Structure and Catalytic Mechanism of Horseradish Peroxidase

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol,262, No. 31, Issue of November 5, PP , by The Amerian Soiety for Biohemistry and Moleular Biology, In Printed in U. S. A. Struture and Catalyti Mehanism of Horseradish Peroxidase REGIOSPECIFIC MESO ALKYLATION OF THE PROSTHETIC Mark A. Ator, Shantha K. David, and Paul R. Ortiz de Montellano* HEME GROUP BY ALKYLHYDRAZINES* (Reeived for publiation, May 11, 1987) From the Demrtment of Pharmneutial Chemistrv. Shool of Phnrmay, and the Liver Center, University of California, "I San Franiso, Californh Horseradish peroxidase is inativated in a time-, HzOz-, and onentration-dependent manner by phenylethyl-, ethyl-, and methylhydrazine. The pseudofirst order kineti onstants for these inativation reations at ph 7 are: phenylethyl (KJ = 115 p ~ kinat, = 1.5 min-l, partition ratio = 11), ethyl (KJ = 145 pm, kinat = 0.08 min", partition ratio = 32), and methyl (KJ = 3000 p ~ k,,,,,, = 0.12 min", partition ratio = 80). At ph 5, the onstants for the phenylethyl reation hange to Kr = 1540 p~ and kinat = 0.86 min". A transient absorbane at approximately 830 nm, suggestive of an isoporphyrin intermediate, is seen during these reations. The prostheti heme is onverted by eah of the three alkylhydrazines into the orresponding 6-rneso-alkylated heme. Complete inativation of the enzymes by methyl-, ethyl-, and phenylethylhydrazine is assoiated with alkylation of 60-70, 70, and 90%, respetively, of the prostheti heme groups. The absene of N-alkylation and the high spe- ifiity for the &meso position, even with agents as small as methylhydrazine, strengthen the proposal that eletron abstration is mediated by the heme edge rather than the ferryl oxygen of horseradish peroxidase. Alkyl- and arylhydrazines inativate many oxidative enzymes, inluding hemoproteins suh as ytohrome P-450 (1, 2), atalase (3), hemoglobin (4, 5), myoglobin (6), and latoperoxidase (7), non-heme' metalloproteins suh as lysyl oxidase (8), soybean lipoxygenase (9), (lo), plasma amine oxidase (ll), and atehol oxidase (12), and flavoproteins suh as monoamine oxidase (13) and trimethylamine dehydrogenase (14). The mehanisms of these inativation reations vary in that some involve alkylation of heme or flavin prostheti groups by atalytially generated radials, some involve reations of the radials or other ativated intermediates with protein residues, and still others involve formation of a hydrazone with the arbonyl group of a quinonoid ofator. The parameters that determine whih of these mehanisms predominates in a given situation remain unlear. * This researh was supported by Grants GM and GM from the National Institutes of Health. Mass spetra were obtained in the Bioorgani, Biomedial Mass Spetrometry Faility of the University of California, San Franiso, supported by Grant RR from the National Institutes of Health. The osts of publiation of this artile were defrayed in part by the payment of page harges. This artile must therefore be hereby marked "advertisement" in aordane with 18 U.S.C. Setion 1734 solely to indiate this fat. 2 To whom orrespondene should be addressed. The abbreviations used are: heme, iron protoporphyrin IX regardless of oxidation and ligation state; HPLC, high pressure liquid hromatography The inativation of horseradish peroxidase by phenylhydrazine, first reported by Hidaka and Udenfriend in 1970 (E), reently was shown to losely parallel the ovalent binding of 2 eq of radiolabeled phenylhydrazine/enzyme moleule (16). The inativation is assoiated with onversion of a fration of the prostheti groups to 6-meso-phenyl heme and 8-hydroxymethyl heme, but the bulk of the heme groups (54%) are reovered intat from the fully inativated enzyme. Enzyme inativation therefore results primarily from reation of a phenylhydrazine metabolite, probably the phenyl radial, with the apoprotein. The lear orrelation between enzyme inativation and binding of two phenylhydrazine moieties annot otherwise be reoniled with the fat that less than half of the prostheti groups are atually modified (16). The high speifiity of the reation for the 6-meso position and the 8-methyl group and the absene of the N-phenyl adduts expeted from reation with the iron or nitrogen atoms of the heme (1-6) led us to propose that phenylhydrazine and pre- sumably most other substrates are oxidized by eletron transfer to the heme edge rather than to the ferryl oxygen. The present studies were undertaken to determine (a) if alkylhydrazines inativate horseradish peroxidase, (b) if substrates smaller than phenylhydrazine also reat only with the heme edge, and () what the relative roles of heme and protein alkylation are in enzyme inativation. MATERIALS AND METHODS Horseradish peroxidase (type VI), bovine liver atalase, hydrogen peroxide, guaiaol, and sodium asorbate were obtained from Sigma. Phenylethylhydrazine hydrohloride and methylhydrazine sulfate were purhased from ICN Pharmaeutials (Plainview, NY) and ethylhydrazine oxalate from Fluka. Methylhydrazine sulfate was rerystallized three times from 80% ethanol. Deuteriohloroform and pyridine-& (100 atom%, 'H) were obtained from Aldrih. Absorption spetra were obtained on a Hewlett-Pakard 8450A diode array spetrophotometer. High pressure liquid hromatography was performed with a system onsisting of Bekman Model lloa pumps, a Bekman Model 420 ontroller, and a Hewlett-Pakard 1040A diode array detetor. NMR spetra were reorded on a General Eletri GN 500 MHz instrument. Chemial shift values are reported in parts per million relative to tetramethylsilane. Mass spetra were obtained on a Kratos MS 50 instrument operating in the liquid matrix seondary ion mode. The onentration of horseradish peroxidase was determined using E,o2 = 95,000 M" m" (17). All peroxidase experiments, unless otherwise speified, were performed at 25 "C in 50 mm sodium phosphate buffer (ph 7.0) that had been passed through a olumn of Chelex 100 (Bio-Rad). Znativation of Horseradish Peroxidase by Alkylhydrazines-The rate of inativation of horseradish peroxidase by phenylethylhydrazine was measured in a reation mixture that ontained 1 pm peroxidase, 125 PM H202, and p~ phenylethylhydrazine. At 1-min intervals after addition of the peroxide and inativator, 5-pl aliquots of the mixture were transferred to uvettes ontaining 1.0 ml of an assay mixture ontaining 50 mm sodium phosphate (ph 7.0), 5 mm guaiaol, and 0.6 mm H202. The inrease in absorbane at 470 nm

2 ~ Horseradish Peroxidase meso-heme Alkylation was reorded as a measure of the peroxidase ativity. The effet of ph on the inativation by phenylethylhydrazine was determined by repeating the inubations in 50 mm sodium itrate buffer (ph 5.0). Analogous experiments were arried out with ethylhydrazine and methylhydrazine, exept that inubations with the former were arried out with 1 mm H20Z and ethylhydrazine onentrations ranging from 0.1 to 1.0 mm, and inubations with the latter with 5 mm Hz02 and methylhydrazine onentrations ranging from 0.7 to 4.4 mm. Partition Ratios and Correlation of Heme Modifiation and Ativity Loss during the Inativation of Horseradish Peroxidase-One-milliliter samples of 10 p~ horseradish peroxidase were inubated for 30 min with 0.1 mm H202 and mm phenylethylhydrazine. Aliquots of the reation mixtures were then removed and assayed for remaining peroxidati ativity as desribed above. Exess Hz02 was destroyed by inubating eah sample for 3 min with 2 pl of a 5.6 mg/ml solution of bovine liver atalase before 2 pl of 50 mm sodium asorbate was added to ensure that the enzyme was redued. The mixtures were plaed on ie until analyzed. Following the ompletion of all of the reations, the heme was isolated from eah sample. The solution was aidified by adding 0.27 mlof glaial aeti aid and the heme extrated with two 1.5-ml aliquots of diethyl ether. Low speed entrifugation was required to separate the aqueous and organi layers in the extration proedure. The organi layers were pooled, washed with H20, and evaporated to dryness under a stream of NP. The omposition of the extrated heme mixture was analyzed by HPLC on a 4.6 X 250-mm Whatman Partisil 5 ODs-3 olumn eluted with 7:3:1 methanol/h20/glaial aeti aid at a flow rate of 1 ml/min. Heme absorbane was monitored at 400 nm with the diode array detetor, and a Hewlett-Pakard Model 3390A integrator was used to determine the relative absorbane of eah peak. Similar experiments were performed with ethylhydrazine and methylhydrazine, exept that the Hz02 onentration was raised to 0.5 mm in the ase of ethylhydrazine and to 1.5 mm in the ase of methylhydrazine. The ethylhydrazine onentration was varied from 0 to 0.4 mm and the methylhydrazine onentration from 0 to 1 mm. The reations with ethylhydrazine and methylhydrazine were allowed to proeed for 1 h before peroxidase ativity was measured and the heme isolated. Isolation and Identifiation of Heme Adduts Generated in the Reation of Horseradish Peroxidase with Alkylhydrazines-Mixtures ontaining 50 pm horseradish peroxidase, 1.5 mm H202, and 1.5 mm phenylethylhydrazine in 60 ml of 50 mm sodium phosphate buffer (ph 7.0) were inubated for 20 min at room temperature. Catalase (10 p1 of a 5.6 mg/ml solution) was then added, followed 5 min later by 0.3 ml of 50 mm sodium asorbate and 16 ml of glaial aeti aid. The heme was extrated with diethyl ether and thethereal solution, after washing with H20, was evaporated to dryness in urnuo. The same proedure was used to isolate the adduts formed in the reations of horseradish peroxidase with ethylhydrazine and methylhydrazine, exept that the onentration of Hz02 was 2.0 and 5.0 mm, respetively, and the onentration of methylhydrazine 2.5 mm rather than 1.5 mm. The modified hemes were purified from the rude heme extrats by two different proedures. The unesterified hemes were diretly hromatographed on a 9 X 250-mm Whatman Partisil 10 ODS-3 HPLC olumn at a flow rate of 4 ml/min (ompound, solvent system, retention time: phenylethyl addut, 72:2810 methanol/h20/aeti aid, 12.4 min; ethyl addut, 6832:lO methanol/hzo/aeti aid, 8.2 min; methyl addut methanol/h20/aeti aid, 11.9 min). The phenylethyl addut from a number of hromatographi runs was rehromatographed on a 4.6 X 250-mm Partisil 5 ODs-3 olumn using a 10-min linear gradient from 641 methanol/hzo/glaial aeti aid to 101 methanol/glaial aeti aid at a flow rate of 1 ml/min. A portion of the mixture was analyzed by mass spetrometry, while the remainder was onverted to the hloroiron(ii1) omplex, dissolved in pyridine-d5, redued with SnC12, and examined by 500 MHz 'H NMR spetrosopy (16). The pyridine peak at 8.70 ppm was used as the referene for hemial shift values. The seond proedure for isolation of the heme adduts involved esterifiation with (CH&OBF, (18) and demetallation with ferrous sulfate as desribed by Fuhrhop and Smith (19). The resulting mixture of dimethyl-esterified porphyrins was hromatographed on a 4.6 X 250-mm Partisil 5 PAC HPLC olumn in 1:2 tetrahydrofuran/ hexane at 1 ml/min with detetion at 410 nm (ompound, retention time: phenylethyl addut, 14 min; ethyl addut, 12.4 min; methyl addut, 15.5 min). The isolated adduts were dissolved in CH2Clz and onverted to the Zn2+ omplexes by treatment with exess Zn(OA)z in methanol at room temperature for 30 min. The porphyrins were extrated into ether, and the ether solutions were washed with satu- rated aqueous NaCl solution, dried over NazSOI, and evaporated to dryness in uauo. The 500 MHz 'H NMR spetra of the porphyrins were obtained in CDCl,. Chemial shift values are given relative to the solvent peak at 7.26 ppm. Nulear Overhauser effets were measured using a 3-9 gated presaturation pulse and a sweep width of 3000 Hz. Saturation of the ethyl methylene (5.223 ppm) of the zin omplex of dimethyl-esterified meso-ethyl protoporphyrin IX required a power level of 40 db. A referene spetrum was obtained with the deoupler set 0.5 ppm downfield from the methylene peak. A line-broadening funtion of 1.0 Hz was applied to the free indution deay signal prior to zero filling the 16K spetrum to 64K. The nulear Overhauser effet was obtained by subtrating the transformed referene spetrum from the experimental spetrum. RESULTS Inativation of Horseradish Peroxidase by Alkylhydrazines-Phenylethylhydrazine is a potent time- and onentration-dependent inativator of horseradish peroxidase. The inativation is haraterized by pseudo-first order kinetis. A replot of the kineti data yields a KI of 115 PM and a kinat of 1.5 min" (Fig. 1, Table I). Horseradish peroxidase is similarly inativated by ethylhydrazine (Fig. 2) and methylhydrazine (Fig. 3). The KI value for ethylhydrazine is omparable to that for phenylethylhydrazine, but the kinat value is 20 times OI E" 30 - P C,o? 20 t 10 / Time (rnin) FIG. 1. Time-dependent inativation of horseradish peroxidase by phenylethylhydrazine at ph 7.0. The onentrations of phenylethylhydrazine are 12.5 (O), 25 (m), and 37.5 p~ (A), A replot of the time required to inativate half of the enzyme (tu) versus the reiproal of the inhibitor onentration is shown in the inset. The details of the inubations are given under "Materials and Methods." TABLE I Kineti onstants for the inativation of horseradishperoxidme by alkvlhvdrazines I Alkylhydrazine KI Partition ratio PM min" Phenylethyl ph ph Ethyl Methyl

3 14956 meso-heme Peroxidase Horseradish a Alkylation b :s , $ 30- P E $ 40 0,.f.f E 30 t? 9)! Kt l/[ethylhydrazine] (n ) 10-1 I r 1 I I I Time (min) FIG. 2. Time-dependent inativation of horseradish peroxidase by ethylhydrazine at ph 7.0. The onentrations of ethylhydrazine are 0.1 (.), 0.15 (B), 0.25 (A), and 0.5 mm (0). A replot of the th values uersus the reiproal of the inhibitor onentration is shown in the inset. Details of the inubations are given under Materials and Methods Time (rnin) FIG. 3. Time-dependent inativation of horseradish peroxidasebymethylhydrazineat ph 7.0. The onentrations of methylhydrazine are 0.71 (O), 0.94 (B), 1.28 (A), 2.1 (O), and 4.4 mm (0). A replot of the tc values versus the reiproal of the inhibitor onentration is shown in the inset. Details of the inubations are given under Materials and Methods. lower (Table I). The KI value for methylhydrazine, in ontrast, is 20 times larger than that for ethylhydrazine, but the kinat value is omparable (Table I). In any ase, inativation of horseradish peroxidase by the alkylhydrazines is muh slower than that mediated by phenylhydrazine, whih ours so rapidly that kineti parameters for the inativation annot be obtained by stati spetrosopi methods (16). No evidene has been found for the formation of alkylhydrazine metabolites omparable to those generated from phenylhydrazine that protet the enzyme from omplete inativation (16). Methylhydrazine differs from the phenyl, phenylethyl, and ethyl analogues in that its inativation is reversible. No ativity is reovered if enzyme inativated with phenylhydrazine, phenylethylhydrazine, or ethylhydrazine is passed through a Sephadex G-25 olumn to remove exess reagent. However, in the ase of methylhydrazine, the enzyme remains inative immediately after passage through a Sephadex G-25 olumn, but slowly reovers at least part of its ativity if it is then inubated at 30 C for an extended period. The mehanism of this reativation, whih is losely paralleled by a derease in l/[peh](mm ) FIG. 4. Time-dependent inativation of horseradish peroxidase by phenylethylhydrazine at ph 6.0. A replot of tc values taken from a plot analogous to that of Figs. 1-3 uersus the reiproal of the inhibitoronentrations isshown. The onentrations of phenylethylhydrazine (PEH) employed in the study were 0.15, 0.25, 0.50, and 1.0 mm. Details of the inubations are given under Materials and Methods. the inhibitor at ph 5.0 therefore reflets, in part, a derease in the onentration of the unprotonated hydrazine. the isoporphyrin absorbane at approximately 830 nm (see The partition ratios for the inativation of horseradish below), is under investigation. peroxidase by methyl-, ethyl-, and phenylethylhydrazine have The inativation of horseradish peroxidase by phenylethyl- been determined by inubating the enzyme with inreasing hydrazine is muh less effiient if the reation is arried out onentrations of eah of these ompounds and then measurat ph 5.0 rather than ph 7.0. The inativation at ph 5.0 also ing the residual atalyti ativity after suffiient time has follows pseudo-first order kinetis but is haraterized by a elapsed for the alkylhydrazine to be ompletely onsumed. KI of 1.54 mm and a kinat of 0.86 min (Fig. 4, Table I). the Complete inativation of 1 mol of the peroxidase requires KI is therefore approximately 13 times larger at ph 5.0 than approximately 11 eq of phenylethylhydrazine (Fig. 5), 32 eq ph 7.0. The pk, of phenylethylhydrazine at 25 C is 6.75 (22). of ethylhydrazine (Fig. 5), and 80 eq of methylhydrazine The onentrations of unprotonated phenylethylhydrazine at (Table I). ph 7.0 and 5.0 an be estimated to be 74 and 26 p ~ respe-, Changes in the Absorbane Spetrum of Horseradish Pertively, if the total onentration of the substrate is assumed oxidase-complex hanges in the absorbane spetrum of the to be equal to the KI value at the given ph (ph 7.0, 115 p ~ ; peroxidase aompany its inativation by the alkylhydrazines. ph 5.0, 1540 PM). The dereased affinity of the enzyme for The Soret absorbane of the enzyme shifts to the red from its

4 \ 100 * Y I \.\ O Alkylation meso-heme Peroxidase Horseradish [Hydrazine]/[HRP] FIG. 5. Partition ratios for the inativation of horseradish peroxidase (HRP) by phenylethylhydrazine (0) and ethylhydrazine (B). Loss of atalyti ativity, as measured by the guaiaol oxidation assay, is plotted against the alkylhydrazine/enzyme ratio. The experimental details are given under Materials and Methods d I I HRP I\ &il Wavelength (nm) FIG. 6. Changes in the horseradish peroxidase (HRP) hromophore aused by inubation of the enzyme with methylhydrazine and HzOa. The spetra are shown for the enzyme before initiation of the reation and after 30 min of reation with H202 and methylhydrazine. The experimental details are given under Materials and Methods. TABLE I1 H NMR proton hemial shifts for meso-phenylethyl heme The spetrum of the unesterified ferrous omplex was taken in pyridine-ds/snc12. Chemial shift values are given in parts/million relative to tetramethylsilane, and oupling onstants are given in hertz. Chemial shift Protons (multipliity) Meso Internal vinyl (dd, J = 11, J = 18) (dd, J = 12, J = 18) Phenyl (t, J = 7) (d, J = 9) (d, J = 8) External vinyl (d, J = 18) (d, J = 18) (d, J = 11) (d, J = 12) C&CH2C (t, J = 7) Methyl CH2CEzC (t, J = 7) to the viinal phenylethylmethylene protons is strengthened by the observation that deoupling of one auses the other to ollapse to a singlet (not shown). No other hanges are observed in the spetrum in these deoupling experiments. All of the other resonanes for both the iron porphyrin in pyridine-d, and the dimethyl-esterified zin porphyrin in CDC1, are onsistent with meso substitution of the porphyrin. The modified heme isolated from the reation of horseradish peroxidase with ethylhydrazine is less polar on reverse phase HPLC than heme itself, and its Soret maximum is at 404 nm. The moleular ion of the unesterified, ferri material is at m/z 643, as expeted for an ethyl heme addut. The H initial position at 402 nm and dereases in intensity. The NMR spetrum of the dimethyl-esterified zin omplex in degree of this shift is both substrate- and time-dependent. The Soret band shift is aompanied by the appearane of an absorbane peak with a maximum at approximately 830 nm (Fig. 6). Isolation and Identifiation of Heme Adduts-HPLC analysis of the prostheti group reovered from phenylethylhydrazine-inativated horseradish peroxidase shows that the heme is onverted in nearly quantitative yield to a single produt. CDC13 onfirms that the modified prostheti group is mesoethyl heme (Table 111). Signals are present for only three of the four meso-protons and the methyl and methylene protons of the ethyl group are readily diserned as a triplet (J = 8 Hz) at ppm and a quartet (J = 8 Hz) at ppm. Irradiation of the protons at ppm results in ollapse of the signal at to a singlet, as required by the strutural assignment. The other signals in the spetrum are typial of The modified heme is less polar than ferri protoporphyrin a meso-substituted porphyrin. I X and has a Soret maximum at 404 nm (1O:l methanol/ The modified heme isolated from the reation of horseradaeti aid) rather than, as in heme, at 398 nm. The mass spetrum of the unesterified produt has a moleular ion at m/z 719, in agreement with its identifiation as a phenylethyl heme addut. The 500-MHz H NMR spetrum of the sample in pyridine onfirms this inferene and learly identifies the isolated material as meso-phenylethyl heme (Table 11). Three rather than four rneso-proton singlets are found in the ish peroxidase with methylhydrazine, whih aounts for 60-70% of the total heme originally present, is again less polar on reverse phase HPLC than heme itself. The Soret maximum of the modified heme is at 404 nm. The moleular ion of the modified ferri heme is found in the mass spetrum at m/z 629, as expeted from addition of a methyl group to the heme. The eletroni absorption spetrum of the dimethyl-esterified, ppm region of the NMR spetrum. The signals for the demetallated porphyrin onsists of a Soret maximum at 412 ortho-, meta-, and para-protons of the phenylethyl moiety are nm and peaks at 512,547,584, and 638 nm with relative found in the region between 7.1 and 7.3 ppm, although no absorbane values, respetively, of100:5.2:2.7:2.4:1. These signals are observed for the orresponding methylene protons, values are very similar to those for the metal-free, dimethylpresumably beause they are obsured by the water peak. esterified, phenylethyl addut, whih has peaks at 410, 508, These resonanes are learly visible in the spetrum of the 542,584, and 634 nm with relative absorbane values, respedimethyl-esterified zin omplex taken in CDC13 (Table 111). tively, of 100:8.4:3.6:3.0:1. The H NMR spetrum of the zin- The methylene adjaent to the porphyrin appears as a triplet omplexed, dimethyl-esterified porphyrin onfirms its iden- (J = 8 Hz) at ppm and that adjaent to the phenyl ring tity as one of the four isomers of meso-methylated protoporas a triplet (J = 8 Hz) at Assignment of these signals phyrin IX (Table 111). Singlets are found at 9.920, 9.808, and

5 14958 Horseradish Peroxidase meso-heme Alkylation ~ TABLE 111 'H NMR proton hemial shifts for the dimethylesterified zin omplexes of meso-alkylporphyrins The 'H NMR spetra of the zin omplexes were taken in deuteriohloroform. Peak positions are reported in parts/million relative to tetramethylsilane. Coupling onstants are in hertz. Proton(s) Methyl - Ethyl Phenylethyl Meso Internal vinyl" External vinyl -C&CHZCO: Methyls Meso group Methyl Methylene Phenvl ' 6.261' 6.200' 6.150' ' 6.280' 6.168' 6.128' d 5.223' ' 6.277" 6.157' 6.134' d 2.990d 7.338' Eah of these signals is a doublet of doublets, J = 11 and 18 Hz. ' This signal is a doublet, J = Hz. e This signal is a doublet, J = Hz. This signal is a triplet, J = 8 Hz. e This signal is a quartet, J = 8 Hz. 'This signal is a multiplet ppm for three of the four meso-protons. The mesomethyl protons appear as an isolated singlet at ppm. This methyl is learly separated from the luster of singlets between 3.74 and 3.53 ppm ontributed by the methyl groups of the protoporphyrin IX skeleton and the esterified arboxyl groups. The other signals in the spetrum are readily assigned to the protons of the vinyl groups and the two propioni aid side hains of protoporphyrin IX. The positions of these protons fully support identifiation of the porphyrin as a meso-substituted protoporphyrin IX derivative. The regiohemistry of heme modifiation was established by nulear Overhauser experiments. Irradiation of the phenylethyl protons adjaent to the porphyrin ring in the Zn2+omplexed meso-phenylethyl addut (ie. at ppm) enhanes the methyl resonanes at and ppm. Likewise, irradiation of the orresponding methylene protons in the meso-ethyl addut (5.223 ppm) enhanes the methyl resonanes at and ppm (Fig. 7). Finally, irradiation of the meso-methyl protons at ppm in the methyl addut enhanes the methyl signals at and ppm. Small bakground nulear Overhauser effets were observed in some instanes due to intermoleular effets, but these were easily differentiated from the signals of interest. The positions of some of the porphyrin methyl signals deviate slightly from those listed in the tables beause pyridine was added to minimize the intermoleular effets. The key finding in all ases is that two methyl signals are enhaned by nulear Overhauser effets when the protons of the meso-alkyl group are irradiated. This is only possible if the alkyl group is on the d-meso position, as this is the only meso position flanked PPM PPM FIG. 7. a, nulear Overhauser signal enhanements observed when the alkyl methylene protons of the dimethyl ester of (Znz+)-mesoethylprotoporphyrin obtained with ethylhydrazine are irradiated and the off-resonane deoupled spetrum is subtrated. b, methyl region of the 'H NMR spetrum of the same porphyrin. The instrumental onditions are given in the text Perent of HEME onverted to meso addut FIG. 8. Correlation between loss of peroxidati ativity and formation of the 8-meso-alkyl-heme in reations of horseradish peroxidase with phenylethylhydrazine (0) and ethylhydrazine (M). Experimental details are given under "Materials and Methods." by two methyl groups. The two methyls that exhibit nulear Overhauser effets are those at positions 1 and 8. Correlation of Peroxidase Inativation with Heme Modifi- ation-complete inativation of horseradish peroxidase by [l4c]pheny1hydrazine orrelates with the binding of approximately 2 eq of phenylhydrazine/mol of enzyme (16). Sine only a minor fration of the heme groups are radiolabeled, the loss of ativity is primarily aused by ovalent binding of phenyl moieties to the protein rather than to the prostheti group. In ontrast, inativation of the enzyme by phenylethylhydrazine losely parallels formation of the meso-phenylethyl heme addut (Fig. 8). A similar orrelation holds for ethylhydrazine, exept that the phenylethyl addut isolated from the fully inativated enzyme aounts for more than 90% of the heme originally present, whereas the ethyl addut only aounts for approximately 70% of the original heme (Fig. 8). The situation in the ase of methylhydrazine is more omplex in that the inativation is reversible, but long term inubation of the enzyme with methylhydrazine onverts approximately 60-70% of the heme into the meso-methyl addut (not shown). Heme modifiation thus appears to be the ritial event in the inativation of horseradish peroxidase by alkylhydrazines. The presene of minor amounts of intat heme in the fully inativated enzyme suggests that the protein is also sometimes modified, but protein modifiation is not, in ontradistintion to phenylhydrazine, the major route of inativation.

6 DISCUSSION Horseradish peroxidase, as shown here, is inativated by phenylethyl-, ethyl-, and methylhydrazine. As previously reported for phenylhydrazine (16), inativation depends on the onentration of the hydrazine and requires atalyti turnover of the enzyme (Figs. 1-3) (16). The reation with phenylhydrazine is too rapid for its kineti properties to be determined by stati spetrosopi methods (16), but lean pseudo-first order kinetis are obtained for the reations of the alkylhydrazines with horseradish peroxidase (Figs. 1-3). The kineti data (Table I) show that the ethyl and phenylethyl analogues bind reversibly to the enzyme with similar affinities, but methylhydrazine binds muh more weakly. The high Kr value for methylhydrazine presumably reflets the fat that it is less lipophili than the ethyl and phenylethyl analogues. Despite the similarities in their KT values, the phenylethyl analogue inativates the enzyme some 20 times more effetively than the ethyl analogue. This differene in inativation rates may inlude differenes in the rates of oxidation of the two hydrazines to the diazenes, differenes in the rates of radial release from the diazenes, and differenes in the partitioning between metabolite formation and enzyme inativation. An independent estimate of the partition ratios for the phenylethyl and ethyl analogues shows that they differ by a fator of approximately 3 (11 for phenylethyl versus 32 for ethyl) (Fig. 5). This differene aounts for some, but not all, of the differene in the inativation rates. Inativation of horseradish peroxidase by the alkylhydrazines orrelates losely with meso alkylation (see Sheme 1) of the prostheti heme group (Fig. 8). Complete inativation of the enzyme by the methyl, ethyl, and phenylethyl analogues oinides, respetively, with meso alkylation of approximately 60-70, 70, and 90% of the prostheti heme groups. Inativation by the alkylhydrazines is thus losely linked, partiularly in the latter instane, to alkylation of the prostheti group. Reations with the apoprotein are required, however, to explain the residual 10-30% of the ativity loss that is not assoiated with heme alkylation. This ontrasts sharply with the finding that inativation by phenylhydrazine orrelates V b 6 P I v v V 6 P SCHEME 1. Mehanism proposed for the inativation of horseradish peroxidase by alkylhydrazines. The fourth struture in the sequene is an isoporphyrin. The final struture is that proposed for the heme adduts (R = phenylethyl, ethyl, or methyl). Horseradish Alkylation Peroxidase meso-heme v with attahment of 2 eq of phenylhydrazine/mol of enzyme. The phenylhydrazine reation also differs in that the mesophenyl addut aounts for only a minor part of the modified heme. The major produt, the 8-hydroxymethyl derivative of heme (16), is not detetably formed with the alkylhydrazines. It is not lear why horseradish peroxidase is primarily inativated by phenylhydrazine by a reation with the protein but by the alkylhydrazines by a reation with the heme. Meso alkylation of the prostheti heme group by the alkylhydrazines, by analogy with the phenylhydrazine reation (16), involves addition of the alkyl radials to the 6-mesoarbon of Compound I1 (Sheme 1). This mehanism is supported, in the ase of phenylhydrazine, by the demonstration that phenyl radials are formed (231, that phenyldiazene inativates the enzyme only if Hz02 is present, and that the ferrous state is not traversed during the atalyti yle (16). If the analogy between the aryl- and alkylhydrazines holds, the first step in the reation is oxidation of the alkylhydrazine (RNHNH,) to the diazene (RN=NH). Transfer of an eletron from the diazene to Compound I, the first step in Sheme 1, produes Compound I1 and the diazenyl radial. Rapid elimination of nitrogen from the diazenyl radial then yields the alkyl radial whih adds, as shown in Sheme 1, to the meso position of Compound 11. Eletron redistribution followed by loss of the meso-proton finally produes the meso-alkylated heme. The alkyl and phenyl radials differ in that abstration of a hydrogen from the 8-methyl group (bottom left methyl in Sheme 1) only ompetes with addition to the meso position in the ase of the phenyl radial. This differene presumably derives from differenes in the intrinsi reativities of the phenyl and alkyl radials. The sp3-hybridized ethyl and phenylethyl radials are less reative than the sp2-hybridized phenyl radial, although the methyl radial is more reative than the other alkyl radials beause it is not stabilized by indutive and hyperonjugative effets. The phenyl radial is also less nuleophili than the alkyl radials. The higher intrinsi reativity of the phenyl radial, whih makes it a less disriminating reatant, and its lower nuleophiliity, whih dereases its rate of reation with the meso position, are probably responsible for its distint reativity profile. The reations of methyl-, ethyl- and phenylethylhydrazine with horseradish peroxidase onfirm and extend the proposal that substrates interat, as shown in Sheme 1, with the edge of the heme between the 6-meso-arbon and 8-methyl group rather than diretly with the ferryl oxygen (16). No nitrogenor meso-alkylated derivatives are obtained exept for the 6- meso addut. The stringeny of this regiospeifiity, even for a moiety as small as the methyl radial, indiates that it is tightly enfored by the protein. These results ontrast sharply with the finding that N-alkyl adduts are the major produts in the reations of the alkylhydrazines with ytohrome P- 450 (1, 20). Nitrogen alkylation of the heme by the alkylhydrazines is thus expeted if the iron and the nitrogens are aessible to the reative speies. The absene of nitrogen alkylation in the reations with horseradish peroxidase therefore suggests that the reation is atively suppressed by the protein struture. Ative regiohemial ontrol of the reation by the apoprotein is furthermore required by the finding that only one of the four essentially equivalent meso positions is alkylated. These results are onsistent with eletron transfer from the substrates to the heme edge assoiated with the 6- meso-arbon, but are more diffiult to reonile with diret reation of the substrate with the ferryl oxygen. The formation of an intermediate with an absorption band at approximately 830 nm in the reations of the alkylhydra-

7 14960 Alkylation meso-heme Peroxidase Horseradish zines with horseradish peroxidase is reminisent of the for- J. Biol. Chem. 257, mation of a similar hromophori speies in the reation of 5. Saito, S., and Itano, H. A. (1981) Pro. Natl. Aud. Si. U. S. A. the enzyme with ylopropanone hydrate (21). The half-life 78, Ringe, D., Petsko, G. A., Kerr, D. E., and Ortiz de Montellano, of the intermediate obtained with the alkylhydrazines de- P. R. (1984) Biohemistry 23, 2-4 pends on the struture of the alkyl group but the intermediate, 7. Allison, W. S., Swain, L.C., Tray, S. M., and Benitez, L.V. in most instanes, is remarkably long-lived. The diret rela- (1973) Arh. Biohem. Biophys. 155, tionship between formation of the 830 nm hromophore and 8. Tank, S.-S., Trakman, P. C., and Kagan, H. M. (1983) J. Biol. loss of atalyti ativity learly impliates the intermediate Chem. 258, as a key speies in the inativation proess. This onlusion Gibian, M. J., and Singh, K. (1986) Biohim. Biophys. Ata 878, is supported by the fat that reation with alkylhydrazines the 10. Fitzpatrik, P. F., and Villafrana, J. J. (1986) J. Biol. Chem. onverts the heme in high yield into meso-heme adduts. It is 261, also onsistent with the observation, in the ase of methyl- 11. Falk, M. C. (1983) Biohemistry 22, hydrazine, that atalyti ativity is reovered as the 830 nm 12. Lerner, H. R., Mayer, A. M., and Harel, E. (1974) Phytohemistry hromophore disappears. It is likely that this hromophore, OX^.) 13, Patek, D. R., and Hellerman, L. (1974) J. Biol. Chem. 249,2373- as proposed for that observed with ylopropanone hydrate, 2380 is due to the isoporphyrin (middle bottom struture in Sheme 14. Nagy, J., Kenney, W. C., and Singer, T. P. (1979) J. Biol. Chem. 1) expeted from addition of the alkyl moiety to the meso- 254, arbon. The preise identity of the intermediate with the Hidaka, H., and Udenfriend, S. (1970) Arh. Biohem. Biophys. nm hromophore, the basis for its unexpeted stability, and 140, its role in the inativation reation are urrently under inves- Ator, M. A,, and Ortiz de Montellano, P. R. (1987) J. Biol. Chem. 262, tigation. 17. Mauk, M. R., and Girotti, A. W. (1974) Biohemistry 13, Aknowledgments-We thank Dr. David Tew for obtaining the 18. Dean, R. T., DeFilippi, L. J., and Hultquist, D. E. (1976) Anal. NMR spetrum and nulear Overhauser data for the meso-methyl Biohem. 76, 1-8 addut. 19. Furhop, J.-H., and Smith, K. M. (1975) in Porphyrins and Metalloporphyrins (Smith, K. M., ed) pp , Elsevier/North- REFERENCES Holland, New York) 20. Ortiz de Montellano, P. R., Kunze, K. L., and Beilan, H. S. (1983) 1. Ortiz de Montellano, P. R., Augusto, O., Viola, F., and Kunze, K. J. Biol. Chem. 258,45-47 L. (1983) J. Biol. Chem. 258, Wiseman, J. S., Nihols, J. S., and Kolpak, M. X. (1982) J. Biol. 2. Jonen, H. G., Werringloer, J., Prough, R. A., and Estabrook, R. Chem. 257, W. (1982) J. Biol. Chem. 257, Krueger, P. J. (1975) in The Chemistry of the Hydruzo, Azo, and 3. Ortiz de Montellano, P. R., and Kerr, D. E. (1983) J. Biol. Chem. Azoxv GFOLLDS (Patai.. S... ed)." DD John Wilev & Sons. 258, Ltd.,"New 9ork 4. Augusto, O., Kunze, K. L., and Ortiz de Montellano, P. R. (1982) 23. Sinha, B. K. (1983) J. Biol. Chem. 258,