Tyrosyl radicals have been detected during
turnover of prostaglandin endoperoxide H synthase (PGHS), and they are
speculated to participate in cyclooxygenase catalysis. Spectroscopic
approaches to elucidate the identity of the radicals have not been
definitive, so we have attempted to trap the radical(s) with nitric
oxide (NO). NO quenched the EPR signal generated by reaction of
purified ram seminal vesicle PGHS with arachidonic acid, suggesting
that NO coupled with a tyrosyl radical to form inter alia
nitrosocyclohexadienone. Subsequent formation of nitrotyrosine was
detected by Western blotting of PGHS incubated with NO and arachidonic
acid or organic hydroperoxides using an antibody against nitrotyrosine.
Both arachidonic acid and NO were required to form nitrotyrosine, and
tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid
chromatography and monitored with a diode array detector. A single
peptide was detected that exhibited a spectrum consistent with the
presence of nitrotyrosine. Consistent with Western blotting results,
both NO and arachidonic acid were required to observe nitration of this
peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.
 |
INTRODUCTION |
Prostaglandin endoperoxide H synthase
(PGHS),1 a bifunctional heme
enzyme, catalyzes the first two steps of prostaglandin and thromboxane biosynthesis. Its cyclooxygenase activity catalyzes the
incorporation of two molecules of dioxygen into arachidonic acid to
form prostaglandin endoperoxide G2, a hydroperoxy
endoperoxide (Reaction R1).
The peroxidase activity of PGHS then catalyzes the two-electron
reduction of prostaglandin G2 to prostaglandin endoperoxide H2, a hydroxy endoperoxide (Reaction
R2)
(1-3).
The mechanism of prostaglandin synthesis by PGHS, particularly the
relationship between the two activities of this enzyme, has been the
subject of intense investigation. The peroxidase and cyclooxygenase
activities of PGHS are separate and distinct (4). The two active sites
are located on opposite sides of the protein and are separated by the
heme prosthetic group. Nonsteroidal antiinflammatory drugs that bind in
the cyclooxygenase active site do not inhibit the peroxidase (3).
Despite this spatial separation the peroxidase and cyclooxygenase
activities are functionally interconnected. Ligands to the distal heme
binding site inhibit both activities (5-7). Scavenging of fatty acid
hydroperoxides with glutathione peroxidase inhibits cyclooxygenase
activity, and protein or heme modifications that reduce peroxidase
activity induce a lag phase in cyclooxygenase activity that can be
overcome by addition of hydroperoxide (8, 9). It is believed that peroxidase turnover activates the oxidizing agent responsible for
cyclooxygenase turnover.
Ruf and co-workers (10, 11) first detected a tyrosyl radical by EPR
spectroscopy upon addition of arachidonic acid or prostaglandin
G2 to PGHS and proposed that the radical is the oxidant
responsible for cyclooxygenase activity. They proposed that ferric PGHS
is oxidized by a fatty acid hydroperoxide by two electrons to yield the
oxoferryl porphyrin
cation radical intermediate, PGHS compound I
(Reaction 3). Compound I oxidizes a
tyrosine residue to a tyrosyl radical and is reduced to PGHS compound
II (Reaction
4). The
tyrosyl radical was postulated to abstract a hydrogen atom from
arachidonic acid to initiate the cyclooxygenase cycle (11). Evidence
supporting this mechanism was provided by the work of Tsai et
al. (12), which demonstrated that under anaerobic conditions, the
spectroscopically detectable PGHS-1 tyrosyl radical (formed by the
reaction of PGHS-1 with ethyl hydroperoxide) oxidized arachidonic acid
to a carbon-centered radical.
Identification of the catalytically active tyrosyl radical has
been problematical. Smith and co-workers (13, 14) showed that mutation
of tyrosine 385 to phenylalanine abolishes cyclooxygenase but not
peroxidase activity. Tyrosine 385 is present in the cyclooxygenase active site in between the heme group and the putative arachidonic acid
binding site. However, reaction of the Y385F mutant with peroxide
produces a tyrosyl radical that is qualitatively similar by EPR
spectroscopy to the radical produced from wild-type enzyme (14, 15).
Either the spectrally detectable radical in wild-type enzyme is not
derived from tyrosine 385 or a different tyrosine is oxidized in the
Y385F mutant. Thus, to this point there is no conclusive evidence that
the radical signal observed by EPR is that of tyrosine 385, and
although it is an attractive hypothesis, there is no direct evidence
that tyrosine 385 is oxidized to a radical during PGHS turnover.
The apparent limitation of site-directed mutagenesis in
combination with EPR for identification of PGHS tyrosyl radicals
prompted us to use a different approach to solve this problem. The
reaction of tyrosyl radicals with nitric oxide (NO) to form
nitrosocyclohexadienone has been demonstrated in other enzymes such as
ribonucleotide reductase (16, 17) and photosystem II (18, 19). It has been shown both in these enzymatic systems as well as with simple phenols that the reaction of NO with phenoxyl radicals to form nitrosocyclohexadienones is reversible (16-22). Recently, it has been
shown that NO quenches the PGHS-2 tyrosyl radical signal, but the
reaction is irreversible due to the eventual formation of nitrotyrosine
(23). We have observed that NO quenches the tyrosyl radical signal
formed by reaction of PGHS-1 with arachidonic acid. The subsequent
oxidation of nitrosocyclohexadienone to nitrotyrosine (presumably by
the peroxidase activity of PGHS) provides a potential marker for
identification of the modified tyrosine. Thus, using this method in
conjunction with peptide mapping techniques, PGHS tyrosyl radicals
generated during arachidonic acid oxidation can be identified.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Arachidonic acid was purchased from Nu-Chek Prep,
Inc. (Elysian, MN). L-1-Tosylamido-2-phenylethyl
chloromethyl ketone (TPCK)-treated trypsin (type XIII) from bovine
pancreas, superoxide dismutase from bovine erythrocytes,
tetranitromethane, and hematin were purchased from Sigma. Diethylamine
NONOate (DEA/NO) was purchased from Cayman Chemical Co. (Ann Arbor,
MI), and proline nonoate (proli/NO) was obtained from Alexis
Biochemicals (San Diego, CA). 3-Chloroperoxybenzoic acid was purchased
from Aldrich. PGHS from ram seminal vesicles was purified as described
previously (24). Apo-PGHS was prepared from purified holo-PGHS as
described previously (25). Its cyclooxygenase activity was determined
by monitoring O2 consumption using a Gilson 6/5 oxygraph
equipped with a Clark-type electrode maintained at 37 °C (Gilson
Medical Electronics, Inc., Middleton, WI). The specific activity of the
apo-PGHS used in these studies was ~17 µmol of arachidonic acid per
mg protein per min.
EPR Spectroscopy--
PGHS samples were prepared by
reconstitution of apo-PGHS with 1 mol of heme per mol of protein. The
resulting stock solution was diluted to a final concentration of 50 µM in 200 µl of 100 mM potassium-phosphate
buffer, pH 8.0, containing 25% glycerol. Samples were stored on dry
ice until immediately before use. For each experiment, the sample was
thawed to
12 °C and placed into 3-mm quartz EPR tubes. Arachidonic
acid was added to a final concentration of 1 mM in a volume
of 20 µl from a stock solution dissolved in ethanol. When used, the
NO generators, Proli/NO or DEA/NO, were dissolved in cold 10 mM NaOH and were allowed to decompose in the PGHS-1
solution for one half-life (2 s in the case of Proli/NO and 180 s
in the case of DEA/NO) before the addition of the arachidonic acid. The
samples were frozen in liquid nitrogen 5 s after the addition of
arachidonic acid and were transferred to a quartz fingertip Dewar that
was inserted into a TM110 cavity of a Bruker ESP 300 EPR
spectrometer. Spectra were acquired using the following instrument
settings: modulation amplitude, 1 G; time constant, 1.3 s; scan
time, 1342 s; receiver gain, 1 × 105; microwave
power, 2 mW; microwave frequency, 9.49 GHz; modulation frequency, 100 kHz.
Nitration of PGHS-1 with NO and Arachidonic Acid--
Due to its
rapid decomposition at neutral pH, DEA/NO was chosen as a source of NO.
Stock solutions of DEA/NO were prepared using 10 mM NaOH.
At this high pH, the DEA/NO was stable for several weeks. Because of
the relatively slow decomposition of DEA/NO at pH 8.0, Centricon-10
concentrators (Amicon) were used to change the apo-PGHS-1 buffer from
80 mM Tris (pH 8.0) to phosphate buffer (pH 7.2). PGHS-1
(1.1 mg in 250-400 µl) was placed on the concentrator, and the
volume was increased to 2 ml using 100 mM phosphate buffer, pH 7.2. Following a 90-min centrifugation (4000 × g),
1.7 ml phosphate buffer was added, and the samples were centrifuged an
additional 60-90 min.
Apo-PGHS-1 was reconstituted by adding heme (1 mol/mol PGHS subunit)
and incubating at room temperature for 5 min. The PGHS-1·indomethacin complex was generated by adding indomethacin (1.2 mol/mol PGHS subunit)
to reconstituted PGHS-1 and incubating at room temperature for 25 min.
Generation of NO was accomplished by adding 500 nmol DEA/NO to PGHS-1
or PGHS-1·indomethacin complex and incubating at room temperature for
10 min. 10 eq of arachidonic acid was then added and allowed to react
for 3 min at room temperature. The entire reaction volume (400 µl)
was then placed on a Bio-Rad 10DG gel filtration column to separate
PGHS from the other reagents. The column was equilibrated with 100 mM Tris buffer (pH 8.0) and 0.1% Tween 20.
Nitration of PGHS-1 with CPBA and NO--
The procedure was
exactly the same as that described for arachidonic acid above except
that 10 eq of CPBA was used in place of arachidonic acid. 3 min after
addition of CPBA, the reaction mixture was passed over a 10DG desalting
column as described above. The concentration of the CPBA stock solution
was determined by spectrophotometrically monitoring the oxidation of
ferric horseradish peroxidase to horseradish peroxidase compound I
according to the method of Bakovic and Dunford (26).
Nitration of PGHS-1 with Tetranitromethane--
Nitration of
PGHS-1 with tetranitromethane was carried out in 80 mM
Tris, pH 8.0, 0.1% Tween 20. Thus, no buffer change was necessary. The
reaction of tetranitromethane with PGHS-1 or PGHS/indomethacin was
carried out by adding tetranitromethane (1 mM final
concentration, 1.7% ethanol) to 38 µM enzyme and
incubating at room temperature for 5 min. Following reaction, PGHS-1
was separated from unreacted tetranitromethane and other small
molecules by passing the mixture through a 10DG gel filtration column
(Bio-Rad) as described above.
Detection of Nitrotyrosine by Western Blot Analysis--
Frozen
aliquots (20 µl) were thawed quickly and heated with Laemmli reducing
sample buffer at 95 °C for 3 min. Samples (1.25 µg) were run on
10% denaturing polyacrylamide gel electrophoresis for 45 min at 160 V
then transferred onto nitrocellulose membranes for 2 h at 70 V. The blot was probed overnight with 2 µg/ml rabbit antinitrotyrosine,
polyclonal IgG (Upstate Biotechnology) and 1 h with donkey
anti-rabbit horseradish peroxidase (Amersham Pharmacia Biotech) as the
secondary antibody. Detection was carried out for 10 s with the
ECL Western blotting system (Amersham).
Tryptic Digestion of PGHS-1 for Peptide Mapping--
Following
the desalting procedure, PGHS-1 prepared under a variety of reaction
conditions was proteolytically cleaved using TPCK-treated trypsin. To
obtain predominantly peptide a (a nitrated PGHS peptide with
~75 min retention time), a PGHS-1 to trypsin ratio of 50:1 (w/w) was
used. The digestion was allowed to incubate for 18 h at 37 °C.
To obtain predominantly peptide b (a longer nitrated PGHS
peptide with ~81 min retention time), a PGHS-1 to trypsin ratio of
10:1 (w/w) was used with an incubation of 1.8 h at 37 °C.
Digestion was stopped by the addition of 20 µl of glacial acetic acid
per ml sample. Each sample was then centrifuged for 5 min, and the
resulting peptides were separated by HPLC.
Separation of PGHS-1 Peptides by HPLC--
PGHS-1 peptides were
separated by reversed-phase HPLC using a Zorbax SBC-18 reversed-phase
column (3.0 × 250-mm) (MAC-MOD Analytical, Inc., Chadds Ford, PA)
at 0.4 ml/min. Buffer A was water, 0.1% trifluoroacetic acid, and
buffer B was 80% acetonitrile, 0.1% trifluoroacetic acid. Peptides
were separated using the following nonlinear gradient: 0-50% buffer
B, 0-75 min; 50-75% buffer B 75-100 min; 75-100% buffer B,
100-115 min. Elution of peptides was monitored using a diode array
detector. Those peptides containing nitrotyrosine were identified by
the unique absorption spectrum of nitrotyrosine at acidic pH
(
max = 360 nm). Peptides containing nitrotyrosine were
collected and further purified by HPLC as described below.
Nitrotyrosine-containing peptides were further purified by HPLC using a
120-min linear gradient from 40-50% buffer B for peptide
a, and 44-54% buffer B for peptide b.
Separation was accomplished using the Zorbax SB-C18 reversed-phase
column (3.0 × 250-mm) at 0.4 ml/min. The nitrotyrosine-containing
peptides (as determined by diode array detection) were collected, and
the volume was reduced to 50 µl by lyophilization. These peptides were then submitted for amino acid sequence analysis.
Peptide Sequencing--
All peptide sequencing was carried out
by Edman degradation in the Center in Molecular Toxicology Protein
Sequencing and Amino Acid Analysis Core Facility. Sequencing was
carried out using a Beckman/Porton LF 3400t N-terminal
protein sequencer, and HPLC separation of phenylthiohydantoin amino
acid derivatives was accomplished using a Beckman System Gold apparatus
equipped with a Beckman 168 diode array detector.
 |
RESULTS |
The reaction of PGHS-1 with arachidonic acid resulted in the
formation of a tyrosyl radical as detected by EPR spectroscopy (Fig.
1A). The detected radical has
a line width of 26 G, corresponding to the narrow singlet spectrum
previously reported (27). The radical intermediate persisted for over 1 min at either
12 or 0 °C (data not shown). No tyrosyl radical was
detected when the reaction was carried out in the presence of 1 mM proli/NO (Fig. 1B). Similarly, no tyrosyl
radical was detected prior to the addition of arachidonic acid (Fig.
1C) These data are consistent with results obtained
previously with ribonucleotide reductase (17) and photosystem II (18,
19) in which protein tyrosyl radicals reacted with NO to form
nitrosocyclohexadienone and/or nitrite esters. These results are also
consistent with those recently obtained by Gunther et al.
(23) using PGHS-2. However, unlike the reaction of PGHS-2 with
arachidonic acid in the presence of NO, no iminoxyl radical intermediate was detected when PGHS-1 was used in place of PGHS-2. Thus, no evidence was provided by EPR to suggest the subsequent oxidation of nitrosocyclohexadienone to iminoxyl radical.

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Fig. 1.
Effect of NO on EPR spectra collected during
the reaction between PGHS-1 and arachidonic acid. Instrument
settings were those described under "Experimental Procedures."
Spectrum A, PGHS-1 after the addition of arachidonic acid.
Spectrum B, PGHS-1 after the additions of proli/NO and
arachidonic acid. Proli/NO was used due to its extremely short
half-life to attempt to detect the iminoxyl radical as was done for
PGHS-2 (23). Identical results were obtained when DEA/NO was used in
place of proli/NO (data not shown). Spectrum C, PGHS-1
before the addition of arachidonic acid.
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To determine whether nitrotyrosine was formed as a result of the
reaction of PGHS-1 with arachidonic acid in the presence of NO, we
utilized antinitrotyrosine antibodies in Western blot analysis to probe
for nitration of PGHS-1 (Fig. 2).
Antinitrotyrosine antibody was observed to bind PGHS following its
reaction with arachidonic acid in the presence of NO (lane
3). Both arachidonic acid and NO were required to detect
nitrotyrosine (lanes 1 and 2). Superoxide
dismutase did not inhibit nitration (lane 4), indicating that peroxynitrite was not the species responsible for the nitration of
tyrosine. Much higher concentrations of superoxide dismutase (800 µM/62,500 units/ml) inhibited nitration to a certain
extent, but this was due to a nonspecific effect of elevated protein
concentration. A similar extent of inhibition was observed with an
equivalent concentration of glutathione transferase in the absence of
glutathione. Turnover of PGHS was also required to observe nitration of
the protein. When PGHS-1 was treated with the inhibitor indomethacin before the addition of DEA/NO and arachidonic acid, no tyrosine nitration was observed (lane 5). The order of addition of
arachidonic acid and NO was also important. If arachidonic acid was
added 3 min before DEA/NO, no nitration was observed (data not shown). The blot shown in Fig. 2 was also probed with anti-PGHS-1 antibody. As
expected, PGHS was detected in all 5 lanes at equal concentrations (data not shown).

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Fig. 2.
Western blot analysis of PGHS-1 reacted with
arachidonic acid in the presence of NO. Blots were reacted with
rabbit antinitrotyrosine IgG. PGHS-1 was reacted with 150 nmol of
arachidonic acid alone (lane 1), 500 nmol DEA/NO alone
(lane 2), arachidonic acid and DEA/NO (lane 3),
arachidonic acid, DEA/NO, and 3.2 nmol of superoxide dismutase (~250
units) (lane 4), or arachidonic acid, DEA/NO, and 15 nmol of
indomethacin (lane 5). Details regarding sample preparation
are given under "Experimental Procedures."
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To obtain more information regarding the number and identity of PGHS
tyrosines nitrated by arachidonic acid/NO, we turned to peptide mapping
using the unique absorption characteristics of nitrotyrosine as a
marker for modified peptides. Tryptic digestion of PGHS-1 prepared by
reaction with arachidonic acid/NO produced many peptides as evidenced
by the HPLC elution profile observed at 230 nm (Fig.
3B). Relatively few peaks were
detected at 360 nm (Fig. 3A). Moreover, many of the peaks
detected at 360 nm did not have spectra resembling nitrotyrosine. Only
two peptides (peptide a and peptide b in Fig.
3A) had spectra consistent with nitrotyrosine at acidic pH.
A typical spectrum of peptide a is shown in Fig. 3
(inset). Peptide a was consistently observed
under these reaction conditions. Peptide b on the other hand
was not consistently detected using these trypsin digestion conditions.
The other peptides with absorbance at 360 nm, particularly those
eluting after peak b, did not have an absorption maximum at
360 nm, but instead showed a maximum at 400 nm. These data suggested
that nitration of PGHS-1 by arachidonic acid/NO occurred predominantly
on a single peptide, peptide a.

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Fig. 3.
HPLC elution profile of PGHS-1 digested with
trypsin following reaction with arachidonic acid in the presence of
NO. Peaks were observed using a diode array detector (200-600
nm). Shown are the elution profiles recorded at 360 nm (A)
and 230 nm (B). The two peaks detected at 360 nm with
spectra consistent with that of nitrotyrosine are indicated as
a and b. A typical spectrum for peak a
is shown in the inset. The conditions for preparation of
nitrated PGHS-1 and trypsin digestion are described under
"Experimental Procedures."
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Similar results were obtained when arachidonic acid was replaced by the
organic hydroperoxide CPBA (data not shown). Other modifications of the
arachidonic acid/NO protocol were also examined. For example, following
the addition of arachidonic acid, another 500 nmol of DEA/NO was added
followed by an additional 20 eq of arachidonic acid added in two
aliquots. In another protocol, phenol (10 enzyme eq) was present as a
reducing substrate during the multiple additions of DEA/NO and
arachidonic acid. These slight modifications did not affect either the
location or extent of PGHS nitration. Consistent with the Western
blotting results, both arachidonic acid and NO were required to observe
the nitrotyrosine-containing peptide a (Fig.
4). In the absence of DEA/NO, no
nitrotyrosine was observed (Fig. 4B). Similarly, when
arachidonic acid was withheld from the reaction very little of this
nitrated peptide was detected (Fig. 4C). It is also
important to point out that reversing the order of addition
(i.e. arachidonic acid added 3 min before DEA/NO) prevented
the appearance of peptide a (data not shown). We observed
that in the absence of a peroxidase-reducing substrate, the added
amount of arachidonic acid (10 eq) was sufficient to cause irreversible
inactivation of PGHS-1. These data suggest that irreversibly
inactivated protein is unable to carry out the nitration reaction.

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Fig. 4.
Effect of arachidonic acid and NO on
nitration of PGHS-1 peptide a. Shown are the elution profiles
(60-82 min.) recorded at 360 nm for PGHS-1 digested with trypsin
following reaction with 150 nmol of arachidonic acid and 500 nmol of
DEA/NO (A), 150 nmol of arachidonic acid alone
(B), or 500 nmol of DEA/NO alone (C). Nitration
of PGHS-1 was accomplished according to the procedure described under
"Experimental Procedures."
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It has been shown previously that nitration of three PGHS tyrosines by
tetranitromethane (Tyr-355, Tyr-385, and Tyr-417) can be blocked by
indomethacin (13). Our results with Western blotting showed that
tyrosine nitration of PGHS with arachidonic acid and NO was also
sensitive to inhibition by indomethacin. Thus, we determined that
indomethacin may provide valuable insight to the identity of the
nitrated tyrosine observed in peptide a (see Figs.
3A and 4A). We observed that the reaction of PGHS
with arachidonic acid/NO or tetranitromethane resulted in nitration of
what appeared to be the same peptide (Fig.
5, A and C).
Indeed, when either tetranitromethane or arachidonic acid/NO were used as nitrating agents, indomethacin was able to block the formation of
nitrated peptide a (Fig. 5, B and D).
To confirm that peak a observed in Fig. 5, A and
C, corresponded to the same peptide, the peptide was
collected from each separation. These fractions were then
rechromatographed separately as described under "Experimental
Procedures." In each case a single peptide with the nitrotyrosine
spectrum was observed confirming that in each case one nitrated peptide
was produced and collected. The nitrated peptide from each
chromatographic reseparation was collected. These peptides were then
pooled and rechromatographed using the conditions described under
"Experimental Procedures." A single peak was observed at 360 nm
confirming that the same PGHS-1 peptide was nitrated using either
tetranitromethane or arachidonic acid/NO. The ability of indomethacin
to block nitration of this peptide by either tetranitromethane or
arachidonic acid/NO suggested that tyrosine 355, 385, or 417 was
nitrated during PGHS reaction with arachidonic acid in the presence of
NO.

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Fig. 5.
Effect of indomethacin on nitration of PGHS
peptide a by tetranitromethane or arachidonic acid/NO. PGHS-1,
prepared as indicated below, was digested with trypsin and separated by
HPLC as described under "Experimental Procedures." Shown are the
elution profiles (60-82 min) observed at 360 nm. Profile A
was obtained by reacting PGHS with 150 nmol of arachidonic acid and 500 nmol of DEA/NO. Profile B was obtained as in profile
A except that PGHS was pretreated with 15 nmol of indomethacin.
Profile C was obtained by reacting PGHS with 1 mM tetranitromethane for 5 min, and profile D
was obtained as in profile C except that PGHS-1 was
pretreated with 15 nmol of indomethacin.
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We also used peptide sequencing to determine the identity of the
tyrosine nitrated during PGHS-catalyzed arachidonic acid oxidation in
the presence of NO. Early attempts at sequencing were performed on
peptides that were not sufficiently purified by HPLC. Consequently, two
peptide sequences were obtained. The first started at tyrosine 254 (YQMLNGEVYPPSVEEA), and appeared to be an unnitrated contaminant.
Indeed, further purification of peptide a eliminated the
appearance of this sequence. The second peptide sequence (MEFNQL) was
of greater interest. This second sequence started with methionine 379 and appeared to end with leucine 384. The next amino acid in the
sequence should have been tyrosine 385; however, no tyrosine was
detected in cycle 7. Instead, a peak that did not correspond to any of
the standard 20 amino acids was detected eluting just after
diphenylthiourea and just before the phenylthiohydantoin derivative of
tryptophan. According to Smith and co-workers (13), the
phenylthiohydantoin derivative of nitrotyrosine should elute at this
point in the profile of derivatized amino acids. This suggested that
the tyrosine labeled during arachidonic acid oxidation in the presence
of NO was tyrosine 385. We also sequenced the nitrated peptide obtained by using the hydroperoxide CPBA in place of arachidonic acid. The same
sequence results were obtained.
As indicated in Fig. 3, two peptides containing nitrotyrosine
were detected, peptides a and b. This suggested the possibility that more than one tyrosine was nitrated during arachidonic acid oxidation in the presence of NO. Therefore, it was
important to determine the identity of peptide b; however, peptide b was not consistently observed. By modifying the trypsin digestion protocol (5-fold higher trypsin concentration and
10-fold shorter digestion time), we detected two peaks containing nitrotyrosine (Fig. 6). The first peak
appeared to correspond to peptide a observed in Figs.
3A, 4A, and 5, A and C. The
second peptide had a longer retention time (about 81.5 min) and
appeared to correspond to peptide b shown in Fig.
3A. Unlike results obtained with the longer digest protocol,
peptide b was consistently observed under these tryptic
digest conditions, and roughly twice as much peptide b was
produced as peptide a.

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Fig. 6.
Production of peptide b using a modified
trypsin digestion protocol. Shown is the elution profile (60-82
min) at 360 nm. Peaks that correspond to peptides with spectra
consistent with nitrotyrosine are labeled as a and
b and correspond to peptides a and b
in Fig. 3A. The inset shows the spectrum
corresponding to peak b. Nitration of PGHS-1 was carried out
as described for Fig. 4. The trypsin digest protocol was modified by
reducing the digest time from 18 h to 1.8 h and increasing
the trypsin concentration by 5-fold.
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Peptide b was collected and purified as described under
"Experimental Procedures" and sequenced. Surprisingly the sequence for peptide b also started with Met-379, and a sequence of
MEFNQLYnitHWH (where Ynit is nitrated tyrosine) was
obtained for the first 10 amino acids of peptide b. As
observed for peptide a, cycle 7 showed no tyrosine as is
predicted by the amino acid sequence. Instead, a peak was detected
following diphenylthiourea (Fig. 7,
panel A). This is the predicted elution time for derivatized nitrotyrosine. Indeed, the spectrum corresponding to this peak was
consistent with that of nitrotyrosine (Fig. 7, panel C).
This spectrum was only detected for this peak. Spectra collected for diphenylthiourea (cycle 7) and derivatized histidine (cycle 8) do not
indicate an absorption maximum at 360 nm. Furthermore, a spectrum
collected at the expected retention time for nitrotyrosine in cycle 8 indicates that very little nitrotyrosine was detected after cycle 7. These data confirm that tyrosine 385 of PGHS-1 is nitrated during the
oxidation of arachidonic acid in the presence of NO.

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Fig. 7.
Peptide sequencing results (cycles 7 and 8)
for peptide b. The HPLC elution profile for cycle 7 as observed at
268 nm is shown in panel A, and the profile for cycle 8 is
shown in panel B. Panel C shows the spectra
corresponding to the peaks indicated in panels A and
B. The phenylthiohydantoin derivatives of nitrotyrosine and
histidine are indicated by nit-Y and H,
respectively. Diphenylthiourea is shown as dptu, and the
expected retention time for the phenylthiohydantoin derivative of
tyrosine is indicated by Y.
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 |
DISCUSSION |
Ruf and co-workers (11) proposed 10 years ago that a tyrosyl
radical was required for prostaglandin synthesis by PGHS, and since
that time, Smith and others (13, 14) have shown that tyrosine 385 is
essential to the cyclooxygenase activity of PGHS. In light of the ideal
position of tyrosine 385 as shown by PGHS crystal structures, many have
believed that tyrosine 385 must be the source of the tyrosyl radical
observed by EPR and the radical responsible for hydrogen abstraction
from arachidonic acid. However, the use of site-directed mutagenesis in
conjunction with EPR has failed to produce clear data to support this
proposal.
Consistent with other tyrosyl radical-containing proteins, the PGHS-1
tyrosyl radical detected during arachidonic acid oxidation is quenched
by NO. As with these other proteins, a likely intermediate in the
reaction is nitrosocyclohexadienone or a nitrite ester. Our Western
blotting and peptide mapping studies both indicate that the
nitrosocyclohexadienone adduct is oxidized to form nitrotyrosine. Furthermore, peptide mapping and subsequent amino acid sequence analysis showed that only one tyrosine, tyrosine 385, is nitrated during arachidonic acid oxidation in the presence of NO. Taken together
these data provide the first direct evidence to show that tyrosine 385 is oxidized to a free radical intermediate during PGHS catalysis.
An alternative mechanism to explain the nitration of PGHS-1 in the
presence of NO is coupling to a tyrosyl radical to nitrogen dioxide,
(·NO2) the product of NO oxidation by molecular
oxygen. This reaction would produce nitrotyrosine directly without the
intermediacy of a nitrosocyclohexadienone intermediate.
Although NO appears to trap all of the spectroscopically detectable
tyrosyl radicals as evidenced by their disappearance from the EPR
spectrum, recent investigations have shown that NO does not appear to
significantly inhibit the rate of arachidonic acid oxygenation by PGHS
(28, 29). This suggests that under the conditions employed in the
present experiments, NO does not compete effectively with arachidonic
acid for the tyrosine 385 radical. Thus, many molecules of arachidonic
acid are oxidized on each enzyme molecule before the catalytically
active tyrosine 385 radical is trapped by NO. Recent investigations
into the effect of NO on arachidonic acid oxidation by PGHS would seem
to support such a proposal. It is important to recognize that NO is an
efficient reducing substrate for the PGHS peroxidase (29) and as such should greatly stimulate cyclooxygenase activity (2). Contrary to this
expectation, NO (added as DEA/NO) has little effect on the initial rate
and inhibits by one-third the extent of arachidonic acid oxygenation
when compared with a reaction that contains no peroxidase-reducing
substrate (29). Moreover, when compared with a reaction containing the
peroxidase-reducing substrate aminopyrine, NO inhibits the extent of
arachidonic acid oxygenation by ~65% (29). It is possible that the
unique inability of NO to stimulate arachidonic acid oxidation
commensurate with its efficiency as a reducing substrate stems from its
role in nitration of tyrosine 385.
Considerable controversy has surrounded the question of whether the
tyrosyl radicals detected spectroscopically during PGHS turnover
represent catalytic intermediates or inactivated enzyme (14, 15,
30-32). Our finding that NO quenches the tyrosyl radical(s) and
produces nitrotyrosine at tyrosine 385 is consistent with the
hypothesis that the EPR signal detected during turnover arises from a
catalytic intermediate. However, this interpretation must be considered
tentative. Our identification of tyrosine 385 as a trapped radical
requires that its tyrosyl radical react with NO and that the
nitrosocyclohexadienone intermediate be further oxidized to
nitrotyrosine. The coupling of NO to protein tyrosyl radicals appears
to be completely reversible. Therefore, if the nitrosocyclohexadienone-trapping product from a particular tyrosine was
not further oxidized to nitrotyrosine, it would decompose to tyrosine
and NO and not be detected by peptide mapping (Reactions R5 and
R6).
Further, any nitrite ester trapping product would not oxidize to
nitrotyrosine and would not be detected. Thus, although the present
study establishes that tyrosine 385 is oxidized to a radical intermediate during arachidonic acid oxygenation, it does not answer
the question of whether the radical signals detected by EPR
spectroscopy represent catalytic intermediates or inactivated enzyme.
In summary, the nitration of PGHS tyrosine 385 by reaction with
arachidonic acid or organic hydroperoxide in the presence of NO appears
to result from the reaction of NO with a tyrosyl radical. This provides
solid evidence that tyrosine 385 is oxidized to a free radical
intermediate during arachidonic acid oxidation. This is consistent with
the proposed requirement for a tyrosyl radical in the cyclooxygenase
catalytic cycle and previous investigations that have shown tyrosine
385 to be essential to the cyclooxygenase activity of PGHS. The
nitration of only one tyrosine is consistent with a role for the
tyrosyl radical of tyrosine 385 in cyclooxygenase catalysis but does
not prove that tyrosine 385 is the only tyrosine oxidized to a radical
intermediate during turnover of the wild-type PGHS. The detection of
tyrosyl radicals during the turnover of the PGHS mutant Y385F indicates
that other tyrosines can be oxidized in mutant and possibly wild-type
enzymes. It will be interesting to determine whether the methodology
developed in the present report is useful for the identification of
tyrosyl radicals generated from site-directed mutants of PGHS.
The authors are grateful to Eric Howard and
the Center in Molecular Toxicology Protein Sequencing and Amino Acid
Analysis Core Facility for performing the peptide sequencing.