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Originally published In Press as doi:10.1074/jbc.M610785200 on April 1, 2007

J. Biol. Chem., Vol. 282, Issue 22, 16681-16690, June 1, 2007
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Unique Peroxidase Reaction Mechanism in Prostaglandin Endoperoxide H Synthase-2

COMPOUND I IN PROSTAGLANDIN ENDOPEROXIDE H SYNTHASE-2 CAN BE FORMED WITHOUT ASSISTANCE BY DISTAL GLUTAMINE RESIDUE*

Shizuo Ichimura{ddagger}§, Takeshi Uchida{ddagger}||, Shuhei Taniguchi§, Shusuke Hira§, Takehiko Tosha§, Isao Morishima§, Teizo Kitagawa1, and Koichiro Ishimori{ddagger}||**2

From the {ddagger}Division of Chemistry, Graduate School of Science, Hokkaido University, Hokkaido 060-0810, Japan, §Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan, Okazaki Institute for Integrative Bioscience, Okazaki 444-8787, Japan, ||Division of Chemistry, Faculty of Science, Hokkaido University, Hokkaido 060-0810, Japan, and **Institute for Molecular Science, Okazaki 444-8585, Japan

Received for publication, November 21, 2006 , and in revised form, March 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H2, but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal glutamine (Gln-189) mutants, Arg (Gln->Arg), Asn (Gln-> Asn), and Val (Gln-> Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574) suggested that the Gln residue might serve as a functionally equivalent residue to Arg, our current results clearly showed that the peroxidase activity of the Val and Asn mutants was comparable with that of the wild-type enzyme. In addition, the Fe-C and C-O stretching modes in the CO adduct were almost unperturbed by the mutation, implying that Gln-189 might not directly interact with the heme-ligated peroxide. Rather, the peroxidase activity of the Arg mutant was depressed, concomitant with the heme environmental change from a six-coordinate to a five-coordinate structure. Introduction of the bulky amino acid residue, Arg, would interfere with the ligation of a water molecule to the heme iron, suggesting that the side chain volume, and not the amide group, at position 189 is essential for the peroxidase activity of PGHS-2. Thus, we can conclude that the O-O bond cleavage in PGHS-2 is promoted without interactions with charged side chains at the peroxide binding site, which is significantly different from that in typical plant peroxidases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin endoperoxide H synthase (PGHS,3 also known as cyclooxygenase or COX) is a membrane-bound heme-containing protein catalyzing the first committed step in prostanoid biosynthesis (1-3) including two sequential enzymatic reactions, bis-oxygenation of arachidonic acid to prostaglandin G2 (PGG2) (cyclooxygenase reaction) and the reduction of PGG2 to prostaglandin H2 (PGH2) (peroxidase reaction). PGH2 is, then, further metabolized to various kinds of prostaglandins, prostacyclins, and thromboxanes by the appropriate synthase (2). Two isoforms of PGHS have been discovered thus far, PGHS-1 and PGHS-2, both of which are composed of ~600 amino acid residues, sharing more than 60% sequence identity (4, 5), and their crystal structures are essentially superimposable (6-8).

Although both the PGHS isozymes function as a homodimer of ~70-kDa subunits and have similar catalytic properties, they have distinctly different biological functions. PGHS-1 is a "housekeeping" enzyme, expressed constitutively in most tissues, and produces prostaglandins to regulate cellular responses to hormonal stimulation and to regulate vascular homeostasis. PGHS-2, in contrast, is inducibly expressed in response to cytokines (9), growth factors (10), tumor promoters (11), and inflammatory stimuli (12) and produce prostaglandins, which play various kinds of roles in mitogenesis and inflammation. PGHS inhibitors have shown promise in the prevention of colon cancer (13), Parkinson disease (14) and Alzheimer disease (15, 16). Although inhibitors of PGHS-2 are effective anti-inflammatory agents that also reduce fever and pain (17), concomitant inhibition of PGHS-1 causes undesirable side effects, such as gastric ulcer formation. Therefore, investigation of the functional property of PGHS-2 is profoundly significant to develop a selective inhibitor of the catalytic activity of PGHS-2.

The catalytic reaction mediated by PGHS is shown in Fig. 1, termed the "branched chain" mechanism, and depicts the relationship between the cyclooxygenase and peroxidase reactions (18). First, the heme group at the peroxidase site of PGHS undergoes a two-electron oxidation by a hydroperoxide, such as PGG2, yielding a corresponding alcohol, such as PGH2, and an oxyferryl heme cation radical (Compound I, Fig. 1a). In the next step, Compound I is reduced by Tyr-371 for PGHS-2 to produce an oxyferryl heme and a tyrosyl radical (Intermediate II) (Fig. 1b). Finally, the tyrosyl radical at Tyr-371 abstracts a 13-pro-S-hydrogen from arachidonic acid to begin the cyclooxygenase cycle of oxygen insertion and cyclization reactions (Figs. 1, c and d). Although neither the identity nor the source of the hydroperoxide necessary to initiate the first heme oxidation in vivo are known, in vitro hydroperoxides contaminating commercial fatty acid substrate can initiate the process, and once started, a newly generated hydroperoxide becomes available to continue the process as necessary. Resting enzyme can be regenerated by reducing Compound I to Compound II and then Compound II with a cosubstrate (Fig. 1). Once the cyclooxygenase reaction is initiated, it can operate independently of the peroxidase cycle (19). Thus, the formation of Compound I in PGHS is essential for initiation of cyclooxygenase activity.


Figure 1
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  		FIGURE 1.
Branched-chain radical mechanism for human PGHS-2 peroxidase and cyclooxygenase catalysis based on the proposal by Ruf and co-workers (18). The heme prosthetic group is oxidized by a hydroperoxide (PGG2) yielding the corresponding alcohol (PGH2) and an oxyferryl heme radical cation (Compound I) (step a). Compound I is reduced by the endogenous Tyr-371 to generate the Intermediate II (oxyferryl and a Tyr radical) (step b). The subsequent Tyr-371 radical abstracts the 13-pro-S-hydrogen of arachidonic acid (AA) (step c), and then the arachidonic acid radical (AA·) undergoes the insertion of two molecules of oxygen insertion and the cyclization reactions resulting in the PGG2 formation (step d).

 
The mechanism of Compound I formation in peroxidases has been thoroughly investigated with a central focus on cytochrome c peroxidase and horseradish peroxidase (HRP) over the years (20-24). The molecular mechanism of H2O2 activation by heme peroxidases was first hypothesized by Poulos and Kraut (25, 26). They proposed the term "Poulos-Kraut model," shown in Fig. 2A, which had been developed by numerous mutational studies in cytochrome c peroxidase and HRP. In their mechanism the conserved His-42 in the distal heme pocket of HRP primarily assists the formation of a putative iron-peroxide complex by deprotonating H2O2 and the heterolysis of the oxygen-oxygen bond by protonating the terminal oxygen atom. In addition, the conserved distal Arg-38 forms a hydrogen bond with the iron-bound H2O2, that is supposed to induce polarization of the oxygen-oxygen bond to facilitate heterolytic cleavage (25).

These two key amino acid residues, His and Arg, in the distal side are conserved in almost all plant peroxidases (27). Interestingly, the distal His is conserved in PGHS-2, whereas the distal Arg is not present in the distal pocket, and instead, Gln-189 is located at the corresponding position (Fig. 2B) (7). Previously, Marnett and co-workers (28) reported that the mutation of Gln-189 to Val showed a drastic decrease in peroxidase activity by 2 orders of magnitude and also a loss of cyclooxygenase activity, proposing that the neutral amide group of Gln-189 is responsible for the peroxidase reaction in PGHS-2. However, a recent resonance Raman study for the CO adduct of PGHS-1 suggested that no hydrogen bond is formed between iron-bound CO and the surrounding amino acid residues (29). In addition, the x-ray structure of PGHS-2 revealed that the amide group in Gln-189 is located relatively far from the heme iron (6.26 Å) (7) compared with the guanidinium group of Arg in HRP (4.61 Å) (30). These results allow us to speculate that Gln-189 would not interact with the iron-bound peroxide and/or that the interaction between Gln-189 and peroxide would not be essential to facilitate the O-O bond cleavage of the peroxide. Therefore, the functional significance of Gln-189 on peroxidase activity of PGHS-2 is still controversial.

In this study, to reinvestigate the role of Gln-189 in the peroxidase activity of PGHS-2, we prepared three mutant PGHS-2s, Q189R, Q189V, and Q189N, in which the amide group replaced, completely removed, or retained the guanidinium group, respectively. Here, we used absorption and resonance Raman spectroscopies along with kinetic and steady state analysis of the peroxidase reaction mediated by PGHS-2 to examine the functional significance of Gln-189. We found that the loss of the amide group of Gln-189 slightly increased the rate of Compound I formation without structural perturbations around the heme environment, implying that PGHS-2 does not need the interaction of Gln-189 with the iron bound peroxide, which is a unique molecular mechanism for the peroxidase reaction.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—General molecular biology supplies were obtained from Invitrogen, Qiagen (Valencia, CA), and Amersham Biosciences. General chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Expression of Human Recombinant His-tagged PGHS-2—A cell line from Spodoptera frugiperda (Sf21) was used for expression of the recombinant His-tagged PGHS-2. Construction of the His-tagged PGHS-2 cDNA, site-directed mutagenesis, and construction of recombinant baculovirus for infection were carried out as described previously (31). The Sf21 cells were cultured at 27 °C in Grace's insect medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 IU/ml penicillin G (Meiji Seika Kaisha), and 100 µg/ml streptomycin (Meiji Seika Kaisha) in tissue culture dishes (10 cm, BD Biosciences, Falcon) or a spinner flask (1 liter, Wheaton Science Products). For the suspension culture, the initial density of the Sf21 cells was 3-5 x 105 cells/ml in the spinner flask, and the spinning speed of the propeller was 150 rpm. When the density of the cells reached 1.5-2 x 106 cells/ml in 500-600 ml of medium after incubation for 2-3 days, the recombinant baculovirus stock solution was added to the medium (31, 32). For the monolayer culture, 9 x 106 Sf21 cells were grown overnight in the tissue culture dishes. Subsequently, the recombinant baculovirus stock solution was added to the dishes. In both the suspension culture and the monolayer culture, the final concentration of the baculovirus was 0.25-0.5% of the stock solution. Because the added volume of the baculovirus depends on the concentration of the stock solution, it is necessary to check the expression of the protein in various baculovirus concentrations before utilizing it for protein expression studies. After infection, Sf21 cells were incubated at 27 °C for 60-72 h. Cells were harvested by centrifugation at 3500 rpm for 5 min and washed with the phosphate buffer once. Cells were stored as a pellet at -80 °C for further purification.


Figure 2
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  		FIGURE 2.
The proposed mechanism of Compound I formation in peroxidases (A) and heme active site structures in HRP and PGHS-2 (B). A, the numbering of the amino acid residues is for HRP, although the original proposal was based on the structure of cytochrome c peroxidase. His-42 and Arg-38 facilitate the heterolytic cleavage of the O-O bond by the formation of a hydrogen bond in Compound 0, an intermediate for Compound I formation (25). His-42 acts as a general acid-base catalyst and translocates a proton from the proximal to the distal oxygen. The positively charged guanidinium side chain of Arg-38 induces polarization of the oxygen-oxygen bond in the iron-ligated peroxide. B, some of the amino acid residues located near the heme are depicted. The structure of HRP (a) and PGHS-2 (b) are based on the x-ray data sets of 1ATJ (59) and 5COX (7), respectively. Both enzymes have His in the distal region (distal His) and another His as a residue coordinated to the heme iron (proximal His). The significant residue proposed for the peroxidase reaction of HRP is Arg in the distal region, corresponding to Gln of PGHS-2.

 
Purification of His-tagged PGHS-2—Sf21 cells were suspended in homogenization buffer (25 mM Tris-HCl, 0.25 M sucrose, pH 8.0, containing Complete EDTA-free Protease Inhibitor Mixture (Roche Applied Science)) and then homogenized by a Dounce homogenizer after soaking in homogenization buffer for 10 min. The volume of homogenization buffer was three times that of the pellet volume. The pellet was collected by centrifugation at 18,000 rpm for 40 min and rehomogenized in the same buffer. CHAPS, a detergent, was added at 1% (w/v) to remove the membrane protein, and the mixture was stirred for 2 h at 4 °C. Phenylmethanesulfonyl fluoride was added to 50 µg/ml from a 50 mg/ml stock solution in methanol every 30 min. The precipitation was discarded after centrifugation at 18,000 rpm for 40 min, and the supernatant was incubated with a Ni-NTA resin (Qiagen) for 3 h at 4 °C with rotation. The Ni-NTA resin was equilibrated with an equilibrating buffer (25 mM Tris-HCl, pH 8.0, 0.4% CHAPS) before incubation. The volume of the Ni-NTA resin was 1 ml per 10 ml of the supernatant. After incubation, the solution was centrifuged at 1000 rpm for 3 min, and then the supernatant was removed. The resin was washed with 10 volumes of the washing buffer (25 mM Tris-HCl, pH 8.0, 20 mM imidazole, 0.4% CHAPS) 10 times (centrifuged at 1000 rpm for 3 min). PGHS-2 was eluted with 2 volume of an elution buffer (25 mM Tris-HCl, pH 8.0, 50 mM imidazole, 0.4% CHAPS) for 1.5 h at 4 °C with rotation. After eluting 3 times, the eluate was concentrated to 1 ml and exchanged with a reaction buffer (50 mM Tris-HCl, pH 7.5, 0.1% CHAPS) by Amicon Ultra-15 Centrifugal Filter Units (nominal molecular weight limit 30 kDa, Millipore). The concentration of the purified enzyme was calculated by using an extinction coefficient of 74.7 mM-1 cm-1 at 280 nm (33), and the purity was assessed by SDS-PAGE.

Reconstitution of ApoPGHS-2 with Hemin—The PGHS-2 was purified as an apoenzyme because the heme group of PGHS-2 is relatively loosely bound to the enzyme (Kd ≥ 1 µM) (34) and released from the protein during purification. To prepare the holoenzyme, the purified protein was reconstituted with hemin chloride (Sigma). A hemin solution (~1mM) was prepared by dissolving in dimethylformamide, and its concentration was calculated exactly by using an extinction coefficient of 58.4 mM-1 cm-1 at 386 nm (35). Then, 1 eq of the hemin solution was added to apoPGHS-2. Excessive heme was removed by passing through a NAP-5 column (Amersham Biosciences). Enzyme concentration was calculated by using an extinction coefficient of 129 mM-1 cm-1 at 407 nm (36).

Spectroscopic Measurements—Electronic absorption spectra of the PGHS-2 were recorded with a UV-visible spectrophotometer (Lambda 950, PerkinElmer Life Sciences) at room temperature. Resonance Raman spectra excited at 413.1 nm were measured with a 75-cm single monochromator (SPEX750M, Jobin Yvon) equipped with 2400 grooves/mm holographic grating and a liquid nitrogen-cooled CCD detector (Spec10:400B/LN, Roper Scientific). The continuous wave light at 413.1 nm was obtained from a krypton ion laser (BeamLok 2060, Spectra Physics). To prevent samples from photo-damage, sample cells were rotated at 2000 rpm. The Raman shifts were calibrated with indene, CCl4, acetone, and an aqueous solution of potassium ferrocyanide, which provides an accuracy of ±1cm-1. The sample concentration was ~30 µM. The reduced enzyme was prepared by adding a small amount of a fresh sodium dithionite solution to the ferric enzyme solution in anaerobic conditions. The ferrous carbonmonoxy (CO)-bound form of PGHS-2 was obtained by flushing CO gas to the ferrous samples.


Figure 3
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  		FIGURE 3.
Resonance Raman spectra in the Fe-C ({nu}Fe-C) and C-O ({nu}C-O) stretching modes for ferrous-12C16O (solid line) and -13C18O (dotted line) forms of wild-type (a), Q189R (b), Q189N (c), and Q189V (d) PGHS-2. A, Fe-C stretching mode ({nu}Fe-CO). B, C-O stretching mode ({nu}C-O). Spectra were recorded with the 413.1 nm excitation at 0.05 milliwatt. Samples were in 50 mM Tris-HCl at pH 7.5 containing 0.1% CHAPS. Sample concentration was ~30 µM.

 
Peroxidase Activities under Steady State Conditions—A steady state peroxidase activity was evaluated by measuring absorbance changes of a reducing cosubstrate, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), whose oxidation reaction was monitored at 414 nm with a UV-visible spectrophotometer (Lambda 950, PerkinElmer Life Sciences) at 20 °C (20). PGHS-2 was added to a solution containing 1 mM ABTS and peroxide (15-hydroperoxyeicosatetraenoic acid (HPETE) or H2O2) in 500 µl of the reaction buffer (50 mM Tris-HCl, pH 7.5, 0.1% CHAPS) until the final concentration of PGHS-2 was 20 nM. The oxidation rate was expressed as µM·s-1 by using a molar absorption coefficient of the oxidation products of ABTS (36.0 mM-1 cm-1) (20). The initial oxidation rate (Vi) was determined by a linear fit of the absorbance change at 414 nm. For the reaction of 15-HPETE, the concentration of the oxidant was 10 µM. For the reaction of H2O2, the kinetics parameters, Vmax and Km were obtained by fitting the plot of Vi against the various concentrations of H2O2 with the Michaelis-Menten equation (Equation 1).

Formula(Eq.1)

The concentration of H2O2 was assayed by using an extinction coefficient of 43.6 M-1 cm-1 at 240 nm (37).

Furthermore, the peroxidase reaction mechanism of PGHS-2 can be described by Scheme 1,

Formula(SCHEME1)

whose pattern mirrors that of other well known mechanisms found for HRP and other hemoprotein peroxidases. This mechanism was used as the basis for quantitative analysis of the reaction kinetics, particularly of the initial reaction of PGHS-2 with H2O2. The reaction mechanism can be approximated by a simple expression using k to represent the rate constant for the rate determining step. The initial velocity (Vi) can be described as Equation 2 by analogy to the Michaelis-Menten formula. The kinetic parameters Vmax and Km are given by Equations 3 and 4, respectively.

Formula(Eq.2)

Formula(Eq.3)

Formula(Eq.4)

Assuming that the value of k-1 is quite small, Equation 4 can be simplified. Consequently, the rate constant of Compound I formation (k1) and kcat was obtained from Equations 5 and 6, respectively (38).

Formula(Eq.5)

where k[ABTS] >> k-1

Formula(Eq.6)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Interaction of Gln-189 with Heme-bound Ligand—Almost all the peroxidases have Arg in the distal heme pocket, which forms a hydrogen bond with the iron-bound peroxide to facilitate the scission of the O-O bond. Intriguingly, PGHS-2 has Gln (Gln-189), not Arg, at the corresponding position even though it also has peroxidase activity. The distal Gln for PGHS-2 is, therefore, supposed to be involved in peroxidase activity similar to distal Arg in peroxidases. To examine the interactions between the iron-bound ligand and surrounding amino acid residues, we used the Fe-C ({nu}Fe-C) and C-O ({nu}C-O) stretching modes as a marker. Polar interactions between CO and protein residues modulate the extent of back-donation from the Fe d{pi} orbital to the CO {pi}* orbital, which can be monitored by the Fe-C and C-O stretching frequencies (39). Fig. 3A illustrates the low frequency region of the resonance Raman spectra with 413.1-nm excitation for the CO adducts of wild-type and mutant PGHS-2. In the spectrum of wild-type PGHS-2, a CO-isotope sensitive band was observed at 496 cm-1 that was downshifted to 490 cm-1 upon substitution of 13C18O(dotted line in Fig. 3A) and can be assignable to the Fe-C stretching mode (Fig. 3Aa). This Raman spectrum was quite different from those reported for PGHS-1 (29, 40). In PGHS-1, two Fe-C stretching modes were observed at 502 and 529 (40) and at 496 and 531 cm-1 (29) that are similar to those observed in HRP (516 and 539 cm-1), indicating the presence of interactions between iron-bound CO and distal amino acid residues.


Figure 4
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  		FIGURE 4.
The initial reaction rate (Vi) of ABTS oxidation of wild-type PGHS-2 (a), Q189R (b), Q189N (c), and Q189V (d) as a function of the concentration of H2O2. Vi was determined from the time courses of absorbance at 414 nm after the mixing of 20 nM concentrations of the enzymes with 50, 100, 200, 500, and 1000 µM of H2O2 at 20 °C in 50 mM Tris buffer, pH 7.5, containing 0.1% CHAPS. The solid curves represent the least squares fits by the Michaelis-Menten equation (Equation 1).

 
A small shoulder appeared at 524 cm-1 for PGHS-2. Even though the spectrum was measured at an extremely low laser power (~0.05 milliwatt), its intensity was dependent on the excitation laser power (data not shown), suggesting that this Raman band is derived from the photo-dissociated species. Because the 5-coordinate (5c) heme-CO species has been observed at 521-524 cm-1 (41, 42) and it is sometimes hardly photo-dissociated (43), we assigned this band to the photo-dissociated 5c-CO species. The Asn and Arg mutants also showed {nu}Fe-C at 496 cm-1, whereas the Val mutant showed a slight downshift in {nu}Fe-C. The mutational effects on the Fe-C stretching mode are so small, implying that the distal amino acid residue has no or only weak interactions with the iron-bound ligand in PSHS-2.

In the high frequency region of the resonance Raman spectra for the CO-bound PGHS-2, a broad band appeared at 1961 cm-1 (Fig. 3B) that can be assigned to the C-O stretching vibration from the same species that gives rise to {nu}Fe-C of 496 cm-1. The observed frequency of {nu}C-O in PGHS-2 is much higher than that of typical hemoproteins having interactions between the ligand and the surrounding amino acid residues such as CO-bound myoglobin (1944 cm-1) (44) or HRP (1906 cm-1) (45) and is rather close to hemoproteins lacking these interactions (46). In the Gln-189 mutant enzymes, the {nu}C-O band showed a low frequency shift, but the deviations of the {nu}C-O bands are small, indicating that the heme environmental structure of these CO-ligated PGHS-2 mutants would be similar to that of the wild-type enzyme.

Peroxidase Activity for Fatty Acid Hydroperoxide—The effect of the Gln-189 mutation on the peroxidase activity of PGHS-2 was investigated using 15-HPETE, which is structurally similar to the native hydroperoxide substrate of PGHS, as an oxidant, and ABTS as a reducing cosubstrate. The kinetic assay was performed under a steady state condition with 10 µM 15-HPETE. The initial rate constant (Vi) was estimated by the change in absorbance at 414 nm, at which the produced ABTS cation radical has an intense absorption band (47); the obtained values are listed in Table 1. Vi of wild-type PGHS-2 was 0.046 µM·s-1, which is almost identical to that previously reported (28). Vi of the Q189R mutant was decelerated by about 10-fold compared with that of wild-type enzyme, which is also qualitatively consistent with a previous report (28). Interestingly, replacement of the distal Gln with Arg inhibits peroxidase activity in PGHS-2, although the distal Arg is conserved in almost all plant peroxidases. In contrast, Vi increased about 10-fold in the Q189N and Q189V mutants. Despite no electrostatic interaction of Val with the iron-bound peroxides, the Q189V mutant has an enhanced peroxidase activity, suggesting that the amide group at position 189 does not affect the acceleration of the peroxidase reaction with the native peroxide substrate in PGHS-2.


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  		TABLE 1
Initial rates (Vi) of the ABTS oxidation by PGHS-2 with 10 µM 15-HPETE

 
Determination of Kinetic Parameters for Peroxide Reaction with Hydrogen Peroxide—Despite changes in the peroxidase activity of the Gln-189 mutant enzymes, the accurate determination of the Vi value in the reaction with fatty acid hydroperoxide was difficult due to the irreversible inactivation of PGHS-2 by the oxidant itself, which prevented a linear increase in the amount of the ABTS cation radical (48). Therefore, to analyze the mutational effects of Gln-189 on the peroxide activity in detail, we used hydrogen peroxide as an oxidant and estimated the kinetic parameters of the Michaelis-Menten constants, Km and kcat, and the rate constant (k1) for Compound I formation. In PGHS-2, the iron (IV) porphyrin radical, Compound I, is rapidly transferred to the cyclooxygenase active center, Tyr-371, which makes the direct determination of the k1 value so difficult. However, Equation 5 can give the k1 value as previously reported (38).

Fig. 4 shows plots of the initial oxidation rate (Vi) under various H2O2 concentrations (50-1000 µM) for wild-type and Gln-189-mutated PGHS-2. The observed data were fit by Equation 1 to give Vmax and Km values, then k1 and kcat were calculated using Equations 5 and 6, respectively. The obtained kinetic parameters are summarized in Table 2. In wild-type PGHS-2, kcat, Km, and k1 are 49 s-1 1.9 mM, and 2.7 x 104 M-1 s-1, respectively. k1 for the wild-type enzyme is much lower than that of HRP (1.6 x 107 M-1 s-1) but close to that of the Arg-38 -> Leu mutant HRP (1.1 x 104 M-1 s-1) (20). Km of wild-type PGHS-2 (1.9 mM) was also similar to that of R38L HRP (8.2 mM) and far from that of wild-type HRP (1.4 x 10-2 mM) (20). These results evidently suggest the absence of the interaction between Gln-189 and heme ligand in PGHS-2.


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  		TABLE 2
The kinetic parameter from the Michaelis-Menten equation, kcat and Km, and the rate constant of Compound I

 


Figure 5
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  		FIGURE 5.
UV-visible absorption spectra for ferric forms wild-type PGHS-2 (a), Q189R (b), Q189N (c), and Q189V (d). Samples were in 50 mM Tris-HCl at pH 7.5 containing 0.1% CHAPS. The CT-band of Q189R was blue-shifted to 644 nm with decreasing the intensity of the Soret peak.

 
As clearly shown in Table 2, kcat and k1 values were lower in the Q189R mutant (8.2 s-1 and 1.2 x 104 M-1 s-1, respectively). In contrast, kcat for the Q189N mutant (52 s-1) is comparable with that of wild-type (49 s-1), and Km (0.67 mM) was slightly lower than that of wild-type enzyme (1.9 mM). k1 of the Q189N mutant increased from 2.7 x 104 to 7.3 x 104 M-1 s-1. This trend is the same as reported previously (28). In contrast to the kinetic parameters of the Q189R and Q189N mutants, the peroxidase activity of the Q189V mutant was quite different from that of a previous study (28). Although the previous study (28) reported a loss of peroxidase activity in the Q189V mutant, the present analysis clearly indicates that the kinetic parameters kcat, Km, and k1 for the Q189V mutant are quite similar to those for the wild-type enzyme.4 Considering that the Q189V mutant has a hydrophobic side chain at position 189 and it cannot form a hydrogen bond with the heme-ligated peroxide, it is safe to assume that the hydrogen bond between the side chain of Gln-189 and heme-ligated peroxide is not formed or does not affect Compound I formation in the oxidation reaction with hydrogen peroxide.

Heme Environmental Structure of Wild-type and Mutant PGHS-2—To assess the mutational effects of Gln-189 on the heme environmental structure, the electronic absorption spectra were measured. The wild-type enzyme has a Soret peak at 407 nm and the CT band at 632 nm as shown in Fig. 5. The spectral pattern of the wild-type enzyme (Fig. 5a) is similar to that of lignin peroxidase (49), manganese peroxidase (50), and PGHS-1 (51), which are known to have a six-coordinate high spin (6cHS) heme in the ferric state, indicating that the ferric wild-type PGHS-2 also has a 6cHS (52) in which a water molecule is coordinated to the heme iron as the sixth ligand. In the Q189N and Q189V mutants, the Soret peak also appeared at 407 nm. Although the CT bands of both mutants had shifted only slightly from that in the wild-type enzyme, the absorption spectra of these mutants suggest that the heme iron is also in the 6cHS. On the other hand, the Soret peak in the Q189R mutant was broad with a decreased intensity, and the CT band was red-shifted to 644 nm by 12 nm. These spectral properties are characteristic of the 5-coordinate high spin (5cHS) state (52), indicating that introduction of Arg at position 189 interferes with the coordination of a water molecule to the heme iron. The positions of the absorption maxima are listed in Table 3.


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  		TABLE 3
Absorption maxima in the Soret and visible regions of PGHS-2 (nm)

 
Resonance Raman spectroscopy is also useful for defining the coordination structure of heme. Fig. 6 displays the high frequency region of the resonance Raman spectra for the resting states of the enzymes with 413.1 nm excitation. The heme iron oxidation state marker ({nu}4) and the ligation state markers ({nu}2 and {nu}3) (53) were used to characterize the heme active site structure in PGHS-2, as reported for PGHS-1 (29, 40). {nu}4 was observed at 1370 cm-1 in the spectrum for the oxidized wild-type enzyme (Fig. 6a), consistent with the ferric heme iron as previously reported (54). The {nu}2 and {nu}3 marker bands appeared at 1556 and 1481 cm-1, respectively, characteristic of the 6cHS heme. In addition to the Raman bands derived from the 6cHS heme, {nu}3 at 1491 cm-1 corresponds to the 5cHS heme. Although the crystal structure of wild-type PGHS-2 shows no exogenous ligand coordinated to the heme iron (7), the absorption and resonance Raman spectra clearly indicate that PGHS-2 contains a significant amount of the 6cHS heme as found in PGHS-1 (29, 40), implying that a water molecule would be coordinated to the heme iron as the sixth ligand in solution. Such inconsistency between crystal structure and Raman spectra was also observed in other hemoproteins (55). The Raman bands observed in the high frequency region for the ferric form of wild-type and mutant PGHS-2 are summarized in Table 4.


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  		TABLE 4
Positions of marker bands in the resonance Raman spectra for ferric and ferrous forms of wild-type and the mutant PGHS (cm–1)

 


Figure 6
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  		FIGURE 6.
High frequency resonance Raman spectra of the ferric forms of wild-type PGHS-2 (a), Q189R (b), Q189N (c), and Q189V (d). Spectra were recorded with the 413.1-nm excitation at 5 milliwatts. Samples were in 50 mM Tris-HCl at pH 7.5 containing 0.1% CHAPS. Sample concentration was ~30 µM.

 
Despite the significant increase in the peroxidase activity using 15-HPETE and ABTS (Table 1), the Q189N and Q189V mutants exhibited almost the same Raman spectra as that of the wild-type enzyme, suggesting that these mutations did not affect the coordination structure of heme. On the other hand, the Q189R mutant showed a different Raman spectrum from that of the wild-type enzyme. The Raman marker bands were observed at 1491 cm-1 ({nu}3) and 1564 cm-1 ({nu}2), both of which can be assignable to the 5cHS state, and no bands from the 6cHS heme were detected. Therefore, in this mutant enzyme the 5cHS heme is the major coordination state that coincides with the result of the absorption spectrum.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Peroxidase Activity of the Gln-189 Mutant PGHS-2—In this study we focus on the role of Gln-189 of PGHS-2 in the peroxidase activity. By use of hydrogen peroxide as an oxidant, three kinetic parameters, k1, kcat, and Km, were obtained as shown in Table 2. Among them, k1 and kcat correspond to the rate of Compound I formation (Scheme 1) and the reactivity of Compound I (kcat = Vmax/[enzyme] = k[ABTS] (Equations 6 and 3, respectively)). k1 for the Q189N mutant increased nearly 3-fold, showing that Compound I is more easily formed by the substitution of Gln-189 for Asn. In contrast, k1 was little affected by the Gln-189 -> Val mutation. The side chain volume of Asn (114.1 Å) (56) is smaller than that of Gln (143.8 Å), whereas the volume of Val (140.0 Å) is almost the same as that of Gln. These results imply that the reduced steric hindrance for the heme ligand in the Q189N mutant results in the acceleration of Compound I formation. The reduced k1 value for the Arg (173.4 Å) mutant also supports this idea. kcat of the Q189N and Q189V mutants is identical to that of wild-type PGHS-2 (Table 2), suggesting that the reactivity of the Compound I is less sensitive to these mutations. In contrast, that of the Q189R mutant decreased by less than 20%. It is likely that the larger side chain at position 189 inhibits the access of the substrate to the active site, thus depressing the reactivity of Compound I in the mutant.

Although the kinetic parameters for the oxidation reaction with hydrogen peroxide are almost insensitive to the replacement of Gln with Val as discussed above (Table 2), the Vi value for the ABTS oxidation with 15-HPETE was enhanced by more than 10-fold from 0.046 to 0.55 µM-1 s-1 in this mutant (Table 1). The Q189N mutant also showed an increase in Vi for 15-HPETE (0.32 µM-1 s-1). Although the detailed kinetic analysis of the oxidation reaction with 15-HPETE has not yet been successful due to the inactivation of the enzyme in the presence of 15-HPETE, the observed changes in Vi can be explained as discussed next.

On the basis of Equation 2, enhanced Vi can be derived from a larger Vmax and/or a smaller Km. Here, Vmax is k[ABTS] [enzyme] (Equation 3) and is correlated with the reactivity of Compound I (Scheme 1); thus, it would be independent of the peroxide used in the experiments. The Vmax values for 15-HPETE of the Q189N and Q189V mutants are supposed to be similar to that of the wild-type enzyme (Vmax = kcat[enzyme] in our experimental conditions (Equation 6)), since kcat for hydrogen peroxide of the Q189N and Q189V mutants are almost the same as that of the wild-type enzyme (Table 2). Accordingly, Vmax would not be a major factor to enhance Vi for 15-HPETE of the Q189N and Q189V mutants. On the other hand, Km is equal to k[ABTS]/k1 under our conditions (Equation 4), and the small Km value in the mutants reflects a large k1 value for 15-HPETE, indicating that the formation of Compound I by 15-HPETE is accelerated by these mutations.

This analysis of the kinetic parameters for the peroxidase reaction clarified that Compound I can be readily formed in the Q189N and Q189V mutant enzymes and suggests that 15-HPETE can be more closely accessible to the heme iron than in the wild-type enzyme and/or that cleavage of the O-O bond is facilitated by the substitutions. The absence of interactions between amino acid residues at position 189 and iron-bound ligand suggests that the latter is less likely, although we cannot rule out the possibility without rapid kinetic studies. In the Q189N mutant, the smaller amino acid residue would have an advantage in the facile access of the larger oxidant to heme. The residue volume of Val is, however, almost the same as that of Gln and larger than that of Asn, thus, steric hindrance cannot explain the enhanced activity. One of the factors that enhances peroxidase activity for 15-HPETE in the Q189V mutant would be a hydrophobic interaction between the peroxide and amino acid residues in the substrate binding site. Because 15-HPETE has a large hydrophobic group, hydrophobic interactions between 15-HPETE and the side chain of Val would increase the affinity for 15-HPETE in the Q189V mutant, leading to the enhanced peroxidase activity.

In sharp contrast to the enhanced peroxidase activity observed in the Q189N and Q189V mutants, the Arg mutant (Q189R) showed a reduced peroxidase activity for the oxidation with both hydrogen peroxide and 15-HPETE (Tables 1 and 2). The decrease in Vi for 15-HPETE could result from a smaller Vmax and/or larger Km as discussed above (Equation 2). Vmax is related with kcat (Equation 6), and it would be independent of peroxides (Equation 3). Because the Arg mutant has a smaller kcat for hydrogen peroxide (Table 2), kcat for 15-HPETE in this mutant is likely to be smaller than that of the wild-type enzyme, which leads to a smaller Vmax and then a smaller Vi for 15-HPETE (Equation 2). Accordingly, we can propose that the larger residue volume (173.4 Å) of Arg would interfere with the proper binding of 15-HPETE or hydrogen peroxide for the peroxidase reaction in this mutant. This is supported by the coordination structure of the Q189R mutant in which a water molecule cannot bind to the heme iron in the resting state as discussed later.

Heme Environmental Structure of PGHS-2—The enzymatic analysis clearly showed that removal of the amide group of Gln-189 (Q189V) does not lead to the drastic changes in the oxidation activity of PGHS-2 and that the presence of the amide group at this position (Q189N) is not essential for the peroxidase reaction. This observation is supported by the resonance Raman spectra of the mutants. The resonance Raman spectra for the resting states of the Q189N and Q189V mutants are almost identical to that of the wild-type enzyme whose heme is in a mixture of 5cHS and water-bound 6cHS state. This implies that the amide group of Gln-189 does not or only weakly, if at all, interacts with the ligand of heme and that the amide group at position 189 does not affect the heme environment structure.

Such weak or no interaction between the ligated peroxide and Gln-189 was also evident by the frequency of {nu}Fe-C in the ferrous CO-bound PGHS-2. Although wild-type PGHS-2 shows a single {nu}Fe-C band at 496 cm-1, HRP has been reported to have two distinct Fe-C stretching modes at 516 and 539 cm-1 (57). For the conformer giving {nu}Fe-C at 516 cm-1, the iron-bound CO is supposed to be hydrogen-bonded to His-42, whereas {nu}Fe-C at 539 cm-1 corresponds to the conformer having a hydrogen bond between CO and Arg-38. A low {nu}Fe-C below 500 cm-1 was observed for the CO adducts of mutant myoglobin in which the distal His is replaced with leucine (58), corresponding to no interaction between ligated CO and the amino acid residues in the ligand binding site. Accordingly, the single Raman band observed at 496 cm-1 in wild-type PGHS-2 also supports the absence of an interaction between heme-ligated CO as was also observed in the x-ray structure (7).

In contrast to the mutation in Asn or Val, replacement of Gln-189 with Arg led to the complete conversion of the mixture of 5cHS and 6cHS species to 5cHS, showing the displacement of the iron-coordinated H2O molecule from the heme iron. In PGHS-2, the distance between the C{delta} atom of Gln-189 and heme iron was 4.98 Å (7), and the position of the C{delta} atom of Gln-189 corresponded to the C{delta} atom of Arg-38 and the distance to the heme iron is 4.92 Å in HRP (59), suggesting that an increase in the side chain volume at position 189 by the mutation of Gln to Arg suppresses the access of H2O to the heme iron. A similar steric effect was observed for the mutation of the distal His, His-64, in myoglobin (60, 61). Mutation of His-64, positioned 4.4 Å away from the heme iron, to Tyr prevented the coordination of H2O to the heme. Thus, Gln-189 would not directly interact with the ligated peroxide, as evident by the current spectroscopic results, but is rather located near the ligand binding site. Furthermore, a bulky side chain at position 189 forms a steric barrier for the binding of peroxides.

It should be noted here that the Fe-C stretching modes of PGHS-2 were significantly different from those of PGHS-1, showing that the interactions between iron bound CO and distal amino acid residues are weak in PGHS-2 and suggesting that the distal His in PGHS-2 is away from the heme iron and the heme cavity would be enlarged in PGHS-2. Such structural differences might be clues for the selective inhibition of PGHS-2 by inhibitors of the peroxidase activity because the interactions of the distal His with inhibitors and the relative position of the inhibitor to the heme iron are crucial for the inhibition activity in plant heme peroxidases (62).

Molecular Mechanism of Peroxidase Reaction in PGHS-2—Despite the lack of structural and electronic factors to promote the heterolytic O-O bond cleavage as discussed above, PGHS-2 showed a rather higher activity for 15-HPETE (36). Although the inactivation in the presence of 15-HPETE prevented us from determining the precise kinetic parameters, Km for 15-HPETE in PGHS-2 can be estimated to be less than 1/100 of that for hydrogen peroxide, and kcat is also more than 100-fold (36). Such preferential high peroxidase activity for 15-HPETE would be derived from the heme environmental structure of PGHS-2. The crystal structure of PGHS-2 revealed that this enzyme has an exposed heme binding site and an enlarged distal cavity (7). Small peroxides such as hydrogen peroxide can bind to the heme iron, but the position of the ligated peroxide would not be fixed due to a lack of interaction with amino acid residues in the distal cavity. 15-HPETE, however, has a large moiety of the fatty acid, and it can interact with some amino acid residues in the substrate binding site, which decreases conformational fluctuation of the peroxide to facilitate the O-O bond cleavage by the distal His and heme iron. A recent theoretical molecular dynamics study showed electrostatic interactions between PGHS-1 and the substrate (63).

In conclusion, the present results from the peroxidase activity and spectroscopic measurements of wild-type and Gln-189 mutated PGHS-2 clearly show that the amide group of the "distal" Gln would not participate in the O-O scission of the peroxide in PGHS-2 and that a steric hindrance at position 189 would be rather essential for the peroxidase reaction. Unlike most plant peroxidases, the distal histidine would be the only amino acid residue that can directly interact with peroxides and assist the O-O bond cleavage of peroxides in PGHS-2. A higher reactivity for the native hydroperoxide substrates in PGHS-2 would be due to the specific interactions of the side chains of the peroxide substrate with some amino acid residues in the substrate binding site, which properly orient the O-O bond of the peroxide to the catalytic amino acid residue, distal His, in PGHS-2.


   FOOTNOTES
 
* This work was supported in part by Grants-in-aid 15350101 (to K. I.) from the Ministry of Education, Culture, Science, Sports, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Toyota Physical & Chemical Research Inst., Nagakute, Aichi 480-1192, Japan. Back

2 To whom correspondence should be addressed: Division of Chemistry, Faculty of Science, Hokkaido University, Hokkaido 060-0810, Japan. Tel.: 81-11-706-2707; Fax: 81-11-706-3501; E-mail: koichiro{at}sci.hokudai.ac.jp.

3 The abbreviations used are: PGHS, prostaglandin-endoperoxide H synthase; PGG2, prostaglandin G2; PGH2, prostaglandin H2; HRP, horseradish peroxidase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); 15-HPETE, 15-hydroperoxyeicosatetraenoic acid; Ni-NTA, nickel-nitrilotriacetic acid; 5c, 5-coordinate; 5cHS, five-coordinate high spin; 6cHS, six-coordinate high spin; CT, charge transfer. Back

4 One possibility of the discrepancy would be attributed to the low heme content of the mutant in the previous study (28). We confirmed reconstitution of heme with all the proteins by using UV-visible absorption spectra as shown in Fig. 5, whereas the sputum was not reported in previous study. Back


   ACKNOWLEDGMENTS
 
We acknowledge the excellent technical assistance of Prof. Kazuhiro Iwai (Osaka City University) in construction of the expression system for PGHS-2. We also thank Prof. Satoshi Takahashi (Osaka University) for helpful comments and fruitful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Garavito, R. M., Malkowski, M. G., and DeWitt, D. L. (2002) Prostaglandins Other Lipid Mediat. 68-69, 129-152[CrossRef][Medline] [Order article via Infotrieve]
  2. Smith, W. L., DeWitt, D. L., and Garavito, R. M. (2000) Annu. Rev. Biochem. 69, 145-182[CrossRef][Medline] [Order article via Infotrieve]
  3. Smith, W. L., and Song, I. (2002) Prostaglandins Other Lipid Mediat. 68-69, 115-128[CrossRef][Medline] [Order article via Infotrieve]
  4. DeWitt, D. L., and Smith, W. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1412-1416[Abstract/Free Full Text]
  5. Hla, T., and Neilson, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7384-7388[Abstract/Free Full Text]
  6. Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249[CrossRef][Medline] [Order article via Infotrieve]
  7. Kurumbail, R. G., Stevens, A. M., Gierse, J. K., McDonald, J. J., Stegeman, R. A., Pak, J. Y., Gildehaus, D., Miyashiro, J. M., Penning, T. D., Siebert, K., Isakson, P. C., and Stallings, W. C. (1996) Nature 384, 644-648[CrossRef][Medline] [Order article via Infotrieve]
  8. Luong, C., Miller, A., Barnett, J., Chow, J., Ramesha, C., and Browner, M. F. (1996) Nat. Struct. Biol. 11, 927-933
  9. Guan, Z., Buckman, S. Y., Pentland, A. P., Templeton, D. J., and Morrison, A. R. (1998) J. Biol. Chem. 273, 12901-12908[Abstract/Free Full Text]
  10. Xie, W., and Herschman, H. R. (1996) J. Biol. Chem. 271, 31742-31748[Abstract/Free Full Text]
  11. Kujubu, D., Fletcher, B., Varnum, B., Lim, R., and Herschman, H. (1991) J. Biol. Chem. 266, 12866-12872[Abstract/Free Full Text]
  12. Zhang, Y., Shaffer, A., Portanova, J., Seibert, K., and Isakson, P. C. (1997) J. Pharmacol. Exp. Ther. 283, 1069-1075[Abstract/Free Full Text]
  13. Williams, C. S., Mann, M., and DuBois, R. N. (1999) Oncogene. 18, 7908-7916[CrossRef][Medline] [Order article via Infotrieve]
  14. Teismann, P., Tieu, K., Choi, D.-K., Wu, D.-C., Naini, A., Hunot, S., Vila, M., Jackson-Lewis, V., and Przedborski, S. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5473-5478[Abstract/Free Full Text]
  15. Nagano, S., Huang, X., Moir, R. D., Payton, S. M., Tanzi, R. E., and Bush, A. I. (2004) J. Biol. Chem. 279, 14673-14678[Abstract/Free Full Text]
  16. McGeer, P., and McGeer, E. (1999) J. Leukocyte Biol. 65, 409-415[Abstract]
  17. Crofford, L. J., Lipsky, P. E., Brooks, P., Abramson, S. B., Simon, L. S., and van de Putte, L. B. (2000) Arthritis Rheum. 43, 4-13[CrossRef][Medline] [Order article via Infotrieve]
  18. Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 321-328[Medline] [Order article via Infotrieve]
  19. Koshkin, V., and Dunford, H. B. (1999) Biochim. Biophys. Acta. 1430, 341-348[CrossRef][Medline] [Order article via Infotrieve]
  20. Rodriguez-Lopez, J. N., Smith, A. T., and Thorneley, R. N. F. (1996) J. Biol. Chem. 271, 4023-4030[Abstract/Free Full Text]
  21. Rodriguez-Lopez, J. N., Smith, A. T., and Thorneley, R. N. F. (1996) J. Biol. Inorg. Chem. 1, 136-142[Medline] [Order article via Infotrieve]
  22. Rodriguez-Lopez, J. N., Lowe, D. J., Hernandez-Ruiz, J., Hiner, A. N. P., Garcia-Canovas, F., and Thorneley, R. N. F. (2001) J. Am. Chem. Soc. 123, 11838-11847[CrossRef][Medline] [Order article via Infotrieve]
  23. Hiner, A. N. P., Raven, E. L., Thorneley, R. N. F., Garcia-Canovas, F., and Rodriguez-Lopez, J. N. (2002) J. Inorg. Biochem. 91, 27-34[CrossRef][Medline] [Order article via Infotrieve]
  24. Shintaku, M., Matsuura, K., Yoshioka, S., Takahashi, S., Ishimori, K., and Morishima, I. (2005) J. Biol. Chem. 280, 40934-40938[Abstract/Free Full Text]
  25. Poulos, T., and Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205[Free Full Text]
  26. Poulos, T., Freer, S., Alden, R., Edwards, S., Skogland, U., Takio, K., Eriksson, B., Xuong, N., Yonetani, T., and Kraut, J. (1980) J. Biol. Chem. 255, 575-580[Free Full Text]
  27. Welinder, K. G. (1992) Curr. Opin. Struct. Biol. 2, 388-393[CrossRef]
  28. Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574[Abstract/Free Full Text]
  29. Lou, B. S., Snyder, J. K., Marshall, P., Wang, J. S., Wu, G., Kulmacz, R. J., Tsai, A. L., and Wang, J. (2000) Biochemistry 39, 12424-12434[CrossRef][Medline] [Order article via Infotrieve]
  30. Berglund, G., Carlsson, G. H., Smith, A. T., Szoke, H., Henriksen, A., and Hajdu, J. (2002) Nature 417, 463-468[CrossRef][Medline] [Order article via Infotrieve]
  31. Taniguchi, S. (2004) Study on the Peroxidase Reaction Mechanism in Prostaglandin-endoperoxide H Synthase-2, Graduate School of Engineering, Kyoto University, Kyoto, Japan
  32. Smith, T., Leipprandt, J., and DeWitt, D. (2000) Arch. Biochem. Biophys. 375, 195-200[CrossRef][Medline] [Order article via Infotrieve]
  33. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) Protein. Sci. 4, 2411-2423[Abstract]
  34. Barnett, J., Chow, J., Ives, D., Chiou, M., Mackenzie, R., Osen, E., Nguyen, B., Tsing, S., Bach, C., Freire, J., Chan, H., Sigal, E., and Ramesha, C. (1994) Biochim. Biophys. Acta 1209, 130-139[CrossRef][Medline] [Order article via Infotrieve]
  35. Beaven, G. H., Chen, S. H., d'Albis, A., and Gratzer, W. B. (1974) Eur. J. Biochem. 41, 539-546[Medline] [Order article via Infotrieve]
  36. Goodwin, D. C., Rowlinson, S. W., and Marnett, L. J. (2000) Biochemistry 39, 5422-5432[CrossRef][Medline] [Order article via Infotrieve]
  37. Nielsen, K. L., Indiani, C., Henriksen, A., Feis, A., Becucci, M., Gajhede, M., Smulevich, G., and Welinder, K. G. (2001) Biochemistry 40, 11013-11021[CrossRef][Medline] [Order article via Infotrieve]
  38. Kulmacz, R. J. (1986) Arch. Biochem. Biophys. 249, 273-285[CrossRef][Medline] [Order article via Infotrieve]
  39. Spiro, T. G., and Wasbotten, I. H. (2005) J. Inorg. Biochem. 99, 34-44[CrossRef][Medline] [Order article via Infotrieve]
  40. Seibold, S. A., Cerda, J. F., Mulichak, A. M., Song, I., Garavito, R. M., Arakawa, T., Smith, W. L., and Babcock, G. T. (2000) Biochemistry 39, 6616-6624[CrossRef][Medline] [Order article via Infotrieve]
  41. Vogel, K. M., Spiro, T. G., Shelver, D., Thorsteinsson, M. V., and Roberts, G. P. (1999) Biochemistry 38, 2679-2687[CrossRef][Medline] [Order article via Infotrieve]
  42. Pal, B., Li, Z., Ohta, T., Takenaka, S., Tsuyama, S., and Kitagawa, T. (2004) J. Inorg. Biochem. 98, 824-832[CrossRef][Medline] [Order article via Infotrieve]
  43. Ohta, T., Pal, B., and Kitagawa, T. (2005) J. Phys. Chem. B 109, 21110-21117[Medline] [Order article via Infotrieve]
  44. Tsubaki, M., Srivastava, R. B., and Yu, N. T. (1982) Biochemistry 21, 1132-1140[CrossRef][Medline] [Order article via Infotrieve]
  45. Uno, T., Nishimura, Y., Tsuboi, M., Makino, R., Iizuka, T., and Ishimura, Y. (1987) J. Biol. Chem. 262, 4549-4556[Abstract/Free Full Text]
  46. Li, T., Quillin, M. L., Phillips, G. N. J., and Olson, J. S. (1994) Biochemistry 33, 1433-1446[CrossRef][Medline] [Order article via Infotrieve]
  47. Goodwin, D. C., Yamazaki, I., Aust, S. D., and Grover, T. A. (1995) Anal. Biochem. 231, 333-338[CrossRef][Medline] [Order article via Infotrieve]
  48. Ple, P., and Marnett, L. (1989) J. Biol. Chem. 264, 13983-13993[Abstract/Free Full Text]
  49. Sinclair, R., Copeland, B., Yamazaki, I., and Powers, L. (1995) Biochemistry 34, 13176-13182[CrossRef][Medline]