A tyrosyl radical generated in the peroxidase
cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial
oxidant for arachidonic acid (AA) in the cyclooxygenase reaction.
Peroxides also induce radical formation in prostaglandin H synthase-2
(PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX
(MnPGHS-1), but the EPR spectra of these radicals are distinct from the
initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line
width of 29-31 gauss (G) was generated by reaction of PGHS-2 with
ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately
after formation of this radical led to its disappearance and emergence
of an AA radical (AA·) with a 7-line EPR, substantiated by
experiments using octadeuterated AA. Subsequent addition of oxygen
resulted in regeneration of the tyrosyl radical. In contrast, the
peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2
mutant lacking cyclooxygenase activity failed to react with AA. The
peroxide-generated radical in MnPGHS-1 exhibited a line width of
36-38G, but was also able to convert AA to an AA· with an EPR
spectrum similar to that found with PGHS-2. These results indicate that
the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve
as immediate oxidants of AA to form the same carbon-centered fatty acid
radical that subsequently reacts with oxygen to form a hydroperoxide.
The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1
could be accounted for by a planar pentadienyl radical with two
strongly interacting
-protons at C10 of AA. These results support a
functional role for peroxide-generated radicals in cyclooxygenase
catalysis by both PGHS isoforms and provide important structural
characterization of the carbon-centered AA·.
 |
INTRODUCTION |
Prostaglandin H synthase
(PGHS)1 plays a key role in
controlling the biosynthesis of various physiologically important
prostaglandins (1, 2). Although two distinct PGHS isozymes, PGHS-1 and PGHS-2, have been discovered, they are both hemoproteins, and it is
assumed that they share the same basic catalytic mechanism (1, 3). Both
PGHS isozymes have two enzyme activities: a cyclooxygenase activity
that converts arachidonic acid to PGG2 and a peroxidase
activity that catalyzes the transformation of PGG2 to
PGH2 (1-4). A branched-chain radical mechanism has been proposed to integrate the two catalytic activities (5, 6). In this
proposal (Scheme 1), a tyrosyl radical
(Fe(IV)Tyr·) is generated by an internal electron transfer in
peroxidase Compound I (Fe(V)), shown as Step 2. The
cyclooxygenase reaction itself begins when the tyrosyl radical reacts
with bound arachidonate (AA) to form a fatty acid radical
(Fe(IV)Tyr/AA·), (Step 3). The bound fatty acid radical
then reacts with molecular oxygen and rearranges to form a
PGG2 radical (Step 4). In the final cyclooxygenase step,
the tyrosyl radical is regenerated and PGG2 is released (Step 5).
The tyrosyl radical mechanism nicely explains the heme dependence
of both enzyme activities and the requirement of the cyclooxygenase for
a hydroperoxide activator (3, 7). It also is consistent with x-ray
crystallographic data that show a tyrosine residue (Tyr-385 in PGHS-1
and Tyr-371 in PGHS-2) positioned between the heme and the arachidonic
acid binding channel (8-10). The peroxidase-associated tyrosyl radical
in PGHS-1 was shown to react with arachidonic acid to form a
carbon-centered radical (11), providing direct evidence for a key step
in the proposed tyrosyl radical mechanism (Scheme 1, Step
3). Peroxides induce a tyrosyl radical in PGHS-2, but its EPR
characteristics, a wide-singlet with 29-30 gauss line width, are
distinct from those of the tyrosyl radical species in PGHS-1 (12, 13).
Replacement of heme in PGHS-1 by mangano protoporphyrin IX gives a
holoenzyme (MnPGHS-1) that retains most cyclooxygenase activity but
only ~4% of the peroxidase activity (14-16). Previous studies
demonstrated that a 35-38 G radical was produced in MnPGHS-1 during
aerobic reactions with either fatty acid peroxide, 15-HPETE, or AA (16,
17). The MnPGHS-1 radical structure was sensitive to the tyrosine
modifying agent, tetranitromethane, and the kinetics of its formation
appeared to correlate with production of
PGG2/PGH2 (16). However, because of the EPR
spectral differences with the tyrosyl radical in FePGHS-1, the roles of
the peroxide-induced radicals in PGHS-2 and MnPGHS-1 in cyclooxygenase
catalysis remained unclear. We have therefore tested the ability of
these radicals to oxidize arachidonate to a fatty acid radical, a key
step in the branched-chain mechanism in Scheme 1. The results indicate that very similar arachidonyl free radicals are formed when the fatty
acid is reacted with the PGHS-2 and MnPGHS-1 protein radicals. Analysis
of the EPR spectrum of the arachidonyl radicals provides insight into
the structure of the enzyme-bound fatty acid radicals in both PGHS
isoforms.
 |
MATERIALS AND METHODS |
AA was purchased from NuChek Preps, Inc., Elysian, MN;
5,6,8,9,11,12,14,15-octadeuterated arachidonic acid (d8-AA)
was a generous gift of Hoffman-La Roche, Nutley, NJ. Polar contaminants
were not detected in either deuterated or unlabeled AA when analyzed by
silica gel thin layer chromatography with hexane/diethyl ether/acetic acid (60:60:1) as the solvent. Mass spectral analysis of the methyl ester of the deuterated arachidonate indicated a composition of 76%
d8 and 24% d7. The d8-AA was not
purified further. Hemin was obtained from Sigma. Ethyl hydroperoxide
(EtOOH) was the product of Polyscience Inc., Warrington, PA, and
15-HPETE was synthesized according to Graff et al. (18).
PGHS-1 was purified from sheep seminal vesicles (19) and PGHS-2
was purified from a baculovirus overexpression system (20). The
holoenzymes were prepared by replenishing with heme or mangano protoporphyrin IX as described previously (16). Cyclooxygenase activity
was calculated from the oxygen consumption rate (19). The batches of
PGHS-1 used in this study had specific activities of 55-80 µmol of
O2/min/mg of protein; those of PGHS-2 had specific activities of 27-42 µmol of O2/min/mg. The Y371F mutant
of PGHS-2 was expressed in a baculovirus system and purified as the
apoenzyme using procedures developed for wild-type PGHS-2 (21). The
Y371 holoenzyme was prepared by the same heme reconstitution procedure used for PGHS-1; the single turnover experimental procedures have also
been described (11, 22).
EPR spectra were recorded at liquid nitrogen temperatures on a Varian
E-6 spectrometer (11). The EPR conditions were: modulation amplitude, 2 G; time constant, 1 s; power, 1 mW; and temperature, 96 K. Radical
concentrations were determined by double integration of the EPR
signals, with reference to a copper standard (11). Computer simulations
of EPR spectra were done on a PC using a modified version of the POWFUN
program (23), kindly provided by Drs. Gerald T. Babcock and Curt
Hoganson, Michigan State University.
 |
RESULTS |
Serial Reaction of PGHS-2 with Hydroperoxide and
Arachidonate--
When PGHS-2 was reacted anaerobically with 1.5 eq of
EtOOH, a 29 G wide singlet (denoted WS2) amounting to about 0.1 spin/heme was routinely detected by EPR (Fig.
1, spectrum a). Similar yields of radical have been found under these conditions with several batches
of PGHS-2; the yield increased to about 0.3 spin/heme with 5 eq of
EtOOH (data not shown). Subsequent anaerobic addition of 1.5 eq of AA
led to the replacement of the WS2 radical with a new radical exhibiting
a 7-line EPR centered at g = 2.0022, with a hyperfine
splitting of 13.5-14 G and a slightly lower intensity (Fig. 1,
spectrum b). Another sample freeze-trapped a minute later exhibited a very similar EPR line shape but with reduced amplitude (Fig. 1, spectrum c). These three samples subsequently were
thawed and mixed with air. The EPR intensity of the WS2 signal in
PGHS-2 reacted only with EtOOH decreased during aerobic incubation
(Fig. 1, spectrum a'). The two samples of PGHS-2 reacted
with EtOOH and AA both reformed the original WS2 tyrosyl radical and
recovered significant intensity. This regeneration of the
hydroperoxide-dependent tyrosyl radical upon aerobic
incubation indicates that the radical formed after AA addition itself
reacted with oxygen to form fresh hydroperoxide, as is expected from
the mechanism (Scheme 1), where the arachidonate radical reacts with
oxygen to form the hydroperoxide, PGG2, and regenerate the
tyrosyl radical (Steps 4 and 5).

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Fig. 1.
EPR spectra during reaction of the
peroxide-induced PGHS-2 radical with AA. Left panel, PGHS-2
holoenzyme (13 µM heme) in 0.1 M potassium
Pi, pH 7.2, containing 0.1% Tween 20 and 10% glycerol was
incubated on ice under anaerobic conditions with 1.5 eq of EtOOH for
23 s before addition of AA (1.5 eq). EPR spectra were recorded
with samples taken 13 s after addition of EtOOH (a),
and 7 s (b) or 68 s (c) after the
subsequent AA addition. Right panel, a second set of EPR
spectra (a'-c') was obtained after the same three
samples used for the spectra in the left panel were thawed
and incubated aerobically on ice for 30 s. The value at
the left side of each spectrum is the signal intensity obtained from double integration (in spins/heme). The heavy
dashed line in spectrum b is the simulation using
the parameters for PGHS-2 and AA listed in Table I.
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The 7-line EPR observed for the putative AA· in the PGHS-2 reaction
(Fig. 1) is distinct from the isotropic AA· found earlier in PGHS-1
(11). To determine if this 7-line radical in the PGHS-2 reaction is
actually derived from the fatty acid, the single-turnover experiments
were repeated with d8-AA. As shown in Fig.
2, spectra b and c,
the radical formed upon addition of d8-AA displayed an EPR
centered at the same g value (2.0022) and had similar
hyperfine splitting (13.5-14 G) as the radical formed with the
unlabeled AA. The main difference is that the radical formed with
d8-AA has five hyperfine lines in the EPR spectrum instead
of the seven lines observed with unlabeled AA. A 5-line EPR pattern was
also observed with PGHS-1 when d8-AA was used as reactant
(11). Conversion from a 7-line to a 5-line EPR spectrum with the
deuterated fatty acid strongly supports the conclusion that these
radicals are derived from the arachidonate substrate. Thawing and
aerobic incubation of the samples with deuterated fatty acid radical
regenerated the WS2 tyrosyl radical (Fig. 2, spectra b' and
c'), consistent with formation of hydroperoxide by reaction
of the d8-AA fatty acid radical with oxygen.

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Fig. 2.
EPR spectra during reaction of the
peroxide-induced PGHS-2 radical with d8-AA. Left
panel, PGHS-2 holoenzyme (13 µM heme) in 0.1 M potassium Pi, pH 7.2, containing 0.1%
Tween-20 and 10% glycerol was incubated on ice under anaerobic
conditions with 1.5 eq of EtOOH for 25 s before addition of
d8-AA (1.5 eq). EPR spectra were obtained for samples taken
15 s after addition of EtOOH (a) and 10 (b)
or 44 s (c) after the subsequent d8-AA addition. Right panel, a second set of EPR spectra
(a'-c') was obtained after the same three samples
used for the spectra in the left panel had been thawed and
incubated aerobically on ice for 30 s. The value at the
left side of each spectrum is the signal intensity obtained
from double integration (in spins/heme). The heavy dashed line
is the simulation using parameters for PGHS-2 and
d8-AA given in Table I.
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Serial Reaction of Y371F PGHS-2 with EtOOH and AA--
Tyr-385 has
been shown to be the likely location of the wide doublet tyrosyl
radical in PGHS-1 (24). The effects of mutating the corresponding
residue in PGHS-2, Tyr-371, to phenylalanine were examined. The
cyclooxygenase activity of the Y371F mutant was undetectable, whereas
the peroxidase activity was 40-60 µmol of
H2O2/min/mg, in the range observed for the
wild-type activity (34-77 mmol of
H2O2/min/mg). Thus, the Y371F mutation in
PGHS-2 led to selective loss of cyclooxygenase activity, as was
observed with the Y385F mutant of PGHS-1 (25).
When Y371F PGHS-2 was reacted with EtOOH, a 21 G radical signal was
observed (Fig. 3, spectrum a).
This signal is very similar to those found with indomethacin-treated
PGHS-1, the Y385F mutant of PGHS-1 (24), and nimesulide-treated PGHS-2
(13), all of which have reduced or undetectable cyclooxygenase
activity. Anaerobic addition of AA to the peroxide-induced radical in
the Y371F mutant did not cause any EPR line shape change (Fig. 3,
spectrum b). Longer incubation with AA led only to the decay
of the 21 G signal (Fig. 3, spectrum c). Subsequent thawing
and aerobic incubation of the samples did not lead to any changes of
the EPR line shape (Fig. 3, spectra a'-c'). These
results showed that although mutation of Tyr-371 in PGHS-2 did not
prevent radical formation, the resulting radical was not able to
oxidize AA.

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Fig. 3.
EPR spectra during reaction of
peroxide-induced Y371F PGHS-2 radical with AA. Left panel,
Y371F PGHS-2 holoenzyme (7.8 µM heme) in 50 mM potassium Pi, pH 7.4, containing 0.02%
octyl glucoside and 10% glycerol was incubated on ice under anaerobic conditions with 1.5 eq of EtOOH for 31 s before addition of AA (1.5 eq). EPR spectra were obtained for samples taken 21 s after addition of EtOOH (a) and 21 (b) or 60 s
(c) after the subsequent AA addition. Right
panel, a second set of EPR spectra (a'-c') was obtained after the same three samples used for the spectra in the
left panel had been thawed and incubated aerobically for 15-20 s. The value at the left side of each
spectrum is the signal intensity obtained from double integration (in
spins/heme).
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Serial Reaction of MnPGHS-1 with 15-HPETE and AA--
A 35 G wide
doublet was generated upon reaction with 1.5 eq of 15-HPETE (Fig.
4, spectrum a). Subsequent
anaerobic addition of 1.5 eq. of AA converted this wide doublet to an
isotropic radical with about the same line width, centered at
g = 2.003 (Fig. 4, spectrum b). A sample
freeze-trapped after another 30 s of reaction with AA
(spectrum c in Fig. 4) displayed the same EPR spectral line
shape but only 50% of the intensity of that in spectrum b. These three
samples were subsequently thawed and incubated in air. Aerobic
incubation had little effect on the intensity or line shapes of
MnPGHS-1 incubated with 15-HPETE alone (spectrum a'). For the MnPGHS-1
samples incubated with 15-HPETE and AA, aerobic incubation led either
to subtle changes of EPR line shape (spectrum b') or a significant
increase in amplitude (spectrum c').

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Fig. 4.
EPR spectra during reaction of
peroxide-induced MnPGHS-1 radical with AA. Left panel,
MnPGHS-1 holoenzyme (25 µM heme) in 50 mM
potassium Pi, pH 7.4, containing 0.02% octyl glucoside and
10% glycerol was incubated on ice under anaerobic conditions with 1.5 eq of 15-HPETE for 21 s before addition of AA (1.5 eq). EPR
spectra were obtained for samples taken 13 s after addition of
15-HPETE (a) and 13 (b) or 42 s
(c) after the subsequent AA addition. Right
panel, a second set of EPR spectra (a'-c')
was obtained after the same three samples used for the spectra in the
left panel had been thawed and incubated aerobically on ice for 16 s. The value at the left side of each
spectrum is the signal intensity obtained from double integration (in
spins/heme). The heavy dashed line is the simulation
obtained using parameters for MnPGHS-1 and AA in Table I.
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To see if the isotropic MnPGHS-1 signals in spectra b and
c in Fig. 4 are derived from arachidonic acid, these
experiments were repeated with d8-AA in place of AA. The
resulting spectra are shown in Fig. 5.
The EPR signal from enzyme reacted with 15-HPETE and d8-AA
exhibited five hyperfine lines with splittings of 13-14 gauss and
centered at g = 2.003 (spectra b and
c, respectively). Thawing and oxygenation of the samples
reacted with 15-HPETE and d8-AA restored the 34-35 G
radical signal found in the initial reaction of MnPGHS-1 with peroxide
(Fig. 5, spectra b' and c'). This change in EPR
line shape, from an isotropic signal with unlabeled AA to a 5-line
pattern with d8-AA, confirmed that the radicals in
spectra b and c in Figs. 4 and 5 were derived
from the fatty acid substrate, similar to the previous observations
with FePGHS-1 (11). In all cases, the radical concentrations peaked at
about 0.1 spin/metalloporphyrin, as was previously observed for
reaction between 15-HPETE and MnPGHS-1 (16).

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Fig. 5.
EPR spectra during reaction of
peroxide-induced MnPGHS-1 radical with d8-AA. Left
panel, MnPGHS-1 holoenzyme (25 µM heme) in 50 mM potassium Pi, pH 7.4, containing 0.02%
octyl glucoside and 10% glycerol was incubated on ice under anaerobic
conditions with 1.5 eq of 15-HPETE for 19 s before addition of
d8-AA (1.5 eq). EPR spectra were obtained for samples taken
12 s after addition of 15-HPETE (a) and 11 (b) or 44 s (c) after the subsequent
d8-AA addition. Right panel, a second set of EPR
spectra (a'-c') was obtained after the same three
samples used for the spectra in the left panel had been
thawed and incubated aerobically on ice for 16 s. The
value at the left side of each spectrum is the
signal intensity obtained from double integration (in spins/heme). The heavy dashed line is the simulation generated using
parameters for MnPGHS-1 and d8-AA in Table I.
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Formation of Carbon-centered Arachidonate Radical under Aerobic
Conditions--
In a separate experiment, concentrated FePGHS-1 (150 µM heme) in air-saturated buffer was reacted with 25 eq
of AA. A radical with a 7-line spectrum centered at g = 2.002 was obtained with hyperfine splitting of 12-16 G and a
concentration of about 0.11 spin/heme (Fig.
6, top). The EPR lineshape of
this radical species is very similar to that of the AA carbon-centered
radical generated by PGHS-2 (Fig. 1, spectra b and
c). Under these experimental conditions, the dissolved
oxygen initially present in the FePGHS-1 reaction mixture (~300
µM) would be enough to sustain only one catalytic cycle
(2 mol of O2/mol of PGG2) for the concentrated enzyme used. With very limited oxygen replenishment by diffusion into
the unstirred solution in the narrow EPR tube, anaerobiosis was
achieved in situ, stopping the cyclooxygenase cycle at the stage of the fatty acid radical (Scheme 1) in this sample.

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Fig. 6.
EPR spectra of reaction between high
concentrations of PGHS-1 and AA. Top spectrum, FePGHS-1 (153 µM heme) in 50 mM potassium Pi,
pH 7.5, containing 0.04% octyl- -D-glucoside and 33%
glycerol was reacted with 25 eq of AA at 0 °C for 7 s before it
was freeze-trapped. EPR conditions were as described under "Materials
and Methods." Bottom spectrum (digitized from spectrum E
in Fig. 3 of Ref. 17), 50 µM MnPGHS-1 was incubated with
5 mM AA at 12 °C for 4 min. The spectrum was recorded
at 200 K with 2 mW power and 4 G modulation. Dashed lines
are the data and solid lines are the simulations. The
parameters used for simulation of both spectra are those for the PGHS-2
AA· (Table I) with a line width of 4.3 G (top spectrum) or
5 G (bottom spectrum).
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 |
DISCUSSION |
Oxidation of AA by Peroxide-induced PGHS Radicals--
Oxidation
of arachidonate to a fatty acid radical is a key step in the proposed
branched-chain mechanism for PGHS cyclooxygenase catalysis (Step
3 in Scheme 1). It has been demonstrated previously that the wide
doublet (33-35 G) tyrosyl radical generated in FePGHS-1 by peroxide
can oxidize AA and produce a carbon-centered AA· (11). The tyrosyl
radical induced by peroxide in PGHS-2 is a wide singlet (29-30 G),
quite distinct from the initial wide doublet tyrosyl radical in
FePGHS-1 (11-13); the peroxide-induced radical in MnPGHS-1 also has an
EPR spectrum distinct from the radical in FePGHS-1 (16). The results of
the present study demonstrate that the peroxide-induced radicals in
PGHS-2 and MnPGHS-1 are both able to oxidize AA to form an AA· (Figs.
1, 2, 4, and 5). This establishes that a peroxide-generated,
protein-linked radical is chemically competent to play a common
functional role as a redox mediator between the metalloporphyrin center
and the AA substrate in FePGHS-1, MnPGHS-1, and FePGHS-2. In this
study, prostaglandin formation is blocked by the lack of oxygen during
the anaerobic portion of the reactions. Subsequent incubation of the
AA· with air for several seconds leads to regeneration of the
original tyrosyl radical(s) as predicted by Scheme 1, consistent with
conversion of the AA· to PGG2. Formation of
prostaglandins during aerobic reaction of FePGHS-1 and MnPGHS-1
with arachidonate on a similar time scale has already been
demonstrated (16, 27).
Several lines of evidence have been previously cited (13) to suggest
that the wide singlet radical observed in PGHS-2 upon reaction with
EtOOH resides on Tyr-371 (corresponding to Tyr-385 in FePGHS-1).
Assignment of the PGHS-2 radical to Tyr-371 and the involvement of this
residue in cyclooxygenase catalysis are strengthened by the present
results with the Y371F mutant of PGHS-2. This mutant lacked
cyclooxygenase activity, demonstrating the requirement for a tyrosine
residue at position 371 of PGHS-2. The Y371F mutant retained peroxidase
catalytic competence, as expected from the proposed mechanism (Scheme
1). Although the Y371F mutant generated a narrow singlet radical when
reacted with peroxide, this radical did not oxidize AA (Fig. 3). The
21G narrow singlet radical in the PGHS-2 Y371F mutant has a line shape
very similar to those in nimesulide-treated PGHS-2 or PGHS-1 inhibited by aspirin or indomethacin, each of which has impaired cyclooxygenase activity and normal peroxidase activity (13, 28). The narrow singlet in
inhibitor-treated PGHS-1 has been proposed to reside on a tyrosine
residue other than Tyr385 (24). The similarities with the present
results suggest that the 21G narrow-singlet found in the PGHS-2 Y371F
mutant is a tyrosyl radical formed on a residue other than Tyr-371. The
narrow singlet tyrosyl radical induced in indomethacin-treated PGHS-1
by EtOOH was not able to oxidize AA (11), but this could be ascribed to
blockage of the cyclooxygenase site by indomethacin. The Y371F PGHS-2
mutant radical is also unable to oxidize AA (Fig. 3), but in this case,
no inhibitor is present in the cyclooxygenase channel. This establishes
clearly that AA is not oxidized by the Y371F mutant radical even though the fatty acid can freely access its binding site, making it likely that the radical in the Y371F mutant is some distance from the fatty
acid site or not properly positioned to interact with the fatty acid.
The residue bearing the radical has yet to be identified.
MnPGHS-1 has been found to resemble FePGHS-1 in overall peroxidase
mechanism and formation of high oxidation-state intermediates (29). In
MnPGHS-1, the radical is generated more efficiently by lipid
hydroperoxides than by EtOOH, as might be expected from the preference
of the MnPGHS-1 peroxidase activity for lipid substrates (16).
Accumulation of the radical correlates roughly with cyclooxygenase catalysis by MnPGHS-1 (16), just as for FePGHS-1 (27). For both
MnPGHS-1 and FePGHS-1, reaction with the tyrosine nitrating agent,
tetranitromethane, leads to formation of a peroxide-induced radical
with a narrower EPR spectrum and loss of cyclooxygenase activity (16,
26). The present results extend the parallels between MnPGHS-1 and
FePGHS-1, showing that the MnPGHS-1 radical is also able to oxidize
arachidonate to a fatty acid radical (Figs. 4 and 5). This implies that
the peroxide-generated radical in MnPGHS-1 is near the fatty acid
binding site. A simple interpretation is that the wide doublet radical
species in both MnPGHS-1 and FePGHS-1 are tyrosyl radicals residing on
Tyr-385. If this is the case, the EPR differences between the MnPGHS-1
and FePGHS-1 radicals must arise from the influence of the different
metal centers, situated about 10 Å away from Tyr-385 in the crystal structure (8). The effect of the metal on the reactivity of the radical
toward the fatty acid remains to be evaluated.
Structure of the Arachidonic Acid Carbon-centered
Radical--
Dramatic changes of EPR line shape, from an isotropic
singlet with unlabeled AA to a 5-line hyperfine pattern, have now been observed for both Fe- and MnPGHS-1 (Ref. 11, and Figs. 4 and 5). The
spectral changes with labeled AA are more subtle for PGHS-2 and
required careful examination of the spectra to conclude that a
transition from a 7- to a 5-line spectrum occurred when
d8-AA replaced AA (Figs. 1 and 2).
Computer simulation has been used to derive a consistent structural
interpretation for the several AA (or d8-AA) radicals observed, with their distinct EPR spectral features. The uniform 5-line
EPR signal found for d8-AA· (Figs. 2 and 5, and Ref. 11) for FePGHS-1, MnPGHS-1, and FePGHS-2 indicates that the carbon-centered radical is most likely a pentadienyl radical (delocalized on five carbons, C11-C15 in Fig. 7) rather than
an allyl radical (delocalized on three carbons, C11-C13 or C13-C15 in
Fig. 7). This is because the latter would give, at most, a 4-line
hyperfine pattern (when d8-AA was the substrate),
i.e. splitting from the C13 proton and two
-protons, at
either C10 or C16. The 7-line AA· EPR found for PGHS-2 reaction with
unlabeled AA indicates that six protons interact with the unpaired
electron delocalized over the C11-C15 pentadienyl structure (Fig. 7).
Ab initio calculation for model pentadienyl radicals indicates that the
electron densities at C12 and C14 are negligible compared with those at
C11, C13, and C15 (30). Therefore, in addition to the three strongly
interacting protons at C13, C11, and C15, there must be 3
-protons
at C10 and C16 which also strongly interact with the unpaired electron (31, 32). Acceptable simulations of the observed AA·EPR spectra were
indeed achieved using a pentadienyl radical model having six strongly
interacting protons, with three of them located at C10 and C16 (Table
I and Figs. 1, 4, 6, and 7). The optimal
values for the A tensors of the
-protons derived from these
simulations allowed calculation of the dihedral angle,
, between the
pz orbital at C11 or C15 with the C10-H or
C16-H bond containing the
-protons in question, using the
conventional McConnell relationship (33) as follows,
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(Eq. 1)
|
where AH, in gauss, is the coupling
constant due to hyperconjugation,
is the unpaired electron density,
and B0 and B1 are constants (assigned values of 4 and 50 for a hydrocarbon-free radical
system). The ratio of the A tensor value measured for the endo proton
of a model pentadienyl radical (9.6 G) to that of an isolated radical
found for a methyl proton in the ethyl radical model (23 G), can be
used to estimate a
value of 0.42 (33). Taken with the PGHS-2 and
MnPGHS-1 EPR data (Table I), two sets of
proton dihedral angles can
be calculated: 32° and 152° for the carbon atom where both protons
interact strongly with the adjacent unpaired electron, and 51° and
71° for the carbon with only one strongly interacting proton. To
decide which set of dihedral angles is associated with
protons on
C10 and which is associated with those on C16, we considered the
geometric conformation of the AA· in the cyclooxygenase site and its
subsequent chemical conversion to PGG2, involving
endoperoxide formation across C9 and C11 and ring closure between C8
and C12. We favor assignment of the 32°/152° pair to C10 and the
51°/
71° pair to C16 because this arrangement places C9 closer to
C11 for endoperoxide formation and also brings C8 closer to C12 for
subsequent ring closure (Fig. 7). These assignments fix the structure
from C9-C17 of the carbon-centered AA· formed in the first step of
cyclooxygenase reaction in PGHS-2 and MnPGHS-1.

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Fig. 7.
Proposed conformation of the carbon-centered,
planar, pentadienyl radical generated by abstraction of the 13-pro(S)
hydrogen from AA. The dihedral angles ( a and
b) between the pz orbitals at C11
or C15 and the protons at C10 or C16 are deduced from the EPR data
(Figs. 1, 2, and 4-6) and calculated using Equation 1 as described
under "Discussion." The double-headed arrow indicates the position of bond formation between C8 and C12 in the product, PGG2.
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Table I
Parameters used to simulate EPR spectra of AA radicals observed in
reactions with unlabeled and deuterated arachidonate
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The values of the parameters which produced optimal EPR simulations for
the AA· EPR of PGHS-2 and MnPGHS-1 are very similar (Table I). The
main difference is a 2 G smaller coupling constant for the C13 proton
for the MnPGHS-1 EPR. The EPR line shape is a sensitive function of the
coupling constants used in the simulation. For example, the change of
A(H13) from 9.5 to 11.5 G and of line width from 5 to 4 G resulted in a
dramatic change of the predicted EPR appearance from a broad isotropic
singlet for MnPGHS to a 7-line hyperfine pattern for PGHS-2 (compare
Fig. 1, spectrum b and Fig. 4, spectrum b).
Obviously, the experimental resolution of hyperfine features is
dependent on many factors such as the inherent line width, temperature,
sample homogeneity, etc. Using d8-AA significantly enhanced
the resolution as the line broadening effects of protons at C11 and C15
were removed from the spectra, leading to a consistent 5-line hyperfine
EPR spectrum originating from interactions of the unpaired electron and
only the four protons at C10, C13, and C16.
In our earlier provisional analysis (11), a 5-proton system was used to
simulate the FePGHS-1 AA· EPR and a 3-proton system for the
d8-AA· EPR, assuming that only one
proton each on C10 and C16 was interacting strongly with the unpaired electron at C11 and
C15. These earlier simulations also did not attempt to use a consistent
set of A tensor values for AA· and d8-AA·, and thus
provided limited information about the fatty acid radical structure.
The current EPR spectra for the AA· generated by PGHS-2, MnPGHS-1,
and concentrated FePGHS-1 at high AA are clearly better interpreted as
originating from a pentadienyl radical with not one but two strongly
interacting
-protons at C10 (Figs. 1, 2, 4-6). We therefore have
reanalyzed the earlier PGHS-1 AA· EPR spectra (11) in light of the
new data. However, the narrow line width of the PGHS-1 AA· EPR, 23 G,
did not permit a satisfactory fit to the EPR data for both AA and
d8-AA radicals with the individual basic parameter sets
tested. Possible explanations for this difference in EPR lineshape are
discussed below.
It is important to note that the radical formed under oxygen-limiting
conditions at high concentrations of enzyme and arachidonate exhibited
a typical 7-line EPR very similar to that found for the AA· in PGHS-2
(Figs. 1 and 6). This 7-line EPR found for concentrated PGHS-1 is
easily simulated using the same parameter values used to simulate the
PGHS-2 AA·, with only a slight increase in line width (4.3 G instead
of 4 G; Table I). Interestingly, a similar radical EPR spectrum was
obtained earlier by Lassmann et al. (17) for concentrated
MnPGHS-1 reacted with a high concentration of AA (Fig. 6) This 7-line
EPR with MnPGHS-1 is also simulated easily by the same set of
parameters used for PGHS-2, with a line width of 5 G (Table I). These
results help to resolve one of the remaining puzzles about the identity
of the radical generated using high enzyme and substrate
concentrations: why was an EPR similar to that found for the AA·
observed instead of a tyrosyl radical? We propose that the oxygen
dissolved in the reaction mixture was exhausted in a very few turnovers
when high concentrations of enzyme and fatty acid are present, thus
trapping the cyclooxygenase reaction at the carbon-centered AA·
stage, as predicted by the mechanism in Scheme 1. This conclusion that
further turnover of the enzyme was blocked by oxygen exhaustion is
supported by the observation that only 1 eq of total eicosanoid
products were formed by MnPGHS-1 (17). As mentioned above, the EPR line
shape of the AA· generated in FePGHS-1 at high concentrations of
enzyme and substrate (Fig. 6) does differ from the singlet observed
earlier at low enzyme and substrate concentration (11). This could be due to different AA· structures under the two sets of reaction conditions. However, the simulated EPR line shape of the AA· is highly sensitive to the effects of small changes in coupling constants (Table I and Figs. 1b and 4b). We thus suggest
that several aspects of the different appearances of AA· spectra
probably result from slight differences in factors that determine the
inherent EPR line width of a common planar pentadienyl radical. It does
remain possible that differences in the kinetics of AA· formation
with high and low enzyme concentrations may lead to trapping of AA· with different conformations. In any case, the AA· structure may not
be the same as that of enzyme-bound AA, because substantial conformational changes accompany pentadienyl radical formation, and
adaptive changes in the protein could occur to stabilize the carbon-centered radical.
In summary, the present results show that the peroxide-induced tyrosyl
radical in FePGHS-2 and the peroxide-induced radical in MnPGHS-1, which
is probably also a tyrosyl radical, are both capable of oxidizing
arachidonate to a carbon centered fatty acid radical. This provides
strong support for a key step in the tyrosyl radical mechanism proposed
for PGHS cyclooxygenase catalysis. Interpretation of the EPR spectra
provides a detailed description of the structures of the arachidonyl
radicals formed in FePGHS-1 and -2 and in MnPGHS-1.
We are very grateful to Dr. Mark J. Nelson at
DuPont Merck Pharmaceutical Company, Wilmington, DE. for valuable
advice on interpretation of the arachidonate EPR data. We also thank
Wei Chen and Liliana Scarafia for their help in enzyme preparation.