From the
The coupling between the peroxidase and cyclooxygenase
activities of prostaglandin H synthase (PGHS) has been proposed to be
mediated by a critical tyrosyl radical through a branched chain
mechanism (Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur.
J. Biochem. 171, 321-328). In this study, we have examined
the ability of PGHS isoform-1 (PGHS-1) tyrosyl radicals to react with
arachidonate. Anaerobic addition of arachidonate following formation of
the peroxide-induced wide doublet or wide singlet tyrosyl radical led
to disappearance of the tyrosyl radicals and emergence of a new EPR
signal, which is distinct from known PGHS-1 tyrosyl radicals. The new
radical was clearly derived from arachidonate because its EPR line
shape changed when 5,6,8,9,11,12,14,15-octadeuterated arachidonate was
used. Subsequent addition of oxygen to samples containing the fatty
acyl radical resulted in regeneration of tyrosyl radical EPR. In
contrast, the peroxide-generated tyrosyl radical in
indomethacin-treated PGHS-1 (a narrow singlet) failed to react with
arachidonate, consistent with the cyclooxygenase inhibition by
indomethacin. These results indicate that the peroxide-generated wide
doublet and wide singlet tyrosyl radicals serve as immediate oxidants
of arachidonate bound at the cyclooxygenase active site to form a
carbon-centered fatty acyl radical, which reacts with oxygen to form a
hydroperoxide. These observations represent the first direct evidence
of chemical coupling between the peroxidase reaction and arachidonate
oxygenation in PGHS-1 and support the proposed role for a tyrosyl
radical in cyclooxygenase catalysis.
Prostaglandin H synthase (PGHS)
Arachidonic acid was purchased from NuChek Preps, Inc. (Elysian,
MN). 5,6,8,9,11,12,14,15-Octadeuterated arachidonic acid was a generous
gift of Hoffman-La Roche (Nutley, NJ). Polar contaminants were not
detected in either deuterated or unlabeled arachidonic acid 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% octadeuteration and 24% heptadeuteration. The deuterated
arachidonate was not purified further. Hemin and indomethacin were
obtained from Sigma. Ethyl hydroperoxide was the product of Polyscience
Inc. (Warrington, PA).
PGHS-1 was purified from sheep seminal
vesicles
(12) . The holoenzyme was prepared by replenishing the
lost heme as previously described
(8) . Cyclooxygenase activity
was assayed by oxygen consumption rate
(12) ; enzyme
preparations used in this study had specific activities ranging from 65
to 80 µmol of O
The glass
titration vessel used in our single turnover experiments
(Fig. SII) was adapted from the original design of Dutton
(13, 14) . The PGHS-1 enzyme solution (1.1-1.5 ml)
was placed in the glass vessel, and the opening was sealed by clamping
down the hard plastic cap lined with a rubber gasket. The bottom
portion of the glass vessel was immersed in ice water, and the enzyme
was constantly stirred with a glass-encased magnet to keep the enzyme
solution at 0 °C. The vessel and contents were made anaerobic by
five cycles of alternating vacuum (30 s) and argon replacement (5 min)
through a glass valve connected to an anaerobic train
(14) .
Arachidonic acid and ethyl hydroperoxide solutions contained in glass
tubes sealed with rubber septa were made anaerobic by bubbling with
argon for 5 min on ice. The substrates were then added in the desired
sequence through the rubber septum plugs on the titration vessel cap
using gas-tight Hamilton syringes.
EPR spectra were recorded at liquid nitrogen or
liquid helium temperatures on a Varian E-6 spectrometer
(8) .
Unless otherwise noted, the EPR conditions for liquid nitrogen
temperature were as follows: modulation amplitude, 2 G; time constant,
1 s; power, 1 milliwatt; and temperature, 96 K. Radical concentrations
were determined by double integration of the EPR signals, with
reference to a copper standard
(15) . Computer simulations of
EPR spectra were done on a PC using a modified version of the POWFUN
program
(16) kindly provided by Dr. Gerald T. Babcock and Dr.
Curt Hoganson (Michigan State University).
Reaction of the WS radical with arachidonate
was also examined. To accomplish this, the hydroperoxide-induced
radical was incubated at 0 °C long enough to allow most of the
transition from the WD to the WS species (Fig. 3 a)
before addition of fatty acid. In this case, addition of arachidonate
resulted in a singlet (Fig. 3 b) with a peak-trough width
of 30 G, intermediate between that of the wide singlet and the new
radical signal shown in Fig. 2, spectrumb. The
singlet narrowed with further incubation (Fig. 3 c) until
it was almost identical with that from the longer reaction of
arachidonate with the WD radical (Fig. 2 c). In the
sample taken before arachidonate addition, the wide singlet
(Fig. 3 a) simply decreased in intensity with aerobic
incubation (Fig. 3 a`). In contrast, for samples taken
after reaction of the WS radical with arachidonate (Fig. 3, b and c), aerobic incubation appeared to regenerate the
original wide singlet tyrosyl radical, with considerably increased
intensity (Fig. 3, b` and c`). As mentioned
above, regeneration of the tyrosyl radical suggests that fresh
hydroperoxide was produced from the new radical species when oxygen was
admitted.
A key step in the mechanism in Fig. SIis the reaction
of the hydroperoxide-induced tyrosyl radical with bound arachidonate to
activate the fatty acid for reaction with oxygen. The reduction
potential of the tyrosyl radical (TyrO/TyrOH) in solution at pH 7 is
0.94 V versus NHE
(19) . Abstraction of hydrogen from
the fatty acid gives a pentadienyl radical; this radical has a
reduction potential (R/RH) of 0.60 V
(20) . A tyrosyl radical is
thus a strong enough oxidant to abstract a hydrogen atom from
arachidonate. The tyrosine residue believed to carry the unpaired
electron in native PGHS-1, Tyr-385
(6, 7) , is located
in the vicinity of both the heme and the putative fatty acid binding
site
(5) . Thus, having a tyrosyl radical in PGHS-1 as the
oxidant for bound arachidonate is quite plausible from the structural
standpoint. The new radical produced by reaction of arachidonate with
tyrosyl radical is established as a fatty acyl radical by the dramatic
change in line shape observed when deuterated arachidonate was used
(Figs. 2-4). This demonstration of the ability of tyrosyl radical
to form fatty acyl radical provides the first direct evidence that
tyrosyl radical in native PGHS-1 actually does react with arachidonic
acid as required by the proposed mechanism (Fig. SI) and is not
just a marker for self-inactivation as suggested by other investigators
(10, 11) .
Given the arrangement of double bonds in
arachidonate and the fact that the fatty acid-derived radical was
trapped in the absence of oxygen, this radical is most likely a
pentadienyl carbon-centered radical, as depicted in Fig. 7. The
neutral radical formed from abstraction of the 13-pro- S hydrogen atom of arachidonate would be expected to have its
electron density delocalized over C-11-C-15, as found in model
systems containing the pentadienyl structure
(21, 22, 23, 24) and in the
substrate-associated purple lipoxygenase radical
(25, 26) . The unpaired electron density is likely to be
higher at C-11, C-13, and C-15 and lower at C-12 and C-14
(21, 22, 23, 24) . Spectral simulations
were performed using the proton hyperfine coupling constants observed
for a planar pentadienyl radical
(21, 22, 23, 24) as initial values and a range of isotropic coupling
constants for the
The simulation for the EPR signal with deuterated arachidonate was
less satisfactory (Fig. 8 B). Based on the chemical
structure shown in Fig. 7, the radical spectrum would be expected
to have proton hyperfine structure caused by three protons: the single
proton at C-13 and the
A tyrosyl radical plays a
central role in the branched chain mechanism proposed for
cyclooxygenase catalysis by PGHS-1 ( Fig. SIand Ref. 4). As
required by the mechanism, reaction of pure PGHS-1 with hydroperoxide
can produce a tyrosyl radical
(9, 28) . In fact,
detailed EPR studies have detected at least three types of
hydroperoxide-induced radical signals in the enzyme, all ascribed to
tyrosyl radicals
(8, 9, 10, 28) . A WD
arising from a tyrosyl radical with a strained ring orientation is
observed early during reaction of native enzyme with hydroperoxide
(9, 17, 28) . PGHS-1, whose cyclooxygenase
activity has been inhibited with agents such as indomethacin, produces
NS EPR characteristic of a tyrosyl radical with a relaxed ring
orientation
(8, 9, 17) . PGHS-1 inactivated
during catalysis also produces an NS EPR but with somewhat less
distinct hyperfine features than observed with the indomethacin-treated
enzyme
(8, 9) . WS EPR signals also are observed
(8, 9, 10, 11) . The EPR of the WS
radical produced under self-inactivation conditions can be accounted
for by the sum of the corresponding WD and NS radicals
(10, 11) but not by the sum of the WD from native enzyme and the NS
from indomethacin-treated enzyme.
It is important to note
that the WD and WS radicals associated with PGHS-1 with active
cyclooxygenase
(8) reacted with arachidonate (Figs. 2-4),
whereas the NS radical associated with inactivated cyclooxygenase
(9) did not react with the fatty acid (Fig. 6). This
demonstrates that reaction with arachidonate requires particular PGHS-1
tyrosyl radicals, and it links the reaction to cyclooxygenase catalysis
rather than to nonspecific fatty acid oxidation. Further, the
observation that the fatty acyl radical is converted back to the
tyrosyl radical upon reaction with oxygen is exactly the behavior
proposed for the arachidonate radical intermediate in cyclooxygenase
catalysis (Fig. SI). The evidence placing the reaction of tyrosyl
radical with arachidonate within the context of cyclooxygenase
catalysis indicates that the observed reaction corresponds to step 3 in
the mechanism in Fig. SI. In summary, the results of the present
study provide strong support for the key initial cyclooxygenase step in
the tyrosyl radical mechanism proposed for PGHS-1 catalysis.
We thank Chunhong Wei and Chris Walker for help in
enzyme preparation and activity assays.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
plays a
key role in controlling the biosynthesis of various physiologically
important prostaglandins
(1) . Although two distinct PGHS
isozymes have been discovered in many different cells or tissues, they
are believed to share the same basic catalytic mechanism
(2) .
Type 1 PGHS (PGHS-1) thus serves as a useful prototype for detailed
mechanistic study. PGHS-1 has two enzyme functions: a cyclooxygenase,
which converts arachidonic acid to PGG
, and a peroxidase,
which catalyzes the transformation of PGG
to PGH
(1, 2, 3) . A branched chain radical
mechanism (Fig. SI) has been proposed to integrate the two
catalytic activities
(4) . In this proposal, 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 acyl radical (Fe(IV)Tyr/AA), shown as step 3. The bound
fatty acyl radical then reacts with molecular oxygen and rearranges to
form a PGG
radical (step 4). In the final cyclooxygenase
step, the tyrosyl radical is regenerated, and PGG
is
released (step 5).
Figure SI:
Scheme IHypothetical
branched chain mechanism for PGHS catalysis (adapted from Ref.
4).
The tyrosyl radical mechanism nicely explains the
heme dependence of both enzyme activities and the hydroperoxide
requirement of the cyclooxygenase activity, and it also is consistent
with x-ray crystallographic data that interposes a tyrosine residue
(Tyr-385) between the heme and the arachidonic acid binding channel
(5) . Results from chemical modification and site-directed
mutagenesis also have suggested a critical role for Tyr-385 in
catalysis and radical formation
(6, 7) . Further, the
kinetic correlation of cyclooxygenase product formation and tyrosyl
radical levels has provided indirect evidence that a specific wide
doublet tyrosyl radical (WD) could serve as the immediate oxidizing
agent in the cyclooxygenase cycle
(8) . However, other PGHS-1
tyrosyl radical species also are observed
(8, 9, 10, 11) , and direct evidence for
reaction of any of the radicals with arachidonate has been lacking. In
the studies described here, we have used single turnover experiments to
demonstrate that the peroxidase-associated WD radical indeed reacts
with arachidonic acid to form a carbon-centered radical, providing
direct evidence for a key step in the proposed tyrosyl radical
mechanism.
/min/mg of protein.
Figure SII:
Scheme
IIDiagram of the anaerobic vessel used. 1, gas-tight Hamilton
syringes; 2, butyl rubber tubing connecting to the anaerobic
train; 3, stainless steel transfer needle (wrapped to minimize
warming from the fingers during manipulation); 4, hollow glass
high-vacuum stopcock; 5, quartz EPR tube; 6, rubber
septa; 7, hard plastic cap; 8, metal clamp;
9, rubber gasket; 10, glass vessel; 11, well
for enzyme solution; 12, stirring
magnet.
Aliquots of the reaction mixtures
were transferred to EPR tubes through a long stainless steel transfer
needle using the positive argon pressure in the vessel. For this, the
transfer needle was first positioned in the titrator headspace, and the
argon venting from the outside end of the needle was used to flush a
prechilled EPR tube for a few seconds. Then, the tip of the needle was
lowered into the enzyme solution to begin transfer. After the EPR tube
was filled to a depth of about 3 cm, transfer was completed by raising
the tip of the needle above the liquid surface. While still being
flushed with argon, the EPR sample was frozen quickly in a dry
ice/acetone bath and stored in liquid nitrogen for EPR analysis.
Further aerobic reaction of individual EPR samples was achieved by
immersion in a 0 °C bath and mixing with a nichrome wire plunger
for the desired time interval before refreezing the sample in the dry
ice/acetone bath.
Control Reactions of PGHS-1 with Arachidonate or
Hydroperoxide
Even at 0 °C, the relatively concentrated
solutions (approximately 10 µM) of PGHS-1 used for the EPR
experiments react quantitatively with arachidonate in air within a few
seconds, producing a large tyrosyl radical EPR signal
(8) .
Thus, a lack of EPR signal after mixing arachidonate with enzyme
provides a sensitive way to verify anaerobic conditions in the titrator
vessel. When argon-saturated arachidonate solution (1.5 eq relative to
heme) was added to enzyme solution in the titrator and incubated for
over 1 min, no EPR signal above background was observed (Fig. 1,
a and b). Thawing and further reaction of the same
samples resulted in large EPR signals accounting for as much as 0.4
spins/heme (Fig. 1, a` and b`), which
corresponded to the expected WD and WS radicals
(8, 9) .
These results established that satisfactory anaerobic conditions were
maintained in the titrator and during sample transfer to the EPR tubes.
Figure 1:
PGHS-1 radicals during incubation with
arachidonate or EtOOH. Leftpanel, PGHS-1 holoenzyme
(10 µM heme) in 50 mM potassium P, pH
7.4, containing 0.02% octyl glucoside and 10% glycerol, was incubated
under anaerobic conditions with 1.5 eq of either arachidonate or EtOOH.
EPR spectra were recorded with samples taken 15 s ( a) or 83 s
( b) after fatty acid addition, and 11 s ( c) or 71 s
( d) after EtOOH addition. Rightpanel, a
second set of EPR spectra ( a`-d`) was obtained after the
same four samples used for the spectra in the leftpanel had been thawed and incubated aerobically for 16-17 s. The
value at the leftside of each spectrum is
the signal intensity obtained from double integration (in spins/heme).
Details are given under ``Materials and
Methods.''
PGHS-1 was also reacted with ethyl hydroperoxide (1.5 eq) using the
same anaerobic procedures, resulting in WD and WS EPR spectra
(Fig. 1, c and d) similar to the
hydroperoxide-induced tyrosyl radicals observed in the presence of air
(8, 9) . Subsequent incubation of these samples under
aerobic conditions resulted in decreased radical intensity
(Fig. 1, c` and d`).
Serial Reaction of PGHS-1 with Hydroperoxide and
Arachidonate
To examine the possibility of reaction between
hydroperoxide-induced tyrosyl radical(s) and arachidonic acid, PGHS-1
was reacted with ethyl hydroperoxide (1.5 eq) before addition of the
fatty acid (1.5 eq), all under anaerobic conditions. As shown in
Fig. 2
, spectruma, the expected WD EPR was
formed by the hydroperoxide. With subsequent addition of the fatty
acid, the WD signal was replaced by a radical that was distinct from
the WD and the WS spectra (Fig. 2 b). This new radical
was a featureless isotropic singlet centered at g =
2.004, with an overall line width of 23.5 G. The same radical was
observed in similar reactions with two other enzyme preparations. The
maximal intensity of the new radical was about 0.26 spins/heme,
approximately half the amount of the wide doublet tyrosyl radical. The
intensity of the new radical was not changed with 5 eq of arachidonate.
The new radical persisted when the incubation was prolonged, although
its intensity decreased (Fig. 2 c). Aerobic incubation of
the original tyrosyl radical sample taken before arachidonate addition
resulted in decreased radical intensity (Fig. 2 a`). In
contrast, introduction of air into the two samples taken after addition
of fatty acid led to regeneration of radical signals characteristic of
PGHS-1 tyrosyl radicals, with a sizable increase in intensity
(Fig. 2, b` and c`). The regeneration of
hydroperoxide-induced tyrosyl radical suggests that fresh hydroperoxide
was formed when the new radical was exposed to oxygen.
Figure 2:
EPR spectra during reaction of WD PGHS-1
radical with arachidonate. Leftpanel, PGHS-1
holoenzyme (10 µM heme) in 50 mM potassium
P, pH 7.4, containing 0.02% octyl glucoside and 10%
glycerol, was incubated under anaerobic conditions with 1.5 eq of EtOOH
for 20 s before addition of arachidonate (1.5 eq). EPR spectra were
obtained for samples taken 12 s after addition of EtOOH ( a),
and 12 s ( b) or 60 s ( c) after the subsequent
addition of arachidonate. Rightpanel, a second set
of EPR spectra ( a`-c`) was obtained after the same three
samples used for the spectra in the leftpanel had
been thawed and incubated aerobically for 20 s. The value at the
leftside of each spectrum is the signal
intensity obtained from double integration (in spins/heme). Details are
given under ``Materials and
Methods.''
Simultaneous
anaerobic addition of hydroperoxide and arachidonate to PGHS-1 produced
EPR spectra similar to b and c in Fig. 2.
Subsequent aerobic incubation of the same samples resulted in spectra
similar to Fig. 2, b` and c`. When arachidonate
was added to the enzyme first, no EPR was observed initially (just as
in Fig. 1, spectruma); upon subsequent
addition of hydroperoxide, a signal with the same line shape as that in
Fig. 2b appeared. Aerobic incubation of the same samples
again resulted in EPR essentially the same as Fig. 2, b` and c`.
Figure 3:
EPR spectra during reaction of WS PGHS-1
radical with arachidonate. Leftpanel, PGHS-1
holoenzyme (10 µM heme) in 50 mM potassium
P, pH 7.4, containing 0.02% octyl glucoside and 10%
glycerol, was incubated under anaerobic conditions with 1.5 eq of EtOOH
for 82 s before addition of arachidonate (1.5 eq). EPR spectra were
obtained for samples taken 71 s after addition of EtOOH ( a),
and 12 s ( b) or 72 s ( c) after the subsequent
addition of arachidonate. Rightpanel, a second set
of EPR spectra ( a`-c`) was obtained after the same three
samples used for the spectra in the leftpanel had
been thawed and incubated aerobically for 13-15 s. The value at
the leftside of each spectrum is the signal
intensity obtained from double integration (in spins/heme). Details are
given under ``Materials and
Methods.''
Serial Reaction of PGHS-1 with Hydroperoxide and
Deuterated Arachidonate
The WD radical was also reacted
with deuterium-labeled arachidonate using the same sequence as for the
spectra in Fig. 2. As expected, the initial WD radical
(Fig. 4 a) was essentially the same as that in
Fig. 2
, spectruma. However, the spectra
obtained after addition of deuterated arachidonate (Fig. 4, b and c) were quite different from the corresponding ones
following reaction with unlabeled fatty acid (Fig. 2, b and c), demonstrating that the new radical resides on the
fatty acid itself. The radical signal produced in reactions with
deuterated arachidonate addition (Fig. 4, b and
c) is centered at g = 2.003, with an overall
line width of 22.5 gauss and recognizable 5-line hyperfine features.
The maximum spin concentration observed was approximately 0.24
spins/heme, essentially the same as obtained with unlabeled arachidonic
acid. A comparison of the power dependence of a typical wide doublet
tyrosyl radical (Fig. 4 a), the new radical observed from
reaction with unlabeled arachidonate (Fig. 2 b), and the
new radical observed in reaction with deuterated arachidonate
(Fig. 4 b) is presented in Fig. 5. The three
species had similar power dependences, with half-saturation power
values of 1-3 milliwatts at 96 K. When the samples used to obtain
spectra b and c in Fig. 4were subsequently
incubated aerobically, a significantly more intense radical resembling
the WS radical was generated (Fig. 4, b` and
c`), just as was observed in corresponding samples with
unlabeled fatty acid (Fig. 2, b` and c`).
Regeneration of hydroperoxide-dependent tyrosyl radical upon aerobic
incubation indicates that the fatty acyl radical reacted with oxygen to
form fresh hydroperoxide. This is just as expected from the mechanism
in Fig. SI, where the arachidonate radical reacts with oxygen to
form PGG and tyrosyl radical ( Steps 4 and 5). The
PGG
released is then available to react at the peroxidase
site of other, unactivated enzyme molecules to accumulate additional
tyrosyl radical.
Figure 4:
EPR spectra during reaction of WD PGHS-1
radical with deuterated arachidonate. Leftpanel,
PGHS-1 holoenzyme (10 µM heme) in 50 mM potassium
P, pH 7.4, containing 0.02% octyl glucoside and 10%
glycerol, was incubated under anaerobic conditions with 1.5 eq of EtOOH
for 20 s before addition of d
-arachidonate (1.5
eq). EPR spectra were obtained for samples taken 13 s after addition of
EtOOH ( a), and 14 s ( b) or 56 s ( c) after
the subsequent addition of d
-arachidonate.
Rightpanel, a second set of EPR spectra
( a`-c`) was obtained after the same three samples used
for the spectra in the leftpanel had been thawed and
incubated aerobically for 20 s. The value at the leftside of each spectrum is the signal intensity obtained from
double integration (in spins/heme). Details are given under
``Materials and Methods.''
Figure 5:
Power saturation behavior of radical
signals. The EPR signal amplitude was measured at power levels of
0.01-128 milliwatts for PGHS-1 samples, which exhibited the WD
radical ( circles and Fig. 2 a), or had been reacted
with unlabeled ( triangles and Fig. 2 b) or
d-arachidonate ( squares and Fig.
4 b). No heterogeneous behavior was observed for any of the
radical signals. Arrows indicate p values.
Serial Reaction of Indomethacin-treated PGHS-1 with
Hydroperoxide and Arachidonate
Addition of hydroperoxide to
indomethacin-treated PGHS-1 produces a distinct NS EPR, which has been
ascribed to yet another tyrosyl radical, one with a more relaxed ring
orientation than that observed in native PGHS-1
(9, 17) . Indomethacin inhibits only the cyclooxygenase
activity
(18) , so it was of some interest to examine the
ability of the NS radical in indomethacin-treated PGHS-1 to react with
arachidonate. Addition of hydroperoxide to indomethacin-treated PGHS-1
under anaerobic conditions produced the expected NS EPR
(Fig. 6 a). Subsequent addition of arachidonate left the
NS unchanged (Fig. 6 b), and further anaerobic incubation
only resulted in a slight decrease in intensity
(Fig. 6 c). Thus, the NS radical did not appear capable
of reacting with arachidonate. When the same samples were incubated in
air, the only change observed was a decrease in intensity
(Fig. 6, a`-c`).
Figure 6:
EPR spectra during reaction of NS
indomethacin-PGHS-1 radical with arachidonate. Leftpanel, PGHS-1 holoenzyme (10 µM heme) in 50
mM potassium P, pH 7.4, containing 0.02% octyl
glucoside and 10% glycerol, was pretreated with indomethacin (1 mol/mol
of subunit) on ice for 1 h. The indomethacin-treated enzyme was
incubated under anaerobic conditions with 1.5 eq of EtOOH for 17 s
before addition of arachidonate (1.5 eq). EPR spectra were obtained for
samples taken 11 s after addition of EtOOH ( a), and 14 s
( b) or 60 s ( c) after the subsequent addition of
arachidonate. Alternatively, arachidonate was added first and incubated
with the enzyme for 20 s before addition of EtOOH. In this case, EPR
spectra were obtained for samples taken 10 s after addition of
arachidonic acid ( d), and 13 s ( e) or 62 s
( f) after the subsequent EtOOH addition. Rightpanel, a second set of EPR spectra ( a`-f`)
was obtained after the same six samples used for the spectra in the
leftpanel had been thawed and incubated aerobically
for 15-20 s. The value at the leftside of each
spectrum is the signal intensity obtained from double integration (in
spins/heme). Details are given under ``Materials and
Methods.''
In a separate experiment, the
unlabeled fatty acid was added to indomethacin-treated enzyme before
the hydroperoxide. In this case, no EPR was observed initially
(Fig. 6 d), as expected from the anaerobic conditions and
the inhibition of the cyclooxygenase. Subsequent addition of the
hydroperoxide produced a rather weak NS EPR, which decayed with further
incubation (Fig. 6, e and f). Aerobic
incubation of the same samples resulted in very minimal changes in line
shape or total spin concentration (Fig. 6, d`-f`).
-protons. It was assumed that the principal axes
of the A and g tensors are parallel and that only one out of the two
-protons at C-10 and C-16 has significant interaction with the
unpaired electron. The optimal spectrum fits achieved for the radicals
from unlabeled and deuterated arachidonate are shown together with the
original EPR spectra in Fig. 8. The values for the parameters
used in the simulations are summarized in , which also
includes data from a model planar pentadienyl radical.
Figure 7:
Proposed structures of planar pentadienyl
radicals generated by abstraction of the 13-pro(S) hydrogen from
unlabeled arachidonate ( upper) or
d-arachidonate
( lower).
Figure 8:
Simulations of EPR spectra of radicals
derived from unlabeled arachidonate ( A) and from
d-arachidonate ( B). The simulated spectra
( dottedlines) were calculated as described in the
text and are compared with the actual spectra ( solidlines).
A
satisfactory simulation was obtained for the radical derived from
unlabeled arachidonic acid (Fig. 8 A and )
using coupling constants of 1 (2H), 9.8 (2H), 5 (1H), 7 (1H), and 12
(1H) G. A line width of 4.1 G was used to reflect the poor resolution
in the original spectrum. The hyperfine coupling constants for the
first two sets of doublet protons can thus be assigned to those
associated with C-12/C-14 and C-11/C-15, respectively, whereas the
other three coupling constants are associated with the protons at C-13
and one each of the -protons associated with either C-10 or C-16
(Fig. 7). The EPR spectrum produced by reaction of PGHS-1 tyrosyl
radical with arachidonate can thus be reasonably ascribed to the
expected carbon-centered pentadienyl radical on the fatty acid. The
radical observed directly in the present experiments may well be the
parent of a spin-trapped adduct previously reported
(27) .
-proton pairs at C-10 and C-16. Such
splitting would produce a quadruplet radical EPR. However, a fifth
splitting was actually observed in samples prepared from three
different batches of PGHS-1 (Fig. 8 B). This additional
splitting may reflect the additional proton in the 24 mol % of
d
-arachidonate present in the deuterated
arachidonate used for the experiments. However, the location(s) of the
additional proton could not be determined from the mass spectral
analyses, and so no attempt was made to account for it explicitly in
the EPR simulation, which was based on
d
-arachidonate. The coupling constants giving the
optimal simulation for the d
-arachidonate radical
structure (Figs. 7 and 8 and ) are 1 (4H), 9.6 (1H), 11.6
(1H), and 13 (1H) G and a line width of 5 G. As the hyperfine
interaction from a deuteron is only one-sixth that of a proton, the
first four equivalent protons thus are the deuterons substituted at
C-11, C-12, C-14, and C-15 (Fig. 7).
(
)
Thus, there
may actually be two types of NS radicals. Comparisons of the kinetics
of the various tyrosyl radicals with the cyclooxygenase kinetics have
indicated that the WD radical is present at appreciable concentrations
during cyclooxygenase catalysis
(8) .
Table: Parameters used to simulate EPR spectra of
radicals observed in reactions with unlabeled and deuterated
arachidonate
,
prostaglandin G
; PGH
, prostaglandin
H
; d
-arachidonate,
5,6,8,9,11,12,14,15-octadeuterated arachidonate; WD, wide doublet
tyrosyl radical; WS, wide singlet tyrosyl radical; NS, narrow singlet
tyrosyl radical.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.