the of biological chemistry vol. no. 5, of …the journal of biological chemistry 0 1987 by the...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 4. Issue of February 5 , pp. 1542-1551, 1987 Printed in U. S. A.

Protein Control of Prosthetic Heme Reactivity REACTION OF SUBSTRATES WITH THE HEME EDGE OF HORSERADISH PEROXIDASE*

(Received for publication, August 18, 1986)

Mark A. Ator and Paul R. Ortiz de Montellano$ From the Department of Pharmaceutical Chemistry, School of Pharmacy, and Liver Center, University of California, San Francisco, California 94143

Incubation of horseradish peroxidase with phenylhydrazine and H202 markedly depresses the catalytic activity and the intensity, but not position, of the Soret band. Approximately 11-13 mol of phenylhydrazine and 25 mol of H202 are required per mol of enzyme to minimize the chromophore intensity. The enzyme re- tains some activity after such treatment, but this activity is eliminated if the enzyme is isolated and reincubated with phenylhydrazine. The prosthetic heme of the enzyme does not react with phenylhydrazine to give a a-bonded phenyl-iron complex, as it does in other hemoproteins, but is converted instead to the b-meso- phenyl and 8-hydroxymethyl derivatives. The loss of activity is due more to protein than heme modification, however. The inactivated enzyme reacts with H202 to give a spectroscopically detectable Compound I. The results imply that substrates interact with the heme edge rather than with the activated oxygen of Com- pounds I and I1 and specifically identify the region around the 6-meso-carbon and 8-methyl group as the exposed sector of the heme. Horseradish peroxidase, in contrast to cytochrome P-450, generally does not catalyze oxygen-transfer reactions. The present results indicate that oxygen-transfer reactions do not occur because the activated oxygen and the substrate are physically separated by a protein-imposed barrier in horseradish peroxidase.

Heme’ serves as the prosthetic group of a large number of functionally diverse proteins. These include electron trans- port proteins (e.g. cytochrome &, cytochrome c), oxygen trans- port proteins (e.g. hemoglobin, myoglobin), one-electron oxi- dants (the peroxidases), oxygen-transfer proteins (the cytochrome P-450 monooxygenases), catalase, and proteins that catalyze relatively complex substrate transformations (e.g. prostaglandin, prostacyclin, and thromboxane synthases). This functional diversity is achieved through control of the inherent reactivity of the prosthetic heme group by the surrounding protein. The mechanisms by which this control is exerted are poorly understood, however, despite their impor-

*This research was supported by Grant GM 32488 from the National Institutes of Health. Equipment support was provided by Grant GM 25515. Mass Spectra were obtained through the University of California, San Francisco, Biomedical Mass Spectrometry Facility supported by Grant RR 01614. 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. The abbreviations used are: heme, iron protoporphyrin IX re-

gardless of the iron oxidation state; HPLC, high performance liquid chromatography.

tance to the development of a comprehensive theory of hemoprotein function.

The parallels between cytochrome P-450 enzymes and the peroxidases have been underscored by recent advances (1). Cytochrome P-450 derives its oxidative power from the catalytic reduction of molecular oxygen but, like the peroxidases, is able to function more or less normally if provided with a peroxide rather than with molecular oxygen and NADPH (1- 3). The oxidative species produced by cytochrome P-450 has not been observed directly, but model studies with ferric porphyrins indicate that the catalytic requirements of the enzyme can be met by an [Fe0I3+ species analogous to that of Compound I of horseradish peroxidase (4, 5). Bioorganic studies indicate that the reactions of cytochrome P-450, like those of horseradish peroxidase, at least sometimes involve sequential electron transfers (3). Despite these similarities, however, the reactions catalyzed by cytochrome P-450 and horseradish peroxidase are sharply differentiated by the fact that the net cytochrome P-450 reaction is generally a two- electron oxidation coupled with transfer of oxygen to the substrate (3, 6, 7), whereas that of horseradish peroxidase is a one-electron oxidation with no oxygen transfer. Indeed, with the possible exception of a recently reported sulfur oxidation (8), there is, to our knowledge, no unambiguously documented example of ferryl oxygen transfer from horseradish peroxidase to a substrate. The incorporation of oxygen into substrates in horseradish peroxidase-catalyzed reactions is known (e.g. Refs. 9-13), but the oxygen has generally been found to derive from water or molecular oxygen rather than from the peroxide. The divergent oxidation of a common substrate by cytochrome P-450 and horseradish peroxidase is graphically illustrated by acetaminophen, which is oxidized by cytochrome P-450 to an iminoquinone but by horseradish peroxidase to a free radical (14) that polymerizes by radical- recombination reactions (15). This categorical reactivity difference cannot be attributed to the lower oxidizing power of the ferryl species in horseradish peroxidase, although the presence of a histidine rather than thiolate iron ligand is undoubtedly of some importance, because model metalloporphyrin complexes analogous to horseradish peroxidase Com- pound I readily promote oxygen-transfer reactions (5,6). The protein matrix of horseradish peroxidase therefore must sup- press the inherent oxygen-transfer activity of the ferryl species.

The structures, regiochemistries, and stereochemistries of the N-alkylated hemes generated when cytochrome P-450 turns over olefinic, acetylenic, and heterocyclic “suicide” substrates have been used to define the orientation of its heme (16) and its active site topology (17). The stable phenyl-iron complex formed in the H20z-dependent reaction of myoglobin with phenylhydrazine has more recently unmasked the route taken by substrates and ligands into the heme crevice (18).

1542

Catalytic Mechanism of Horseradish Peroxidase 1543

Analogous phenyl-iron complexes are formed in the reactions of catalase (19) and cytochrome P-450 with phenylhydrazine (20), but preliminary spectroscopic studies suggest that a phenyl-iron complex is not formed in the reaction with horseradish peroxidase even though the enzyme is inactivated (19, 21). We have therefore investigated the reaction of horseradish peroxidase with phenylhydrazine in detail to elucidate the structural and mechanistic reasons for this difference in reactivity. We provide evidence here that substrate oxidation by horseradish peroxidase involves the transfer of electrons to the heme periphery between the 6-meso-carbon and 8-methyl substituent. The results imply that the oxygen of Compounds I and I1 is sequestered in a site to which substrates have little or no access and which therefore cannot be transferred to the substrates.

MATERIALS AND METHODS

Horseradish peroxidase (type VI), bovine liver catalase, hydrogen peroxide, guaiacol, and sodium ascorbate were obtained from Sigma. Phenylhydrazine hydrochloride, azobenzene, and pyridine-d5 were purchased from Aldrich. Solutions of phenylhydrazine hydrochloride, which was recrystallized from ethanol, were prepared in 0.01 N HCI and were used within 8 h. [U-'4C]Phenylhydrazine hydrochloride was obtained from ICN Radiochemicals (Irvine, CA). Phenyldiazene was generated in situ from potassium phenyldiazene carboxylate prepared by the method of Huang and Kosower (22). H2I80 (50 atom %) was purchased from Icon Services, Inc. (Summit, NJ). Buffers were made with glass-distilled deionized water and were passed through a column of Chelex 100 (Bio-Rad).

The concentration of horseradish peroxidase was determined using E,,, = 95,000 M" cm" (23) or, where indicated, by Lowry et al. (24) protein assay. Hydrogen peroxide solutions were standardized by the peroxidase-catalyzed oxidation of iodide to iodine, utilizing an extinction coefficient of 23,000 M" cm" at 353 nm (25). All peroxidase experiments were performed in 50 mM sodium phosphate buffer (pH 7.0) a t 25 "C.

Absorption spectra were obtained on a Hewlett-Packard 8450A diode array spectrophotometer. Scintillation counting was done on a Searle Mark 111 scintillation counter in Aquasol (New England Nu- clear). High pressure liquid chromatography was performed with a system consisting of Beckman Model llOA pumps, a Model 420 controller, and a Hewlett-Packard 1040A diode array detector. Gas- liquid chromatography was carried out on a Hewlett-Packard 5890A capillary instrument equipped with flame ionization detectors and a Hewlett-Packard 3390A integrator. NMR spectra were recorded on a General Electric GN 500 MHz instrument in deuterochloroform. Chemical shifts are reported in parts/million relative to tetramethyl- silane. Mass spectra were obtained on a Kratos MS 50 instrument operating in the liquid matrix secondary ion mode.

Inactivation of Horseradish Peroxidase by Phenylhydrazine-The rate of inactivation of peroxidase by phenylhydrazine was measured using a reaction mixture which contained 1 p M peroxidase, 0.2 mM H202, and 10-150 p~ phenylhydrazine in a volume of 0.2 ml. At 0, 1, 5, and 10 min after the addition of inactivator and peroxide, 5-pl aliquots of the mixture were transferred to quartz cuvettes containing 1.0 ml of an assay solution composed of 50 mM sodium phosphate (pH 7.0), 5 mM guaiacol, and 0.6 mM H202. Peroxidase activity was measured by the increase of absorbance a t 470 nm. Activity values were corrected for the small amount of inactivation observed in control incubations without phenylhydrazine.

Metabolite Identification and Quantitation-Five aliquots of hydrogen peroxide, each of them sufficient to make the incubation solution 2 mM in peroxide, were added a t 0.5-min intervals to 1 ml of a solution of phenylhydrazine (5 mM) and horseradish peroxidase (0.1 mM). After a total of 5 min a t room temperature, 10 pl of a 0.2 M ether solution of toluene was added and the incubation extracted with 1 ml of ether. The organic layer was washed with brine, dried over anhy- drous sodium sulfate, and placed on ice until analyzed by gas-liquid chromatography on a DB-5 capillary column. The injector and detector temperatures were 200 and 250 "C, respectively, and the column was programmed to rise from 30 to 50 "C a t 2 "C/min and then from 50 to 250 "C at 20 "C/min.

Decrease in Soret Absorbance of Peroxidase following Treatment with Phenylhydrazine-The decrease in the Soret absorbance of horseradish peroxidase caused by phenylhydrazine was measured as

a function of the hydrogen peroxide concentration in mixtures that contained 12.5 p~ peroxidase, 250 p~ phenylhydrazine, and 0 to 500 p~ H202 in a volume of 0.7 ml. After 10 min, the solutions were chromatographed a t 4 "C on columns of Sephadex G-25 (1.5 x 25 cm) equilibrated with 50 mM sodium phosphate (pH 7.0). Fractions of 1.0 ml were collected and the absorbance spectrum of each fraction recorded.

The amount of phenylhydrazine required to maximally decrease the Soret absorbance was determined as above, except that the phenylhydrazine concentration was varied from 0 to 275 p M with the peroxide concentration set at 625 p ~ . In these experiments, 4.5 pg of catalase was added after 10 min to destroy residual hydrogen peroxide, and the peroxidase was returned to the ferric state by adding 25 pM sodium ascorbate. The enzyme was then chromatographed on Seph- adex G-25.

Heme Adduct Analysis and Radiolabeling by [14ClPhenylhydra- zine-Peroxidase was radiolabeled in a mixture containing 125 p~ peroxidase, 3.75 mM H2O2 (added in three portions a t 0, 1, and 2 min), and 2.5 mM [U-14C]phenylhydrazine (specific activity: 1 X lo6 cpm/pmol) in a volume of 3.2 ml. After 10 min at room temperature, the mixture was chromatographed on Sephadex G-25 as already described. Fractions of 1.0 ml were collected and their peroxidase content determined by the Lowry procedure with horseradish peroxidase as the standard. Aliquots (0.3 ml) of each fraction were analyzed for bound radioactivity by liquid scintillation counting.

To determine if phenyl heme adducts were present in the radiolabeled peroxidase, the protein-containing fractions from the Sephadex column were combined and concentrated to a volume of 3.0 ml by centrifugation in a Centricon 10 (Amicon) apparatus at 4 "C. The protein solution was acidified with 1 ml of glacial acetic acid and the heme extracted into two 3-ml portions of ether. An emulsion formed during the extraction, which was broken by low speed centrifugation. The combined organic layers were washed with water, dried over sodium sulfate, and evaporated to dryness under vacuum. The isolated heme fraction was then chromatographed on a 4.6 X 250-mm What- man Partisil5 ODS-3 reverse phase HPLC column (solvent A 6:4:1 methanol/H20/glacial acetic acid; solvent B: 1 0 1 methanol/glacial acetic acid. Flow rate: 1 ml/min, linear gradient from 0 to 100% solvent B over 10 min. Metalloporphyrin absorbance was monitored at 400 nm and 1-min fractions were collected. Aliquots (0.3 ml) of each fraction were combined with 0.7 ml of H20 and analyzed by liquid scintillation counting.

To determine the extinction coefficient for meso-phenyl heme, the two fractions containing meso-phenyl heme were combined, concentrated, and rechromatographed isocratically on the same column with a 4:l mixture of solvents A and B (1 ml/min flow rate). The fractions containing meso-phenyl heme were isolated and put twice through the gradient elution chromatography described in the preceding par- agraph. The specific activity of the radiolabeled fraction, determined in 10:1 methanol/acetic acid, remained unchanged after each of the latter chromatographic steps.

Isolation and Identification of Modified Hemes-The modified hemes generated during the inactivation of peroxidase by phenylhydrazine were obtained on a preparative scale in reactions that contained 30 pM peroxidase, 0.6 mM phenylhydrazine, and 0.9 mM H,O, in a volume of 70 ml. After 15 min a t room temperature, the mixture was identified and extracted as described above. The mixture of hemes was separated by isocratic reverse phase high pressure liquid chromatography on a Partisil5 ODS-3 column eluted a t a flow rate of 1 ml/min with 65:35:10 methanol/H20/glacial acetic acid. Fractions containing residual heme (retention time: 11.5 min) and the two major heme derivatives (retention times: 7.7 and 26.8 min) were separately pooled and taken to dryness in vacuo. Analogous incubations were carried out on a smaller scale under an argon atmosphere to determine the role of oxygen in heme modification. Negative ion mass spectra were obtained of the underivatized hemes, and NMR spectra were recorded in pyridine-& containing an excess of SnClz (26). In order to dissolve the hemes in pyridine it was necessary to convert them from the acetatoiron(II1) to the chloroiron(II1) form by partitioning them between ether and a solution of 0.1 N DC1 in NaCI- saturated D,O.

The mixture of hemes was esterified with (CH,),OBF, (27) and was then demetallated by the procedure of Smith and Fuhrhop (28). The resulting porphyrins were separated by HPLC on a 4.6 x 250- mm Whatman Partisil-5 PAC column in 1:2 tetrahydrofuran/hexanes at a flow rate of 1 ml/min. The phenyl-substituted porphyrin was characterized by its electronic absorption spectrum in CH,Cl, (wavelength (relative absorbance)): 410 (loo), 508 (8.4), 544 (4.5), 578 (3.7),

1544 Catalytic Mechanism of Horseradish Peroxidase

632 (1.5) and mass spectrometric molecular ion (m/z = 667). The zinc complex of the porphyrin was prepared by adding a small amount of Zn(OAc)z in methanol to the free base dissolved in CHzCl2, followed by washing with HzO and brine and drying over NazSO,. The spectrum of the zinc complex in CHZClZ has peaks at (wavelength (relative intensity)) 418 (loo), 548 (6.3), and 584 nm (4.0).

Origin of the Oxygen in the Hydroxylated Heme-The incubation mixture was prepared by lyophilizing 2 ml of 50 mM sodium phosphate buffer (pH 7.0) to dryness in a 10-ml pear-shaped flask and adding 2 ml of H2I80 (50 atom %), 16 mg of horseradish peroxidase, and 1.2 mg of solid phenylhydrazine.HC1. The reaction was initiated by adding 1.2 p1 of 30% HZ02 in 5 aliquots over a period of 3 min. After 10 min, the mixture was frozen and the solvent removed by bulb-to- bulb distillation for use in a second incubation. The prosthetic heme was isolated from the combined incubations and analyzed by mass spectrometry as described above.

RESULTS

Inactivation of Horseradish Peroxidase by Phenylhydra- zine-Phenylhydrazine inactivates horseradish peroxidase within 1 min in the presence of 0.2 mM HzOz, in contrast to the 30 min required to inactivate half the enzyme in the absence of added peroxide (21). The much slower inactivation observed in the absence of added peroxide probably depends on generation of the required peroxide by autooxidation of phenylhydrazine, as found for the inactivation of hemoglobin by this agent (29). The inactivation is concentration-dependent, but the enzyme is not completely inactivated by a large excess of the hydrazine even if the H202 concentration is raised to ensure it is not the limiting reagent (Fig. 1). The activity of horseradish peroxidase is not restored by Sephadex G-25 chromatography and is thus irreversibly lost.

The failure of even a large excess of phenylhydrazine to completely inactivate the enzyme could, as suggested by Sil- verman and Zieske (30) for the inactivation of monoamine oxidase by 1-phenylcyclobutylamine, result from competitive binding of a metabolic product. If tight but reversible binding of a metabolite protects horseradish peroxidase from inactivation, removal of the metabolite by chromatography should make the enzyme vulnerable to complete inactivation. Indeed, the residual catalytic activity was eliminated if peroxidase that was 89% inactivated by exposure to 55 eq of phenylhydrazine and 80 eq of H202 was chromatographed through Sephadex G-25 and reincubated with the same ratio of re- agents (Fig. 2). Control experiments established that the chromatographic procedure did not alter the catalytic activity of the intact or partially inactivated enzyme.

%$ 0 20 40 60 EO 100 120 140 160

Phenylhydrazine/HRP ratio

FIG. 1. Inactivation of horseradish peroxidase as a function of the phenylhydrazine to enzyme ratio. Horseradish peroxidase (HRP) (1 PM) was incubated with 0.2 mM H202 and increasing concentrations of phenylhydrazine. Aliquots were withdrawn after 10 min, and the peroxidase activity was measured by the guaiacol oxidation assay as described under “Materials and Methods.”

1 oo-p

I- 0 1 2 3 4 5 0 1 2 3 4 5

Time (min)

FIG. 2. Inactivation of horseradish peroxidase isolated from an incubation with phenylhydrazine and HzOz by a second incubation with these agents. The loss of activity caused by incubating horseradish peroxidase with 55 eq of phenylhydrazine and 80 eq of H202 is shown on the left. The enzyme was then passed through Sephadex G-25 (G-25), and the loss of activity caused by reincubation with the same ratio of phenylhydrazine and peroxide is shown on the right (0). The activity observed after chromatography if the enzyme is incubated with HzOz in the absence of phenylhydrazine is also indicated on the right (A). The data on the right have been normalized to allow for dilution of the enzyme during the chromatographic step.

The incubation products were extracted, analyzed by gas- liquid chromatography, and shown by coelution with authentic standards to be azobenzene, benzene, and a trace of biphenyl (not shown). Azobenzene, the principal metabolite, is not obtained if any of the reaction components is omitted, but benzene is generated even if horseradish peroxidase is omitted from the incubation, probably because no metal che- lating agents were added to the incubation. Addition of high concentrations of authentic azobenzene, biphenyl, or benzene to incubations of phenylhydrazine and horseradish peroxidase, however, did not protect the enzyme against inactivation (not shown).

Decrease of the Soret Absorbance-Incubation of horseradish peroxidase with phenylhydrazine and H202 causes a rapid change in the absorbance spectrum of the mixture that is complete within 1 min. The absorbance of the peroxidase is obscured by a product of the reaction that absorbs near the Soret region. This product may be trans-azobenzene (Amax (MeOH) 228 ( e = 13,400) and 314 ( e = 18,200)) (31), but its identity has not been pursued further. Removal of this product by Sephadex G-25 chromatography allowed determination of the spectrum of the inactivated enzyme (Fig. 3). The intensity of the Soret band is decreased without a detectable shift in wavelength, and subtle changes occur in the visible region of the spectrum. Measurement of the concentration of the modified peroxidase by the Lowry protein assay demonstrated that its extinction coefficient at 402 nm was 70% of that of the native enzyme, while no significant change occurred in the extinction coefficient at 280 nm. The ratio of absorbance at 402 nm to that at 280 nm (A402/A280), which decreases from 3.05 to an end point of 2.0, therefore serves as a convenient internal indicator of the extent of the decrease of Soret absorbance.

The loss of Soret intensity, measured as a decrease in the A402/A280 ratio, was quantitated as a function of the phenylhydrazine and H202 concentrations to determine the partition ratios for the inactivation reaction. Titration of the peroxidase with phenylhydrazine in the presence of excess (50 eq) H2O2 established that 11-13 eq of the hydrazine are required to maximally decrease the absorbance ratio (Fig. 4). A more


FIG. 3. Electronic absorption spectra of native horseradish peroxidase (-) and phenylhydrazine- treated horseradish peroxidase (- - -). Peroxidase was incubated with a 20- fold excess of phenylhydrazine and a 30- fold excess of HZ02 and reisolated on a column of Sephadex G-25. The spectra were normalized to the same absorbance at 280 nm.

3.2,

0 1 2 4 6 8 1 0 1 2

I I 14 16 18 20 22

PhenylhydrazinelHRP ratio

FIG. 4. Decrease in the Soret absorbance of horseradish peroxidase (HRP), measured as a change in the A402/A280 ratio, as a function of the phenylhydrazine concentration. A fixed 50:l ratio of H202/heme was employed in these incubations.

complex result is obtained when the enzyme is titrated with H202. In the presence of excess phenylhydrazine (20 eq), approximately 25 eq of peroxide are required to achieve max- imum Soret loss, but a lag is observed in chromophore loss at low peroxide/heme ratios (Fig. 5). The ratio of 2 eq of H202 per eq of phenylhydrazine suggests that phenylhydrazine must be catalytically oxidized to phenyldiazene at the expense of 1 eq of peroxide and that a second equivalent of peroxide is necessary to convert the phenyldiazene to the species that decreases the Soret absorbance. This conclusion is strength- ened by data on the decrease in the A402/A280 ratio when phenylhydrazine is replaced by phenyldiazene generated in situ by decarboxylation of potassium phenyldiazene carboxylate (22). Measurement of A402/A280 as a function of the HzOz to enzyme ratio in the presence of excess (20 eq) phenyldiazene yields a plot that only deviates from linearity at high peroxide/enzyme ratios. Extrapolation of the linear segment indicates that maximal absorbance loss requires approximately 11 molecules of H202 per enzyme molecule (Fig. 6).

Complexes of the Inactivated Enzyme-The absorption spectrum of the phenylhydrazine-treated enzyme on addition

Wavelength lnrnl

3.2 1

0 1 I I I 5 10 15 20 25 30 35 40 45

HZ02/HRP raho

FIG. 5. Decrease in the Soret absorbance of horseradish peroxidase (HRP), measured as a change in the A402/A280 ratio, as a function of H202/enzyme ratio. A fixed 20:l phenylhydrazine/ heme ratio was employed in these incubations.

of 1.1 eq of Hz02 is very similar to that of Compound I of the native enzyme (Fig. 7). The inactive enzyme therefore appears to still process hydrogen peroxide in the normal manner. Furthermore, dithionite reduction of the inactive enzyme in the presence of carbon monoxide yields a complex with a spectrum that is essentially identical to that of the ferrous. CO complex of the parent enzyme. The carbon monoxide complex is not detected spectroscopically, however, if horseradish peroxidase is incubated with phenylhydrazine and H2OZ under an atmosphere of carbon monoxide, even though control experiments show it is stable enough to be observed under the reaction conditions. The enzyme therefore does not appear to pass through the ferrous state in the inactivation process.

Radiolabeling of Horseradish Peroxidase by ['4CJPhenylhy- drazine-The Soret loss observed when horseradish peroxidase is treated with phenylhydrazine requires modification of the heme or the apoprotein in a manner that attenuates the heme chromophore. To determine if the substrate is cova- lently bound to the enzyme, horseradish peroxidase was incubated with [14C]phenylhydrazine and the inactivated en-

1546

0

Y w 2 cu 0

3.0 1 Catalytic Mechanism of Horseradish Peroxidase

0 , 1 l I l l l l l l l I f

2 4 6 8 10 12 H202/HRP ratio

FIG. 6. Decrease in the Soret absorbance, measured as a change in the A402/A280 ratio, as a function of the HzOz/enzyme ratio in the presence of 20 eq of phenyldiazene.

zyme was chromatographically separated from labeled phenylhydrazine. Simultaneous measurements of the loss of catalytic activity, decrease in A402/A280 ratio, and covalent binding of radiolabeled phenylhydrazine to the protein indicate that all three phenomena are closely correlated (Fig. 8). Minimal activity and maximal chromophore loss coincide with covalent binding of two phenylhydrazine moieties to the enzyme.

Isolation and Characterization of Modified Hemes-The prosthetic groups of hemoglobin, myoglobin, and catalase are converted to N-phenylprotoporphyrin IX in the reaction with phenylhydrazine (19, 29). However, no trace of N-phenylprotoporphyrin IX could be detected spectrophotometrically or by high pressure liquid chromatography when the prosthetic group of phenylhydrazine-inactivated horseradish peroxidase was extracted with acidic methanol (data not shown). The prosthetic group was therefore extracted under conditions that do not esterify the carboxyl groups, and the extract was subjected to reverse phase high pressure liquid chromatography. Three major heme-derived products were observed (Fig. 9). The same products are obtained in approximately the same ratio if horseradish peroxidase is inactivated with phenylhydrazine under anaerobic conditions. If the partially inactivated enzyme is purified and is reincubated with phenylhy-

0.30 I I I I I I 1

A.

L I

drazine, the recovery of heme products is decreased, the proportion of heme in the mixture is decreased, and the two minor peaks that elute just after heme are slightly increased.

The middle peak (Fig. 9, 13.2 min) was identified by its retention time and mass and 'H NMR spectra as intact heme. Radioactivity from labeled phenylhydrazine was associated with the third peak at 16.2 min and with a substance that trailed off the column, but not with the first peak at 10.5 min (Fig. 9). Chromatographic purification of the radiolabeled substance to constant specific activity yielded an absorbance value for the Soret band of 1.6 X lo5 M" cm", a value essentially unchanged from that determined for heme itself (1.7 X lo5 M" cm"). An independent estimate of the molar absorbance value of the substance in the peak at 10.5 min is not available. If we assume that it has an absorbance identical to that of heme, an assumption warranted by its structure (see below), the three products are present in the extract in a 34:54:12 ratio. Comparison of the amount of heme-derived products extracted from intact and phenylhydrazine-inactivated enzyme indicates that only 70% as much of the heme is recovered after reaction with the hydrazine. The 54% of heme in the isolated mixture of heme products thus represents approximately 30% of the original heme.

Structural elucidation of the substance in the third peak is relatively straightforward. The incorporation of radiolabel in a 1:l ratio with the heme points to covalent attachment of a phenyl moiety. This is confirmed by the observation that the demetallated, and esterified porphyrin exhibits a molecular ion in the mass spectrum a t m/z 667, as expected for a monophenyl adduct of dimethylesterified protoporphyrin IX. The porphyrin chromophore is not greatly perturbed by the phenyl group, however, since the absorption spectrum of the porphyrin only differs from that of protoporphyrin IX in that the Soret band is at 410 rather than 406 nm and the etio pattern of the protoporphyrin IX bands gives way to a phyllo pattern in which bands I1 and I11 are of nearly equal intensity. The Soret band shift and the change in band pattern are consistent with meso-substitution of the porphyrin (32).

The 'H NMR spectrum of the SnC12-reduced dipyridine complex unambiguously identifies the derivative in the third peak as a meso-phenyl derivative of heme (Fig. 10). Three rather than four meso-proton signals are observed in the NMR spectrum (at 10.34, 10.24, and 10.04 ppm). The meso signal that is missing, when the spectrum is compared to that of heme, is that of the 6-meso-proton at 9.93 ppm (26). The inference that the phenyl moiety is bound exclusively to the 6-meso-carbon is confirmed by the finding that two of the

I I I I I I I I I I 1 I I I I I 300 400 500 600 700 300 400 500 600 700

Wavelength (nm)

FIG. 7. Electronic absorption spectra of native horseradish peroxidase and its Compound I (A) and phenylhydrazine-treated horseradish peroxidase (HRP) and its Compound I (B). The protein concentration was the same for the native and phenylhydrazine-treated enzyme.

Catalytic Mechanism of Horseradish Peroxidase 1547 I:m:: 6 8 2.5 1.0

$ 2.3

2.1

1.9 0 0 20 40 60 80 100

T 0.5

2.0 I I

3.1 2.9 2.7 2.5 2.3 2.1 1.9

Percent Activity Lost A4Q2/A200

FIG. 8. Left, correlation of chromophore loss with activity loss (0) and covalent binding of radiolabeled phenylhydrazine to the protein with loss of activity (0). and right, covalent binding of radiolabeled phenylhydrazine to the protein with loss of the chromophore. These data were obtained from a single set of incubations to minimize experimental variability. The peroxidase concentration was 10 p ~ , the ['4C]phenylhydrazine concentration 10-300 pM and the HzOz concentration twice that of phenylhydrazine. The incubation time was 5.5 min. Aliquots of 1 p1 were analyzed for activity by the guaiacol assay. The mixtures were chromatographed on a Sephadex G-25 column in 50 mM potassium phosphate buffer prior to absorbance measurements and radioscintil- lation counting.

800

400

o / , l l l , l , l l l l l l _..- 10 20

Tlme (min)

FIG. 9. Reverse phase high pressure liquid chromatogram of the metalloporphyrins extracted from ['4C]phenylhydrazine-treated horseradish peroxidase. Peroxidase was inactivated and heme was isolated as described under "Materials and Methods." The mixture was chromatographed on a reverse phase HPLC column and the absorbance at 400 nm was monitored. Fractions of 1 min were collected and 0.3-ml aliquots of each fraction were analyzed for radioactivity hy liquid scintillation counting.

--T-l-T"~ 18.1 10.0 8.0 7.6 3.t 3.1 2.6 2.5 ppn

FIG. 10. Partial 500 MHz 'H NMR spectrum of the meso- phenyl heme adduct isolated from phenylhydrazine-inactivated horseradish peroxidase. The three meso protons (10.34, 10.24, and 10.04), five phenyl protons (8.01, 7.67, and 7.59), four external propionic acid methylene protons (3.53 and 3.44), and 12 methyl protons (3.56, 3.46, 2.54, and 2.49) are shown. The vinyl and propionic acid methylene protons are not shown.

-

protoporphyrin IX methyl groups are shifted approximately 1 ppm upfield relative to the other two methyl groups and to the position in which they are normally found (Fig. 10). This shift, caused by the ring current of the phenyl group, can only

involve two methyls if the phenyl group is located at the 6- meso position. The signals of the ortho-, para-, and meta- protons of the phenyl group are found, respectively, at 8.01 (doublet, J = 7.5 Hz), 7.67 (triplet, J = 7.5 Hz), and 7.59 ppm (triplet, J = 7.5 Hz). The phenyl group assignments have been confirmed by decoupling experiments. Irradiation of the proton at 8.01 thus collapses the triplet at 7.59 ppm to a doublet without altering the signal a t 7.67, whereas irradiation of the proton at 7.59 collapses the doublet at 8.01 and the triplet at 7.67 to two singlets. All of the other signals in the spectrum are consistent with the 6-meso-phenyl structure.

The substance associated with the first peak exhibited a molecular ion under negative ion conditions at m/z 631. The molecular ion of heme under the same conditions is found at m/z = 615:616. The polar product thus appears to be a heme with one additional oxygen atom. The molecular ion of the polar material isolated from reactions of peroxidase with phenylhydrazine in H2180 (50 atom %) shifts from 631 to 633, indicating that the additional oxygen atom is provided by the aqueous medium (Fig. 11). Proton NMR indicates that the purified product contains one major and one minor compo- nent (Fig. 12). The variation in the ratio of these two components with workup and storage conditions suggests that the minor product derives, at least in part, from decomposition of the major product. The most informative signals due to the minor product are the two singlets at 11.61 and 11.09 ppm (Fig. In), which appear at virtually the same positions as the aldehyde and 6-meso-protons of heme a (11.63 and 11.03 ppm, respectively). The 6-meso-proton of heme a, in which the 8- methyl group is replaced by a formyl moiety (26), is shifted downfield by the deshielding effect of the vicinal carbonyl group. This suggests that the decomposition product is the 8- formyl derivative of heme and, therefore, that the primary biological product is the 8-hydroxymethyl derivative. This inference is confirmed by the mass spectrometric data and a number of other observations. Four meso-proton signals are observed (10.39, 10.39, 10.22, 10.10), which rules out meso substitution. The internal methylene protons of the propionic acid side chains appear as a set of clearly resolved signals at 4.68 and 4.48 ppm (Fig. 12) rather than as the single multiplet at 4.50 ppm found in heme. This is consistent with the fact that the 7-methylene protons of heme a are also present as clearly resolved signals. A singlet at 6.33, which belongs to the major product, can be attributed to the 8-hydroxymethyl

A B

uz w4 FIG. 11. Molecular ion region of the mass spectrum of the

polar product isolated from a normal incubation ( A ) and an incubation carried out in H,"O (50 atom %) (B).


FIG. 12. Partial 500 MHz ‘H NMR spectrum of the 8-hydroxymethyl derivative of heme containing a small amount of the 8-formyl derivative.

A B R=CH20H c R=CHO

FIG. 13. Structures of the meso-phenyl- (A) , %hydroxymethyl- (B) , and 8-formyl (C) derivatives of heme isolated from phenylhydrazine-inactivated horseradish peroxidase.

group. This assignment is supported by the presence of three rather than four methyl singlets (at 3.60, 3.50, and 3.45 ppm). Minor changes in the vinyl (8.3-8.6 and 5.9-6.3 ppm) and external methylene (3.5-3.7 ppm) proton regions are consistent with introduction of additional asymmetry into the heme molecule. The spectroscopic data thus argues convincingly that the polar product generated in the reaction of horseradish peroxidase with phenylhydrazine is the 8-hydroxymethyl derivative and that this alcohol slowly autooxidizes to the 8- formyl derivative (Fig. 13). Aerobic incubation of the product mixture in pyridine gradually shifts the Soret band toward longer wavelengths, as expected from oxidation of the alcohol to the aldehyde. It is possible, however, that the 8-formyl moiety is produced, in part, by an enzymatic event.

DISCUSSION

Catalytic turnover of phenylhydrazine by horseradish peroxidase rapidly decreases the catalytic activity of the enzyme (Fig. 1) and the intensity of its Soret band (Fig. 3). In the presence of excess HzOz, 11-13 mol of phenylhydrazine are required per mol of enzyme to decrease the chromophore to the extent possible in a single incubation (Fig. 4). Conversely, in the presence of excess phenylhydrazine, approximately 25 mol of HzOz are required per mol of enzyme to achieve the same goal (Fig. 5). These relationships indicate that the inactivation of each molecule of horseradish peroxidase is associated with the oxidation of 11-13 molecules of phenylhydrazine and the consumption of approximately two molecules of H202 per phenylhydrazine. The stoichiometry for the inactivation of horseradish peroxidase thus differs from that for the inactivation of hemoglobin and myoglobin, which requires 6 mol of phenylhydrazine (19,ZO) and 6 mol of HzOZ2

D. E. Kerr and P. R. Ortiz de Montellano, unpublished result.

II 7-

per mol of heme, or for the inactivation of catalase, which requires 3 mol of phenylhydrazine and 52 mol of hydrogen peroxide per mol of heme (19). The much higher HzOz to phenylhydrazine ratio in the case of catalase presumably reflects the fact that Compound I of catalase is normally discharged by a second molecule of H202, whereas reducing substrates are required to complete the catalytic cycle of horseradish peroxidase.

The absence of a decrease in the Soret band of horseradish peroxidase at low HzOz/enzyme ratios (Fig. 5) implies that an intermediate is generated in the reaction of phenylhydrazine with horseradish peroxidase that is free to dissociate from the enzyme and is eventually responsible for the observed chromophore loss. The nonlinear dependence of chromophore loss on the peroxide/heme ratio is otherwise difficult to explain. The oxidation of phenylhydrazine by hemoglobin yields phenyldiazene, which decomposes in a separate step to the phenyl radical (29). If phenylhydrazine is similarly oxidized by horseradish peroxidase (see below), 1 eq of H202 will be required to generate the diazene. The requirement for the second equivalent of H202 is then rationalized if release of the phenyl radical from the diazene and modification of the chromophore are promoted by Compound I rather than by the ferric enzyme. This mechanistic scheme is substantiated by the find- ings that chemically generated phenyldiazene alters the enzyme chromophore in the same way as phenylhydrazine, that the partition ratios for the inactivation of horseradish peroxidase by phenyldiazene (1O: l by extrapolation of the data in Fig. 6) and phenylhydrazine (11:l) are approximately the same (Figs. 4 and 6), and that one HzOz is required per phenyldiazene (Fig. 6) rather than the 2 eq required per phenylhydrazine. Competitive binding of phenyldiazene and phenylhydrazine prior to the oxidation that terminates in chromophore loss readily explains the resistance of the chromophore at low peroxide/heme ratios (Fig. 5).

The formation of azobenzene as the principal metabolite is consistent with the proposed mechanism. This product can result from coupling of the phenyl radical with the phenyldiazenyl or phenylhydrazyl radicals, although the latter appears more likely due to the probable instability of the phenyldiazene radical. The former reaction would yield the observed metabolite directly, whereas the latter reaction would yield 1,2-diphenylhydrazine which would have to be oxidized to azobenzene in a second step (Scheme 1) (33). Control experiments confirm that horseradish peroxidase does, in fact, oxidize 1,2-diphenylhydrazine to azobenzene (not shown). Spin-trapping evidence for formation of the phenyl radical in the phenylhydrazine-horseradish peroxidase system has been reported (34). The high flux of radicals generated by horseradish peroxidase presumably is responsible for the fact that azobenzene is the predominant metabolic product.


-"+ -e@

N2

SCHEME 1. Pathway proposed for formation of the metabolites isolated from incubations of phenylhydrazine with horseradish peroxidase.

The reactions of hemoglobin, myoglobin, and catalase with phenylhydrazine yield protein-stabilized phenyl-iron complexes that rearrange to N-phenylprotoporphyrin IX adducts when the proteins are denatured in the presence of oxygen (18, 19, 29). Similar reactions are observed with iron porphyrins, except that the N-phenyl adducts are directly obtained due to the instability of unprotected iron-aryl complexes (35). The characteristic chromophore of a phenyl-iron complex is not observed in the reaction of horseradish peroxidase with phenylhydrazine nor is N-phenylprotoporphyrin IX isolated from the phenylhydrazine-treated enzyme. The absence of this reaction pathway in the case of horseradish peroxidase, in view of its predominance where the iron is accessible, suggests that the protein structure of horseradish peroxidase actively suppresses the reaction that leads to the iron- and nitrogen-alkylated products. This influence is supported by the finding that the prosthetic heme is converted into periph- erally modified derivatives (Fig. 13). The phenyl moiety is bound to the 6-meso-carbon in one product and the 8-methyl group is oxidized to an 8-hydroxymethyl function in the other. The bulk of the prosthetic group is recovered as these two products, although approximately 30% of the heme is recovered unchanged and a number of minor products, including the 8-formyl derivative, are also formed (Fig. 9).

The meso-phenyl adduct is explained by addition of the phenyl radical to the d-meso position and subsequent elimi- nation of the meso-hydrogen as a proton. Addition of the phenyl radical to the porphyrin radical cation of Compound I is most favored because it represents the recombination of two radicals, but addition to Compound I1 is still quite favor- able because it results in reduction of the hypervalent Fe(IV)=O complex to the ferric state. Addition of the phenyl radical to the ferric enzyme is least favored because it reduces the enzyme to the ferrous state. Our failure to detect the spectrum of the ferrous.CO complex in incubations carried out under an atmosphere of carbon monoxide specifically argues against this last mechanism. The 2:l peroxide/phenylhydrazine stoichiometry also favors reaction with Com- pound I or I1 because the first peroxide equivalent would be required to generate the phenyldiazene and the second to generate the hypervalent hemoprotein states. Electron transfer from the diazene to Compound I, decomposition of the phenyldiazenyl radical to the phenyl radical, and addition of the phenyl radical to the concomitantly formed Compound I1 most satisfactorily explain the data (Scheme 2).

The mechanism in Scheme 2 also readily explains formation of the 8-hydroxymethyl derivative because the phenyl radical can abstract a hydrogen atom from the 8-methyl group instead

V V V

P

1 V

P V P P V P

P P

SCHEME 2. Hypothetical mechanism for formation of the 6- meso-phenyl and 8-hydroxymethyl derivatives of heme during catalytic turnover of phenylhydrazine by horseradish peroxidase.

of adding to the vicinal meso position. Reaction of the carbon radical with oxygen, hydrogen abstraction from another hydrazine, and reduction of the hydroperoxide provide one route to the 8-hydroxymethyl structure, but the finding that oxygen is not required for formation of the 8-hydroxymethyl compound argues against this alternative. Oxidation of the carbon radical to the cation and addition of water, the theoretically preferred route because oxidation of the methylene radical only requires internal transfer of the unpaired electron to the porphyrin radical cation of Compound I or the ferry1 iron of Compound 11, is confirmed by the finding that the oxygen derives from water and not molecular oxygen (Fig. 11). Hy- drogen abstraction from the ferric enzyme is again unlikely because it would take the enzyme to the ferrous state. The reaction with Compound I1 is illustrated in Scheme 2.

The key to the relationship between structure and mechanism in horseradish peroxidase is provided by the finding that only the 6-meso-carbon and the 8-methyl group react signifi- cantly with the phenyl radical. The protons of these two carbons are so close to each other that they give rise to nuclear Overhauser effects (36). It is clear from the high regiaspeci- ficity of the observed heme transformations that only the small sector of the heme perimeter defined by these atoms is accessible to the phenyl radical and, by inference, to the phenylhydrazine from which the radical is catalytically generated. The existence of such a solvent-exposed heme edge is suggested by the observation that Compound I and ferric horseradish peroxidase disproportionate to two molecules of Compound I1 (37). NMR studies suggest that the environment in the vicinity of the 1- and 8-methyls varies in horseradish peroxidase isoenzymes, whereas that in the vicinity of the 3- and 5-methyls is relatively constant (38). The environment of the 1- and 8-methyls has furthermore been suggested to influence the catalytic activity of the isoenzymes (38). The changes in the environment of these methyl groups have been


interpreted in terms of “clamping” of the vinyl substituents in different orientations, but could equally well be explained by structural changes surrounding the sector of the heme periphery crucial for oxidation of substrates. A schematic of the active site of horseradish peroxidase suggested by these results is presented in Fig. 14. This model is based on the catalytic behavior of the activated enzyme and is thus valid for Compounds I and 11, but may not be appropriate for the ferric or other forms of the enzyme if they are separated from Compounds I and I1 by substantial conformational changes.

The catalytic turnover of cyclopropanone hydrate (39) and nitromethane (40) by horseradish peroxidase has been shown to result in meso-alkylation of the prosthetic heme. The 6- meso-carbon was shown to be the site alkylated by cyclopropanone hydrate. The mechanistic information provided by phenylhydrazine could not be extracted from the earlier studies, however, because no other hemoproteins are known to be alkylated by cyclopropanone hydrate or nitromethane. The present results suggest that cyclopropanone hydrate and nitromethane may also be oxidized by electron transfer to the heme edge rather than, as originally proposed, by direct interaction with the ferryl oxygen. Reversible addition of sulfite to the meso position, based on the observation of a transient intermediate with a broad Soret absorbance and a band at 850 nm, has been proposed to explain the unusual two-electron oxidation of this substrate (41). An analogous mechanism has been proposed for the two-electron oxidation of iodide, although the evidence for a spectroscopically ob- servable transient intermediate is more controversial (42,43). The 6-meso position, as shown by the present results, is likely to be the site of such reversible additions.

A number of literature observations are consistent with the proposal that phenylhydrazine does not react directly with the ferryl oxygen. NMR relaxation and other spectroscopic studies suggest that indolepropionic acid, p;cresol, and ami- notriazole are bound some distance (6-10 A) from the iron atom in the ferric enzyme (44, 45). The hyperfine shifts of the heme protons in the substrate-free and substrate-bound forms, and the fact that most substrates bind equally well to the cyanide-chelated enzyme, suggest that substrates are not bound along the heme axial position (46, 47). Finally, the finding that the oxidation of phenols by protohemin mono- methyl ester mono+[ 1-imidazolyl]propylamide and perox- ides proceeds through outer sphere electron transfer confirms the chemical feasibility of edge-electron transfer (48).

The direct correlation that exists between loss of catalytic activity, chromophore depression, and the binding of two

Peroxide Channel FIG. 14. Model of the active site proposed for horseradish

peroxidase showing the exposed sector of the prosthetic heme to which electrons are transferred from the substrate.

radiolabeled phenyl moieties per enzyme monomer suggests that modification of the protein is probably more important than heme modification for inactivation of the enzyme. The partial protection afforded the enzyme by a chromatographically removable substance (Fig. 2) remains unexplained because azobenzene, biphenyl, and benzene, the metabolites identified in this study, afford no protection whatever against inactivation. The inactivation of the enzyme is not due to destruction of the protein machinery required to generate Compound I because this species is formed when the inactive enzyme reacts with H202 (Fig. 7). The failure of heme modification to block the channel used by peroxide to reach the heme suggests the mouth of the channel is some distance from the alkylated meso position, as shown schematically in Fig. 14. The fact that the modified Compound I does not oxidize guaiacol also indicates that substrates do not reach the active oxygen of Compound I via the peroxide channel, either because it is inherently too narrow or because it is constricted by a conformational change in Compound I. The inactivity of modified Compound I argues strongly that modification of the heme or protein interferes with the binding of substrates or with electron transfer to the heme edge.

The fact that cytochrome P-450 does not generally catalyze the one-electron oxidation of phenols can only be reconciled with the present results if the heme periphery is buried in the protein framework and is therefore not accessible for electron transfer reactions. This interference is consistent with the finding that the closest distance between the heme edge and the surface in the recptly reported crystal structure of cytochrome P-450,., is 8 A (49).

In summary, the present results suggest that horseradish peroxidase catalyzes one-electron oxidations rather than oxygen-transfer reactions because substrates interact with a small sector of the heme periphery rather than directly with the ferryl oxygen. Cytochrome P-450, in contrast, not only has a stronger electron donor as the trans ligand but requires direct interaction of the substrate with the activated oxygen species.

Acknowledgments-The help of the personnel of the University of California, San Francisco, Mass Spectrometry Facility in obtaining the negative ion mass spectra is gratefully acknowledged.

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