. 1A, lane2) the purified
cytochrome b showed a major band at 22 kDa and a
diffuse major band at 90-95 kDa. The latter represents the
glycosylated large subunit, which is known to behave in this manner.
Two minor bands were also present at around 75 and 24 kDa,
respectively. The two main protein bands were identified as the small
and large subunits of cytochrome b
by Western
blot analysis (Fig. 1B, lane2). The
antibody raised to the synthetic peptide corresponding to the COOH
terminus of the small subunit detected the 22 kDa band of cytochrome b
, whereas that raised to the synthetic
COOH-terminal peptide of the large subunit gave diffuse staining around
92-kDa region and also appeared to stain the minor 75 kDa band,
suggesting that the latter is a degradation (proteolysis or partial
loss of carbohydrates) product of the large subunit. The reduced minus
oxidized difference absorption spectrum of the purified preparation had
absorption maxima at 427, 530, and 558 nm, which are characteristic of
cytochrome b
(data not shown). The final
preparation eluted from an ion-exchange column (HiTrap Q) contained
16.2 nmol heme/mg of protein, but less than 0.01 nmol FAD/mg of
protein.
Figure 1:
SDS-PAGE pattern and immunoblot
analysis of purified cytochrome b. The purified
cytochrome b
(1.2 µg) was loaded onto
SDS-PAGE, followed by silver staining to examine the purity (laneA-2). Major protein peaks corresponded to apparent
molecular masses of 22 kDa (I) and 92 kDa (II). After
transfer to an Immobilon-P membrane, the membrane was incubated with a
mixture of rabbit polyclonal antibodies (3.0 µg/ml) against two
synthetic carboxyl-terminal peptides corresponding to the human
cytochrome b
small and large subunits as
described under ``Experimental Procedures'' (laneB-2). LaneA-1is
silver staining of detergent-solubilized plasma membrane (10.7 µg). LaneM shows prestained SDS-PAGE standards
(phosphorylase b, 106 kDa; bovine serum albumin, 80 kDa; ovalbumin, 50
kDa; carbonic anhydrase, 32.5 kDa; soybean trypsin inhibitor, 27.5 kDa;
lysozyme, 14.5 kDa).
The superoxide-generating activity of the purified
cytochrome b was initially measured in a
cell-free assay system consisting of cytosol, arachidonate, and
GTP
S with or without FAD in the assay mixture. In the presence of
5 µM FAD, the purified FAD-depleted cytochrome b
showed low superoxide-generating activity
(1.34 mol O
/s/mol of cytochrome b
)(40) , but superoxide production was
negligible in the absence of FAD (0.05 mol
O
/s/mol of cytochrome). These results
confirm previous reports (26, 27) that added flavin is
required for superoxide generation. Flavin-depleted cytochrome b
was also incubated with FAD or 8-SH FAD in the
presence of phosphatidylcholine, and an aliquot was used for the assay
for superoxide generation. In the presence of phospholipids and either
FAD or 8-thio-FAD added to the cell-free assay, somewhat higher
superoxide generation was seen. The highest value (11.7 mol/s/mol of
cytochrome b
seen with heme) was only 20% of
that seen when native plasma membranes were used as the source of the
intact cytochrome. 8-Mercapto-FAD exhibited almost the same ability to
reconstitute superoxide-generating activity using this protocol. The
low activity is incomplete probably due to in the reflavination of
cytochrome b
under these conditions or possibly
due to dissociation of flavin upon dilution of the flavocytochrome in
the assay mixture.
exhibits varying levels of
O
-producing activity depending upon the
nature of phospholipids serving for lipidation and flavination. Our
present studies confirm that binding of flavin to cytochrome b
is affected by the composition of
phospholipids. Flavin-depleted cytochrome b
was
incubated with phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w))
and either native FAD or 8-mercapto-FAD as detailed under
``Experimental Procedures,'' and then subjected to gel
filtration to separate flavin-bound cytochrome from free flavin (Fig. 2, A and B, respectively). An initial
peak of flavin fluorescence coeluted with the purified cytochrome b
, which migrated identically in the absence of
added flavin (Fig. 2C). In the absence of the
cytochrome, but with all other components present (Fig. 2D), the early eluting peak of flavin fluorescence
was not detected. The weak fluorescence seen in the first peak of Figs.
2, C and D, was not due to flavin but to scattering
due to the presence of phospholipids in the fractions. The major
fractions with high flavin fluorescence and heme absorbance were pooled
to examine the ability of either native FAD or 8-mercapto-FAD to
reconstitute NADPH oxidase activity, and the results are summarized in . The FAD-reconstituted cytochrome b
exhibited O
-producing activity
amounting to 70% of the original activity of the plasma membranes on a
per heme basis. Likewise, 8-mercapto-FAD-reconstituted cytochrome b
indicated a greatly increased NADPH oxidase
activity, which corresponded to about 61% of the original activity of
the plasma membranes. These results suggest that the
flavin-reconstituted cytochrome b
has a high
affinity for 8-mercapto-FAD as well as native FAD, and stabilized
binding of flavin to the protein leads to high catalytic activity. The
difference in the redox potentials of native FAD (-219 mV) (47) and 8-SH FAD (-290 mV) (48) does not appear to
change significantly the oxidase activity of the flavin-reconstituted
cytochrome b
.
Figure 2:
Reconstitution of cytochrome b with either native FAD or 8-mercapto-FAD.
FAD-depleted cytochrome b
(194 pmol as heme) was
incubated (3 h, 3 °C) with 0.64 mg of phospholipids
(PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)) in total of 0.3 ml of 0.05 M Tris acetate buffer, pH 7.45, containing 0.05 M
KCl, 10% glycerol, 1 mM EGTA, 1 mM PMSF, and 1
µg/ml TLCK (Buffer I). This sample was then mixed with 0.2 ml of
either 7.1 µM native FAD (panelA) or
7.0 µM 8-SH FAD (panelB) or with 0.2 ml
of Buffer I instead of flavin (panelC). As a
control, 0.64 mg of phospholipids in 0.3 ml of Buffer I was mixed with
0.2 ml of 7.0 µM 8-SH FAD in the absence of cytochrome (panelD). After keeping the mixture at 3 °C for
12 h, it was applied to a Sephadex G-25 column (12
200 mm)
equilibrated with Buffer I to separate bound from free flavin.
Fractions (0.85 ml) were collected, and each fraction was used for the
measurements of absorbances at 280 and 414 nm. For emission
measurements of native FAD, an aliquot (0.20 ml) of each fraction was
mixed with 0.1% SDS and heated for 3 min in a boiling water bath. The
supernatant (6,000
g, 15 min) was used for the assay
of flavin content. The excitation and emission wavelengths were 450 and
525 nm, respectively. For determination of 8-SH FAD, the supernatant
was reacted with 10 mM iodoacetamide for 30 min at pH 7.8. The
fluorescence at 519 nm was measured when excited at 473 nm (bandwidth
of 3 nm). The peak fractions (fraction number 9, 10, and 11) indicated
by the horizontalbars in panelsA and B were individually pooled, and aliquots were
subjected to the assay for the cell-free NADPH oxidase activity and
spectral studies.
Spectrophotometric Changes of 8-Mercapto-FAD Induced by
Binding to Cytochrome b
When 8-mercapto-FAD is
mixed and incubated with the flavin-depleted cytochrome b at pH 7.45, there was a large red-shift in the
visible absorption spectrum (Fig. 3), with the peak shifting from
about 520 nm to about 560 nm. No detectable absorbance or fluorescence
change was seen with native FAD binding to the cytochrome. A stable
spectrum was reached within 3 h at 3 °C, and no further change
occurred over this period. A major peak at 560 nm with a weak residual
shoulder at about 520 nm was seen using a 1.7-fold excess of
flavin-depleted cytochrome b
(heme) to the
8-mercapto-FAD. Although difficult to quantify precisely, these data
indicate that most of the 8-mercapto-FAD was bound to protein under
these conditions.
Figure 3:
Effect of flavin-depleted cytochrome b on the absorption spectrum of 8-mercapto-FAD.
The 8-SH FAD (0.21 µM in 1 ml) was titrated with 0.5, 0.9,
and 1.1 ml of 0.32 µM cytochrome b
in Buffer I containing 0.64 mg/ml phospholipids, and spectra D, E, and F were recorded, respectively. The
molar ratios of the heme to 8-SH FAD were 0.76, 1.37, and 1.68,
respectively. Curves B and C, respectively, show the
absorption of the cytochrome (0.32 µM as heme) and of 8-SH
FAD (0.21 µM) measured in the presence of phospholipids
(0.64 mg/ml), and curve A is a base line containing the
phospholipids alone in both sample and reference cuvettes. The titrated
mixture was allowed to stand for 3 h at 3 °C and then each spectrum
was scanned at the same temperature. Dry nitrogen gas was circulated
through the cell compartments.
Consistent with the titration data, the
8-mercapto-FAD-reconstituted cytochrome b chromatographed on Sephadex G-25 column (Fig. 2B)
also showed a broad peak with the absorption maximum around 560 nm,
indicating that on binding to the protein, the 8-SH flavin is
stabilized in its p-quinoid state (Fig. 4). Heme and
8-mercapto-FAD concentrations of the flavin-reconstituted cytochrome b
were determined from its absorption spectrum.
Their concentrations were 0.15 µM (heme) and 0.085
µM (8-SH FAD), respectively, indicating a molar ratio of
FAD analog to protoheme of 0.57. The flavin of
8-mercapto-FAD-reconstituted cytochrome b
was
also quantified by fluorimetric assay, and its content (8-SH FAD/heme,
0.51) was almost the same as the value obtained by the
spectrophotometric method.
Figure 4:
Absorption spectrum of
8-mercapto-FAD-reconstituted cytochrome b. The
cytochrome was incubated with a 7.4-fold molar excess of 8-thio-FAD,
and the reconstituted cytochrome was resolved from free flavin by gel
filtration, as in Fig. 2. The spectrum of the flavinated cytochrome b
, which subtracted a base line containing the
phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)) alone, was
recorded from 350 to 650 nm at 3 °C.
Solvent Accessibility of the 8-SH Position
We
examined the reactivity of iodoacetamide with 8-mercapto-FAD in order
to explore the effect of the protein environment on the reaction
kinetics. The 8-mercapto-FAD reacts readily with this small
electrophilic reagent producing a large increase in
fluorescence(33) . Fig. 5shows typical time courses of
fluorescence increase at 519 nm for the reaction of both free and
cytochrome b-bound 8-mercapto-FAD with excess
iodoacetamide. Plotting the observed initial rate constant as a
function of iodoacetamide concentration gave a straight line passing
through the origin with a slope equal to the second-order rate
constant, which was calculated to be 48.5 M
min
for free 8-SH
FAD at pH 7.45 (Fig. 6), a value that is comparable with the rate
reported previously(33) . The reaction of the thiol reagent with
8-mercapto-FAD bound to the cytochrome occurred with a second order
rate constant of 38.8 M
min
,
approximately 20% slower than that found for free 8-mercapto FAD. This
relatively large rate constant suggested that position 8 of the
protein-bound flavin was freely available to solvent. Second-order
kinetics was also seen upon reacting iodoacetic acid with
8-mercapto-FAD bound to cytochrome b
(k = 12.1 M
min
). The rate
constant was close to 16.4 M
min
, which was
calculated for the reaction of free 8-SH FAD with iodoacetic acid (data
not shown). This water-soluble electrophile also appears to indicate
that the position 8 of isoalloxazine ring is accessible to solvent.
Figure 5:
Time course for the reaction of
iodoacetamide with free 8-mercapto-FAD and 8-SH FAD-reconstituted
cytochrome b. Free 8-SH FAD (0.075
µM, curve A), and 8-SH FAD-reconstituted
cytochrome b
prepared as in Fig. 2 (0.070
µM as flavin, curve B) were incubated in 0.2 ml
of Buffer I containing 0.16 mg of phospholipids
(PC/PE/PI/SM/cholesterol, 4:2:1:3:3 (w/w)). The reaction was initiated
by adding 9.2 mM CH
ICONH
to the
incubation mixture. Fluorescence was monitored using excitation and
emission wavelengths of 473 and 519 nm, respectively. In a control
reaction, 9.2 mM iodoacetamide was added to the reaction
mixture without added 8-SH FAD (curve C).
Figure 6:
Rate constants for the reaction of
iodoacetamide with free 8-mercapto-FAD and 8-SH FAD-reconstituted
cytochrome b. The apparent rate constant (K
) obtained in experiments as in Fig. 5 was
plotted as a function of CH
ICONH
concentration,
and the second-order rate constant was determined from the slope (see
``Experimental Procedures''). Opencircles show the reaction with free 8-SH FAD, and filledcircles are that with 8-SH FAD-reconstituted cytochrome b
.
is a flavocytochrome. As purified,
the cytochrome loses flavin so that in its purified form it lacks both
measurable flavin and activity. Either native FAD or 8-substituted FAD
analog can readily be bound to the cytochrome in such a way as to
reconstitute activity. As was found by other
groups(26, 27) , reconstitution of activity with FAD
requires the presence of phospholiplids. Our data are thus consistent
with the proposal that FAD binding requires phospholipids or a membrane
environment and that detergent solubilization results in a cytochome
conformation from which flavin is readily lost. In the presence of
excess FAD or analog in the assay medium, the activity that can be
maximally achieved was only about 20% of that seen with the cytochrome
in its native plasma membrane environment (based on activity/heme). It
may be either that flavin reconstitution is incomplete under these
conditions or that the native and active conformation of the cytochrome
has not been completely restored (or both), for example due to a
nonoptimal phospholipid composition. In this regard it is worth noting
earlier studies (45, 46, 49) in which
phospholipid composition has significant effects on activity in
detergent solubilized preparations. The most effective flavination was
found using cytochrome b
reconstituted with a
mixture of PC, PE, PI, SM, and cholesterol (4/2/1/3/3 (w/w)). Under
these conditions, the FAD:heme ratio was found to be 0.4-0.5
mol/mol, consistent with the previously proposed ratio of two
hemes/flavin. Binding of 8-SH FAD to cytochrome b
was demonstrated by co-elution of flavin and heme by gel
exclusion chromatography, and also by the perturbation of the
8-thioflavin spectrum by the apoflavocytochrome. The high degree of
reflavination of cytochrome b
in the presence of
the above lipids was accompanied by a corresponding increase in
O
producing activity, but this was
slightly less than the activity seen in native plasma membranes
suggesting that the plasma membrane provides a more optimal environment
than does the defined lipid mixture.
Table: Superoxide-generating activity of
FAD-reconstituted cytochrome b in a cell-free
assay system
(0.73 µg) or FAD-reconstituted cytochrome b
(0.70 µg) was incubated with 0.22 mg of
cytosol, 0.25 mg of phospholipids (PC/PE/PI/SM/cholesterol, 4:2:1:3:3
(w/w)), 10 µM GTP
S, and 0.25 mM arachidonate
in 0.2 ml of medium A as described under ``Experimental
Procedures.'' The assay was carried out in the absence of added
flavin in the reaction medium. FAD-reconstituted cytochrome b
was obtained from Sephadex G-25 column
chromatography as described in Fig. 2. Flavin content of
FAD-reconstituted cytochrome b
was estimated by
fluorimetric method.
, superoxide; GTP
S, guanosine
5`-(
-thio)triphosphate; PAGE, polyacrylamide gel electrophoresis;
PMSF, phenylmethylsulfonyl fluoride; TLCK, N
-tosyl-L-lysine chloromethyl ketone;
PIPES, 1,4-piperazinediethanesulfonic acid; PC, L-
-phosphatidylcholine; PE, L-
-phosphatidylethanolamine; PI, L-
-phosphatidylinositol; SM, sphingomyelin; HPLC,
high-performance liquid chromatography; P-450 reductase, microsomal
NADPH-cytochrome P-450 reductase from rabbit liver.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.