Stabilized Isoporphyrin Intermediates in the Inactivation of ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 16, Issue of June 5, pp. 9250-9267,

1989

Printed in U.S.A.

Stabilized Isoporphyrin Intermediates in the Inactivation of Horseradish Peroxidase by Alkylhydrazines* (Received for publication, December 27, 1988)

Mark A. Ator, ShanthaK. David, and PaulR. Ortiz deMontellano$ From the Departmentof Pharmaceutical Chemistrv. Schoolof Pharmacy, University of California, S a n Francisco, California94143-0446 “ I

The reaction of horseradish peroxidase with alkyl- Compound I, a ferryl porphyrin radical cation that can be hydrazines results in 6-meso-alkylation of the pros- representedas (protein)(porphyrint)Fe” = 0 (1, 2).Cytothetic heme group and enzyme inactivation (Ator, M. chrome c peroxidase differs from horseradish peroxidase in A., David, S. K., and Ortiz deMontellano, P. R. (1987) that the protein rather than the porphyrin is oxidized to a J. Biol. Chem. 262, 14954-14960). As reported here, radical to give a catalytic species best represented as (proenzyme inactivation is associated with the accumula- tein.)(porphyrin) Fe” = 0 (3, 4). Formation of the ferryl tion of intermediates that absorb at approximately 835 complex and the porphyrin or protein radical are now well nm. The properties of these intermediates, including established even though the identity of the aminoacid residue their collapse to give meso-alkylhemes, identify them c peroxidase that carries the unpaired electron cytochrome in as isoporphyrins. The tnhvalues for inactivation and is still ambiguous (3, 4). Little is known about the structural formation of the isoporphyrin intermediate at 25 “C and electronic factorsthatdeterminewhetherthe second are, respectively, 11.6 and 12.5 min for methylhydra- oxidation equivalent is located on the porphyrin or the prozine (2.0mM), 8.7 and 7.2 min for ethylhydrazine (1.0 tein. mM), and 30 and 25 s for phenylethylhydrazine (50 We have recently demonstrated that the reactions of horsePM). The isoporphyrin intermediates are surprisingly radish peroxidase with phenylhydrazine ( 5 ) , alkylhydrazines long-lived, with half-lives (35“C, pH 7.0)of 9,28,96, (6), and sodium azide (7) result in addition of the phenyl, and 450 min for, respectively, the phenylethyl, methyl, alkyl, or azido moiety, respectively, to the 6-meso-carbon of n-butyl, and ethyl analogues. pH studiesshow that the prosthetic heme group. Meso-alkylation of the prosthetic protonation of a group withpK. = 5.0-6.5 accelerates group by catalytic turnover of cyclopropanone hydrate (8) isoporphyrin decay and decreases steadystate isopor- and nitromethane (9) has been independently reported and, phyrin accumulation. Horseradish peroxidase reconinthe formercase,proposed to also involve the 6-mesostituted with 6-meso-methylheme, unlike horseradish position. In no instance has addition to one of the other three peroxidase with a heme that hasa larger meso-substitmeso carbons or to the iron or nitrogens of the heme group uent, is catalytically active but is more sensitive to been detected. The reactions of the fungal peroxidase from HzOz-mediated degradation of theprostheticgroup Coprinus macrorhizuswith phenylhydrazine and sodium azide than is the native enzyme. The 6-meso-methylheme parallel those of horseradish peroxidase (10). The peroxidases prosthetic groupis converted in the reaction with Hz02 C. macrorhizus thus differ from hemofrom horseradish and to a biliverdin-likeproduct.Theresultsimplicate highly stabilized isoporphyrin intermediates in the in- globin ( l l ) , myoglobin (12), catalase(13), and cytochrome Pactivation of horseradish peroxidase by alkylhydra- 450 (14, 15) in that all of the latter react with aryl- and zines and indicate that inactivationby the meso-alkyl alkylhydrazines togive iron and nitrogen alkylated prosthetic heme groups. These findings have led us to postulate that groups is due to steric interference with electron delivery to theheme edgerather thanto intrinsic electronic substrates interactexclusively with the hemeedge near the6consequences of meso-alkylation. The structural fea- meso-carbon of the peroxidasesfrom horseradishand C. macrorhizus ( 5 , 16). Meso-alkylation irreversibly inactivates tures that stabilize the cationic isoporphyrins may also be involved in stabilization of the Compound I porphy- horseradish peroxidase except for meso-methylation by methylhydrazine, which results in peroxide- and time-dependent rin radical cation. but reversible inactivation of the enzyme (6). Inactivation of the enzyme by methylhydrazine (6) and cyclopropanone hydrate (8) is associated with the appearanceof an absorption The catalytic turnover of horseradish peroxidase involves peak at 835 nm suggestive of an isoporphyrin intermediate. H202-mediated oxidation of the prosthetic heme’ group to An isoporphyrin intermediate is consistent with the mechanism proposed for meso-heme alkylation (Fig. 1) ( 5 , 6, 16). * This work was supported by Grant GM 32488 from the National Isoporphyrins have been isolated from the oxidative reacInstitutes of Health. Mass spectra were obtained in the Biomedical, tions of meso-tetrasubstitutedmetalloporphyrinswithnuBioorganic Mass Spectrometry Facility of the University of California, San Francisco (A. Burlingame, Director), supported by Grants cleophiles (17, 18) and alkyl peroxides (19, 201, but the mesoonly been RR 01614 and P-30 DK 26743. The costs of publication of this article unsubstituted analogues are very unstable and have were defrayed in part by the payment of page charges. This article indirectlycharacterized.Thepresence of a secondmesomust therefore behereby marked “aduertisement” in accordance with substituent stabilizes isoporphyrins by preventing the depro18 U.S.C. Section 1734 solely to indicate this fact. tonation-rearomatizationsequence responsible fortheir insta$ To whom correspondence should be addressed. only are key ‘The abbreviations usedare:heme, iron protoporphyrin IX re- bility. We report here that isoporphyrins not gardless ofthe ironoxidation and ligation states; HPLC,high pressure intermediates in the inactivation of horseradish peroxidase by alkylhydrazines but are also remarkably stabilized by the liquid chromatography. 9250

Isoporphyrins in Peroxidase Heme Meso-alkylation

9251

The solution was then added via a dropper funnel toa stirred, argonpurged solution of a 10-fold excess of ferrous chloride in methanol. The mixture was refluxed under argon for 30 min, a t which point the absorption spectrum established that metallation was complete. The solution was partitioned between CH2C12and watm and theorganic layer was dried over anhydrous Na2S04 and was concentrated on a rotary evaporator. The residue was dissolved in 1% (w/v) methanolic KOH and was refluxed 1 h to complete the hydrolysis of the methyl ester functions. The mixture was acidified with acetic acid, the heme extracted into P P CH2C12,and 2 volumesof methanol were added to theCHZC12 solution. The mixture was concentrated to remove the CH2Cl2, leaving the heme in approximately 3 ml of methanol. The heme was purified by preparative high pressure liquid chromatography on a 10-pm ODs-3 reverse phase column eluted with68:32:10 methano1:water:acetic acid. The heme eluted a t 8.0 min and a minor impurity, probably partially v V esterified heme, at 9 min. The purified heme was partitioned between diethyl ether and NaC1-saturated 'HCk2H20 to form the ferric chloride complex. The ether was then removed in vacuo from the ether layer. 'H NMR analysis in [2H6]pyridine after reduction with SnC12 showed approximately 95% deuteriation of the 6-meso-position. The other meso-positions and the vinylgroups, as expected, were also partially deuteriated. Reconstitution of Apo-horseradish Peroxidase with Heme, Mesodeuteriated Heme, or 6-Meso-methylheme-Heme was removed from P P horseradish peroxidase and new prosthetic groups were incorporated into the apoprotein as described previously for the reconstitution of FIG. 1. Mechanismproposed for meso-alkylation of the sperm whale myoglobin with zinc protoporphyrin IX (24). The proprosthetic heme group of horseradish peroxidase (6).The cedure differs from commonly used procedures (25) in that a buffer isoporphyrin is the cationicspecies in the centerof the bottom ro'ow. of pH 9.2 was used for the reconstitution step in order to maximize the solubility of the heme group. Measurement of the Rates of Inactivation and Zsoporphyrin Forprotein structure. We demonstrate, furthermore,that enzyme inactivation by meso-alkylation of the heme group is due to mation-Reaction mixtures containing10 p~ horseradish peroxidase, 2.0 mM methylhydrazine, and 2.0 mM H202in 50 mM sodium phossteric interference with the approach of substrates to the phate buffer (pH 7.0) were maintained a t 25 "C in the spectrophotomheme edge rather than to anelectronic effect of meso-substi- eter. Changes in the absorbances a t 835 nm (isoporphyrin) and 416 tution. It ispossible that the structuralfeatures that stabilize nm (Soret) were monitored simultaneously. Analogous experiments the positively charged isoporphyrins also stabilize the porphy- with ethylhydrazine (1.0 mM) contained 10 p M enzyme and 1.0 mM rin radical cation of Compound I with respect to the protein H202, and those with phenylethylhydrazine (50p M ) contained 1 p M enzyme and 100 p~ H202. The loss of enzyme activity was determined radical alternative. under the same conditions by removing aliquots at the appropriate times and assaying the residual catalytic activity. Enzyme activity EXPERIMENTALPROCEDURES was measured in an assay mixture that contained 5 mM guaiacol and Materials-Horseradish peroxidase (type VI), 30% hydrogen per- 0.6 mM H202 in1.0 ml of 50 mM sodium phosphate (pH 7.0) buffer. oxide,bovineliver catalase, guaiacol, andsodiumascorbate were Measurement of the Zsoporphyrin Decay and the Returnof Peroxiobtained fromSigma. Phenylethylhydrazine hydrochloride and meth- dase Activity-A solution (1 ml) of horseradish peroxidase (20 p ~ ) , ylhydrazine sulfate(recrystallized three timesfrom 80% ethanol prior methylhydrazine (4.0 mM), and H202 (4.0 mM) was incubated a t 25 'C to use) were purchased from ICN Pharmaceuticals (Plainview, NY) until theenzyme activity haddecreased to less than 10% of the initial and ethylhydrazine oxalate and n-butylhydrazine oxalate from Fluka value. The incubation mixture was then passed through a 1 X 10-cm (Ronkonkoma, NY). 6-Meso-methylheme was purified from the recolumn of Sephadex G-25 equilibrated with 50 mM potassium phosaction of horseradish peroxidase with methylhydrazine as reported phate (pH 7.0) buffer. The catalytic activity, absorption spectrum, previously (6). and protein concentration of the protein fraction were determined. Analytical Methods-Absorption spectra were obtained on a Hew- The protein fraction was then incubated in the spectrophotometer at lett-Packard 8450A diode array spectrophotometer or, when temper- 35 "C and the increase in the Soret hand at 404 nm or the decrease ature control or wavelengths greater than 800 nm were required, on in the isoporphyrin band a t 835 nm was monitored. Aliquots were a Beckman DU-7 spectrophotometer. High pressure liquid chroma- periodically removed and their peroxidatic activity assayed. Native tography was performed on a system consisting of two Beckman horseradish peroxidase was incubated under identical conditions and model llOA pumps, a Beckman model 420 controller, anda Hewlett- its activityperiodically assayed to ensure that it did not lose activity Packard 1040A diode array detector. The concentration of horse- when incubated at 35 "C. Analogous experiments were carried out radish peroxidase was determined using c402 = 95,000 M" cm" (21). with ethylhydrazine, n-butylhydrazine, and phenylethylhydrazine. All peroxidase experiments were performed with buffers that had Characterization of the Prosthetic Group from Methylhydrazinebeen passed through a column of Chelex 100 (Bio-Rad) to remove treatedHorseradish Peroxidase after Decay of the Isoporphyrintrace metal ion contaminants. Protein concentrations were deter- Horseradish peroxidase (10 PM) was inactivated to varying extents mined by the Lowry method using intact horseradish peroxidase as by incubating 1-ml aliquots for 30 min with methylhydrazine and the standard (22). Hz02 concentrations thatranged from 0.1 to 4.0 mM. Each incubation Preparation of Meso-deuteriated Heme-Protoporphyrin IX was was quenched by adding 2 p1of a 5.6 mg/ml solution of catalase deuteriated essentially as described by Smith et al. (23). Deuteriated followed by 2 p1 of a 0.1 M solution of ascorbic acid. The 1.0-ml p-toluenesulfonic acid was prepared by dissolving 1 g of the undeu- aliquots were then acidified with 0.3 ml of glacial acetic acid and were teriated material in 3 ml of 'Hz0 and removing the water ata rotary extracted with two 1.5-ml portions of diethyl ether. The pooled ether evaporator. The procedurewas repeated twice to ensure complete layers were washed with water andwere evaporated to dryness under proton exchange. The dimethyl ester of protoporphyrin IX (30 mg) a stream of argon. The heme residues were analyzed by reversephase and the deuteriated p-toluenesulfonic acid were heated a t 90 'C in HPLC on a PartisilODs-35-pm column elutedwith a 64:36:10 dry, freshlydistilled1,2-dichlorobenzene for 4days. The reaction methano1:water:glacial acetic acid mixture a t a flow rate of 1 ml/min. mixture was partitioned several times between CH2CL and water to A separate experiment was performed in which the heme was exremove the acid, and the organic phase was removed under vacuum tracted after the isoporphyrin absorbance had completely decayed. a t 80 'C. Thin layer chromatography revealed substantial (30-40%) Measurement of the pH Dependence of Zsoporphyrin Formationhydrolysis of the methyl ester groups. The crude residue was taken Horseradish peroxidase (20 p ~ was ) incubated a t 25 "C with methup in dry tetrahydrofuran and was bubbled with argon for 30 min. ylhydrazine (2.0 mM) and H202(2.0 mM) in 50mM sodium phosphate V

V

9252

Isoporphyrins in Meso-alkylation Peroxidase Heme

buffer of various pH values. The rate of isoporphyrin formation was followed at 835 nm. The absorbance changes in analogous experiments with phenylethylhydrazine were too rapid to accurately measure rates without a stopped flow system. Only the percent of the isoporphyrin formed at various pH values could be determined. Measurement of the pH Dependence ofzsoporphyrin Decay-Horseradish peroxidase (50 p M ) in 10 mM sodium phosphate (pH 7.0) buffer was incubated with methylhydrazine (4.0 mM) and H202 (4.0 mM) until less than 10% of the original activity remained. The incubation mixture was then passed through a Sephadex G-25 column to remove excess methylhydrazine and peroxide. Equal 200-pl aliquots of the enzyme solution were added to 800 pl of buffer of various pH values that had been preequilibrated to 35 'C in the spectrophotometer. Reappearance of the Soretband a t 404 nmwas monitored as a function of time. Loss of the isoporphyrin absorbance a t 835 nm was also monitored but the values were less accurate due to the smaller magnitude of the optical density changes. The pHof the final enzyme solutions was checked in control experiments by adding 1 ml of the pH 7.0 buffer to 4 mlof the buffers of different pH values. An analogous experiment was carried out with the phenylethylhydrazineinactivated enzyme. Reaction of Meso-methylHorseradish Peroxidase with HZOZHorseradish peroxidase reduced to less than 10%of its control activity by incubation with methylhydrazine and Hz02 was filtered through a Sephadex G-25 column and was allowed to stand until the 404 nm Soret absorbance completely recovered. The meso-methyl horseradish ) incubated with H202(10 PM) peroxidase thus obtained (10 p ~ was in the absence of methylhydrazine and aliquots were periodically removed and their catalytic activity determined. In a different experiment, the recovered meso-methylated enzyme was incubated with HZ02 until its activity was partially lost. Glacial acetic acid (0.3 ml) was then added and the heme was extracted into diethyl ether (1.5 ml). The ether was evaporated under a stream of nitrogen and theprosthetic group residue was analyzed on a Partisil ODs-3 5-pm column eluted with a 15 min gradient of 100% solvent Ainto 100% solvent B at a flow rate of1.0 ml/min (solvent A, 60:4010 methano1:water:acetic acid; solvent B, 1O:l methanokacetic acid). The prosthetic group was similarly extracted from a separate control incubation of meso-methylated horseradish peroxidase to which no peroxide had been added. RESULTS

Spectra of the Isoporphyrin Intermediates-The spectra of the isoporphyrin intermediates generated in the reactions of horseradish peroxidase with methyl-, ethyl-,n-butyl-, 450 and 7.2 phenylethylhydrazine are qualitatively very similar. All 4.2 exhibit a near-infrared absorbance band and an attenuated and slightly red-shifted Soret maximum (Fig. 2). The position of the long wavelength band is slightly different for each of the hydrazines although the precise position of each maximum is difficult to define due to thebroad nature of the peaks (Table I). Addition of cyanide to the isoporphyrin obtained with methylhydrazine produces a cyanide complex in which the Soret band sharpens and shifts from 416 to 421 nm but the long wavelength band remains unaltered (Fig. 2). Reduction of the isoporphyrin with dithionite in the presence of carbon monoxide sharpens the Soret band and shifts it to longer wavelengths and also attenuates the long wavelength band (Fig. 2). Isoporphyrin Formation and the Inactivation of Horseradish Peroxidase-The rates of Soret band loss and isoporphyrin formation in incubations of horseradish peroxidase with alkylhydrazines have been measured under identical conditions. Subsaturating alkylhydrazine concentrations and a temperature of 25 "C were employed in order to measure the rates with greater accuracy. Linear semilog plots were obtained when the optical density changes at 835 and 416 nm were plotted against the time of incubation of horseradish peroxidase with the alkylhydrazines. The datafor methylhydrazine, a typical example, is presented in Fig. 3. The tlh values for isoporphyrin formation determined from these plots for methyl-, ethyl-, n-butyl-, andphenylethylhydrazine are given

I

250

I

510

380

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640

770

900

Wavelength(nm)

FIG.2. Spectrum of native horseradish peroxidase (-), horseradish peroxidase 85% inactivated by reaction with ethylhydrazine:H20~and passed through a Sephadex G-25 column to remove excess hydrazine (, . .) and the 85% ethylhydrazine-inactivated enzyme after addition of KCN (- The peak above 800 nm is shown as -.-. - because it is identical in the inactivated enzyme and the inactivated enzyme plus KCN. The spectrum of 85% ethylhydrazine-inactivatedenzyme after bubbling with carbon monoxide and adding dithionite is also shown (0).

-).

TABLE I Half-lives for inactivation, isoporphyrin formation, and isoporphyrin decay in incubations of horseradish peroxidase with alkylhydrazines atpH 7.0 t,, values

Alkylhydrazine (concentration)

Activity lossb

Soret IsopoWhyrin loss' Formation* Decay' rnin

nm

Methyl (2.0 mM) 83110.0 12.5 11.6 28 8.7Ethyl840 (1.0 mM) 842n-Butyl mM) (0.4 4.2 96 Phenylethyl (50 p M ) 825 0.5 0.3 0.4 9 Approximate Soret maximum of the isoporphyrin intermediate. *Experiments were carried out at 25 "C with 10 p M horseradish peroxidase except with phenylethylhydrazine, for which a 1p M horseradish peroxidase concentration was used. e At 35 "C.

0

20 30 Time Imln)

10

40

FIG. 3. Percent remaining absorbance at 416 nm due to the Compound I1 Soret band (El) and percent isoporphyrin absorbance at 835 nm to be formed).( as a function of the time of incubation of 10 p~ horseradish peroxidase with 2.0 mM H102 and 2.0 mM methylhydrazine. The latter value is obtained by subtracting the observed absorbance at 835 nm from the absorbance observed when the isoporphyrin is fully formed. Each value has been corrected for the absorption remaining at maximum inactivation.

Isoporphyrins in Peroxidase Meso-alkylation Heme in Table I. The rates of isoporphyrin formation, Soret loss, and loss of catalytic activity are very similar for each of the four alkylhydrazines (Table I). The rate of isoporphyrin formation could not be measured accurately with phenylethylhydrazine (50 p ~ using ) 10 ~ L Mhorseradish peroxidase but could be measured when the enzyme concentration was decreased to 1 p~ in order to increase the phenylethylhydrazine:horseradish peroxidase ratio. The internalconsistency in the rates for each of the alkylhydrazines suggests that enzyme inactivation and isoporphyrin formation are causally related. Decay of Zsoporphyrin Absorbance and Recoveryof Catalytic Activity-Methylhydrazine-inactivated horseradish peroxidase passed through Sephadex G-25 to remove excess methylhydrazine retains the attenuatedSoret band at 404 nm and the absorbance at 835 nm. The rate of conversion of the isoporphyrin to the meso-methylated heme group calculated from the rise in the 404 nm absorbance as a function of time at 35 "C in pH 7.0 buffer indicates that the isoporphyrin has a half-life (tlh)of 31 min (Table I). The rise in the Soret absorbance is paralleled by recovery of guaiacol oxidation activity, although measurements of guaiacol oxidation by the modified enzyme are not linear with time due to the greater sensitivity of the modified enzyme to degradation by H202 (see later discussion). However, if the increase in absorbance at 470 nm due to the guaiacol oxidation product during the first 5 s is used to estimate the initial catalytic activity, a good correlation is found between the increase in Soretabsorbance, decay of the isoporphyrin, and recovery of catalytic activity (Fig. 4). The activity of the fully regenerated enzyme is estimated to be 170% of the activity of the native enzyme if it is assumed that theSoret bandsof the modified and control enzymes have equal extinction coefficients. The behavior of horseradish peroxidase inactivated by alkylhydrazines other than methylhydrazine is quite different. Incubation of horseradish peroxidase inactivated by ethyl-, nbutyl-, or phenylethylhydrazine after passage through Sephadex G-25 to remove excess inactivatingagent results in complete decay of the isoporphyrin absorption without a significant return of catalytic activity. The tlhvalues for decay of the isoporphyrin intermediates obtained with ethyl-, nbutyl-, andphenylethylhydrazine are given in Table I. Definitive evidence that themeso-ethyl prosthetic group formed in the reaction with ethylhydrazine is not catalytically active is provided by experiments in which intact apo-horseradish peroxidase was reconstituted with 6-meso-ethylheme. The reconstituted enzyme had no detectable catalytic activity even though enzyme similarly reconstituted with authentic heme was fully active. 1

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9253

Relationship of ProstheticHemeModification to Isoporphyrin Formation-The recovery of catalytic activity associated with decay of the methyl isoporphyrin chromophore suggests that the unmodified hemeis regenerated asthe isoporphyrin decays or that the meso-methylated prosthetic group is also catalytically active. Reverse phase HPLC analysis of the prosthetic group extracted from methylhydrazineinactivated horseradish peroxidase prior to decay of the isoporphyrin shows that the only metalloporphyrin products present are heme and 6-meso-methylheme (Fig. 5 ) . The chromatogram is essentially identical to that obtained when the prosthetic group is extracted after complete decay of the isoporphyrin. The acidic extraction procedure thus causes immediate collapse of the isoporphyrin intermediate to 6meso-methylheme. 6-Meso-ethylheme and 6-meso-phenylethylheme are similarly obtained fromenzyme inactivated with, respectively, ethylhydrazine and phenylethylhydrazine (6). To define the relationship between enzyme inactivation and 6-meso-methylheme formation, horseradish peroxidase was inactivated to varying degrees with methylhydrazine and the prosthetic group was extracted before the isoporphyrin decayed. HPLC analysis of the extracted prostheticgroup and correlation of the percent remaining heme with the percent remaining catalytic activity shows that residual activity correlates closely with the presence of unmodified heme groups (Fig. 6). Conversely, enzyme inactivation correlates well with 6-meso-methylheme formation and therefore with isoporphyrin formation. p H Dependence of Zsoporphyrin Decay-The effect of pH on the rate of decay of the isoporphyrin has been examined to explore the factors responsible for the remarkable stability

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100 200 Time (min)

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FIG. 4. Recovery of the ferric enzyme Soret absorbance at 404 nm (m) and the guaiacol peroxidase activity (0)of methylhydrazine-inactivated horseradish peroxidase as a function of time after separation of the inactivated enzyme from excess methylhydrazine by Sephadex G-26 chromatography. The guaiacolperoxidase activity is given in units of change of optical density ( O D ) a t 470 nm.

2

4

6

8

10 12 14 16 18

Time (min)

FIG. 5. HPLC of the prosthetic group extracted from methylhydrazine-inactivated horseradish peroxidase. The minor peak with a retention time of approximately 9 min is heme and the major peak with a retention time of approximately 10.5 min is 6meso-methylheme.

Isoporphyrins in Peroxidase Heme Meso-alkylation

9254

of the isoporphyrin. The isoporphyrin was generated at pH 7.0 inall the experiments to avoid difficulties caused by differences in the rate of formation of the intermediate at different pH values. Aliquots of the inactivated enzyme were then diluted into buffers of the appropriate pH, and therates of decay of the isoporphyrin peak at 835 nm and recovery of the modified heme Soret band at 404 nm were monitored at 35 "C. The results obtained by both methods agree well but the data obtained at 404 nm are more accurate because the amplitude of the response is larger. The tlh values calculated from first order plots of the increase in the404 nm absorbance indicate that the isoporphyrin formed with methylhydrazine decays more rapidly as the pH decreases (Fig. 7). A plot of the tK values from Fig. 7 versus the pHof the medium shows a break with a half-maximal value at approximately pH 6.2 (Fig. 8). These results indicate that simple deprotonation of the isoporphyrin is not the key to itsdecay because deprotonation is favored by higher rather than lower pH values. The data suggest, in fact, that protonation of a residue with a pKa value of approximately 6.2 accelerates isoporphyrin decay. Analogous data on decomposition of the isoporphyrin produced with phenylethylhydrazine (Fig. 8) suggest that decay of the corresponding isoporphyrin is facilitated by protonation of a group with a pKa of approximately 5.4. The same group 100

.2 > .c

may be involved in both cases if its pK, value is sensitive to the structure of the meso-alkyl group in the isoporphyrin. pH Dependence of Isoporphyrin Formation-The isoporphyrin is formed more slowly and reaches a lower maximal concentration in incubations of horseradish peroxidase with methylhydrazine asthepH of the incubation medium is decreased (Fig. 9). The exact magnitude of the effect is difficult to quantitate, however, because it is not known if the extinction coefficient of the isoporphyrin chromophore is pHdependent. A plot of the percent isoporphyrin in the steady state as a function of pH assuming that the extinction coefficient is pH-independent exhibitsa break at pH6.3 (Fig. 10). Formation of the methylisoporphyrin intermediate is thus favored by pH values above 6.3 and itsdecomposition by pH values below that value. The steady state concentration of the isoporphyrin in the reaction of horseradish peroxidase with phenylethylhydrazine is also dramatically altered by pH, but the rapidity of the reaction has prevented us from obtaining comparable titration curves. It is nevertheless clear that the phenylethyl isoporphyrin intermediate also forms more slowly and reaches a lower steady state concentration as the pH is decreased (not shown). Effect of Cyanide on Isoporphyrin Formtion and DecayAddition of 1 mM KCN to 10 pM horseradish peroxidase results in formation of the ferric cyano complex of the enzyme with a Soret maximum at 421 nm. Addition of methylhydrazine and Hz02to thiscomplex givesa Compound II spectrum with a Soret maximum at 416 nm. Decay of the Compound 11 spectrum to give the isoporphyrin spectrum occurs with a tx6 of 5.6 min. This is to be compared with the tu of 9.8 min obtained in theabsence of cyanide. Isoporphyrin formation is therefore accelerated slightly by cyanide but the reason for this acceleration is not known. In contrast, cyanide does not detectably alter the rate of isoporphyrin decay. Addition of cyanide to methylhydrazine-inactivated,Sephadex G-25-filtered enzyme did not detectably change the rate of disappearance of the 837 nm isoporphyrin peak with respect to a parallel incubation without cyanide. The Soret band, however, shifts from 404 to 421 nm when cyanide is added (Fig. 2). The ferryl oxygen is therefore no longer bound to the iron or is readily displaced by cyanide. This contrastswith the fact that cyanide does not displace the ferryl oxygen of Compounds I or I1 (1). Isotope Effect on Isoporphyrin Decay-Comparison of the activity of enzyme reconstituted with meso-deuteriated heme

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YO Native Heme FIG.6. Correlation of remaining guaiacol peroxidase activ-

ity with remaining intact heme. The same correlation holds for loss of peroxidase activity u e r s u extent of isoporphyrin formation because the isoporphyrin decays to 6-meso-methylheme and 6-mesomethylheme is the only significant reaction product.

100 A

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FIG.7. pH dependence of decay of the isoporphyrin from methylhydrazine-inactivatedhorseradish peroxidase. Isoporphyrindecay as a function of time is shown for pH 5.26 (A), 5.70 (*), 6.27 (H),6.60 (D),8.22 (A).

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9255

Isoporphyrins in Peroxidase Heme Meso-alkylation 100

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FIG.8. Replot of the tu values for decay of the isoporphyrin as a function of pH for methylhydrazine-inactivated(0)and phenylethylhydrazine-inactivated (B) horseradishperoxi-

0.30

1

0

50

100 Time (min)

150

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FIG.11. Decay of the isoporphyrin intermediate from the reaction of methylhydrazine with horseradish peroxidasereconstituted with either normalheme (B) or meso-deuteriated heme (0).

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Time (min) FIG.9. Isoporphyrin formation as a function of time of incubation of horseradish peroxidase with methylhydrazineat pH 6.26 (B), 5.70 (A),6.27 (A), 6.60 (+), 8.22 (0).

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1 0

Time (mln)

FIG.10. pH dependence of isoporphyrin accumulation in the FIG.12. Peroxide-dependentdegradation of the mesoreaction of horseradish peroxidase with methylhydrazine demethylated heme group. The bottom tracing shows an HPLC of termined by replotting the percent of the maximum isoporphyrin formed at each pH value from Fig. 9 as a function of theprosthetic group extracted from horseradish peroxidase after partial inactivation with methylhydrazine (retentiontimes: unknown pH.

(7.5 min), heme (8.0 min), 6-meso-methylheme (8.5 min)). Theupper tracing shows the prosthetic group after the inactivated enzyme was shows that it has approximately 90% of the activityof native allowed to regainfull activity and was then incubated with Hz02 horseradish peroxidase and is inactivated by methylhydrazine alone. A new peak a t approximately 6.8 min is seen to be formed a t at the same rate as native horseradish peroxidase (not shown). the expense of the 6-meso-methylheme peak.

Decay of the isoporphyrin to give thecatalytically active enzyme with a meso-methylheme prosthetic group is markedly slower, however, for enzyme reconstituted with mesodeuteriated heme (tH = 160 min) than for enzyme reconstituted with undeuteriated heme ( tlh = 28 min) (Fig. 11).The

isotope effect on decay of the isoporphyrin is therefore k H / k D = 5.7. Sensitivity of 6-Meso-methyl Horseradish Peroxidase to HzOz-The reactivated, meso-methylated enzyme recovered

9256

Isoporphyrins inPeroxidase Heme Meso-alkylation

from an incubation of horseradish peroxidase with methylhydrazine was reincubated with methylhydrazine in an effort to generate the bk-meso-methylisoporphyrin adduct. The meso-methylated enzyme, however, has proven to be much more sensitive to peroxide than thenative enzyme and is very rapidly inactivated even by low peroxide:enzyme ratios in the absence of methylhydrazine. Incubation of the modified enzyme with a 1:l ratio of H202 results in loss of approximately 50% of the activity within 5 min and 70% after prolonged incubation (not shown). Extraction of the prosthetic group from this enzyme indicates that the residual 30% activity is due to native, unmodified heme (Fig. 12). HPLC analysis of the prosthetic group following 40% loss of the activity of the modified enzyme is paralleled by approximately a 40% decrease in the sum of the recovered modified and unmodified heme groups. The disappearance of meso-methylheme is directly related to the appearance of a more polar species with a retention time of 6.8 min (Fig. 12). The spectrum of this green substance recorded with a diode array detector during HPLC purification of the sample has a maximum at 404 nm, but thespectrum of the residue obtained after solvent removal under a stream of nitrogen and dissolution in methanol has, in addition, a shoulder at 380 nm and a peak at 680 nm. The product co-elutes with a commercial mixture of the four biliverdin isomers and has anearly identical absorption spectrum. It appears, therefore, that meso-methyl substitution renders the prosthetic group highly unstable toward ring cleavage to give biliverdin-like products, although the actual structure of the 6-meso-methylheme cleavage product(s) has not yet been established. It is to be noted that the enzyme with the meso-methylisoporphyrin group is much more resistant to degradation by H2O2than the meso-methylated enzyme obtained from it. Meso-methylheme was recoveredin good yield when the isoporphyrin (inactive) enzyme was incubated with 1 equivalent of H2O2for 10 min.

is therefore the key to loss of catalytic activity. This finding, in conjunction with the fact that reaction of meso-alkylated horseradish peroxidase with H202produces a typical Compound I or Compound I1 spectrum, strongly supportsour earlier proposal that meso-alkyl groups inactivate the enzyme by sterically preventing the approach of substrates to the heme edge to which electrons are delivered (5, 6, 16). The rates of isoporphyrin decay, and therefore the stabilities of the isoporphyrins, exhibit no correlation with the size of the alkyl group. The phenylethylisoporphyrin thus decays most rapidly but the ethyl rather than methylisoporphyrin most slowly (Table I). A protein residue with a pK.of approximately 6.2 appears to play a critical role in stabilizing the methylisoporphyrin because isoporphyrin decay is markedly accelerated by its protonation (Figs. 7 and 8). Analogous data on the isoporphyrin obtained with phenylethylhydrazine show that itis destabilized by protonation of a residue with a pK, of approximately 5.3 rather than6.2 (Fig. 8). It is probable that both isoporphyrin intermediates are destabilized by protonation of the same residue but that the pKa of the residue is modified by differences in the active site caused by the greater bulk of the phenylethyl moiety. This pH effect on isoporphyrin decay is not the result of protein denaturation because the catalytic activity of the horseradish peroxidase isozymes used in this study is actually higher at low pH (2). The steady state concentration of the isoporphyrin in the reaction of horseradish peroxidase with methylhydrazine, which is determined by the relative rates of isoporphyrin formation and decay, increases with increasing pH (Fig. 9). The increase in the steady state concentration of the methylisoporphyrin caused by deprotonation of a group with pK. = 6.5 (Figs. 9 and 10) closely mirrors the finding that decay of the methylisoporphyrin is accelerated by protonation of a group with pK, = 6.2. The similarity in these two pKa values suggests that thepH-dependent variable that determines the steady state isoporphyrin concentration is the rate of isoporDISCUSSION phyrin decay. A corollary of this is that the rate of isoporPrior studies of the inactivation of horseradish peroxidase phyrin formation is relatively insensitive to pH(or coincidenby alkylhydrazines established that the enzyme is primarily tally has a pHprofile nearly identical to thatfor decay of the inactivated by 6-meso-alkylation of its prostheticheme group isoporphyrin). It is surprising that conversion of the isoporand suggested that meso-alkylation was preceded by an iso- phyrin- to the meso-substituted-enzyme is accelerated by inporphyrin intermediate with an absorption maximum at ap- creases in the hydrogen ion concentration because conversion proximately 830 nm (Fig. 2) (6). Methylhydrazine, however, of the isoporphyrin to the meso-substituted heme requires has now been found to differ from ethyl-, n-butyl-, and deprotonation of the 6-meso-carbon. The kinetic importance phenylethylhydrazine in that its inactivation of horseradish of the deprotonation reaction is delineated by the finding that peroxidase is reversible. The close, inverse relationship be- replacement of the meso-proton by a deuterium markedly tween loss of the Compound I1 Soret band at 416 nm and decreases the rate of isoporphyrin decay (Fig. 11). The isotope growth of the isoporphyrin band at 835 nm (Fig. 3), and the effect ( kH/kD= 5.7) estimated from the datain Fig. 11 clearly similar relationship between disappearance of the isopor- indicates that removal of the meso-proton is the primary rate phyrin absorbance and recovery of catalytic activity (Fig. 4), limiting step in conversion of the isoporphyrin to the mesosuggest that the isoporphyrin form of the enzyme is inactive methylheme at pH 7. Protonation of a residue with pK, = 6.3 but gives rise to catalytically active enzyme as the isopor- thus facilitates meso-deprotonation of the methylisoporphyrin decays. HPLC analysis of the prosthetic group ex- phyrin intermediate. The isoporphyrins from meso-tetrasubstituted porphyrins, tracted from the isoporphyrin form of the enzyme showsthat which cannot rearomatize by simple deprotonation, can be the only heme products are 6-meso-methylheme andunreacted heme (Fig. 5). A plot of remaining catalytic activity detected and even isolated. Isoporphyrins derived from mesoversus heme not converted to the isoporphyrin shows that unsubstituted porphyrins, however, are generally not detectenzyme inactivation is directly proportional to isoporphyrin able. Stabilization of the isoporphyrin by the protein is thereformation (Fig. 6). Thus, the methylisoporphyrin form of the fore required to rationalize the startling finding that theethyl enzyme is inactive whereas the enzyme with a meso-methyl- and n-butylisoporphyrin forms of the enzyme have half-lives heme is active. This is confirmed by the demonstration that on the order of several hours. If the isoporphyrins are stabi6-meso-methylheme-reconstitutedhorseradish peroxidase is lized by simple lipophilic interaction of the meso-alkyl groups catalytically active. In contrast, the ethyl,n-butyl, and phen- with protein residues, their stabilities should increase in the ylethylisoporphyrin forms of horseradish peroxidase and the order: methyl c ethyl < butyl c phenylethyl. The fact that corresponding 6-meso-alkylhemederivatives are inactive. The the ethylisoporphyrin is the most stable shows that simple size of the 6-meso-substituent rather than itssimple presence lipophilic interactions are not dominant in isoporphyrin sta-

Isoporphyrins in Meso-alkylation Peroxidase Heme

FIG. 13. Schematic view of possible electrostatic stabilization of the cationic isoporphyrin intermediate and Compound I porphyrin radical cation.

bilization. The same observation indicates that the isoporphyrin is not stabilized by simple steric interference of the meso-substituent to the approach of a deprotonating base. The finding that cyanide binds to theiron without decomposing the isoporphyrin suggests, furthermore, that the isoporphyrin is not stabilized by specific ligation of the iron atom (Fig. 2). In principle, the isoporphyrin may be stabilized by electrostatic interactions with negatively charged active site residues because isoporphyrins bear one more positive charge than the parent heme group. The positive charge can be neutralized by electrons from one of the anionic nitrogens to which the iron is coordinated, but this simply transfers the positive charge from the carbon skeleton to the iron atom. Electrostatic stabilization is consistent with the observation that isoporphyrin decay is accelerated by a protonation event because this could switch off the stabilizing charge. A simplistic view of isoporphyrin stabilization by electrostatic interactions is given in Fig. 13. The information in hand, however, does not differentiate between direct electrostatic stabilization of the isoporphyrin and pK,-dependent protein conformational changes that result in non-electrostatic differential stabilization of the isoporphyrin and porphyrin forms of the prosthetic group. Stabilization of the isoporphyrin by the protein may have implications for the normal catalytic function of horseradish peroxidase. The structural parameters that determine whether the Compound I ferry1 species is associated with a porphyrin radical cation, as in horseradish peroxidase, or a protein radical, as in cytochrome c peroxidase, remain unknown. Nevertheless, the factors that stabilize the isoporphyrin may also stabilize the porphyrin radical cation because the salient feature of both of these prosthetic functionsis an increase in the positive charge on the porphyrin ring. Stabilization of the radical cation would favor oxidation of the porphyrin rather than the protein. Support for stabilization of porphyrin radical cations by vicinal negative charge is provided by the observation that chemical polarization of porphyrin radical cations by soft anions produces phlorin-like spectra (26,27).Araiso and Dunford (28) suggested some time ago that changes in electric charge at the hemin periphery could alter theproperties of the chromophore. It is interesting in this context to note that the ionization potential of horseradish peroxidase isozyme c increases with decreasing pH (29), as this i s the behavior expected for an electrostatically stabilized porphyrin radical cation. It may also be relevant

9257

that an aspartate carboxyl group (Asp-64) is found in the crystal structure of catalase close to the &edge of the prosthetic heme group (30), although it is not known whether it plays any role in stabilizing the catalase Compound I porphyrin radical cation. Horseradish peroxidase with a 6-meso-methylheme prosthetic group, unlike the enzyme with a &meso-ethyl-, mesobutyl-, or meso-phenylethylheme, is catalytically active. The enzyme with a 6-meso-methylheme group, however, is much more sensitive to inactivation by H202 than native horseradish peroxidase. Catalytic activity is lost within 5-6 min when the modified enzyme is incubated with H202 in the absence of other substrates (not shown) due to H202-dependent degradation of the meso-methylheme group to anunidentified product (Fig. 12). The retention time and absorption spectrum of the new product resemble those of authentic biliverdin butitsactualstructure is not yet known. The structure of the degradation product, the mechanism by which it is formed, and the basis for the increased sensitivity of the meso-substituted prosthetic group to degradation should provide useful insights into the mechanisms of normal and abnormal heme degradation. REFERENCES 1. Marnett, L. J., Weller, P., and Battista, J. R. (1986) in Cytochrome P-450:

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