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J. Biol. Chem., Vol. 279, Issue 23, 24131-24140, June 4, 2004

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Horseradish Peroxidase Mutants That Autocatalytically Modify Their Prosthetic Heme Group

INSIGHTS INTO MAMMALIAN PEROXIDASE HEME-PROTEIN COVALENT BONDS^*

Christophe Colas and Paul R. Ortiz de Montellano

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280

Received for publication, February 16, 2004 , and in revised form, March 23, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The mammalian peroxidases, including myeloperoxidase and lactoperoxidase, bind their prosthetic heme covalently through ester bonds to two of the heme methyl groups. These bonds are autocatalytically formed. No other peroxidase is known to form such bonds. To determine whether features other than an appropriately placed carboxylic acid residue are important for covalent heme binding, we have introduced aspartate and/or glutamic acid residues into horseradish peroxidase, a plant enzyme that exhibits essentially no sequence identity with the mammalian peroxidases. Based on superposition of the horseradish peroxidase and myeloperoxidase structures, the mutated residues were Leu³⁷, Phe⁴¹, Gly⁶⁹, and Ser⁷³. The F41E mutant was isolated with no covalently bound heme, but the heme was completely covalently bound upon incubation with H₂O₂. As predicted, the modified heme released from the protein was 3-hydroxymethylheme. The S73E mutant did not covalently bind its heme but oxidized it to the 8-hydroxymethyl derivative. The hydroxyl group in this modified heme derived from the medium. The other mutations gave unstable proteins. The rate of compound I formation for the F41E mutant was 100 times faster after covalent bond formation, but the reduction of compound I to compound II was similar with and without the covalent bond. The results clearly establish that an appropriately situated carboxylic acid group is sufficient for covalent heme attachment, strengthen the proposed mechanism, and suggest that covalent heme attachment in the mammalian peroxidases relates to peroxidase biology or stability rather than to intrinsic catalytic properties.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mammalian peroxidases share a unique feature, the formation of two or three covalent bonds to their prosthetic heme¹ group, not found in the peroxidases of other organisms. The two common bonds in all mammalian peroxidases are ester links between a conserved glutamate and aspartate and the heme 1-methyl and 5-methyl substituents, respectively. In MPO a third bond is forged between a methionine and the carbon of the 2-vinyl substituent (1). Mutagenesis studies with LPO have demonstrated that at least one of the two ester bonds is required for catalytic activity (2). Interestingly, a single covalent link between the heme and protein is also found in most members of the CYP4A, -4B, and -4F classes of cytochrome P450 enzymes (3–7). In contrast to LPO, however, mutagenesis studies show that the heme-protein covalent bond is not essential for the catalytic activity of at least some of these P450 enzymes (8, 6). A long known example of heme covalent binding is provided by cytochrome c, in which a cysteine residue is covalently bound to each of the two porphyrin vinyl groups. Although many hypotheses have been formulated concerning the functional advantages of the links in cytochrome c, including bending of the heme, increasing the heme affinity (9), and increasing the stability of the methionyl-Fe(II) coordination (10), the question has yet to be definitively answered.

Despite mutagenesis studies of MPO and LPO (for a review, see Ref. 11), both the mechanism by which the heme covalent bonds are formed in the mammalian peroxidases and their functional advantage remain unclear. One reason for the slow progress in this area is the relative complexity and relatively low yield of the expression systems required to obtain recombinant mammalian peroxidases, as well as the sensitivity and intransigence of these peroxidases to mutations. In addition, the expression systems only yield partially or completely processed enzymes. It has therefore not been possible to study the bond-forming reaction commencing with an enzyme from which the bond is largely absent. Nevertheless, it has been unambiguously demonstrated that the ester covalent links are formed via an autocatalytic mechanism (12). It has also been shown in MPO that only a carboxylate can trigger covalent tethering of the heme to the protein (13), and in LPO that the length of the carboxylate side chain is critical for the same bond to be formed (2).

It is not known, however, whether there are requirements other than the presence of an appropriate carboxyl side chain for ester bond formation. Despite their common function and the presence of some structural homologies, mammalian peroxidases share very little sequence identity with peroxidases from other organisms (14, 15). Is the uniqueness of covalent heme attachment in the mammalian peroxidases simply a result of the presence of appropriately positioned active site carboxylic side chains, for which there is no counterpart in other peroxidases, or is the participation of other residues required? To address this question, we have introduced a carboxylate into the active site of HRP, one of the best studied peroxidases and one that does not have such a group. This monomeric 44-kDa protein is obtained in good expression yields (16, 17), is quite stable, and shares virtually no sequence identity with MPO. Furthermore, its crystal structure has been determined (18), making it possible to rationally select sites for introduction of the carboxylic acid side chain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Restriction enzymes were purchased from New England Biolabs. Hemin, H₂O₂, ABTS, guaiacol, Pronase, bis-tris propane, trifluoroacetic acid, and tissue culture grade water were purchased from Sigma. All other chemicals, including buffer components and HPLC solvents, were from Fisher Scientific. Native HRP was purchased from Roche. The monohydroxylated hemin derivatives used as HPLC standards were isolated from recombinant HRP (19) and LPO (2) according to the published procedures. H ¹⁸₂O (95% ¹⁸O) was obtained from Cambridge Isotope Laboratories (Andover, MA). High-Five^TM cells were grown in supplemented Express Five SFM^TM medium, both purchased from Invitrogen. The Sf9 cells (Invitrogen) were grown in Excell 420^TM medium purchased from JRH Biosciences (Lenexa, KS). Penicillin/streptomycin and Fungizone were purchased from the UCSF Cell Culture Facility. UV-visible absorption measurements were undertaken on a Hewlett Packard 8452A diode array detector. Analytical and preparative HPLC separations were performed on a Hewlett Packard 1090 or 1090-II apparatus equipped with a diode array detector using a Vydac protein-C4 5-µm 150 x 46-mm column or a MetaChem Kromasil C4 5-µm 100 x 46-mm column, both fitted with the appropriate guard column. LC/MS analyses were carried out either on a Perseptive Biosystems Mariner Biospectrometry work station coupled to an Applied Biosystems HPLC apparatus (solvent delivery system 1408, programmable absorption detector 785A) and connected to a 10-cm Applied Biosystems Aquabore C18 7-µm 100 x 1-mm column, or a Waters Micromass ZQ coupled to a Waters Alliance HPLC system (separation module 2695, dual wavelength absorbance detector 2487) connected to an Xterra C18 3.5-µm 21 x 50-mm column. Stopped flow kinetic measurements were made on a HighTech^TM device, equipped with an SF-61 DX2 double mixing apparatus, a CU-61 central unit, a xenon lamp powered by a PS-678 power supply, a M300 monochromator, and an SSU-60 control unit. The mixing chambers and the syringes were thermostated at 25 °C, and the data were analyzed with Kaleidagraph 3.5. Modeling was carried out with Swiss-PDB Viewer 3.7.

Cloning and Expression—The sequence GGA TCC CGG GCC CAC CAC CAC CAC CAC CAT, encoding a BamHI site and Arg-Ala-(His)₆, was added directly to the 5' end of the HRP gene, thus modifying the previously employed construct (17), and the sequence TAA TAA GGA TCC was added at its 3' end. The resulting construct was subcloned into the BamHI site of pUC19. 35-mer primers were designed for the mutagenesis with the nucleotide substitutions in the center of the sequences; only the regions with changed codons are shown (actual substitutions underlined, numbering starting at the ATG codon of HRP): L37D, ¹⁰⁵TCG ATC GAC, introducing a silent PvuI site; L37E, ¹⁰⁵TCG ATC GAA, introducing a silent PvuI site; F41E, ¹²⁰CAC GAG CAT; G69E, ²⁰⁴TTC GAG AAC; S73E, ²¹⁶AAC GAG GCC. Mutagenesis was undertaken with the QuikChange^TM kit (Stratagene, La Jolla, CA). After transformation in XL-1 Blue^TM, DNA was isolated from positive clones with the Promega Wizard Plus SV Miniprep^TM kit, and the mutated genes isolated from their pUC19 plasmids through a BamHI digestion and purified from agarose gels with the QiaEX II^TM kit (Qiagen, Valencia, CA). Ligation was accomplished at 4 °C into a pAcGP67B plasmid (Pharmingen, BD Biosciences) preliminarily digested by BamHI, dephosphorylated, and purified with the Promega Wizard DNA clean-up^TM kit. The plasmids were transformed into DH5^TM, and their sequences were verified. Medium sized cultures allowed for the isolation of larger amounts of endotoxin free plasmids though the use of the Purefection^TM purification kit (Promega) following the instructions from the manufacturer. Transfection in Sf9 cells was done with the Baculogold transfection kit (Pharmingen, BD Biosciences). A plaque assay on the transfection supernatants allowed for the isolation of recombinant virus that was subsequently amplified to a titer of 1 x 10⁸ plaque-forming units/ml using standard baculovirus protocols (20).

Expression and Purification—High-Five^TM cells at a density of 2 x 10⁶ cells/ml were supplemented with hemin (final concentration 6 µM), 1x penicillin/streptomycin, and 1x Fungizone before being infected at a multiplicity of infection of 5. At 3.5 days after infection, the cultures were harvested and centrifuged at 4 °C and 10,000 x g for 1 h. The resulting supernatant was then filtered successively through 1.2-, 0.64-, and 0.44-µm regenerated cellulose filters to remove remaining cell debris. The clarified liquid was then concentrated 12-fold by ultrafiltration over a spiral-wound regenerated cellulose membrane (molecular mass cut-off = 10 kDa, Millipore) connected to an Amicon TCF10 peristaltic pump equipped with a 2l RA2000S reservoir. The resulting solution was then made 0.4 M with solid NaCl and 20 mM with KH₂PO₄/K₂HPO₄ at pH = 8, and ultracentrifuged for 30 min at 4 °C and 100,000 x g. The supernatant was loaded onto a Ni²⁺ column (nickel-nitrilotriacetic acid-agarose, Qiagen) pre-equilibrated with 20 mM KH₂PO₄/K₂HPO₄, pH = 8, containing 0.4 M NaCl; the resin was then washed with 10 volumes, then with 5 volumes of the same buffer containing imidazole (20 mM imidazole for the wild type, and 5 mM for the mutants). Elution was achieved by increasing the imidazole concentration to 200 mM for the wild type and 100 mM for the mutants and switching to pH = 6. The colored fractions were pooled and dialyzed against 20 mM KH₂PO₄/K₂HPO₄, pH = 8, buffer for at least 14 h with at least three bath changes. The dialyzed fractions were then concentrated 12-fold by ultrafiltration over an Amicon YM10 membrane, and passed through a pre-equilibrated QFF-Sepharose (Amersham Biosciences) column. The colored fractions were concentrated again over an Amicon Ultra-4^TM centrifugal filter device (10 kDa) to 1–2 ml.

Spectrophotometric Measurements—ABTS oxidation was measured in 50 mM acetate buffer, pH = 4.5, in the presence of 250 µM H₂O₂ and 250 µM ABTS, both from freshly prepared solutions. Absorbance changes were monitored at 414 nm and specific activities were calculated using = 38,000 cm M^–1 liter^–1 for the oxidized ABTS product. For the oxidation of guaiacol, activities were measured in 50 mM KH₂PO₄/K₂HPO₄, pH = 7, in the presence of 600 µM H₂O₂ and concentrations of guaiacol up to 20 mM. The absorption at 470 nm, = 26,600 cm M^–1 liter^–1, was used to calculate guaiacol product formation. The concentration of H₂O₂ in the stock solution was assessed by measuring the absorption at 240 nm ( = 39.4 cm M^–1 liter^–1) (21). Enzymes were diluted to the appropriate concentration in bovine serum albumin (1 mg/ml) when necessary. UV-visible spectra were recorded at 21 °Cin50 mM KH₂PO₄/K₂HPO₄, pH = 7, and the RZ calculated as A_Soret/A_{278 nm}.

H₂O₂ Treatment—To the enzymes (10 µM) in 50 mM KH₂PO₄/K₂HPO₄, pH = 6.7, was added H₂O₂ (6 mM) in 2 equivalent portions every 5 min. The solutions were further incubated for another 10 min before being subjected to analysis.

HPLC Analysis—Separations were achieved using water containing 0.1% trifluoroacetic acid (solvent A) and acetonitrile containing 0.085% trifluoroacetic acid (solvent B) at a flow rate of 1 ml/min. For the analysis of undigested samples, the Vydac column was eluted with a 3-min isocratic wash of 33% solvent B, followed by a 0.5%/min linear gradient to 95% of B. For isolation of the 3-monohydroxyheme derivative, the Vydac column was eluted with a 3-min isocratic wash of 15% solvent B, followed by a 0.9%/min linear gradient to 43% of B (see above). For analysis of the 8-monohydroxyheme derivative, the Kromasil column was eluted with a 3-min isocratic wash of 15% solvent B, followed by a 0.9%/min linear gradient to 43% of B.

Characterization of the F41E Mutant Porphyrin—The F41E mutant was treated with 6 x 2 equivalents of H₂O₂ in 100 mM bis-tris propane buffer, pH = 8.2, over a period of 35 min. Disodium EDTA, pH = 8, was added to a final concentration of 2 mM, and the resulting mixture was incubated for 15 min at room temperature before being transferred to a siliconized glass vessel (2). A freshly made 2.5 mg/ml solution of Pronase was then added, and the digestion was allowed to proceed for 36 h at 37 °C. The digest was then injected into an HPLC using the same eluant (water/acetonitrile with trifluoroacetic acid) as above with the following gradient: 0–3 min at 15% acetonitrile, and then a linear gradient of 1%/min. The porphyrin peaks were repeatedly collected in a siliconized glass tube and concentrated under a stream of N₂ with gentle heating. Mass spectrometric analyses were undertaken on the Mariner instrument with the following settings: positive ion mode, spray potential 2500 V, nozzle potential 125 V and 20 scans/min, inlet flow rate 5 µl/min.

Characterization of the S73E Mutant Porphyrin—The S73E mutant was treated with 7 x 2 equivalents of H₂O₂ as described above. The sample was made up to 0.5% trifluoroacetic acid and 33% acetonitrile before being subjected to HPLC separation. The peak eluting at 6.4 min in each HPLC run was collected in a siliconized glass tube and concentrated under a stream of N₂ with gentle heating. LC/MS analysis was undertaken with a Waters Alliance chromatographic system coupled with a ZQ mass analyzer. The solvents used were water and methanol containing 0.1% formic acid; a linear gradient of 2.5%/min starting at 30% of methanol was started after a 2-min wash. The settings of the mass spectrometer were as follows: capillary voltage, 3500 V; cone voltage, 30 V; desolvation temperature, 350 °C; source temperature, 120 °C.

Labeling with H ¹⁸₂O—A 10 mM KH₂PO₄/K₂HPO₄, pH = 7, buffer in H ¹⁸₂O (95% ¹⁸O) was prepared by diluting the concentrated buffer (in H ¹⁸₂O water) 100-fold with H ¹⁸₂O. An S73E mutant sample was concentrated 10-fold over a Millipore Ultrafree centrifugal filter device (Biomax, 10 kDa) and rediluted 10 times with the phosphate buffer in H ¹⁸₂O. The solution was allowed to stand overnight at 4 °C. Three new cycles of concentration/dilution as described above were then undertaken, and the resulting solution was treated with 6 x 2 equivalents of H₂O₂ as previously described. After reaction, the solution was concentrated again 10-fold and re-diluted 2-fold in water/methanol (9/1). Two volumes of 100 mM disodium EDTA, pH = 8, were then added, and the mixture was allowed to stand for 5 min. An identical volume of 4:6 (v/v) water/methanol was finally added, resulting in the formation of a dense precipitate. The supernatant was harvested after centrifugation, diluted with one volume of 0.5% formic acid in water, and directly analyzed by LC/MS. This procedure is required to allow a thorough denaturation of HRP that the methanol used in the LC/MS does not achieve. The solvents, gradient, and MS settings used were the same as described in the previous paragraph.

Pre-steady State Kinetic Measurements—The experiments were done at 25 °C in 50 mM KH₂PO₄/K₂HPO₄, pH = 6.7. All the H₂O₂ and guaiacol solutions were made daily and kept on ice until used. The integration time was set at 2 ms in the diode array detector mode. For all measurements, at least five different concentrations were used and each measurement was undertaken at least twice. For the measurement of compound I velocity constants, the apparatus was set in single mixing mode and a fixed concentration of enzyme (4 µM prior mixing) was mixed with varying concentrations of H₂O₂. Only initial time points were used for the calculations. For concentrations below a 5-fold excess, the apparent velocity constants were deduced from the slope of the variation of the OD at the compound I/compound II isosbestic point versus time. For concentrations above a 5-fold excess, a single exponential decrease model in the form of a + b x [exp(–k x t)] was used to calculate the apparent velocity constant from the variation of the OD at the compound I/compound II isosbestic point versus time (396 nm for the commercial enzyme, 398 nm for the wild type, 397 nm for both mutants before H₂O₂ treatment, and 400 nm after treatment). For the measurement of compound II velocity constants, the apparatus was set in double mixing mode; a fixed concentration of enzyme (8 µM prior mixing) was mixed with 1 equivalent of H₂O₂. The aging time was set at 300 ms except for the F41E mutant before H₂O₂ treatment, for which a 5-s aging time was employed. The resulting compound I solution was then mixed with varying concentrations of guaiacol and the variation of the OD at the compound II/resting state isosbestic point (410 nm for the commercial enzyme and the wild type, 412 nm for the S73E mutant, 405 nm for F41E mutant before and 417 nm after H₂O₂ treatment) versus time was plotted. An equation of the form OD = a x [exp(–k x t) + b x (1 – exp(–k x t))] was used to model the reduction of compound I, and only the first time points were used because the excess of guaiacol was less than 10-fold.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Structure Superposition and Choice of Mutations—MPO is the reference enzyme for the mammalian peroxidases because its crystal structure has been determined (1, 22). No other mammalian peroxidase structure is available. The MPO structure makes it possible to define the precise location and arrangement of each of the three bonds that link the protein to the heme. To determine which residues in HRP might be suitable candidates for the introduction of active site carboxylate side chains, the x-ray structure of HRP (18) was superimposed on that of MPO. The only constraint applied was conservation of the average heme plane. As noted elsewhere for MPO/prostaglandin H synthase and yeast cytochrome c peroxidase/lignin peroxidase (14), the superposition revealed striking similarities in the secondary structures immediately surrounding the iron porphyrin (Fig. 1, a and b). Helix H2 (residues 84–97) of MPO and helix B (residues 31–44) of HRP, which bear the corresponding distal histidines, coincided almost perfectly. Likewise, MPO helix H8 (residues 326–338) superimposed very well on HRP helix F and the residues immediately following it (161–171); both segments bear the proximal histidines. On the same side, the first segment of helix H12 of MPO (417–423) was found in a similar position to helix H of HRP (244–250), even if the length and orientation of the two helices differed as the distance from the heme increased. It is to be noted that these two segments include Asn⁴²¹ of MPO and Asp²⁴⁷ of HRP, respectively, which confer a partial imidazolate character to their respective proximal histidine. At the approximate position of the MPO 234–243 segment bearing two of the three heme covalent bonds, we found in HRP a similar poorly defined loop (residues 67–78), a region that is among the most variable in class III peroxidases (23). Finally, and to a lesser degree, two additional helical segments further away from the heme were found in comparable positions: part of helix H6 of MPO (287–300) and helix D of HRP (97–112), and part of helix H17 of MPO (493–504) and helix J of HRP (273–283). The HRP segments identified by this superposition of structures are almost the same as the ones invoked as being the remnants of an ancient gene duplication (24). As previously noted (14), the heme in HRP was "flipped" by a 180° rotation about the -meso/-meso axis relative to that in MPO. The result of this was an exchange of pyrrole rings A and B of MPO with pyrrole rings D and C of HRP.

View larger version (23K): [in this window] [in a new window]   FIG. 1. View of the common secondary structures and spatially overlapping residues of MPO and HRP. a, view from the -meso carbon of MPO and the -meso carbon of HRP; b, view from the distal cavities; c, spatially overlapping residues, as viewed from the -meso carbon of MPO and the -meso carbon of HRP. Dark gray, MPO; light gray, HRP.

We surveyed the residues in HRP closest to the methyl heme substituents in the superimposed MPO structure, reasoning that a covalent ester link might be formed between any of them if it were correctly located relative to a protein carboxylate group. We limited our search to the distal cavity, as the covalent heme-protein links in the mammalian peroxidases appear to be located on this side of the heme. The closest residue lying under the 1-methyl is Gly⁶⁹, a residue that has been implicated as part of the substrate-binding site (26). When mutation of this residue into a glutamate was simulated, the calculated configurations with the acidic side chain pointing toward the heme gave a best distance of 5 Å between the closest oxygen and the 1-methyl carbon atom (Fig. 2c). Interestingly, the carboxylate group of this simulated G69E mutant was also found within 5 Å of the 8-methyl group. Because a glutamate gave a quite long calculated distance, an aspartate substitution was not modeled. Next, the closest and most ideally placed residue relative to the 3-methyl was Phe⁴¹. This residue has been shown to impede access of reducing substrates to the compound I ferryl oxygen, thus suppressing the oxygenase activity of HRP (19, 27). Computer modeling of a Phe Glu mutation this time gave much shorter distances, with the distance between the -oxygen and 3-carbon being as short as 2.5 Å (Fig. 2b). Taking into account the pyrrole ring "swapping" between HRP and MPO mentioned earlier, position 41 turned out to be the strict spatial equivalent of position 94 in MPO. The only reasonable candidate for a bond with the 5-methyl was Leu³⁷. This residue has not been assigned any function in the HRP active site, but directed evolution experiments have shown that its mutation into an isoleucine improves enzymatic activity (28). Modeling of a glutamate or an aspartate (an isoleucine steric analog) at this site gave the nearest approach distances of 1.5 and 2.1 Å, respectively (Fig. 2a). Both mutations were therefore selected. Finally, the closest and best oriented residue with respect to the 8-methyl group is Ser⁷³. This serine may help to stabilize one of the heme propionates in both soybean seed coat peroxidase and HRP (23) and also interacts with Arg³⁸ (29). However, an isoleucine is found at this position in cytochrome c peroxidase, ascorbate peroxidase, Arthromyces ramosus peroxidase, and lignin peroxidase (18). Again, because of the pyrrole ring nonequivalence between MPO and HRP, this position corresponds to that of Glu²⁴² in MPO. Simulating the replacement of Ser⁷³ by a glutamate gave distances to the methyl group of approximately 5 Å (Fig. 2d). Hence, five mutations were chosen for this study: L37D, L37E, F41E, G69E, and S73E.

View larger version (39K): [in this window] [in a new window]   FIG. 2. The HRP sites selected for mutagenesis in this study. a, position 37; b, position 41; c, position 69; d, position 73. Dark gray residues depict the wild-type residues/conformations, light gray one of the conformations calculated for a glutamate side chain when substituting the corresponding residue.

The mutants and the wild-type enzyme were first expressed in High-Five cells at an analytical scale to determine the kinetics of the expression. In the case of the L37D and G69E mutants, no significant ABTS oxidizing activity could be detected in the cell supernatant even after 5 days following infection. The L37E, F41E, and S73E mutants, however, showed a gradual increase in ABTS activity in the cell supernatant that peaked at day 4 after infection. These screening results showed that the L37D and G69E mutants, in contrast to the other three, probably yielded inactive or unstable protein. Medium scale expressions confirmed that the L37D and G69E constructs gave no isolable protein. The results of medium scale expression of the L37E, F41E, and S73E constructs are presented in Table I. An SDS-PAGE analysis of the proteins is also presented in Fig. 3. The wild-type protein exhibited good expression yields, and the protein had an RZ value and a specific activity for ABTS oxidation slightly higher than those of commercial HRP. In the case of the L37E mutant, the yields were low, the heme content of the protein was only half of that of the wild type, and the position of one of the charge transfer bands could not determined. Furthermore, a pure preparation of the protein could not be obtained (see Fig. 3), and even limited freeze/thaw cycles triggered extensive protein denaturation. Work on this mutant was therefore discontinued. The F41E mutant gave goods expression yields, but the ABTS specific activity was 10 times lower than that of the wild type. The Soret band was also slightly red-shifted, sharper, and shoulderless, and the newly introduced carboxylate had a strong effect on the molar absorption coefficient, as previously reported for other mutations at this position (16, 30, 31). Finally, the properties of the S73E mutant were quite similar to those of the wild type, except for the specific activity, which decreased by a factor of 3.

View larger version (37K): [in this window] [in a new window]   FIG. 3. SDS-PAGE analysis of the isolated HRP variants. Lane 1, wild type; lane 2, F41E; lane 3, L37E; lane 4, S73E; lane 5, commercial HRP.

View larger version (18K): [in this window] [in a new window]   FIG. 4. HPLC analysis of the isolated HRP variants. Right scale, OD in mAU at 400 nm (solid trace); left scale, OD in mAU at 278 nm (dotted trace). a, commercial enzyme; b, wild type; c, F41E; d, S73E.

View larger version (25K): [in this window] [in a new window]   FIG. 5. SDS-PAGE analysis of the isolated variants after H2O2 treatment. Lane 1, wild type; lane 2, F41E; lane 3, S73E; lane, 4 commercial HRP.

View larger version (24K): [in this window] [in a new window]   FIG. 6. HPLC analysis of isolated HRP variants after H2O2 treatment. Right scale, OD in mAU at 400 nm (solid trace); left scale, OD in mAU at 278 nm (dotted trace). a, commercial enzyme; b, wild type; c, F41E; d, S73E. Arrow, see "Results."

View larger version (11K): [in this window] [in a new window]   FIG. 7. HPLC and MS characterization of the F41E iron porphyrin after H2O2 incubation and digestion. a, chromatogram of the digestion medium; b, MS/ESI analysis of the 22-min peak from a.

Analysis of the Porphyrins in the S73E Mutant—Even though a heme-protein covalent bond was not formed with this mutant, a more polar species absorbing strongly at 400 nm was formed in its reaction with H₂O₂ (Fig. 6d). The compound eluting at 6.7 min in Fig. 6d was isolated from a large incubation of the S73E mutant with H₂O₂ (Fig. 8a), and, upon co-injection with 1-hydroxy-, 3-hydroxy-, and 5-hydroxyheme was shown to co-elute with the 8-hydroxy derivative (data not shown). LC/MS gave a mass of 632.1 amu for the new product (Fig. 8b), consistent with its identification as 8-hydroxymethylheme. Interestingly, although the predicted formation of a covalent bond to the 8-methyl group was not observed, the carboxylate in the mutant nevertheless triggered a partial hydroxylation of that position.

View larger version (12K): [in this window] [in a new window]   FIG. 8. HPLC and MS characterization of the S73E porphyrin after H2O2 incubation. a, 400 nm chromatogram of the incubation medium; unmodified hemin is indicated by h; b, MS/ESI analysis of the 11.5-min peak (arrow) from a.

View larger version (13K): [in this window] [in a new window]   FIG. 9. LC/MS analysis of the S73E iron porphyrin content after incubation with H2O2 in buffered 18O-labeled water. a, 11.5-min peak (8-hydroxyheme); b, control unmodified heme.

View larger version (21K): [in this window] [in a new window]   FIG. 10. Influence of the H2O2 with ratio. Both mutants were incubated various amounts of oxidant, added as an aliquot of 2 equivalents every 5 min. The incubation medium was then analyzed by HPLC, and the proportion of the material absorbing at 400 nm for the peak associated with the enzyme (F41E, gray dotted line) or for the peak eluting at 6.7 min (S73E, black solid line) was quantitated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Superposition of the structure of MPO with that of HRP revealed significant similarities in their three-dimensional structures in the immediate vicinity of the heme despite the fact that they belong to different superfamilies and share little sequence identity. Five helices or helical segments and one loop were found at spatially equivalent positions. More interestingly, both catalytic histidines and the proximal carboxyl side chain responsible for increasing the proximal histidine basicity were found at almost identical spatial positions. The MPO catalytic arginine was located at a position where HRP has a key auxiliary residue hydrogen-bonded to the distal histidine, and the HRP arginine at a site that corresponds to a key component of the MPO active site hydrogen-bonding network. As far as the heme is concerned, the general plane of the tetrapyrrolic assembly is conserved, but not the positions of the heme substituents. This conservation of active site structure in MPO and HRP validates our use of superposition in identifying potential HRP residues to mutate in efforts to introduce a heme-protein covalent bond.

Four positions were thus identified, which, if mutated to a carboxyl-bearing amino acid, might result in covalent bonding of the heme to the protein: Leu³⁷, Phe⁴¹, Gly⁶⁹, and Ser⁷³. From these, only Phe⁴¹ and Ser⁷³ yielded mutants that could be isolated and studied, as the mutations at the other two sites did not yield detectable proteins. The failed mutants show, as might be expected, that the introduction of a negatively charged carboxylic acid residue into the active site can cause serious structural perturbations. The introduction of a carboxylate into the active site of HRP has been achieved earlier for other purposes, notably the substitution of His⁴² by a glutamate in efforts to prepare a chloroperoxidase mimic (29, 32, 36) or to disrupt the active site hydrogen-bonding network associated with a bound benzhydroxamic acid molecule (37). Asn⁷⁰, which interacts with His⁴², has also been successfully replaced by a glutamate (17, 38, 39). The H42E mutation decreased the catalytic activity 300-fold (32) and the N70D mutation 2–6-fold (17). Finally, a glutamate has been introduced at position 143, in the postulated aromatic reductant binding site, leading to an enzyme with reduced activity (40).

Mutagenesis of Ser⁷³ has not been reported, but Phe⁴¹ has been replaced by a variety of residues: glycine (41), alanine (16, 31, 41, 42), valine (30, 31, 41), leucine (41, 43), threonine (43), tyrosine (41), histidine (40, 42), and tryptophan (31, 41). Indeed, Phe⁴¹ has emerged as a key residue in HRP. In all the sequence-related plant peroxidases (24), an aromatic side chain is found at the corresponding position: a tryptophan in cytochrome c peroxidase and ascorbate peroxidase and a phenylalanine in the others. Phe⁴¹ restricts access of reducing substrates to the oxoferryl moiety and thus limits the peroxygenase activity of the enzyme (19, 27). We examined the ability of our mutants to epoxidize styrene as a test for peroxygenase activity (43), but in none of the variants, before of after H₂O₂ treatment, could we detect any styrene oxide formation (data not shown). Replacement of Phe⁴¹ by a glutamate therefore does not increase access to the ferryl oxygen. This aromatic position governs the resting state spin state in both cytochrome c peroxidase and A. ramosus peroxidase and the stability of compound I in cytochrome c peroxidase. A red shift of the HRP Soret band and an increase in its molar absorption coefficient have been observed when Phe⁴¹ is mutated to a Val or Ala (30, 31, 37). This shift has been attributed to an increase in 6-coordinate high spin character at the expense of the 5-coordinate high spin character of the wild-type enzyme. Our observations for the F41E mutant are consistent with the literature; introducing a glutamate at position 41 sharpens the Soret band, shifts it by 3 nm, and increases its molar absorption coefficient. Introducing a glutamate at position 73, further away from both the heme and the iron, only causes minor changes in the UV-visible spectrum.

Not surprisingly, the mutations alter the catalytic properties of the enzyme. Under steady state conditions, the mutant enzymes as isolated from the insect cell cultures exhibited a moderately reduced ABTS oxidizing activity (5 and 2 times lower for F41E and S73E, respectively), but the influence on the guaiacol oxidation activity was stronger (Table II, before H₂O₂). This difference in sensitivity between ABTS and guaiacol has been attributed to the existence of additional limiting association/dissociation steps in the case of ABTS (30). However, we found a much stronger decrease of the guaiacol oxidation rates, by a factor of 100 rather than 8, which was paralleled by a similar reduction of the rate of compound I formation (see below). In contrast, the mutation at position 73 only modestly reduced the specific activity of the enzyme despite the fact that Ser⁷³ has been postulated to interact with Arg³⁸ and one of the heme propionates (29).

Analysis of the individual catalytic steps is informative. In the case of the F41E mutant, a decrease by a factor of 100 of the second order rate constant for the formation of compound I is observed, whereas in the case of the S73E mutant the decrease is only 2–3-fold. After preincubation with H₂O₂, the velocity constant for oxidation of F41E to compound I increases greatly, reaching a value in the same range as that for the wild type. In contrast, the value for the S73E mutant is barely changed. The rates for the single electron reduction of compound I by guaiacol are less affected by the mutations, because the measured variations among all the recombinant enzymes do not exceed a factor of 3. These results tend to show that the major change brought about by the substitutions is a decrease of the ease of reaction with H₂O₂, whereas the subsequent reduction of the oxidized states is relatively unaffected. In the case of the F41E mutant, one could envision either an ionic or steric interaction between the distal histidine at position 42 and the glutamate at position 41. In the case of the H₂O₂ pretreated F41E mutant, we were unable to detect the characteristic red shift upon conversion of compound I to compound II. The two obvious possibilities are that either no compound II forms, or it forms but has spectral properties that cannot be differentiated from those of the resting state. The first possibility seems unlikely, however, as it implies simultaneous reduction of the two oxidizing equivalents of compound I. These reducing equivalents would have to come from two different sources, either two substrate molecules or one substrate molecule and the protein itself. Because in the absence of added reductant, compound I of the H₂O₂-treated F41E mutant is stable (data not shown), the second hypothesis requires the improbable assumption that reaction with guaiacol (or p-aminobenzoic acid) triggers simultaneous oxidation of the protein. We have therefore proceeded on the assumption that compound II of the H₂O₂-treated F41E mutant has spectral characteristics indistinguishable from those of the resting state. In the case of the S73E mutant, the glutamate side chain is further away from the iron center and its perturbations of the catalytic cycle are understandably less pronounced.

The most important result of this work, however, is the finding that both mutants differ from the wild-type and native enzymes in their reactions with H₂O₂. In the absence of any other factor, the F41E mutant underwent nearly quantitative covalent linking of its heme 3-methyl group to the protein, presumably via Glu⁴¹, and the S73E mutant partially hydroxylated the 8-methyl substituent of its prosthetic heme group. Thus, as in the mammalian peroxidases, heme covalent binding occurs via an autocatalytic mechanism (2, 11, 12). Both of these modifications most likely arise via a common mechanism (12). After formation of compound I, a carbocation is formed either by transient oxidation of the carboxylate and subsequent hydrogen abstraction from the methyl, or via direct deprotonation of the methyl acidified by the porphyrin radical cation (11). The resulting cation is then quenched by a nearby nucleophile, either the carboxylate anion or a nearby molecule of water (Fig. 11). In these mechanisms, it is the very presence of the carboxylate that allows the transfer of oxidizing equivalents from the iron and the -system to the methyl substituent. In the F41E mutant, for which modeling predicted a distance of 2.5 Å between the glutamate oxygen and the 3-methyl carbon, quenching gives the ester. In the S73E mutant, where the distance to the 8-methyl carbon was predicted to be 5 Å, the carboxylate is unable to compete with an active site water molecule in trapping of the cation, so that hydroxylation of the heme rather than covalent binding to the protein is observed. In accord with this postulated mechanism for the formation of 8-hydroxymethylheme, the hydroxyl oxygen is shown by incubation in ¹⁸O-labeled water to derive from the solvent. The same type of competition between a carboxylate and a water molecule was postulated in the cytochrome P450 system (3, 5) and was later confirmed for CYP4B1 (6). It is to be noted that the heterogeneity of heme species in the S73E mutant after H₂O₂ treatment does not appear to affect its catalytic properties. This is consistent with the previous finding that HRP with an 8-hydroxymethylheme group has the same catalytic properties of the enzyme with a conventional heme group (44). Finally, we undertook the same type of H₂O₂ treatment in the presence of guaiacol, reasoning that if an oxidized form of the enzyme is indeed needed for the iron porphyrin to be modified, then a competition between porphyrin and guaiacol oxidation should take place. Indeed, we found that 20 equivalents of guaiacol were enough to completely inhibit covalent linking of the heme to the protein during the H₂O₂ pretreatment (data not shown). This finding emphasizes the close similarities between the bond-forming process (porphyrin oxidation) and the normal catalytic cycle (exogenous substrate oxidation).

View larger version (20K): [in this window] [in a new window]   FIG. 11. Radical mechanism, starting with the compound I obtained by reaction with H2O2, proposed to explain autocatalytic covalent binding of the glutamic acid to the 3-methyl group in the F41E HRP mutant. The structure of the 8-hydroxyheme formed with the S73E mutant because of effective competition of water for trapping of the carbocation analogous to that shown for the F41E mutant is shown on the bottom far right.

The implications of covalent bond formation on the properties of HRP remain to be clarified. The UV-visible spectrum, the ABTS and guaiacol oxidation efficiencies, and the second order rate constants for the elemental reactions of the catalytic cycle resemble those of wild-type HRP. No clear advantage or disadvantage is conveyed by the presence of the new ester bond. This suggests that the reasons for the ester links to the heme in the mammalian peroxidases are likely to be related to stability, specificity, or peroxidase biology rather than to intrinsic catalytic properties.

	FOOTNOTES

 
^* This work was supported by Grant GM32488 from the National Institutes of Health. 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.

To whom correspondence should be addressed. Fax: 415-502-4728; E-mail: ortiz{at}cgl.ucsf.edu.

¹ The abbreviations used are: heme, iron protoporphyrin IX regardless of the iron oxidation and ligation state; 1-hydroxymethyl, iron 1-hydroxymethylprotoporphytin IX; iron 5-hydroxymethyl, 1-hydroxymethylprotoporphytin IX; 1,5-dihydroxy heme, iron 1,5-dihydroxymethyl-protoporphyrin IX; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); AU, absorbance unit(s); HPLC, high pressure liquid chromatography; HRP, horseradish peroxidase; LC, liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; LPO, lactoperoxidase; MPO, myeloperoxidase; RZ, Reinheitzahl; bis-tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; amu, atomic mass unit(s).

	ACKNOWLEDGMENTS

 
We thank Jane M. Kuo for making early constructs of the HRP mutants.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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