©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Spectroscopic Evidence for Reaction of Prostaglandin H Synthase-1 Tyrosyl Radical with Arachidonic Acid (*)

Ah-lim Tsai (1)(§), Richard J. Kulmacz (1), Graham Palmer (2)

From the (1) Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030 and the (2) Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Prostaglandin H synthase (PGHS)() 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.


MATERIALS AND METHODS

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/min/mg of protein.

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.


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.

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).


RESULTS

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`.

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.


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`).


DISCUSSION

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 -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) .

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 -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).

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.() 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) .

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.

  
Table: Parameters used to simulate EPR spectra of radicals observed in reactions with unlabeled and deuterated arachidonate



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants GM44911 and GM 21337 and Welch Foundation Grant C636. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Hematology, Dept. of Internal Medicine, University of Texas Health Science Ctr., 6431 Fannin St., Houston, TX 77030. Tel.: 713-792-5450; Fax: 713-794-4230.

The abbreviations used are: PGHS-1, prostaglandin H synthase isoform-1; PGG, 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.

A.-L. Tsai, unpublished results.


ACKNOWLEDGEMENTS

We thank Chunhong Wei and Chris Walker for help in enzyme preparation and activity assays.


REFERENCES
  1. Samuelsson, B., Goldyne, M., Granström, E., Hamberg, M., Hammarström, S. and Malmsten, C. (1978) Annu. Rev. Biochem. 47, 997-1029 [CrossRef][Medline] [Order article via Infotrieve]
  2. Smith, W. L., Eling, T. E., Kulmacz, R. J., Marnett, L. J., and Tsai, A.-L. (1992) Biochemistry 31, 3-7 [Medline] [Order article via Infotrieve]
  3. Miyamoto, T., Ogino, N., Yamamoto, S., and Hayaishi, O. (1976) J. Biol. Chem. 251, 2629-2636 [Abstract]
  4. Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 321-328 [Abstract]
  5. Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249 [CrossRef][Medline] [Order article via Infotrieve]
  6. Shimokawa, T., Kulmacz, R. J., DeWitt, D. L., and Smith, W. L. (1990) J. Biol. Chem. 265, 20073-20076 [Abstract/Free Full Text]
  7. Tsai, A., Hsi, L. C., Kulmacz, R. J., Palmer, G., and Smith, W. L. (1994) J. Biol. Chem. 269, 5085-5091 [Abstract/Free Full Text]
  8. Tsai, A.-L., Palmer, G., and Kulmacz, R. J. (1992) J. Biol. Chem. 267, 17753-17759 [Abstract/Free Full Text]
  9. Kulmacz, R. J., Ren, Y., Tsai, A.-L., and Palmer, G. (1990) Biochemistry 29, 8760-8771 [Medline] [Order article via Infotrieve]
  10. Lassmann, G., Odenwaller, R., Curtis, J. F., DeGray, J. A., Mason, R. P., Marnett, L. J., and Eling, T. E. (1991) J. Biol. Chem. 266, 20045-20055 [Abstract/Free Full Text]
  11. DeGray, J. A., Lassmann, G., Curtis, J. F., Kennedy, T. A., Marnett, L. J., Eling, T. E., and Mason, R. P. (1992) J. Biol. Chem. 267, 23583-23588 [Abstract/Free Full Text]
  12. Kulmacz, R. J., and Lands, W. E. M. (1987) in Prostaglandins and Related Substances: A Practical Approach (Benedetto, C., McDonald-Gibson, R. G., Nigam, S., and Slater, R. F., eds) pp. 209-227, IRL Press, Washington D. C.
  13. Dutton, P. L. (1971) Biochim. Biophys. Acta 226, 63-81 [Medline] [Order article via Infotrieve]
  14. Tsai, A.-L., and Palmer, G. (1983) Biochim. Biophys. Acta 722, 349-363 [Medline] [Order article via Infotrieve]
  15. Wertz, J. E., and Bolton, J. R. (1986) Electron Spin Resonance, pp. 462-464, Chapman and Hall, New York
  16. Hoganson, C. W., and Babcock, G. T. (1992) Biochemistry 31, 11874-11880 [Medline] [Order article via Infotrieve]
  17. Barry, B. A., El-Deeb, M. K., Sandusky, P. O., and Babcock, G. T. (1990) J. Biol. Chem. 265, 20139-20143 [Abstract/Free Full Text]
  18. Mizuno, K., Yamamoto, S., and Lands, W. E. M. (1982) Prostaglandins 23, 743-757 [CrossRef][Medline] [Order article via Infotrieve]
  19. DeFelippis, M. R., Murthy, C. P., Faraggi, M., and Klapper, M. H. (1989) Biochemistry 28, 4847-4853 [Medline] [Order article via Infotrieve]
  20. Koppenol, W. H. (1990) FEBS Lett. 264, 165-167 [CrossRef][Medline] [Order article via Infotrieve]
  21. Sustmann, R., and Schmidt, H. (1979) Chem. Ber. 112, 1440-1447
  22. Hinchliffe, A., and Cobb, J. (1974) J. Mol. Struct. 23, 273-279 [CrossRef]
  23. Bascetta, E., Gunstone, F. D., Scrimgeour, C. M., and Walton, J. C. (1982) J. Chem. Soc. Chem. Commun. 110-112
  24. Davies, A. G., Griller, D., Ingold, K. U., Lindsay, D. A., and Walton, J. C. (1981) J. Chem. Soc. Perkin Trans. 2, 633-641
  25. Nelson, M. J., Seitz, S. P., and Cowling, R. A. (1990) Biochemistry 29, 6897-6903 [Medline] [Order article via Infotrieve]
  26. Nelson, M. J., Cowling, R. A., and Seitz, S. P. (1994) Biochemistry 33, 4966-4973 [Medline] [Order article via Infotrieve]
  27. Schreiber, J., Eling, T. E., and Mason, R. P. (1986) Arch. Biochem. Biophys. 249, 126-136 [Medline] [Order article via Infotrieve]
  28. Karthein, R., Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 313-320 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.