©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Horseradish Peroxidase His-42 Ala, His-42 Val, and Phe-41 Ala Mutants
HISTIDINE CATALYSIS AND CONTROL OF SUBSTRATE ACCESS TO THE HEME IRON (*)

(Received for publication, May 19, 1995; and in revised form, June 12, 1995)

Sherri L. Newmyer Paul R. Ortiz de Montellano (§)

From the Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Polyhistidine-tagged horseradish peroxidase (hHRP) and its F41A, H42A, and H42V mutants have been expressed in an insect cell system. Kinetic studies show that the rates of Compound I formation and peroxidative catalysis are greatly decreased by the His-42 mutation. Furthermore, Compound II is not detected during turnover of the His-42 mutants. Compounds I and II are the two- and one-electron oxidized intermediates, respectively, of hHRP. In peroxygenative catalysis, the F41A and H42A mutants catalyze thioanisole sulfoxidation 100 and 10 times faster, respectively, than hHRP. Styrene epoxidation is catalyzed by both the Phe-41 and His-42 mutants but not by wild-type hHRP. The higher peroxygenase activity of the mutants reflects increased accessibility of the ferryl species. This is indicated by the finding that, contrary to the reaction with wild-type hHRP, reaction of phenyldiazene with the F41A mutant yields a new and unidentified product, and the same reaction with the His-42 mutants yields phenyl-iron complexes. Phe-41 and His-42 thus shield the iron-centered catalytic species, and His-42 plays a key catalytic role in the formation of Compound I. The peroxygenase activities of the Phe-41 and His-42 mutants approach those of cytochrome P450.


INTRODUCTION

Catalytic turnover of classical peroxidases with a histidine as the ligand to the heme (^1)iron involves reaction with H(2)O(2) to give a two-electron oxidized species known as Compound I(1, 2) . The iron atom in Compound I is present as a ferryl (Fe=O) complex and is therefore 1 oxidation equivalent above the resting ferric state. The 2nd oxidation equivalent provided by the peroxide is stored either as a porphyrin or protein radical cation. HRP is the prototype for peroxidases in which the 2nd oxidation equivalent is located on the porphyrin(3) , and cytochrome c peroxidase for peroxidases in which the 2nd oxidation equivalent resides on the protein(4) . Two single-electron transfers from substrate molecules reduce Compound I, first to Compound II and then to the resting state.

A high resolution crystal structure is not available for HRP. However, the similarities among the known primary sequences of peroxidases (5, 6, 7) and among the four available peroxidase crystal structures (8, 9, 10, 11) indicate that the catalytic machinery required to activate H(2)O(2) is highly conserved. The key catalytic residues are a histidine and an adjacent arginine in the distal heme site. The histidine is thought to facilitate formation of the initial iron-peroxide complex by deprotonating the peroxide and subsequently to promote cleavage of the oxygen-oxygen bond by protonating the distal oxygen (Fig. 1)(12) . The arginine is proposed to facilitate oxygen-oxygen bond scission by electrostatic stabilization of dioxygen bond cleavage(12) . Experimental support for these catalytic roles comes from site-specific mutagenesis of cytochrome c peroxidase, which demonstrates that replacement of the catalytic histidine (His-52) or arginine (Arg-48) by a leucine decreases the rate of formation of Compound I by a factor of 10^5(13) or 10^2(14) , respectively. Initial data on the corresponding HRP site-specific mutant suggest that the distal arginine (Arg-38) is also important for catalysis(15) , but the function of the distal histidine in HRP has not yet been directly tested.


Figure 1: Proposed roles of the distal histidine and arginine residues in peroxidase catalysis.



In addition to the catalytic histidine and arginine, a phenylalanine or tryptophan is found adjacent to the histidine in the active sites of the four peroxidases for which crystal structures are available. Sequence alignments suggest that an aromatic residue is adjacent to the catalytic histidine in all plant and fungal peroxidases (Fig. 2)(5, 6, 7, 8, 9, 10, 11) . The reason for conservation of the aromatic residue is unclear, but mutation of the tryptophan in cytochrome c peroxidase (16, 17) and replacement of the phenylalanine (Phe-41) in HRP by a valine(18) , leucine(19, 20) , or threonine (19, 20) indicate that the aromatic residue is not essential for catalysis. We have proposed in earlier studies that the ferryl species of Compounds I and II are partially or fully shielded from direct interaction with substrates(2, 21, 22) . We have furthermore advanced the hypothesis that sequestration of the ferryl species is responsible, at least in part, for the fact that peroxidases catalyze electron abstraction (peroxidase) rather than ferryl oxygen transfer (peroxygenase) reactions(2, 23) . The evidence marshaled in support of this argument includes the finding that reaction of HRP with phenylhydrazine (PhNHNH(2)), via its oxidation product phenyldiazene (PhN=NH), results in either addition of the phenyl moiety to the -meso-carbon of the heme group or abstraction of a hydrogen atom from the 8-methyl group to give, eventually, the 8-hydroxymethyl heme derivative(21) . Similar reactions have been observed with other peroxidases(24, 25) . In contrast, the reaction of phenyldiazene with hemoproteins in which the iron is known to be within an open active site produce -bonded phenyl-iron (Fe-Ph) complexes. This has been observed with myoglobin(26) , hemoglobin, (27) catalase (28) , and cytochrome P450 (29, 30) and is unambiguously confirmed by crystal structures of the myoglobin and cytochrome P450 complexes(26, 29) . Denaturation of the phenyl-iron complexes under acidic, aerobic conditions results in migration of the phenyl group from the iron to the nitrogens of the porphyrin to yield N-phenylheme. This is followed by loss of the iron to give N-phenylprotoporphyrin IX(26, 27, 28, 29, 30, 31) .


Figure 2: Superimposed images of active site residues in the crystal structures of cytochrome c peroxidase and lignin peroxidase. Panel a, side view with the -meso edge of the heme facing the viewer. The residues shown, in addition to the heme and the proximal histidine iron ligand, are the catalytic histidine (His-52 in cytochrome c peroxidase, His-47 in lignin peroxidase), catalytic arginine (Arg-48 in cytochrome c peroxidase, Arg-43 in lignin peroxidase), and the aromatic residue (Trp-51 in cytochrome c peroxidase, Phe-46 in lignin peroxidase). These residues correspond to Phe-41, His-42, and Arg-38 in HRP. Panel b, top view with the -meso edge at the bottom showing the same distal residues as the side view.



The aromatic residue adjacent to the catalytic histidine, and the histidine itself, may be part of the proposed barrier that restricts access of substrates to the ferryl species. Evidence that the aromatic residue plays such a role is provided by our finding that mutation of Phe-41 to a leucine or threonine enhances the peroxygenase activity of HRP(19, 20) . These two mutants oxidize aryl alkyl thioethers to their sulfoxides at higher rates and, in the case of the F41L mutant, with higher enantioselectivity than native HRP. Furthermore, these mutations convey on HRP the ability to epoxidize styrene(20) . These results agree with our finding that replacement of the distal tryptophan in cytochrome c peroxidase by an alanine enhances both the accessibility of the iron and the peroxygenase activity of that enzyme (32) .

To investigate the catalytic role of the distal histidine and to determine if the histidine and the adjacent phenylalanine shield the ferryl species we have developed a new system for the heterologous expression and purification of HRP mutants and have expressed the F41A, H42A, and H42V mutants of HRP. We have examined the activities of these enzymes in the peroxidation of guaiacol, the sulfoxidation of thioanisole, and the epoxidation of styrene and have identified major differences in the catalytic activities of the mutants. The reactions of the three mutants with phenyldiazene have then been used to rationalize the differences in their peroxygenase activities in terms of differences in both the rate of Compound I formation and the accessibility of the heme iron.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes were obtained from Boehringer Mannheim, New England Biolabs, and Life Technologies, Inc. Native HRP and bovine serum albumin were purchased from Boehringer Mannheim. Hemin, 30% H(2)O(2), styrene, trans-beta-methylstyrene, (R)-styrene oxide, racemic styrene oxide, thioanisole, acetophenone, mCPBA, and ABTS were from Aldrich; guaiacol and Sephadex G-25 from Sigma; and methyl phenyldiazene carboxylate azoester from Research Organics (Cleveland, OH). Modified heme derivatives were prepared by reaction of phenyldiazene with HRP as described previously(21) . H(2)O(2) and mCPBA solutions were quantitated by titration with iodide ( = 25,900 M cm for I(3))(33) . In the H(2)O(2) titration, trace amounts of HRP were added to promote complete reaction. Styrene was purified by passage through neutral alumina. In quantitating HRP, the Soret absorbance value for the HRP mutants was assumed to be the same as that for the native protein ( = 95,000 M cm). All assay buffers were treated with Bio-Rad Chelex 100 resin to remove trace metals.

Tissue Culture

Spodoptera frugiperda (Sf9) cells were maintained in spinner flasks at 27 °C (75 rpm) in Hink's TNM-FH insect cell medium supplemented with Grace's medium (JRH Biosciences) containing 10% heat-inactivated, 0.2 µm-filtered fetal calf serum (University of California, San Francisco Cell Culture facility). Trichoplusia ni cells (Invitrogen) were maintained in suspension in Sf900-II SFM (Life Technologies, Inc.) in a rotary shaker incubator at 27 °C (130 rpm).

Site-directed Mutagenesis and Subcloning

Plasmids were transformed into and maintained in Escherichia coli strain DH5alpha grown in LB. Transformed vectors were selected by ampicillin resistance. Plasmid preparation was afforded through Qiagen (Chatsworth, CA) or Promega (Madison, WI) Wizard minipreparations. Plasmid fragments from restriction enzyme digests were typically isolated by subjecting the digest to electrophoresis on an agarose gel and extracting the DNA with a gel extraction kit (Bio101 GeneClean or Qiagen gel extraction kit). DNA sequencing either was carried out with the Sequenase kit, version 2.0 (U. S. Biochemical Corp.) or was performed by the University of California, San Francisco Biomolecular Resource Center. The construct pUC-HRP, containing a synthetic version of the HRP gene cloned into pUC19 (British Biotechnologies), was used for subcloning and site-directed mutagenesis. To append the sequence encoding the N-terminal polyhistidine tag from the pET-19b vector (Novagen) to the 5` end of the HRP gene, the HRP gene was isolated from pUC-HRP by NdeI/EcoRI digestion and subcloned into pET-19b. The resulting hHRP insert was then subcloned into the baculovirus vector pACGP67B (Pharminogen). This vector encodes a sequence for the glycoprotein 67 leader peptide at the 5` end of the multiple cloning site which targets the translated HRP gene product for secretion. NcoI digestion of the pET-HRP construct yielded a fragment containing the polyhistidine sequence and a portion of the HRP sequence. This fragment was ligated into baculovirus vector pACGP67B that had been digested with NcoI. Isolated plasmid containing the correctly oriented gene fragment (GPNcoIHis HRP) was used as the construct for introducing the SacI/EcoRI-cut pUC-HRP, restoring the full length of the HRP gene. SacI/EcoRI fragments from mutated pUC-HRP were similarly ligated into the baculovirus vector. pUC-HRP mutants were generated through cassette mutagenesis. Sense and antisense oligonucleotides that corresponded to the region of the HRP sequence flanked by NdeI and SacI sites were synthesized by the University of California, San Francisco Biomolecular Resource Center. Individual cassettes were generated for the F41A, H42A, and H42V HRP mutations. The annealed cassette was ligated to NdeI/SacI-cut pUC-HRP, and the resulting mutant was subcloned into SacI/EcoRI-digested GPNcoIHisHRP, as outlined above.

Production of Recombinant Virus

Recombinant virus production, purification, and amplification were performed in Sf9 cells. The sequence encoding hHRP was introduced into wild-type Autographica californica nuclear polyhedrosis virus with the Baculogold transfection kit (Pharminogen). The resulting transfection medium was isolated by centrifugation (1,000 g, 10 min) and subjected to one round of plaque purification. Plaques that represent infection by a single virus particle were individually isolated and mixed with 1 ml of complete medium. Viral amplification of this initial stock was achieved through successive rounds of infection. To begin amplifying the virus for expression purposes, a 900-µl volume was used to infect a monolayer of Sf9 cells in a 60-mm Petri dish (10^6 cells/ml, 3 ml final volume). After a 4-day incubation, the medium was isolated through centrifugation (5 min, 1,000 g), and the existence of the hHRP insert was verified by performing polymerase chain reaction on the isolated viral DNA. In a second round of amplification, 250 µl of this viral stock was used to infect a monolayer of Sf9 cells seeded on a 100-mm Petri dish (5 10^6 cells, 10 ml final volume). After incubating the cells for 4 days, the medium was harvested by centrifugation as above. A final round of amplification used 500 µl of this latter viral stock to infect 100 ml of Sf9 in suspended culture (2 10^6 cells/ml). After a 3-day incubation the medium was harvested by centrifugation. This series of amplification steps gave a viral stock with a viral titer of 1-3 10^8 plaque-forming units/ml. Expression was performed in T. ni cells. Typically a 1-liter culture of cells (1.5-2 10^6 cells/ml) was inoculated with virus (multiplicity of infection 1). A 10-ml volume of 0.2 µm filtered hemin/bovine serum albumin solution was added at the time of inoculation (0.24 mM hemin, 0.4 mM bovine serum albumin, in 75 mM Na(2)HPO(4), pH 7.2)(34) . The expression was harvested after 60-64 h.

Protein Purification

The expression medium was separated from the cells by centrifugation (1,000 g, 10 min). The supernatant was concentrated and ultrafiltered at room temperature with an Amicon Spiral-Wound Cartridge Concentrator CH2PRS (S1Y10, 10,000 molecular weight cutoff spiral membrane) to a 150-200-ml volume in 20 mM Na(2)HPO(4) buffer (pH 8.0) containing 500 mM NaCl (binding buffer). Particulate material was removed from the ultrafiltrate by centrifugation (17,000 g, 30 min). The supernatant was stirred with 7.5-10 ml of Ni(II) nitrilotriacetic acid (Invitrogen) at 4 °C. After 1.5-2 h, the resin was collected in a 1.5-cm-diameter column support. The resulting column was washed at 75 ml/min with binding buffer until the eluent ran clear. In a similar manner the resin was washed with binding buffer adjusted to pH 6.0. Successive washes included the pH 6.0 buffer containing 0.1 and 1 M imidazole. The latter wash was dialyzed against 20 mM Na(2)HPO(4) buffer (pH 8.0). The dialysate was run through a 1.5 10-cm Pharmacia Sepharose QFF (gravity flow) column. The total amount of protein was measured after each purification step by the Bradford assay (Bio-Rad) using bovine serum albumin as a standard.

Spectroscopic Characterization of Compound I and Compound II Formation

Compound I was generated by adding 1 equivalent of H(2)O(2) and Compound II by adding 1 equivalent of K(4)Fe(CN)(6) to Compound I. Excess H(2)O(2) or mCPBA was required to obtain Compound I in the case of the His-42 mutants. Attempts to detect Compound II species for H42A hHRP and H42V hHRP involved adding 1 equivalent of K(4)Fe(CN)(6) to Compound I and allowing the Compound I formed with mCPBA to decay spontaneously.

Determination of the Rate of Compound I Formation by Stopped Flow Spectroscopy

The rate of Compound I formation was determined at 25 °C by following the decay of the absorption at 411 nm (an isosbestic point between Compound II and the resting state) using a stopped flow spectrophotometer (Applied Photophysics Limited, model SF.17MV) with a slit width of 0.25 mm. To observe Compound I formation under pseudofirst-order conditions, the enzyme (0.25 µM) in 20 mM Na(2)HPO(4) buffer (pH 7.0) was allowed to react with excess levels of H(2)O(2) (2.5, 5.0, 8.0, 11.0, 15.0, and 25.0 µM). The following set of equations, based on the kinetic scheme in the Introduction, was used to derive the rate constant for Compound I formation.

The decay in absorption was fit to the equation A(t) = A(0)e + C. The second-order rate constant was determined by taking the slope of the plot, kversus the H(2)O(2) concentration. Increased H(2)O(2) concentrations (0.025-1.5 M) were required with H42A hHRP to see the 411 nm decay.

Steady-state Kinetics of the H42A hHRP and H42V hHRP Reactions

Steady-state kinetics were studied by measuring the initial rates for the oxidation of ABTS at 25 °C as the concentrations of ABTS (1.25, 1.67, 2.5, 5.0, and 10.0 mM) and H(2)O(2) (65, 85, 125, 250, and 500 mM) were varied. These H(2)O(2) levels did not oxidize ABTS detectably in the absence of the enzyme. The final assay volume was 1 ml and contained 500 nM His-42 hHRP mutant in 100 mM KH(2)PO(4) buffer (pH 6.0). ABTS oxidation was followed at 414 nm ( = 3.6 10^4M cm). describes the native HRP activity. Typically, the rate of conversion of Compound II to resting state is slower than the rate of conversion of Compound I to Compound II (k(3) < k(2)), which reduces to .

Guaiacol Assay

Guaiacol activity was measured to monitor the activity of hHRP during purification and to compare the peroxidase activity of the native plant and recombinant enzymes. Sample activity was determined spectrophotometrically at 20 °C using the value = 5.53 cm mM. This absorbance value, derived from a plot of the enzyme-catalyzed change in absorbance at 470 nm versus H(2)O(2) concentration, was used to calculate the amount of guaiacol consumed. In a typical assay, the enzyme (10 pmol) was mixed with 5.1 mM guaiacol in 20 mM Na(2)HPO(4) buffer, pH 7.0 (1 ml final volume), and H(2)O(2) (0.5 mM, final) was added to initiate the reaction.

Determination of the Rate and Stereochemistry of Thioanisole Sulfoxidation

The reaction of a solution of the enzyme (25 µM) and thioanisole (5 mM) in 20 mM sodium phosphate buffer (pH 7.0) on ice was initiated by the addition of H(2)O(2) (683 µM, added every 2 min). As found earlier with the native enzyme(35) , product formation is not linear with respect to time if peroxide is not supplemented periodically. At various time points, 500-µl aliquots of the reaction mixture were taken, 50 nmol of acetophenone internal standard was added to each aliquot, and each of the mixtures was extracted with 500 µl of CH(2)Cl(2). The organic layer was concentrated under a stream of argon to a volume of 10 µl, diluted with 50 µl of 80% hexanes, 20% isopropyl alcohol, and loaded onto a Chiracel OD HPLC column (Daicel Chemical Industries). The (R)- and (S)-thioanisole sulfoxides were eluted isocratically with the same solvent mixture at a flow rate of 0.5 ml/min on a Hewlett Packard model 1040A instrument equipped with a diode array detector set at 454 nm with a 6-nm bandwidth(35) . The retention times of acetophenone and the (S)- and (R)-thioanisole sulfoxides were 20.9, 27.0, and 46.2 min, respectively(35) .

Determination of the Rate and Stereochemistry of Styrene Epoxidation

To a solution of the enzyme (25 µM) and styrene (10 mM) in 20 mM sodium phosphate buffer (pH 7.0) on ice was added H(2)O(2) (683 µM every 2 min). At various time points, 500-µl aliquots were taken, and 4.6 nmol of trans-beta-methylstyrene was added to each as an internal standard. Each of the mixtures was then extracted with 500 µl of CH(2)Cl(2), and the organic layer was concentrated to a volume of 10 µl under a stream of argon. The concentrated extracts were analyzed by gas chromatography on a Hewlett Packard model 5890 instrument equipped with a DB-1 column (0.25 mm 30 m, J & W Scientific) programmed to run at 80 °C for 3 min and then rise at 4 °C/min to 150 °C. The retention times for trans-beta-methylstyrene and styrene oxide were 9.0 and 10.0 min, respectively. The stereochemistry of styrene epoxidation was determined by isocratic gas chromatography (90 °C) on a 0.25-mm 30-m Chiraldex G-TA capillary column (Advanced Separation Technologies, Whippany, NJ). The retention times for the (S)- and (R)-styrene oxides were 26.8 and 28.1 min, respectively. The enantiomer configurations were established by comparison with authentic samples of the racemic and (R)-styrene oxides.

Reaction of HRP Mutants with Phenyldiazene

HRP, hHRP, and the three mutants in 100 mM K(2)HPO(4) buffer (pH 7.4) were allowed to react at 25 °C with phenyldiazene prepared by diluting 2 µl of 5.67 M methyl phenyldiazene carboxylate azoester in 200 µl of 0.1 M KOH solution. A 2-µl aliquot of the phenyldiazene solution was added every 5 min until no further changes were observed spectrophotometrically (for 2 ml of 10 µM enzyme, 10 µl of the phenyldiazene solution was sufficient). After incubating for an additional 10 min, 50 µl of a 50 mM ascorbate solution or a few grains of ascorbate were added, and the resulting solution was filtered through a 1.5 10-cm Sephadex G-25 column. The eluted protein was acidified with glacial acetic acid (final 25% v/v) and was then extracted with three 1-ml volumes of ether. The extract was concentrated to dryness on a rotary evaporator, and the residue was resuspended in 100 µl of 6:4:1 MeOH:H(2)O:AcOH (solution A). The sample was analyzed by HPLC on a Partisil ODS-3.5 µm Alltech column eluted with 80% solution A, 20% solution B (10:1 MeOH:AcOH) at a flow rate of 1 ml/min. The eluent was monitored at 416 nm.


RESULTS

Expression and Purification of hHRP and Its Mutants

A polyhistidine tag was fused to the N terminus of the HRP gene to facilitate enzyme purification and to circumvent possible difficulties in the purification of mutant enzymes. The specific activity and R(Z) value obtained after each step in the purification of wild-type hHRP (Table 1) and a sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified protein (Fig. 3) demonstrate the utility of the polyhistidine tag. Nevertheless, even though the chelating column purified hHRP 351-fold, there is a significant (67%) loss of total hHRP activity. The majority of the hHRP activity is retained by the resin, but a significant amount elutes with the 0.1 M imidazole wash used to elute contaminants from the column. Less protein loss is generally observed in the purification of hHRP mutants. The overall yields of the purified, expressed proteins range from 5 to 15 mg/liter of expression medium. N-terminal sequencing of hHRP yielded the sequence ADLGS encoded by the 5` end of the multiple cloning site. This confirmed that the glycoprotein 67 leader sequence was removed from the mature protein.




Figure 3: Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of the purified hHRP, F41A hHRP, H42A hHRP, and H42V hHRP proteins. Lane A, molecular mass standards; lane B, native HRP; lane C, wild-type hHRP; lane D, F41A hHRP; lane E, H42A hHRP; and lane F, H42V hHRP.



Spectroscopic Characterization of Recombinant Proteins

The spectra of the resting ferric states of wild-type and His-42 hHRP mutants, like that of the native enzyme, exhibit asymmetric Soret peaks with maxima at (max) = 402 nm. However, F41A hHRP has a 4 nm red-shifted, shoulderless Soret absorption at (max) = 406 nm. The Compound I and Compound II spectra of hHRP and F41A hHRP are indistinguishable from the corresponding spectra of native HRP. In the case of the H42A and H42V mutants, 1,000 rather than 1 equivalent of H(2)O(2) is required to observe the decrease in Soret absorption characteristic of Compound I. A fully developed Compound I spectrum can also be obtained within 4 min by allowing the His-42 mutants to react with 5 equivalents of mCPBA. Following the formation of Compound I by reaction with excess H(2)O(2) (500 equivalents), the Soret band intensity decreases in a manner suggestive of heme degradation. In contrast to the Soret loss observed when Compound I is generated with an excess of H(2)O(2), the Compound I species generated with mCPBA decays slowly (1 h) to a protein with a resting ferric state spectrum (Fig. 4). This ferric spectrum is also immediately obtained when 1 equivalent of K(4)Fe(CN)(6) is added to either the H42A or H42V Compound I species generated with mCPBA. In no instance was the red-shifted increase in Soret absorbance characteristic of the conversion of Compound I to Compound II detected.


Figure 4: Formation of Compound I of H42A hHRP and reversion to the ferric state on standing. A, resting state; B, 4 min after adding 5 equivalents of mCPBA; and C, 11 min, D, 18 min, E, 30 min, and F, 60 min after initiation of the reaction. A Compound II spectrum is not detected as Compound I reverts to the ferric state.



Rate of Compound I Formation

Stopped flow spectrophotometry was used to determine the rates of formation of Compound I. A typical stopped flow spectroscopic trace of the formation of Compound I exhibited a decay at 411 nm which leveled off within 20 ms. Secondary plots of the stopped flow data for native, hHRP, and F41A hHRP were similar and gave similar second-order rate constants (Table 2). These results clearly establish that neither the polyhistidine tag, altered glycosylation pattern, nor F41A mutation appreciably influences the rate of Compound I formation.



As already noted, formation of Compound I from the H42A mutant requires a large excess of H(2)O(2). Addition of the required excess of peroxide to H42A, as shown by UV/visible spectroscopy, results in rapid formation of Compound I followed within 30 s by formation of a species with a Compound III-like spectrum and a species with a Soret maximum at 680 nm (possibly verdoheme). It is therefore not surprising that the secondary plot of the rate data for this mutant shows a nonlinear dependence of k on the peroxide concentration (Fig. 5). Compound I, Compound III, and a 680 nm-absorbing species are also formed in the reactions of native HRP with excess H(2)O(2), hydroperoxides, or peroxybenzoic acids (36, 37, 38) . One consequence of multiple product formation in the reaction of H42A hHRP is that the measurements of the change in absorbance at 411 nm used to determine the rate constant for Compound I formation may not truly reflect formation of that single species. It is nevertheless of interest that, although the behavior of H42A hHRP differs greatly from that of hHRP with regard to Compound I formation, the H42A mutant still forms species with excess peroxide spectroscopically similar to those obtained under comparable conditions with native HRP.


Figure 5: Plot of k[obs] versus the H(2)O(2) concentration for the H42A hHRP mutant. The rate was determined by measuring the rate of decrease of the absorbance at 411 nm as a function of peroxide by stopped flow methods. The H(2)O(2) concentrations are 0.025, 0.05, 0.125, 0.250, 0.375, 0.5, 0.75, 1.0, 1.25, and 1.5 M.



Steady-state Kinetics of H42A and H42V hHRP

The rates of substrate oxidation and Compound I formation for H42A and H42V hHRP were determined under steady-state conditions to circumvent the experimental difficulties associated with direct determination of the rates in a reaction that gives multiple products. Lower levels of peroxide were required for this approach compared with those required for direct determination of the rate of Compound I formation. Furthermore, in the presence of a substrate, reduction of Compound I by electron transfer from the substrate suppresses secondary reactions and results in normal cycling of the enzyme.

The time-dependent increase in absorbance at 414 nm observed during the oxidation of ABTS trails off in a nonlinear manner, presumably because of enzyme inactivation similar to that seen with native HRP at H(2)O(2) concentrations above 1 mM(39) . Initial rates were therefore measured to circumvent the apparent inactivation at higher peroxide concentrations. The resulting data, plotted as 2[hHRP](0)/v versus 1/[ABTS], give a series of lines analogous to those observed with the wild-type enzyme (Fig. 6a). The lines at low H(2)O(2) concentrations are parallel but deviate from this relationship at the higher H(2)O(2) concentrations. The deviation probably reflects competitive formation of Compound III(39) . The rate of Compound I formation (k(1)) was determined from the inverse of the slope taken from a secondary plot of the y intercept values from the primary plot versus 1/[H(2)O(2)] (Fig. 6b). The value thus obtained is k(1) = 19.4 M s for H42A hHRP and k(1) = 10.0 M s for H42V hHRP (Table 2). These values are 10^6-fold lower than the rate constant for wild-type hHRP.


Figure 6: Steady-state kinetics for the oxidation of ABTS by H42A hHRP. Panel a, the ABTS concentrations used for the primary plot of the rate versus the inverse of the ABTS concentration are 1.25, 1.67, 2.5, 5, and 10 mM. Panel b, secondary plot of the rate constants determined from the y intercepts of the lines from the primary plot versus the inverse of the peroxide concentration. The H(2)O(2) concentrations are 65, 85, 125, and 250 mM.



In HRP and hHRP, the reduction of Compound II is slower than that of Compound I, so that Compound II is observed during steady-state catalytic turnover. A consequence of the fact that k(2) is larger than k(3) is that simplifies to and analysis of HRP steady-state turnover yields a value for k(3). However, we do not spectrally detect Compound II under steady-state turnover conditions with the distal histidine mutants. This suggests that the situation is reversed for these mutants so that reduction of Compound II is more rapid than reduction of Compound I (i.e.k(3) > k(2)). In this case simplifies to .

The average of the inverse of the slopes of the lines in Fig. 6a and of a parallel independent set of data gives values of 1.9 ± 0.3 10^3M s for H42A hHRP and 1.6 ± 0.3 10^3M s for H42V hHRP which, on the basis that Compound II is not detected under steady-state conditions, can be tentatively assigned to k(2) rather than k(3). These values are smaller than the values of both k(3) (0.81 10^6M s) and k(2) for the native enzyme (18) . Although the value of k(2) for the reaction of ABTS with the native enzyme is not known, it must be larger than k(3) because the reduction of Compound II limits the turnover rate of the native enzyme under steady-state conditions. It appears, therefore, that the distal histidine is important for both the formation and the reduction of Compound I.

Peroxidase Activity

hHRP and F41A hHRP exhibit guaiacol peroxidation activities very similar to that of the native enzyme (Table 3). In contrast, guaiacol peroxidation by both H42A hHRP and H42V hHRP is much lower than that catalyzed by the native enzyme. This marked decrease in the peroxidase activity of the H42A mutant reflects the 10^6-fold decrease in the rate of Compound I formation caused by mutation of the catalytic histidine.



Thioanisole Sulfoxidation

The sulfoxidation activities were assayed at 0 °C because the rates measured at 25 °C leveled off quickly due to enzyme inactivation. F41A hHRP was the most sensitive of the recombinant proteins to inactivation, and time-dependent loss of activity was observed with F41A hHRP during both thioanisole sulfoxidation and guaiacol peroxidation. Loss of activity is the result of a turnover-dependent process because the protein is not unusually thermally unstable. A possible precedent for this observation is provided by the inactivation of native HRP during catalytic turnover of phenol(39) . Linear production of thioanisole sulfoxide was observed at 0 °C, however, when the F41A hHRP reaction was monitored for 5 min and the reactions with the other enzymes for 15 min. Control experiments indicate that thioanisole is oxidized nonenzymatically at a rate of 2.5 pmol s at 0 °C. This background oxidation level has been subtracted from the experimentally determined values in calculating the enzymatic rates and enantiomeric excesses listed in Table 3.

The sulfoxidation activities of the wild-type and native proteins are similar, although the commercially available enzyme is slightly more active (Table 3). A difference is observed in the sulfoxidation enantiospecificity of the native (83% ee) and wild-type recombinant (66% ee) enzymes. This minor difference presumably stems from the differences in glycosylation pattern or the presence of the N-terminal polyhistidine tag in hHRP. Asparagine 186, 198, and 214 of HRP are normally glycosylated. In the sequence alignment of lignin peroxidase with HRP, a gap is inserted into this region in lignin peroxidase. The residues that flank this gap, Val-184 and Asp-185, lie within the substrate channel in the crystal structure of lignin peroxidase. It is therefore probable that the unique HRP sequence that includes asparagine 186, 198, or 214 constitutes a portion of the HRP solvent accessible channel, and differences in glycosylation resulting from expression in insect cells might affect substrate docking in the active site.

F41A hHRP is a much better sulfoxidation catalyst than the native or wild-type enzymes, but the enantiospecificities of the three enzymes are comparable (Table 3). The distal histidine mutants also exhibit improved thioanisole sulfoxidation activity relative to the wild-type protein (Table 3), but the improvement is less than that found with the F41A mutant. Decreasing the size of the aliquot of peroxide added every 2 min to the reaction 5-fold causes the rate of thioanisole sulfoxidation catalyzed by His-42 mutants to decrease 2-3-fold but does not alter the sulfoxidation rates for hHRP and F41A hHRP. This suggests that Compound I formation is partially rate-limiting for the distal histidine mutants. The enantioselectivities of the distal histidine mutants are similar and relatively high compared with the enantioselectivities of the wild-type and F41A proteins (Table 3).

Styrene Epoxidation

F41A, H42A, and H42V hHRP are much more active as styrene epoxidation catalysts than the native or wild-type proteins, neither of which catalyzes more than a trace of styrene epoxidation (Table 3). The rates of styrene epoxidation were determined at 0 °C to minimize the sensitivity of the mutants to inactivation. Nonenzymatic styrene epoxidation is not observed under these conditions. Styrene oxide production was linear over a period of 10 min for the H42A and H42V mutants and 20 min for the F41A mutant. The preferred S-enantioselectivities of styrene epoxidation by the His-42 mutants are opposite that of the F41A mutant, which preferentially forms the R-enantiomer (Table 3).

Reaction of Wild-type and Mutant hHRP with Phenyldiazene

As indicated in the Introduction, the reactions of phenyldiazene with the prosthetic heme group provide information on the active site topologies of hemoproteins. The reactions of phenyldiazene with native HRP, wild-type hHRP, and F41A hHRP cause similar decays in the Soret band intensities of the proteins, although the reaction with F41A hHRP occurs more rapidly (<5 min versus 20 min) and to a greater extent (Fig. 7). The reactions of phenyldiazene with the His-42 hHRP mutants also cause a decrease in the Soret band intensity, but the spectra obtained are distinct from those obtained with the native, wild-type, or F41A mutant in that they exhibit multiple maxima, including one at (max) = 434 nm. Gel filtration to remove phenyldiazene-derived metabolites reduces absorption in the near UV region of the spectra without altering the Soret region.


Figure 7: Changes in the absorption spectra caused by reaction hHRP and its mutants in sodium phosphate buffer, pH 7.0, with phenyldiazene. Panel a, hHRP (10 nmol) before (A) and after the addition of 57 (B), 113 (C), and 170 (D) nmol of phenyldiazene. Panel b, the F41A mutant before (A) and after the addition of 57 (B) and 113 nmol (C) of phenyldiazene. Panel c, the H42A mutant before (A) and after the addition of 57 (B), 113 (C), 227 (D), and 340 (E) nmol of phenyldiazene.



HPLC analysis of the prosthetic group extracted from phenyldiazene-treated HRP and hHRP, as reported for native HRP (21) , reveals the presence of three heme-derived products (Fig. 8). Comparison of the retention times and absorption spectra of these products with authentic standards show that the substances eluting at 5.5 and 7 min are, respectively, the 8-hydroxymethyl derivative of heme and heme itself. The third peak with a retention time of 12 min is -meso-phenylheme. As found before for the native protein(21) , no trace is detected of the N-phenylprotoporphyrin IX isomers expected from rearrangement of a -bonded phenyl-iron complex.


Figure 8: HPLC analysis of the heme groups extracted after reaction with phenyldiazene from wild-type hHRP, F41A hHRP, and H42A hHRP. The details of the extraction and chromatographic methods are given under ``Experimental Procedures.''



In contrast to the native and wild-type proteins, HPLC analysis of the prosthetic group from phenyldiazene-treated F41A hHRP does not show peaks for heme or either the 8-hydroxymethyl or meso-phenyl derivatives of heme (Fig. 8). Peaks indicative of the formation of the N-phenylprotoporphyrin IX isomers are also not observed. The absence of a heme peak and the relative intensities of the product peaks suggest that substantial heme degradation has occurred. The major peak observed in the chromatogram corresponds to a new product with a retention time of 9 min and (max) = 400 nm (Fig. 8). The identity of this material has not yet been established.

HPLC analyses of the prosthetic groups of phenyldiazene-treated H42A hHRP and H42V hHRP give similar chromatograms with a major peak attributable to intact heme (Fig. 8). The amplitude of the heme peak is double that of the heme peaks found in the analyses of the wild-type and native proteins. Moreover, for each of the two His-42 mutants, denaturation of the protein complex under acidic conditions gives small amounts of N-phenylprotoporphyrin IX, as confirmed by direct comparison of the retention time and absorption spectra of the product with that of an authentic standard (not shown).


DISCUSSION

Fusion of a polyhistidine tail to the N terminus of HRP greatly simplifies purification of the protein without causing significant alterations in the observable spectroscopic and catalytic properties of the enzyme. Not only are the ferric spectra of HRP and hHRP identical but there are only small differences between these two proteins in terms of their rates of Compound I formation (Table 2), guaiacol peroxidation (Table 3), and thioanisole sulfoxidation (Table 3), or their lack of styrene epoxidizing activity (Table 3). These results establish that the N-terminal polyhistidine extension does not significantly alter the catalytic function of HRP and therefore need not be removed from the protein for mechanistic studies.

The active sites of the four peroxidases for which crystal structures are available suggest that the catalytic histidine and the adjacent aromatic residue act as part of a barrier that restricts interaction of the ferryl oxygen with the substrate. The changes in the catalytic properties of the enzyme caused by the F41A mutation support this inference. Thus, the F41A mutation causes a small (4 nm) shift in the Soret maximum and small percentage increases in the rate of Compound I formation (50%) and the ability of the mutant to catalyze guaiacol peroxidation (30%) ( Table 2and Table 3). Nevertheless, the F41A mutation increases the rate of thioanisole sulfoxidation 10-fold (Table 3). Replacement of Phe-41 by leucine or threonine, residues that are intermediate in size between a phenylalanine and an alanine, increases the V(max) for thioanisole sulfoxidation 1.9- and 1.6-fold, respectively(40) . The 10-fold increase observed here in the thioanisole sulfoxidation rate when Phe-41 is replaced by an alanine is considerably higher, as might be expected, than that obtained when Phe-41 is replaced by groups of intermediate size. The large increase in sulfoxidation activity in the face of minor changes in the rates of Compound I formation and guaiacol oxidation supports the conclusion that the F41A mutation makes the ferryl oxygen more accessible to thioanisole and thereby facilitates ferryl oxygen transfer to the sulfur. The conclusion that the F41A mutation increases access to the ferryl oxygen is confirmed by the observation that F41A hHRP (Table 3), like the F41L and F41T mutants(40) , catalyzes the epoxidation of styrene whereas the wild-type and native enzymes are virtually inactive in this regard (Table 3).

The increase in substrate access to the ferryl oxygen provided by the F41A mutation occurs without significantly enlarging the active site cavity directly above the iron atom. The evidence for this is provided by the finding that reaction of F41A hHRP with phenyldiazene results in modification of the heme group without the detectable formation of a phenyl-iron complex. The failure to form a phenyl-iron complex is indicated by the absence of a shift of the Soret to 430 nm in the reaction with phenyldiazene and by the absence of N-phenylprotoporphyrin IX from among the modified heme products extracted from the protein. In fact, a modified heme is formed in good yield which does not correspond to the normally observed -meso-phenyl- or 8-hydroxymethyl derivatives of heme (Fig. 8)(21) . The absence of these two products and the formation of a new heme product are consistent with a modification of the active site topology in the vicinity of the heme.

Mutations of His-42, the putative HRP distal histidine, have not been reported previously. Replacement of His-42 by an alanine or valine compromises the catalytic machinery required for activation of H(2)O(2). This is clearly demonstrated by a decrease of 10^6 in the rate (k(1)) of Compound I formation (Table 2). This agrees well with the decrease of 10^5 observed in the rate of Compound I formation when the distal histidine of cytochrome c peroxidase is replaced by a leucine(13) . The rate constant for Compound I formation was determined under steady-state conditions due to complications introduced by the requirement for a large excess of H(2)O(2) for catalytic turnover of the H42A and H42V mutants. The 10^6-fold decrease in k(1) confirms that His-42 is, indeed, the distal residue in HRP and establishes that it plays a direct catalytic role in peroxide activation (Fig. 1). The decrease is due, in part, to the fact that the peroxide anion (HOO) is probably required for uncatalyzed binding to the iron of H42A and H42V hHRP in the first step of Compound I formation, and this peroxide species is present in 10^6-fold lower concentration than H(2)O(2) at pH 6.0 (the pK of H(2)O(2) is 11.6)(41) .

A further consequence of replacing His-42 by an alanine or valine appears to be destabilization of Compound II with respect to Compound I. Thus, Compound I of the H42A and H42V mutants formed by reaction with mCPBA is reduced by 1 equivalent of ferrocyanide or by spontaneous decay to the ferric species without the detectable formation of Compound II. The distal histidine residue in HRP apparently stabilizes Compound II, probably by hydrogen bonding to the ferryl oxygen, and loss of this interaction destabilizes the ferryl intermediate with respect to reduction to the ferric state. Resonance Raman studies suggest the existence in native HRP Compound II of a hydrogen bond between a protein residue, presumably the distal histidine with a pK = 8.5-8.8, and the ferryl oxygen(42, 43) . The H42A and H42V mutants show some resemblance to the Arthromyces rhamosus peroxidase. At neutral pH, Compound I of the A. rhamosus peroxidase is reduced by ascorbate or hydroquinone to the ferric state without the apparent accumulation of Compound II, although spectroscopic analysis suggests that Compound II is formed under steady-state conditions at neutral pH, and Compound II formation is readily observed at pH 10(44) . However, Compound II is not observed at pH 10 with the H42A and H42V mutants.

The decreased rates of Compound I formation and changes in the reactivities of Compounds I and II of the H42A and H42V mutants are reflected in the overall rate of guaiacol oxidation, which decreases by approximately 5 orders of magnitude (Table 3). Comparison of the kinetic sensitivity of guaiacol oxidation with the H42A and H42V mutations with the insensitivity of this reaction to mutations of Phe-41 indicates that the rate effects of the His-42 mutations are not simply due to enlargement of the active site cavity or increased exposure to solvent water.

In contrast to the large decreases in the rate of Compound I formation and the peroxidase activity caused by the His-42 mutations, the peroxygenase activities of the H42A and H42V mutants actually increase (Table 3). Thus, the thioanisole sulfoxidation rate increases 13-fold for the H42A mutant and 9-fold for the H42V mutant with respect to wild-type hHRP. The increase in the rate of thioanisole sulfoxidation does not match that obtained with the F41A mutation, but this is not surprising given that the rate of Compound I formation is increased by the F41A mutation but drastically decreased by the H42A mutation. It may be that Compound I formation becomes partly rate-limiting in the thioanisole sulfoxidation reaction catalyzed by the His-42 mutants. The same trend is not observed for styrene epoxidation, a reaction that requires intimate contact of the ferryl oxygen with both carbons of the -bond rather than simply with a sulfur electron pair and is therefore more sterically demanding than thioanisole sulfoxidation. If access to the ferryl oxygen is particularly critical for epoxidation, the finding that the His-42 mutants are approximately three times more active than the F41A mutant (Table 3) suggests that the distal histidine mutations are more effective than those of Phe-41 in providing this access.

The changes in reactivity observed with the mutants are consistent with the topological data obtained in their reactions with phenyldiazene. The reactions of phenyldiazene with the H42A and H42V mutants, in contrast to the reactions with native, hHRP, or F41A hHRP, produce a phenyl-iron complex. The formation of a phenyl-iron complex is signaled by the observation of an absorption maximum at 434 nm (Fig. 7) and is confirmed by the isolation of small amounts of N-phenylprotoporphyrin IX when the prosthetic group is extracted from the phenyldiazene-treated protein under aerobic, acidic conditions (not shown). The high recovery of unmodified heme in this reaction suggests that the H42A and H42V phenyl-iron complexes are unstable and revert to a considerable extent to unmodified heme during the workup procedure. The formation of an iron-phenyl complex is a hallmark of hemoproteins in which the iron atom is accessible to exogenous substrates. Analysis of the location of the histidine and the conserved adjacent aromatic residue in the four peroxidases for which crystal structures are available shows that the histidine sits directly above the iron, whereas the aromatic residue is positioned to one side (Fig. 2). The results obtained with phenyldiazene provide direct experimental evidence that the histidine and phenylalanine are similarly disposed in the active site of HRP.

The enantioselectivities of both thioanisole sulfoxidation and styrene epoxidation are higher for the distal histidine mutants than for F41A hHRP (Table 3). Preferred formation of the (R)-thioanisole sulfoxide and (S)-styrene oxide enantiomers by the F41A mutant requires the same spatial orientation of the substrate phenyl group and the side chain with respect to the ferryl oxygen, whereas the favored formation of the S-enantiomers of both products by the H42A and H42V mutants requires that the aryl substituents assume opposite orientations with respect to the ferryl oxygen. The basis for this difference in substrate orientation cannot be deduced from the available structural information.

The present studies provide direct experimental evidence that His-42 is a key catalytic residue, strengthen the evidence that both His-42 and Phe-41 sterically insulate the ferryl species of Compound I from interaction with the substrate, and demonstrate that reductions in the size of these residues greatly increase the peroxygenase activity of HRP. In fact, the F41A, H42A, and H42V mutants compare favorably with cytochrome P450 as thioanisole sulfoxidation catalysts even if they are still 1 order of magnitude weaker than cytochrome P450 as styrene epoxidation catalysts (Table 3). To improve the peroxygenase properties of HRP, it will be necessary to construct mutants that retain a high rate of Compound I formation but have active sites that allow substrates unhindered access to the ferryl oxygen.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM32488. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 415-502-4728 or 415-476-0688.

(^1)
The abbreviations used are: heme, iron protoporphyrin IX regardless of oxidation and ligation state; HRP, horseradish peroxidase isozyme c; hHRP, polyhistidine-tagged recombinant horseradish peroxidase; mCPBA, meta-chloroperbenzoic acid; ABTS, 2,2`-azinobis-3-ethylbenzothiazoline-6-sulfonic acid; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Christa Hartmann for assistance with expression of HRP in the baculovirus/S. frugiperda cell system.


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