Comparison of Peroxidase Reaction Mechanisms of Prostaglandin H Synthase-1 Containing Heme and Mangano Protoporphyrin IX*

(Received for publication, January 22, 1997)

Ah-lim Tsai Dagger §, Chunhong Wei Dagger , Haesun K. Baek par , Richard J. Kulmacz Dagger and Harold E. Van Wart par

From the Dagger  Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030 and the  Department of Chemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Prostaglandin H synthase (PGHS) is a heme protein that catalyzes both the cyclooxygenase and peroxidase reactions needed to produce prostaglandins G2 and H2 from arachidonic acid. Replacement of the heme group by mangano protoporphyrin IX largely preserves the cyclooxygenase activity, but lowers the steady-state peroxidase activity by 25-fold. Thus, mangano protoporphyrin IX serves as a useful tool to evaluate the function of the heme in PGHS. A detailed kinetic analysis of the peroxidase reaction using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), EtOOH, and other peroxides as substrates has been carried out to compare the characteristics of PGHS reconstituted with mangano protoporphyrin IX (Mn-PGHS) to those of the native heme enzyme (Fe-PGHS). The rate constant describing the reaction of Mn-PGHS with 15-HPETE to form the oxidized, Mn(IV) intermediate with absorption at 420 nm, exhibits saturable behavior as the 15-HPETE concentration is raised from 10 to 400 µM. This is most likely due to the presence of a second, earlier intermediate between the resting enzyme and the Mn(IV) species. Measurements at high substrate concentrations permitted resolution of the absorbance spectra of the two oxidized Mn-PGHS intermediates. The spectrum of the initial intermediate, assigned to a Mn(V) species, had a line shape similar to that of the later intermediate, assigned to a Mn(IV) species, suggesting that a porphyrin pi -cation radical is not generated in the peroxidase reaction of Mn-PGHS. The rate constant estimated for the formation of the earlier intermediate with 15-HPETE is 1.0 × 106 M-1 s-1 (20 °C, pH 7.3). A rate constant of 400 ± 100 s-1 was estimated for the second step in the reaction. Thus, Mn-PGHS reacts considerably more slowly than Fe-PGHS with 15-HPETE to form the first high-valent intermediate, but the two enzymes appear to follow a similar overall reaction mechanism for generation of oxidized intermediates. The difference in rate constants explains the observed lower steady-state peroxidase activity of Mn-PGHS compared with Fe-PGHS.


INTRODUCTION

Prostaglandin H synthase (PGHS)1 catalyzes the first irreversible step in the biosynthesis of many important prostanoids. PGHS exhibits two enzymatic activities: a cyclooxygenase activity which converts arachidonic acid to PGG2, and a peroxidase activity which transforms PGG2 to PGH2. Lambeir et al. (1) observed that intermediates similar to horseradish peroxidase Compounds I and II are formed during the peroxidase reaction of PGHS. The subsequent discovery of a tyrosyl radical intermediate (2-5) suggested that the PGHS peroxidase reaction resembles a hybrid of the reactions of horseradish peroxidase and cytochrome c peroxidase. In this hybrid mechanism, PGHS first interacts with a peroxide substrate to generate Intermediate I, equivalent to Compound I of horseradish peroxidase. Intermediate I then converts to Intermediate II, equivalent to Complex ES of cytochrome c peroxidase, through an intramolecular electron transfer from a tyrosine residue to the oxidized porphyrin. Generation of this tyrosyl radical has been proposed to be necessary for cyclooxygenase activity (2-5).

Replacement of the heme group of PGHS by mangano protoporphyrin IX was originally reported to result in an almost complete loss of peroxidase activity, whereas the cyclooxygenase activity was largely preserved (6). This finding suggested that there might be a peroxidase-independent cyclooxygenase reaction mechanism. However, later studies showed that Mn-PGHS has true peroxidase activity and formed a high-valence peroxidase intermediate, but at a much slower rate than Fe-PGHS (7-9). Interestingly, only one intermediate was observed in the Mn-PGHS peroxidase cycle (7-9), suggesting that the mechanism might be fundamentally different from Fe-PGHS. The present work describes a detailed comparison of peroxidase kinetics of Fe- and Mn-PGHS. The results demonstrate that the peroxidase reaction of Mn-PGHS proceeds through two intermediates that are analogous to Intermediates I and II found with Fe-PGHS. Thus, the overall Fe-PGHS peroxidase reaction mechanism appears to be conserved in Mn-PGHS, despite the change in prosthetic group.


EXPERIMENTAL PROCEDURES

Hydrogen peroxide, Fe-PPIX, aspirin, and indomethacin were purchased from Sigma. Mn-PPIX was obtained from Porphyrin Products (Logan, UT). Arachidonic acid was from NuChek Preps (Elysian, MN). [1-14C]Arachidonic acid was from Amersham Corp. Ethyl hydroperoxide was purchased as a 5% aqueous solution from Polysciences Inc. (Warrington, PA) and trans-5-phenyl-4-pentenyl hydroperoxide (PPHP) was the product of Cayman Chemical Co. (Ann Arbor, MI).

15-Hydroperoxyeicosatetraenoic acid (15-HPETE) was prepared from [1-14C]arachidonic acid with soybean lipoxygenase as described by Graff et al. (10) and purified by silica gel chromatography. The purity of 15-HPETE was assessed chromatographically (10) and quantitated from oxidation of TMPD in the presence of excess Fe-PGHS as described previously (9).

PGH synthase was purified from ram seminal vesicles as described previously (11) except that 5 mM glutathione was included during isoelectric focusing to increase heme depletion. Protein was determined either by the Lowry method (12) or by absorbance at 279 nm (116 mM-1 cm-1). Residual heme was removed by treatment with 5 mM glutathione and DEAE-cellulose (9). Fe-PGHS was prepared by addition of a slight excess of heme to the apoenzyme and incubation at room temperature for 30 min. Nonspecifically bound heme was then removed by treatment with DEAE-cellulose and gel filtration through a Bio-Rad 10DG column. The resulting Fe-PGHS was typically >90% in the holoenzyme form, as assayed by cyclooxygenase activity in the presence and absence of 1 µM hemin. Mn-PGHS was prepared by adding a stoichiometric amount of Mn-PPIX and incubation for 30 min at room temperature. No DEAE treatment was applied to Mn-PGHS as this procedure removed both functional and nonspecifically found Mn-PPIX. The reconstituted holoenzymes were checked for their enzymatic stability and found to be comparable to the apoenzyme when 0.02% octyl beta -D-glucoside and 0.1% Tween 20 were included in the buffer. Acetylation of Fe- and Mn-PGHS by aspirin was performed as described previously (13). Briefly, the holoenzyme was incubated with 0.3 mM aspirin at room temperature for at least 1 h, followed by gel filtration to remove excess aspirin and salicylate. Residual cyclooxygenase activity was less than 3% of the control value. Cyclooxygenase activity was assayed polarographically at 30 °C, and peroxidase activity was measured using spectroscopic detection of the change at 611 nm in the presence of excess TMPD at 25 °C as described previously (11).

Stopped-flow kinetic studies were conducted with two separate instruments. Kinetic measurements below 0 °C were carried out in a custom-built low-temperature stopped-flow machine (14). The entire flow channels were encased in brass blocks which were cooled with 50% methanol circulated by a Neslab ULT-80 cooling unit. All valves and gaskets were spring loaded to prevent leakage at low temperature. Custom software was used for control of data collection and analysis. All other kinetic data were collected with a Bio-Sequential DX-17MV stopped-flow instrument (Applied Photophysics, Leatherhead, United Kingdom). Temperature-dependent kinetic measurements were conducted over the range of -10 °C to 35 °C, limited by the freezing point of the buffer and the enzyme thermostability. For single wavelength kinetic data, the built-in software was used for rate analysis. For data obtained in the kinetic scan mode at serial wavelengths, the data sets were first manipulated by the singular value decomposition (SVD) method (15), and then fitted to kinetic models using the Global analysis package (16). The Global package uses a Marquardt-Levenberg algorithm for least square analysis. The SCoP program (Simulation Resources Inc., Berrien Springs, MI) was also used for simulation and for fitting of single wavelength kinetic data to the same mechanistic model. The default numerical integrator used for solving ordinary differential equations in our kinetic simulations, Adrunge, is a Runge-Kutta algorithm with automatic adjustment of time increment. The "GRID" method and principal axis method, or PRAXIS, were used to carry out fitting procedures.


RESULTS

A detailed analysis of the reaction of Fe-PGHS with several peroxide substrates was conducted as a benchmark for comparison with Mn-PGHS. Reactions with a lipid hydroperoxide substrate, 15-HPETE, and a water-soluble peroxide, EtOOH, were carried out at reduced temperatures (0 or -10 °C) to slow down the overall rates of reaction and maximize the range of substrate concentrations amenable to stopped-flow observation. Reaction progress was monitored at two wavelengths. Absorbance changes at 410 nm (the Soret maximum of resting PGHS) were used to monitor the conversion of the resting, ferric enzyme to Intermediate I. Observations at 424 nm (the reported isosbestic point for the resting enzyme and Intermediate I (17)) were used to measure formation of Intermediate II.

The reaction of 0.5 µM Fe-PGHS with excess 15-HPETE in 30% glycerol buffer solution was very rapid even at 0 °C (data not shown). The rate of decrease in A410 was faster than that of the increase in A424, with the latter exhibiting a brief lag phase. The changes in A410 and A424 (after the initial lag phase) were fitted by a single exponential function to provide estimates of the pseudo first-order rate constants, k1,obs and k2,obs, which describe the rates of formation of Intermediates I and II, respectively. The dependence of k1,obs and k2,obs on the 15-HPETE concentration is shown in Fig. 1 (top panels). Similar data were acquired using EtOOH as the substrate (Fig. 1, bottom panels).


Fig. 1. Dependence of the rates of formation of Fe-PGHS Intermediate I, k1,obs (left two panels), and Intermediate II, k2,obs (right two panels), on the concentrations of 15-HPETE and EtOOH. Fe-PGHS (0.5 µM heme) in 50 mM potassium phosphate, pH 7.2, with 0.02% octyl beta -D-glucoside and 30% glycerol was used throughout. Stopped-flow measurements were performed at -10 °C. Vertical error bars show the standard deviations for two to four determinations. The solid lines were obtained by fitting the data to linear or simple hyperbolic functions.
[View Larger Version of this Image (21K GIF file)]


The values of k1,obs and k2,obs are much lower at equivalent concentrations of EtOOH compared with 15-HPETE. The value of k1,obs increases linearly with 15-HPETE concentration over the range of 0-40 µM concentration range studied (Fig. 1, top left). The y intercept is very close to zero, indicating that the formation of Intermediate I is essentially irreversible. This behavior is consistent with the following mechanism,
<UP>Fe-PGHS</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB>[<UP>ROOH</UP>]</UL></LIM> <UP>Intermediate I</UP> (Eq. 1)
where
k<SUB>1,<UP>obs</UP></SUB>=k<SUB>1</SUB>[<UP>ROOH</UP>] (Eq. 2)
The value of the apparent second-order rate constant, k1, for the formation of Intermediate I calculated from the slope of the line in Fig. 1, top left, is 1.1 × 107 M-1 s-1. Similar behavior was observed for the reaction of Fe-PGHS with the water-soluble hydroperoxide, EtOOH (Fig. 1, bottom left). The value of k1,obs also increases linearly with EtOOH concentration, yielding a second-order rate constant, k1, of 1.2 × 106 M-1 s-1.

The value of k2,obs also increases with increasing concentrations of both 15-HPETE and EtOOH (Fig. 1, right panels). However, the value starts to level off at high concentrations of both peroxides, indicating that the conversion of an earlier intermediate to Intermediate II becomes rate-limiting. Two interpretations for this curvilinear dependence of k2,obs on peroxide concentration are considered here. The first interpretation is based on a two-step, sequential formation of Intermediate II, where the second step is unimolecular,
<UP>Fe-PGHS</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB>[<UP>ROOH</UP>]</UL></LIM> <UP>Intermediate I</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>2</SUB></UL></LIM><UP>Intermediate II</UP> (Eq. 3)
Here, the curvilinear dependence on peroxide concentration is attributable to the first step being rate-limiting at low peroxide concentration, and the second step being rate-limiting at high peroxide concentration. In the version of this mechanism proposed by Ruf and co-workers (2, 17) for reaction of Fe-PGHS with PGG2, Intermediate I is the oxyferryl pi -cation radical and Intermediate II is the oxyferryl tyrosyl radical.

Alternatively, the curvilinear dependence on peroxide concentration could be a saturation kinetic phenomenon associated with a pre-equilibrium complex formation between Intermediate I and a second molecule of peroxide prior to reduction of the heme center of Intermediate I to Intermediate II, as proposed by Bakovic and Dunford (18),
<UP>Fe-PGHS</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB>[<UP>ROOH</UP>]</UL></LIM> <UP>Intermediate I </UP><LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB><UP>−</UP>2</SUB></LL><UL>k<SUB>2</SUB>[<UP>ROOH</UP>]</UL></LIM> <UP>I-ROOH</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>3</SUB></UL></LIM> (Eq. 4)
<UP>Intermediate II</UP>+<UP>ROO<SUP>•</SUP></UP>
After a lag phase due to formation of Intermediate I and the Intermediate I-ROOH complex, the appearance of Intermediate II is described by,
k<SUB>2,<UP>obs</UP></SUB>=k<SUB>3</SUB>[<UP>ROOH</UP>]<UP>/</UP>([<UP>ROOH</UP>]+K<SUB>2</SUB>) (Eq. 5)
where K2 = k-2/k2.

In this mechanism, the formation of the Intermediate I-ROOH complex from Intermediate I and ROOH is fast. Thus, k1,obs essentially reflects the formation of the complex, whereas k2,obs reflects conversion of the complex to Intermediate II. At high [ROOH], it is the complex, and not Intermediate I, that accumulates. It is also important to note that Intermediate II in this mechanism is only one oxidizing equivalent above the resting enzyme, and in this sense, is equivalent to peroxidase Compound II, which lacks an amino acid radical. This is distinct from Intermediate II in the two-step mechanism (Equation 3), which, as an oxyferryl tyrosyl radical, remains two oxidizing equivalents above the resting enzyme, making it analogous to Compound ES of cytochrome c peroxidase.

The k2,obs data were fitted empirically to a hyperbolic function to estimate k2 (Equation 3) or k3 (Equation 4) to be 110 s-1 for the reaction with 15-HPETE and 25 s-1 for the reaction with EtOOH (Fig. 1, right panels). Validation of the use of a hyperbolic function for estimation of k2 in the first mechanism (Equation 3) is detailed below. Equation 5 directly predicts a hyperbolic response to [ROOH] for the second mechanism. An average value of 39 ± 0.5 s-1 for k2 (or k3 in the second mechanism) was obtained for EtOOH in three separate experiments at 1 °C, considerably lower than that observed with 15-HPETE, indicating that the rate of conversion from Intermediate I to II was dependent on the identity of the peroxide.

To examine further the influence of hydroperoxide structure on the rate of Intermediate II formation, the rate constants k1 and k2 (k3 in Equation 4) were also evaluated for reaction of Fe-PGHS with PPHP and HOOH. As summarized in Table I, the values of k1 and k2 (k3) for PPHP were 9 × 106 M-1 s-1 and 280 s-1, respectively, at 1 °C. These values are similar to those obtained with 15-HPETE. With HOOH, the rates of change at 410 and 424 nm were slow and equal, indicating that the first step remained rate-limiting even at the highest substrate concentration tested. Because of this, only a rough estimate for the value of k1, 1 × 103 M-1 s-1 at -10 °C, could be obtained (Table I).

Table I.

Fe-PGHS peroxidase rate constants


Substrate k1a k2 or k3a Temperature Reference

M-1s-1 s-1 °C
PGG2 1.4  × 107 65 1 17
PPHP 1.3  × 107 1.2  × 106M-1 s-1b 5 1
PPHP 9.0  × 106 280 1 This study
15-HPETE 1.0  × 108c 950c 20 This study
15-HPETE 1.8  × 107 250e 1 This study
15-HPETE 1.1  × 107 108  -10 This study
15-HPETE(Indo)d 9.9  × 106 72  -10 This study
15-HPETE(ASA)e 1.1  × 107 146  -10 This study
EtOOH 1.4  × 107c 320c 20 This study
EtOOH 2.7  × 106 68c 1 This study
EtOOH 1.2  × 106 25  -10 This study
EtOOH(Indo) 1.2  × 106 28  -10 This study
EtOOH(ASA) 1.3  × 106 NDf  -10 This study
HOOH 1  × 104 ND 1 1
HOOH 1  × 103 ND  -10 This study

a k1 and k2 (or k3) are defined in the text.
b Reported to be a bimolecular reaction.
c Estimated from the Arrhenius relationship (Fig. 6).
d PGHS pretreated with indomethacin.
e PGHS pretreated with aspirin.
f Not determined.

The effects of two cyclooxygenase inhibitors, indomethacin and aspirin, on the individual steps in Fe-PGHS peroxidase catalysis were also examined. The values of k1 and k2 (k3 in Equation 4) for the reaction of Fe-PGHS pretreated with aspirin and indomethacin with 15-HPETE and EtOOH were measured (Table I). These values were very similar to those for the untreated enzyme, indicating that these two nonsteroidal anti-inflammatory agents do not affect these steps in the peroxidase reaction of Fe-PGHS.

Only one optical intermediate, with an absorbance maximum near 420 nm, was observed earlier in the reaction of peroxides with Mn-PGHS (7, 8). Thus, the reaction was monitored at this wavelength. The lower peroxidase activity of Mn-PGHS permitted kinetic measurements with a wide range of substrate concentrations and at higher temperatures than was possible with Fe-PGHS. Surprisingly, the observed rate constant for the absorbance change at 420 nm, kobs, was found to be a saturable function of the 15-HPETE concentration (Fig. 2, left). Similar results were obtained with two other batches of Mn-PGHS (data not shown), showing that the saturable response at higher levels of peroxide is a reproducible characteristic of the Mn-PGHS peroxidase reaction. This behavior points to the existence of at least one earlier intermediate in the Mn-PGHS peroxidase pathway prior to the formation of the intermediate whose formation is being monitored at 420 nm. The recognition that there are two intermediates in the Mn-PGHS peroxidase reaction pathway suggests that it closely parallels that for Fe-PGHS. Thus, we refer to these two intermediates as I and II, respectively. As with Fe-PGHS, two variants of the mechanism for Mn-PGHS were considered. The first is a simple, two-step sequential mechanism analogous to that shown in Equation 3 for Fe-PGHS,
<UP>Mn-PGHS </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB>[<UP>ROOH</UP>]</UL></LIM> <UP>Intermediate I </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>2</SUB></UL></LIM> <UP>Intermediate II</UP> (Eq. 6)
In this mechanism, kobs is determined by k1[ROOH] at low peroxide concentration and approaches k2 at high peroxide concentration. The second mechanism postulates that there is a reaction of Intermediate I with a second molecule of peroxide, and is analogous to that shown in Equation 4 for Fe-PGHS,
<UP>Mn-PGHS </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB>[<UP>ROOH</UP>]</UL></LIM> <UP>Intermediate I </UP><LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB>−2</SUB></LL><UL>k<SUB>2</SUB>[<UP>ROOH</UP>]</UL></LIM> <UP>I-ROOH </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>3</SUB></UL></LIM> (Eq. 7)
<UP>Intermediate II</UP>+<UP>ROO<SUP>•</SUP></UP>
In this mechanism,
k<SUB><UP>obs</UP></SUB>=k<SUB>3</SUB>[<UP>ROOH</UP>]<UP>/</UP>([<UP>ROOH</UP>]+K<SUB>2</SUB>) (Eq. 8)
where K2 = k-2/k2. The second-order rate constant for formation of Intermediate I, k1, is the same in both mechanisms (Equations 6 and 7) and was estimated from the initial slope of the dependence of kobs on substrate concentration to be 1.0 × 106 M-1 s-1. Values of k3 (or k2) and K2 (Equations 6 and 7) obtained from fitting the data from three individual Mn-PGHS batches were 400 ± 100 s-1 and 0.38 ± 0.02 mM, respectively.


Fig. 2. Dependence of the rate of formation of Mn-PGHS peroxidase intermediate(s) on peroxide concentration. Mn-PGHS in 50 mM KPi, pH 7.3, 0.02% octyl beta -D-glucoside, and 10% glycerol at 20 °C was used throughout. Reactions with 15-HPETE (left) and EtOOH (right) used 1.0 and 1.8 µM Mn-PGHS, respectively. Vertical bars show the standard deviations for three to four measurements. The solid lines were obtained by fitting the data to a simple hyperbolic function. EtOOH concentrations were varied from 17.5 µM to 201 mM, with the data at concentrations below 5 mM enlarged in the inset.
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The reaction of EtOOH with Mn-PGHS was much slower than with Fe-PGHS. The observed rate was linearly dependent on EtOOH concentration up to about 20 mM but showed signs of being saturable at higher levels (Fig. 2, right). The second-order rate constant for the formation of Intermediate I (k1 in Equations 6 and 7) calculated from the data in Fig. 2 (inset in right panel) is 6.8 × 102 M-1 s-1. Estimation of the values of k2 (Equation 6) and k3 (Equation 7) for reaction with EtOOH was not attempted because of the potential for Mn-PPIX destruction at the high EtOOH concentrations needed to approach saturation. As observed with Fe-PGHS, pretreatment of Mn-PGHS with indomethacin or aspirin to inhibit the cyclooxygenase activity had only marginal effects on the rate of the peroxidase reaction with either 15-HPETE or EtOOH (Table II).

Table II.

Mn-PGHS peroxidase rate constants


Substrate k1a k2 or k3b Temperature Reference

M-1s-1 s-1 °C
PGG2 104 22 8
PPHP 4  × 104 22 8
15-HPETE 1.0  × 106 400c 20 This study
15-HPETE(Indo) 0.7  × 106 20 This study
15-HPETE(ASA) 0.8  × 106 20 This study
EtOOH 6.8  × 102 20 This study
EtOOH(Indo) 8.5  × 102 20 This study
EtOOH(ASA) 5.7  × 102 20 This study

a Apparent second-order rate constant in Equations 6 or 7 for formation of Intermediate I.
b Rate constant for conversion of Intermediate I to II (k2 in Equation 6 or k3 in Equation 7).
c Average from three separate experiments.

Stopped-flow kinetic measurements at multiple wavelengths were used to resolve the electronic absorbance spectra of Mn-PGHS peroxidase Intermediates I and II. The analytical approach was first validated with Fe-PGHS, where the intermediate spectra are known. The Fe- or Mn-PGHS and 15-HPETE concentrations were carefully selected to maximize the transient concentration of Intermediate I, and yet minimize the fraction of the reaction occurring in the instrumental dead time (~3 ms). Under the experimental conditions used, computer modeling with the rate constants described above predicts a large difference between Fe- and Mn-PGHS in the peak accumulation of Intermediate I (~60% of total enzyme for Fe-PGHS and ~8% for Mn-PGHS) (data not shown). Kinetic scan data obtained at individual wavelengths in the reactions under optimized conditions were processed through a SVD routine to identify the important kinetic and wavelength components and to remove the background noise. These SVD-simplified data were then fit to a sequential model (Equations 3 or 6) by a nonlinear least square regression in the Global (or Glint) analysis package to obtain resolved spectra for the individual intermediates, which are shown in Fig. 3.


Fig. 3. Reconstructed optical spectra of Fe-PGHS and Mn-PGHS peroxidase intermediates. Stopped-flow observations for reactions with 15-HPETE were made for Fe-PGHS at 2-nm increments (390-450 nm) and for Mn-PGHS at 5-nm increments (340-480 nm). Fe-PGHS (1 µM heme) was reacted with 10 µM 15-HPETE in 50 mM KPi, pH 7.3, 0.02% octyl beta -D-glucoside, and 30% glycerol at 2 °C. Mn-PGHS (2.5 µM subunit, 1.25 µM Mn-PPIX) was reacted with 62.5 µM 15-HPETE in 50 mM KPi, pH 7.3, 0.02% octyl beta -D-glucoside, and 10% glycerol at 20 °C. The raw data was analyzed by the SVD procedure (15). A nonlinear least square fitting routine was then used to fit the multivariate absorption data to a sequential model with two first-order stages (Equation 3). The resulting fitted parameters were used to calculate the spectrum of each intermediate. Additional details are described in the text. Circles, resting enzyme; squares, Intermediate I; triangles, Intermediate II. The lines in the figure were smoothed by a Fast Fourier Transform/Reverse Fast Fourier Transform routine (D. W. Myers, Duke University).
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The spectra of Fe-PGHS and its peroxidase intermediates (Fig. 3) are very similar to those reported by Lambeir et al. (1) and Dietz et al. (17). The Soret band maxima are at 410, 412, and 416 nm for resting Fe-PGHS, Intermediate I and II, respectively. The isosbestic point between resting Fe-PGHS and Intermediate I is 428 nm, that between resting Fe-PGHS and Intermediate II is 422 nm, and those between Intermediate I and Intermediate II are 408 and 448 nm. These isosbestic points are only slightly different from those values reported by Dietz et al. (17). These results with Fe-PGHS confirmed the reliability of the SVD analytical approach.

The spectra obtained for Mn-PGHS and its peroxidase intermediates exhibit quite different line shapes from the corresponding Fe-PGHS intermediates (Fig. 3, right). The maxima for resting Mn-PGHS are at 372 and 468 nm, while the spectra of Intermediates I and II both peak at 420 nm. Mn-PGHS Intermediate II shows two additional peaks at 368 and 464 nm. The relatively high noise level apparent in the spectrum of Mn-PGHS Intermediate I (Fig. 3) is consistent with a smaller contribution to the total absorbance during the kinetic measurements. The isosbestic points between resting Mn-PGHS and Intermediate I are 364, 400, and 446 nm, those between resting enzyme and Intermediate II are 352 and 444 nm, and those between Intermediates I and II are 368, 406, and 433 nm.

The validity of SVD data processing and the fitting method used in the Global (Glint) package was cross-checked as follows. The SCoP numerical program was used to conduct a quantitative prediction of the original kinetic data at individual wavelengths, employing the experimentally determined rate constants for the first two peroxidase steps shown in Equations 3 and 6 with 15-HPETE as substrate (Figs. 1 and 2, and Table I) and the molar absorbance coefficients from the resolved spectra in Fig. 3. Very good fitting to experimental stopped-flow traces was obtained at 410 and 420 nm for Fe-PGHS (Fig. 4, left). Fitting to the stopped-flow data at 430 and 424 was not as good (not shown), with the major discrepancy occurring in the deflection area, perhaps reflecting an under-representation of data points in this time range by the SVD treatment. On the other hand, the Mn-PGHS kinetic data were fitted well at each of the three wavelengths examined in detail, 380 (not shown), 420 and 470 nm (Fig. 4, right).


Fig. 4. Fitting of stopped-flow data using molar absorption coefficients obtained from resolved spectra of reaction intermediates. Least square fittings were conducted for absorbance at 410 and 420 nm for Fe-PGHS, or for 420 and 470 nm for Mn-PGHS, as described in the text. The best fit for Fe-PGHS at 410 nm was achieved using k1 = 2.2 × 107 M-1 s-1 and k2 = 61 s-1. The best fit for the Fe-PGHS 420 nm data was with k1 = 4.2 × 107 M-1 s-1 and k2 = 43 s-1 (compare with observed k1 = 1.7 × 107 M-1 s-1 at 2 °C for this batch of Fe-PGHS). The best fit for the Mn-PGHS data for both wavelengths was obtained with k1 = 3.9 × 105 M-1 s-1 and k2 = 211 s-1 (compare with observed k1 = 4.4 × 105 M-1 s-1 and k2 = 269 s-1 at 20 °C for this preparation of Mn-PGHS).
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With these data in hand, we return to the question of whether the two-step mechanism (Equation 3) can explain the saturation behavior observed for Intermediate II formation (Fig. 1, right panels; Fig. 2, left panel). To examine this issue for Fe-PGHS, a set of predicted kinetic data for Intermediate II formation (424 nm) at different 15-HPETE levels was generated by numerical integration based on Equation 3, using the experimental rate constants and the extinction coefficients at 424 nm for the three enzyme species obtained from the resolved spectra. The predicted kinetic data are shown in Fig. 5. The portion beyond the initial lag phase of each simulated kinetic trace was fitted to a single-exponential function to estimate a rate constant. These pseudo first-order rate constants were found to approximate a hyperbolic dependence on the 15-HPETE level (Fig. 5, inset). Thus, the predicted k2,obs values do indeed show saturable behavior, and the hyperbolic fit used in Fig. 1 provides a reasonable empirical estimate for the upper limit of k2. In addition, the rates obtained from the simulations are similar to those obtained from actual measurements (compare Fig. 5, inset, with Fig. 1, top right).


Fig. 5. Simulated peroxidase reaction kinetics of Fe-PGHS. Changes in 424 nm absorbance were predicted by numerical integration based on the sequential mechanism (Equation 3) using the SCoP program. The enzyme concentration was set at 0.5 µM, k1 at 1.1 × 107 M-1 s-1, k2 at 108 s-1, and the peroxide concentration was varied between 0 and 300 µM. Single-exponential fitting (lines) of the simulated kinetic data (open symbols) after the initial lag phase for each reaction was used to estimate rate constants. These rate constants are presented as a function of peroxide concentration in the inset, along with the nonlinear least squares fit to a hyperbolic equation.
[View Larger Version of this Image (26K GIF file)]


The temperature dependences of k1 and k2 (or k3 in Equations 4 and 7) for the reaction of Fe-PGHS and Mn-PGHS with 15-HPETE and EtOOH were measured to calculate thermodynamic activation parameters for these steps. With Fe-PGHS, the Arrhenius plots were linear for both k1 and k2 or k3 (Equations 3 and 4) for both peroxide substrates (Fig. 6, top panels).


Fig. 6. Temperature dependence of Fe- and Mn-PGHS peroxidase kinetics. The rate constants for formation of Intermediate I (k1; 410 nm observations for Fe-PGHS and 420 nm observations for Mn-PGHS) and Fe-PGHS Intermediate II (k2 or k3; 424 nm observations) in reactions with 15-HPETE (left) or EtOOH (right) were measured at temperatures between -10 °C and 25 °C. Solid lines represent the linear regressions. Vertical bars indicate the standard deviations for two to four measurements.
[View Larger Version of this Image (20K GIF file)]


The activation energy, Ea, for each step was calculated from the Arrhenius equation,
k=A·e<SUP><UP>−Ea/</UP>RT</SUP> (Eq. 9)
where A is the frequency factor. The results are summarized in Table III. Very similar activation energies were found for the two Fe-PGHS peroxidase steps when 15-HPETE was the substrate, whereas the second step displayed a larger temperature dependence than the first step when EtOOH was the substrate. The same data were used to calculate the enthalpy and entropy of activation according to the Eyring equation,
<UP>ln</UP>(k/T)=<UP>ln</UP>(&kgr;/h)+&Dgr;S<SUP><UP>+</UP></SUP><UP>/R</UP>−&Dgr;H<SUP><UP>+</UP></SUP>/(RT) (Eq. 10)
where k is the rate constant in question (either k1, k2, or k3), kappa  is the Boltzmann constant, h is Planck's constant, and Delta S+ and Delta H+ are the entropy and enthalpy of activation, respectively. As shown in Table III, Delta S+ is smaller for the first peroxidase step than the second step with EtOOH as substrate, whereas the second step had a lower Delta S+ with 15-HPETE as substrate. The values of Delta H+ for the two peroxidase steps were comparable for 15-HPETE and EtOOH (Table III).

Table III.

Thermodynamic parameters describing the reaction of PGHS-1 with EtOOH and 15-HPETE


Enzyme Substrate Rate constanta  Delta E+  Delta H+  Delta S+

kcal/mol e.u.
Fe-PGHS EtOOH k1 9.8 9.2 5.8
k2 or k3 13.2 12.2 12.4
Fe-PGHS 15-HPETE k1 11.7 11.2 15.9
k2 or k3 11.1 10.6 10.7
Mn-PGHS EtOOH k1 12.0 11.2  -5.7
Mn-PGHS 15-HPETE k1 10.9 10.4 4.6

a See Equations 3, 4, 6, and 7.

In the case of Mn-PGHS, the temperature dependence was only examined for the k1 values. The Arrhenius plots with both peroxide substrates showed a discontinuity near 4-5 °C (Fig. 6, bottom panels). The linear dependence of ln k on 1/T above 5 °C was used to calculate the thermodynamic parameters. As apparent from Table III, both the activation energies and enthalpies of activation, using either peroxide, were similar to those found for Fe-PGHS. However, the Delta S+ values for Mn-PGHS were substantially lower than those for Fe-PGHS. This is seen most dramatically for the reaction of Mn-PGHS with EtOOH, which had a Delta S+ of -5.7 e.u., in sharp contrast with the value of +5.8 e.u. obtained for Fe-PGHS.


DISCUSSION

The studies reported here enable us to compare in greater detail than reported previously the steps in the peroxidase cycles of Fe- and Mn-PGHS. Both enzymes exhibit a curvilinear dependence of k2,obs on peroxide concentration that has led us to consider two alternative mechanisms, represented by Equations 3 and 4 for Fe-PGHS and Equations 6 and 7 for Mn-PGHS. Both mechanisms can account for the saturation behavior of k2,obs, but differ significantly in other details. These two mechanisms predict the formation of Intermediate II species containing different numbers of oxidation equivalents. However, since an oxyferryl state of the heme is predicted for Intermediate II in both mechanisms, the optical spectrum of this intermediate (Fig. 3) cannot be used to distinguish between the two mechanisms. This would require the use of EPR techniques that could assess whether the Intermediate II formed at different [ROOH] contained a tyrosyl radical.

There are certain observations that favor one mechanism over the other. The three-step mechanism shown in Equation 4 predicts that the values of k2,obs would depend on the identity of the peroxide, since its redox potential should affect the efficiency of this step. On the other hand, the requirement for 2 equivalents of hydroperoxide to form Intermediate II in the three-step mechanism is in conflict with the observation that stoichiometric amounts of EtOOH generate substantial amounts of Intermediate II and tyrosyl radical (19). We have recently found that substoichiometric amounts of peroxide also generate tyrosyl radical in proportion to the amount of peroxide.2 This efficient formation of Intermediate II with limiting levels of hydroperoxide is difficult to reconcile with the stoichiometry of the three-step model.

A possible explanation for these apparently conflicting observations is that the reaction can proceed by both mechanisms, with the proportion of Intermediate I decomposing by the two alternate routes being determined by the hydroperoxide concentration. Specifically, at low peroxide concentrations, the two-step mechanism described by Equations 3 and 6 would reasonably dominate, leading to formation of the oxyferryl tyrosyl radical Intermediate II. At high concentrations, the Intermediate I-ROOH complex is able to form and react intramolecularly to quench the pi -cation radical and reduce Intermediate I to an oxyferryl intermediate before its conversion to a tyrosyl radical intermediate. This reducing action of peroxide may be a laboratory artifact associated with the high peroxide concentrations needed to observe saturation.

An important aspect of this study is the establishment that the reaction of Mn-PGHS with peroxides also proceeds through at least two intermediates. The saturation behavior observed for k2,obs strongly supports the existence of an earlier peroxidase intermediate (I) in Mn-PGHS that precedes intermediate II. This finding resolves a puzzle raised by earlier studies which failed to find an intermediate with an oxidation state two equivalents higher than the Mn(III) resting enzyme (7-9). Intermediate I was not observed in previous studies using relatively low 15-HPETE concentrations because k1 is relatively slow and Intermediate I accumulates only when Mn-PGHS is reacted with rather high 15-HPETE concentrations (>100 µM; Fig. 2).

Fe-PGHS exhibits a 100-fold larger value of k1 than Mn-PGHS for reaction with 15-HPETE at 20 °C (Tables I and II). In contrast, the values of k2 for Fe-PGHS and Mn-PGHS differ by only a factor of two at 20 °C (Tables I and II). Therefore, it is clearly the decrease in the rate of Intermediate I formation that is responsible for the marked decrease in steady-state peroxidase activity in Mn-PGHS. Similar results have been found with other heme-dependent peroxidases. Replacement of the heme by Mn-PPIX in cytochrome c peroxidase and horseradish peroxidase decreases the value of kcat (a lower limit estimation for k1) in steady-state assays by 2-3 orders of magnitude (20, 21).

The values of k1 for both Fe- and Mn-PGHS decrease markedly as the substrate is changed from 15-HPETE to EtOOH to HOOH (Tables I and II). Although HOOH is still a reasonably reactive substrate for Fe-PGHS (k1 of 103 M-1 s-1 at -10 °C), its reactivity with Mn-PGHS was too slow to be measured readily. Other kinetic studies of Fe-PGHS and Mn-PGHS also reveal a clear preference for lipid peroxides over water-soluble peroxides (1, 7, 9, 18). This suggests that binding of larger hydrophobic hydroperoxides involves interaction with a lipid binding subdomain near the heme pocket. As the aliphatic chain of the substrate shortens, the major binding contribution shifts from the secondary hydrophobic site to the metalloporphyrin, revealing the intrinsic difference in reactivity between Fe-PPIX and Mn-PPIX toward the peroxy function itself. The x-ray crystallographic data indicate the heme pocket in PGHS is rather open, without a defined hydroperoxide channel like that found for the cyclooxygenase site (22), so it is not clear what parts of this region make up the hydrophobic peroxide-binding site.

It is of interest to consider why Mn-PPIX substitution causes the consistently decreased activity in peroxidases. Water proton relaxation measurements on native and Mn(III)-PPIX-substituted horseradish peroxidase indicate that the heme iron is predominantly penta-coordinate, whereas the metal in Mn-PPIX-substituted horseradish peroxidase is six-coordinate with water or hydroxide as the distal ligand (23). The existence of six-coordinate Mn(III) in horseradish peroxidase has also been supported by resonance Raman data (23). A slowly exchanging water or hydroxyl distal ligand in the Mn(III)-PPIX-substituted peroxidases could substantially decrease activity and even become the rate-limiting step in Intermediate I formation; once this ligand is displaced by hydroperoxide, conversion to Mn(V) proceeds rapidly. This hypothetical reaction sequence for Mn-PGHS is shown below,
<UP>E-H<SUB>2</SUB>O</UP>+<UP>ROOH</UP> <LIM><OP><ARROW>↔</ARROW></OP><LL>k<SUB><UP>−b</UP></SUB></LL><UL>k<SUB><UP>b</UP></SUB></UL></LIM> <UP>E-ROOH</UP>+<UP>H<SUB>2</SUB>O</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>1</SUB></UL></LIM> (Eq. 11)
 <UP>Intermediate I </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>2</SUB></UL></LIM> <UP>Intermediate II</UP>
If the substrate binding and chemical reaction occur simultaneously, Equation 11 becomes essentially identical to Equations 6 or 7.

The Kd of Fe-PGHS for 15-HPETE can be estimated to be submicromolar by extrapolation of the apparent Km values to zero co-substrate (24, 25). A similarly low Kd value is likely for Mn-PGHS (9). These Kd values are much smaller than the 15-HPETE levels observed to produce linear increases in kobs in the kinetic experiments (Fig. 1, top, and Fig. 2, left). The value of k1,obs at the y axis intercept was found to be near zero (Fig. 1, top, and Fig. 2, left), indicating a negligible rate for the reverse reaction. Thus an initial equilibrium binding is not likely to be established between 15-HPETE with either Fe- or Mn-PGHS, similar to the situation with horseradish peroxidase (1, 17, 26).

Formation of Intermediate II is an intramolecular electron transfer process in the two-step sequential mechanism (Equation 3), so the rate constant should be independent of the hydroperoxide used. However, as discussed above, the peroxidase region can interact with hydrophobic compounds. It is possible that binding of various peroxides or the product alcohols leads to some persistent perturbation of the structure of Intermediate I, and influences the rate of electron transfer from the specific tyrosine in the cyclooxygenase site to the porphyrin pi -cation radical. The distance between the porphyrin edge and the phenyl moiety of Tyr-385 are within 5 Å (22), so that perturbation of the heme orientation by variations in fatty acid peroxide or alcohol structure is plausible. Such a perturbation may be reflected in the much larger entropy change of this reaction step when 15-HPETE was the substrate as compared with EtOOH (Table III). Perturbation by product of this early intramolecular redox step would not be detected in steady-state assays, where the rate-limiting step is the regeneration of resting enzyme by reducing cosubstrate.

The similarities in the peroxidase kinetics reported here for Fe- and Mn-PGHS suggest that the two enzymes have similar mechanisms and intermediates. Turning attention to the intermediates in the Mn-PGHS reaction, observations from the literature allow us to hypothesize on their structures. Mn(V) and Mn(IV) metalloporphyrin model compounds have been studied in aqueous and organic/aqueous solvent systems (27, 28). The optical spectra of Mn(V) model compounds exhibit a Soret band at 418 nm (27, 28). The spectral line shape of Mn(IV) compounds are similar, but have a more intense Soret band. Accordingly, the spectra reported here for Intermediates I and II are consistent with those expected for Mn(V) and Mn(IV) species, respectively. It is unlikely that Mn-PGHS Intermediate I contains a porphyrin pi -cation radical, since it would be expected to give a broad and undefined spectrum (29), quite unlike that observed here (Fig. 3). Thus, Intermediate I in the Mn-PGHS peroxidase reaction is probably a Mn(V)=O species whose formal +5 charge is localized on the metal, rather than a Mn(IV) pi -cation radical. Intermediate II is most likely the corresponding Mn(IV)=O species obtained by one-electron reduction at the metal center. Presumably, since the manganese enzyme has cyclooxygenase activity, this reduction is intramolecular and produces the tyrosyl radical needed for activity.

It should be noted that the present studies concern the formation of Intermediates I and II, and not with the conversion of these intermediates back to resting enzyme. Although parallel mechanisms were found for the initial peroxidase steps in Mn-PGHS and Fe-PGHS, it remains possible that reformation of resting enzyme proceeds by different mechanisms in the two enzymes. Evidence to support divergent reductive pathways back to resting enzyme comes from the finding that Mn-PGHS can oxidize the product alcohol to a keto compound, whereas Fe-PGHS does not (7, 9, 30). In summary, our observations indicate that Mn-PGHS follows the same overall peroxidase reaction steps as Fe-PGHS, as depicted in Fig. 7. Both form an initial high oxidation state Intermediate I with a formal charge of +5 and a subsequent Intermediate II with a formal charge of +4. However, the rate of formation of the first intermediate is much slower in Mn-PGHS, possibly due to a more tightly bound water molecule as the sixth ligand in Mn-PGHS. For 6-coordinate Fe-PGHS, in contrast, the weakly bound water or histidine distal ligands are easily displaced by the peroxide (31). Intermediate I for Mn-PGHS does not appear to be a porphyrin pi -cation radical species, unlike the Fe(V) PGHS species, but is more consistent with a Mn(V) configuration. As with Fe-PGHS, Mn-PGHS forms a peroxidase-associated radical (9). Kinetic correlation of the initial radicals with PGG2/PGH2 formation has been demonstrated for both Fe- and Mn-PGHS (9, 19). The structure of the Mn-PGHS radical is sensitive to pretreatment with tetranitromethane, just as found with the Fe-PGHS radical (4, 9). The significant reduction in the rate of the first step in the peroxidase cycle after replacement of iron by manganese can account for the 25-fold decrease of peroxidase activity observed in steady-state assays (9). Taken with the slower reaction with hydroperoxides, the increased sensitivity to cyclooxygenase inhibition by glutathione peroxidase after substitution with Mn-PPIX (7, 9) also fits well with the current hypothesis that the first two steps of PGHS peroxidase catalysis are integral parts of initiating cyclooxygenase catalysis. This detailed comparison of the peroxidase kinetics of Mn- and Fe-PGHS has provided valuable insights into how this physiologically important enzyme harnesses the energy released in hydroperoxide decomposition to initiate fatty acid oxygenase catalysis.


Fig. 7. Comparison of changes in active site in the peroxidase reaction mechanism of Fe- and Mn-PGHS. The metalloporphyrin is depicted edge-on, with the proximal ligand below, and the distal ligand above, the metal center. The redox state of the metal is indicated. The heavy dots depict free radicals localized on the porphyrin (Fe-PGHS Intermediate I) or on tyrosine or another amino acid residue (Fe-PGHS and Mn-PGHS Intermediate II).
[View Larger Version of this Image (13K GIF file)]



FOOTNOTES

*   This work was supported by United States Public Health Service Grants GM44911 (to A.-L. T.), GM30509 (to R. J. K.), and GM27276 (to H. E. V. W.). A preliminary report of part of this study was presented at the American Society for Biochemistry and Molecular Biology 85th Annual Meeting, May 21-25, 1994, Washington, D. C.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Div. of Hematology, University of Texas Health Science Center, P. O. Box 20708, Houston, TX 77225. E-mail: atsai{at}heart.med.uth.tmc.edu.
par    Present address: Roche Bioscience, Mail Stop S3-1, 3401 Hillview Ave., Palo Alto, CA 94304.
1   The abbreviations used are: PGHS, ovine prostaglandin H synthase-1; Mn-PPIX, mangano protoporphyrin IX; Fe-PGHS, PGHS reconstituted with heme; Mn-PGHS, PGHS reconstituted with MnPPIX; 15-HPETE, 15-hydroperoxyeicosatetraenoic acid; EtOOH, ethyl hydrogen peroxide; PGG2, prostaglandin G2; PGH2, prostaglandin H2; PPHP, trans-5-phenyl-4-pentenyl-1-hydroperoxide; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; SVD, singular value decomposition.
2   A.-L. Tsai and R. J. Kulmacz, unpublished results.

ACKNOWLEDGEMENTS

We thank Dr. Graham Palmer and Dr. John S. Olson for very helpful discussions.


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