Phenylethylhydrazine hydrochloride and methylhydrazine sulfate were purchased .... hydrazine is much less efficient if the reaction is carried out at pH 5.0 rather ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc
Vol ,262, No. 31, Issue of November 5, PP. 14954-14960,1387 Printed in U.S. A.
Structure and Catalytic Mechanism of Horseradish Peroxidase REGIOSPECIFIC MESO ALKYLATION OF THEPROSTHETIC
HEME GROUP BY ALKYLHYDRAZINES* (Received for publication, May 11, 1987)
Mark A. Ator, Shantha K. David, and Paul R. Ortiz de Montellano* From the Demrtment of Pharmnceutical Chemistrv. School of Phnrmacy, and the Liver Center, University of California, San Francisco, Californh 94143 " I
Horseradish peroxidase is inactivated in a time-,
The inactivation of horseradish peroxidase by phenylhy-
HzOz-, and concentration-dependent manner by phen- drazine, first reported by Hidaka andUdenfriend in 1970 (E),
ylethyl-,ethyl-, and methylhydrazine. The pseudofirst order kinetic constants for these inactivation reactions at pH 7 are: phenylethyl (KJ= 115 p ~ kinact , = 1.5 min-l, partition ratio = 11), ethyl (KJ= 145 p M , kinact= 0.08 min", partition ratio = 32), and methyl (KJ= 3000 p ~ k,,,,,, = 0.12 min", partition ratio = 80). At pH 5, the constants for the phenylethyl reaction change to Kr = 1540 p~ and kinact= 0.86 min". A transient absorbance at approximately 830 nm, suggestive of an isoporphyrin intermediate, is seen during these reactions. The prosthetic heme is converted by each of the three alkylhydrazines into the corresponding 6-rneso-alkylatedheme. Complete inactivation of the enzymes by methyl-, ethyl-, and phenylethylhydrazine is associated with alkylation of 6070, 70, and 90%,respectively, of the prosthetic heme groups. The absence of N-alkylation and the high specificity for the &mesoposition, even with agents as small as methylhydrazine, strengthen the proposal that electron abstraction is mediated by the heme edge rather than the ferryl oxygenof horseradish peroxidase.
Alkyl- and arylhydrazines inactivate many oxidative enzymes, including hemoproteins such as cytochrome P-450 (1, 2), catalase (3), hemoglobin (4, 5 ) , myoglobin (6), and lactoperoxidase (7), non-heme' metalloproteins such as lysyl oxidase (8), soybean lipoxygenase (9), dopamine @-hydroxylase (lo), plasma amine oxidase (ll),and catechol oxidase (12), and flavoproteins such as monoamine oxidase (13) and trimethylamine dehydrogenase (14). The mechanisms of these inactivation reactions vary in that some involve alkylation of heme or flavin prosthetic groups by catalytically generated radicals, some involve reactions of the radicals or other activated intermediates with protein residues, and still others involve formation of a hydrazone with the carbonyl group of a quinonoid cofactor. The parameters that determine which of these mechanisms predominates in a given situation remain unclear. * This research was supported by Grants GM 32488and GM 25515 from the National Institutes of Health. Mass spectra were obtained in the Bioorganic, Biomedical Mass Spectrometry Facility of the University of California, San Francisco, supported by Grant RR 01614 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. 2 To whom correspondence should be addressed. The abbreviations used are: heme, iron protoporphyrin IX regardless of oxidation and ligation state; HPLC, high pressure liquid chromatography.
recently was shown to closely parallel the covalent binding of 2 eq of radiolabeled phenylhydrazine/enzyme molecule (16). The inactivation is associated with conversion of a fraction of the prosthetic groups to 6-meso-phenyl heme and 8-hydroxymethyl heme, but the bulk of the heme groups (54%) are recovered intact from the fully inactivated enzyme. Enzyme inactivation therefore results primarily from reaction of a phenylhydrazine metabolite, probably the phenyl radical, with the apoprotein. The clear correlation between enzyme inactivation and binding of two phenylhydrazine moieties cannot otherwise be reconciled with the fact that less than half of the prosthetic groups are actually modified (16). The high specificity of the reaction for the 6-meso position and the 8-methyl group and the absence of the N-phenyl adducts expected from reaction with the iron or nitrogen atoms of the heme (1-6) led us to propose that phenylhydrazine and presumably most other substrates areoxidized by electron transfer to the heme edge rather than to the ferryl oxygen. The present studies were undertaken to determine ( a ) if alkylhydrazines inactivate horseradish peroxidase, ( b ) if substrates smaller than phenylhydrazine also react only with the heme edge, and ( c ) what the relative roles of heme and protein alkylation are in enzyme inactivation. MATERIALSANDMETHODS
Horseradish peroxidase (type VI), bovine liver catalase, hydrogen peroxide, guaiacol, and sodium ascorbate were obtained from Sigma. Phenylethylhydrazine hydrochloride and methylhydrazine sulfate were purchased from ICN Pharmaceuticals (Plainview, NY) and ethylhydrazine oxalate from Fluka. Methylhydrazine sulfate was recrystallized three times from 80% ethanol. Deuteriochloroform and pyridine-& (100 atom%, 'H) were obtained from Aldrich. Absorption spectra were obtained on a Hewlett-Packard 8450A diode array spectrophotometer. High pressure liquid chromatography was performed with a system consisting of Beckman Model llOA pumps, a Beckman Model 420 controller, and a Hewlett-Packard 1040A diodearray detector. NMR spectra were recorded on aGeneral Electric GN 500 MHz instrument. Chemical shift values are reported in parts per million relative to tetramethylsilane. Mass spectra were obtained on a KratosMS 50 instrument operating in theliquid matrix secondary ion mode. The concentration of horseradish peroxidase was determined using E,o2 = 95,000 M" cm" (17). All peroxidase experiments, unless otherwise specified, were performed a t 25 "C in 50 mM sodium phosphate buffer (pH 7.0) that had been passed through a column of Chelex 100 (Bio-Rad). Znactivation of Horseradish Peroxidase by Alkylhydrazines-The rate of inactivation of horseradish peroxidase by phenylethylhydrazine was measured in a reaction mixture that contained 1 pM peroxidase, 125 PM H202, and 10-50 p~ phenylethylhydrazine. At 1-min intervals after addition of the peroxide and inactivator, 5-pl aliquots of the mixture were transferred to cuvettes containing 1.0 ml of an assay mixture containing 50 mM sodium phosphate (pH 7.0), 5 mM guaiacol, and 0.6 mM H202. Theincrease in absorbance at 470 nm
14954
Horseradish Peroxidase meso-Heme Alkylation was recorded as a measure of the peroxidase activity. The effect of pH on the inactivation by phenylethylhydrazine was determined by repeating the incubations in 50 mM sodium citrate buffer (pH 5.0). Analogous experiments were carried out with ethylhydrazine and methylhydrazine, except that incubations with the former were carried out with 1 mM H20Z andethylhydrazine concentrations ranging from 0.1 to 1.0 mM, and incubations with the latter with 5 mM Hz02 and methylhydrazine concentrations ranging from 0.7 to 4.4 mM. Partition Ratios and Correlation of Heme Modification and Activity Loss during the Inactivation of Horseradish Peroxidase-One-milliliter samples of 10 p~ horseradish peroxidase were incubated for 30 min with 0.1 mM H202 and 0-0.1 mM phenylethylhydrazine. Aliquots of the reaction mixtures were then removed and assayed for remaining peroxidatic activity as described above. Excess Hz02 was destroyed by incubating each sample for 3 min with 2 pl of a 5.6 mg/ml solution of bovine liver catalase before 2 pl of 50 mM sodium ascorbate was added to ensure that the enzyme was reduced. The mixtures were placed on ice until analyzed. Following the completion of all of the reactions, the heme was isolated from each sample. The solution was acidified by adding 0.27 mlof glacial acetic acid and the heme extracted with two 1.5-ml aliquots of diethyl ether. Low speed centrifugation was required to separate the aqueous and organic layers in the extraction procedure. The organic layers werepooled, washed with H20, and evaporated to dryness under a stream of NP. The composition of the extracted heme mixture was analyzed by HPLC on a 4.6 X 250-mm Whatman Partisil 5 ODs-3 column eluted with 7:3:1 methanol/H20/glacial acetic acid at a flow rate of 1 ml/min. Heme absorbance was monitored at 400 nm with the diode array detector, and a Hewlett-PackardModel 3390A integrator was used to determine the relative absorbance of each peak. Similar experiments were performed with ethylhydrazine and methylhydrazine, except that the Hz02 concentration was raised to 0.5 mM in the case of ethylhydrazine and to 1.5 mM in the case of methylhydrazine. The ethylhydrazine concentration was varied from 0 to 0.4 mM and the methylhydrazine concentration from 0 to 1 mM. The reactions with ethylhydrazine and methylhydrazine were allowed to proceed for 1h before peroxidase activity was measured and theheme isolated. Isolation and Identification of Heme Adducts Generatedin the Reaction of Horseradish Peroxidase with Alkylhydrazines-Mixtures containing 50 p M horseradish peroxidase, 1.5 mM H202, and1.5 mM phenylethylhydrazine in 60 ml of 50 mM sodium phosphate buffer (pH 7.0) were incubated 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 ascorbate and 16 ml of glacial acetic acid. The heme was extracted with diethyl ether and theethereal solution, after washing with H20, was evaporated to dryness in urnuo. The same procedure was used to isolate the adducts formed in the reactions of horseradish peroxidase with ethylhydrazine and methylhydrazine, except that the concentration of H z 0 2 was 2.0 and 5.0 mM, respectively, and theconcentration of methylhydrazine 2.5 mM rather than 1.5 mM. The modified hemes were purified from the crude heme extracts by two different procedures. The unesterified hemes were directly chromatographed on a 9 X 250-mm Whatman Partisil 10 ODS-3 HPLC column a t a flow rate of 4 ml/min (compound, solvent system, retention time: phenylethyl adduct, 72:2810 methanol/H20/acetic acid, 12.4 min; ethyl adduct, 6832:lO methanol/HzO/acetic acid, 8.2 min; methyl adduct 643610 methanol/H20/acetic acid, 11.9 min). The phenylethyl adduct from a number of chromatographic runs was rechromatographed ona 4.6 X 250-mm Partisil 5 ODs-3 column using a 10-min linear gradient from 6 4 1 methanol/HZO/glacial acetic acid to 1 0 1 methanol/glacial acetic acid at a flow rate of 1 ml/min. A portion of the mixture was analyzed by mass spectrometry, while the remainder was converted to thechloroiron(II1) complex, dissolved in pyridine-d5, reduced with SnC12, and examined by 500 MHz 'H NMR spectroscopy (16). The pyridine peak at 8.70 ppm was used as the reference for chemical shift values. The second procedure for isolation of the heme adducts involved esterification with (CH&OBF, (18) and demetallation with ferrous sulfate as described by Fuhrhop and Smith (19). The resulting mixture of dimethyl-esterified porphyrins was chromatographed on a 4.6 X 250-mm Partisil 5 PAC HPLC column in 1:2 tetrahydrofuran/ hexane at 1 ml/min with detection at 410 nm (compound, retention time: phenylethyl adduct, 14 min; ethyl adduct, 12.4 min; methyl adduct, 15.5 min). The isolated adducts were dissolved in CH2Clzand converted to the Zn2+complexes by treatment with excess Zn(OAc)z in methanol at room temperature for 30 min. The porphyrins were extracted into ether, and the ethersolutions were washed with satu-
14955
rated aqueous NaCl solution, dried over NazSOI, and evaporated to dryness in uacuo. The 500 MHz 'H NMR spectra of the porphyrins were obtained in CDCl,. Chemical shift values are given relative to the solvent peak a t 7.26 ppm. Nuclear Overhauser effects 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 zinc complex of dimethyl-esterified meso-ethyl protoporphyrin IX required a power level of 40 db. A reference spectrum was obtained with the decoupler set 0.5 ppm downfield from the methylene peak. A line-broadening function of 1.0 Hz was applied to thefree induction decay signal prior to zero filling the 16K spectrum to 64K. The nuclear Overhauser effect was obtained by subtracting the transformed reference spectrum from the experimental spectrum. RESULTS
Inactivation of HorseradishPeroxidasebyAlkylhydra-
zines-Phenylethylhydrazine is a potenttime- and concentration-dependent inactivator of horseradish peroxidase. The inactivation is characterized by pseudo-first order kinetics. A replot of the kinetic data yields a KI of 115 PM and a kinactof 1.5 min" (Fig. 1,Table I). Horseradish peroxidase is similarly inactivated by ethylhydrazine (Fig. 2 ) and methylhydrazine (Fig. 3). The KI value for ethylhydrazine is comparable to that for phenylethylhydrazine, but the kinactvalue is 20 times
100
90 80 70
60
.-3
50
-0 40 .-
OI
.E .E
E"
-P
30
C
,o c?
20
10
0
1
2
3 4 Time (rnin)
t
I
/
5
6
7
FIG. 1. Time-dependent inactivation of horseradish peroxidase by phenylethylhydrazine at pH 7.0. The concentrations of
phenylethylhydrazine are 12.5 (O),25 (m), and 37.5 p~ (A),A replot of the time required to inactivate half of the enzyme ( t U )versus the reciprocal of the inhibitor concentration is shown in the inset. The details of the incubations are given under "Materials and Methods."
TABLE I Kinetic constants for the inactivation of horseradishperoxidme by alkvlhvdrazines ~
Alkylhydrazine
Phenylethyl pH 7.0 pH 5.0 Ethyl Methyl
KI
Partition ratio
PM
min"
115 1540 145 3000
0.86 0.08
1.50 0.12
11
32
80
meso-Heme Peroxidase Horseradish
14956 100-
Alkylation
a
100
9080 -
90 80
70 -
70
60 -
60
b 50 -
3 50
:s
‘5 .-
c
0
c
$ 40
40-
0,
0,
.f .f
.E
.E
$
P
E
30-
t?
30
c
c
E
9)
c!
20
30
Kt
1
20
2 4 6 8 1 0 l/[Ethylhydrazine] ( n ” )
10-1
I
r
1
I
I
I
1
0
5
10
15
20
25
30
Time (min) FIG. 2. Time-dependent inactivation of horseradish peroxidase by ethylhydrazine at pH 7.0. The concentrations of ethylhydrazine are 0.1,).( 0.15 (B),0.25 (A),and 0.5 mM (0).A replot of the tH values uersus the reciprocal of the inhibitor concentration is shown in the inset. Details of the incubations are given under “Materials and Methods.”
10
0
10
20
30 Time (rnin)
40
50
FIG. 3. Time-dependent inactivationof horseradish peroxidasebymethylhydrazineat pH 7.0. The concentrations of methylhydrazine are 0.71(O),0.94 (B),1.28 (A),2.1 (O),and 4.4 mM (0).A replot of the tCcvalues versus the reciprocal of the inhibitor concentration is shown in the inset. Details of the incubations are given under “Materialsand Methods.”
lower (Table I). The KI value formethylhydrazine, in contrast, is 20 times larger than that for ethylhydrazine, but the kinact value is comparable (Table I). In any case, inactivation of horseradish peroxidase by the alkylhydrazines is much slower thanthatmediated by phenylhydrazine, whichoccurs so rapidly that kinetic parameters for the inactivation cannotbe obtained by static spectroscopic methods (16). No evidence has been found for the formation of alkylhydrazine metabolites comparable to those generated from phenylhydrazine 0 1 that protect theenzyme from complete inactivation (16). 1 2 3 4 5 6 7 Methylhydrazine differs from the phenyl, phenylethyl, and l/[PEH](mM’) ethyl analogues in thatits inactivation isreversible. No activFIG. 4. Time-dependent inactivation of horseradish peroxity is recovered if enzyme inactivated with phenylhydrazine, idase by phenylethylhydrazine atpH 6.0. A replot of tCcvalues phenylethylhydrazine, or ethylhydrazine is passed througha taken from a plot analogous to that of Figs. 1-3 uersus the reciprocal Sephadex G-25 column to remove excess reagent. However, of the inhibitorconcentrations isshown. The concentrations of phenylethylhydrazine ( P E H ) employed in the study were 0.15,0.25, in the caseof methylhydrazine, the enzyme remains inactive 0.50, and 1.0 mM. Details of the incubations aregiven under “Mateimmediately after passage through a Sephadex G-25 column, rials and Methods.” but slowly recovers a t least part of its activity if it is then incubated at 30 “C for an extendedperiod. The mechanismof the inhibitor at p H 5.0 therefore reflects, in part, a decrease this reactivation, which is closely paralleled by a decrease in in the concentrationof the unprotonatedhydrazine. the isoporphyrin absorbance at approximately 830 nm (see The partition ratios for theinactivation of horseradish below), is under investigation. peroxidase by methyl-, ethyl-, and phenylethylhydrazine have The inactivationof horseradish peroxidase by phenylethyl- been determined by incubating the enzyme with increasing hydrazine is much less efficient if the reaction is carried out concentrations of each of these compounds and then measurat pH5.0 rather than pH7.0. The inactivation at p H 5.0 also ing the residual catalytic activity after sufficient time has follows pseudo-first order kinetics but is characterized by a elapsed for the alkylhydrazine to be completely consumed. KI of 1.54 mM and a kinactof 0.86 min” (Fig. 4, Table I). the Complete inactivation of 1 mol of the peroxidaserequires KI is therefore approximately 13 times larger at p H 5.0 than approximately 11 eq of phenylethylhydrazine (Fig. 5), 32 eq pH 7.0. The pK, of phenylethylhydrazine at 25 “C is6.75 (22). of ethylhydrazine (Fig. 5),and 80 eq of methylhydrazine The concentrationsof unprotonated phenylethylhydrazine at (Table I). pH 7.0 and 5.0 can be estimated tobe 74 and 26 p ~ respec, Changes in the Absorbance Spectrum of Horseradish Pertively, if the total concentration of the substrate is assumed oxidase-Complex changes in the absorbance spectrum of the to be equal to the KI value at the given pH (pH 7.0, 115 p ~ ;peroxidase accompanyits inactivationby the alkylhydrazines. p H 5.0, 1540 PM). The decreased affinity of the enzyme for The Soret absorbanceof the enzyme shifts to the red from its
100
Alkylation meso-Heme Peroxidase Horseradish
*
14957 TABLEI1
’HNMR proton chemical shifts for meso-phenylethyl heme I
Y
The spectrum of the unesterified ferrous complex was taken in pyridine-ds/SnC12. Chemical shift values are given in parts/million relative to tetramethylsilane, and coupling constants are given in hertz.
.\
\
Chemical shift (multiplicity)
Protons
Meso \
O 5
10
15
20
25
30
35
40
Internal vinyl
[Hydrazine]/[HRP]
FIG. 5. Partition ratios for the inactivation of horseradish peroxidase (HRP) by phenylethylhydrazine (0)and ethylhydrazine (B).Loss of catalytic activity, as measured by the guaiacol oxidation assay, is plotted against the alkylhydrazine/enzyme ratio. The experimental details are given under “Materials and Methods.”
0.9
0.8 0.7
1
44
External vinyl
8.366 (dd, J = 11,J = 18) 8.213 (dd,J = 12, J = 18) (t, J = 7) 7.195 (d, J = 9) 7.112 (d, J = 8) 6.234 (d, J = 18) 6.117 (d, J = 18) 5.985 (d,J = 11) 5.901 (d, J = 12)
d
II I\
Phenyl 7.273
10.014 9.933 9.845
HRP
3.739
C&CH2C02 Methyl
4.400 (t, J = 7) 3.664 3.514
3.412 CH2CEzC02
&il-
3.484 (t, J = 7)
to the vicinal phenylethylmethylene protons is strengthened by the observation that decoupling of one causes the other to collapse to a singlet (not shown). No other changes are ob0.1 served in the spectrum in these decoupling experiments. All 200 300 400 500 600 700 800 900 of the other resonances for both the iron porphyrin in pyriWavelength (nm) dine-d, and the dimethyl-esterified zinc porphyrin in CDC1, FIG. 6. Changes in the horseradish peroxidase ( H R P )chro- are consistent with meso substitution of the porphyrin. mophore caused by incubation of the enzyme with methylhyThe modified heme isolated from the reaction of horseraddrazine and HzOa. The spectra are shown for the enzyme before initiation of the reaction and after 30 min of reaction with H202 and ish peroxidase with ethylhydrazine is less polar on reverse methylhydrazine. The experimental details are given under “Mate- phase HPLC than heme itself, and its Soret maximum is at 404 nm. The molecular ion of the unesterified, ferric material rials and Methods.” is at m/z 643, as expected for an ethyl heme adduct. The ’H initial position at 402 nm and decreases in intensity. The NMR spectrum of the dimethyl-esterified zinccomplex in CDC13 confirms that the modified prosthetic group is mesodegree of this shift is both substrate- and time-dependent. The Soret band shiftis accompanied by the appearance of an ethyl heme (Table 111). Signals are present for only three of absorbance peak with a maximum at approximately 830 nm the four meso-protons and themethyl and methylene protons of the ethyl group are readily discerned as a triplet( J = 8 Hz) (Fig. 6). Isolation and Identification of Heme Adducts-HPLC anal- at 1.631 ppm and a quartet ( J = 8 Hz) at 5.223 ppm. Irradiaysis of the prosthetic group recovered from phenylethylhydra- tion of the protons at 5.223 ppm results in collapse of the zine-inactivated horseradish peroxidase shows that the heme signal at 1.631 to a singlet, as required by the structural is converted in nearly quantitative yield to a single product. assignment. The other signals in the spectrum are typical of The modified heme is less polar than ferric protoporphyrin a meso-substituted porphyrin. The modified heme isolated from the reaction of horseradI X and has a Soret maximum at 404 nm (1O:l methanol/ acetic acid) rather than, as in heme, at 398 nm. The mass ish peroxidase with methylhydrazine, which accounts for 60spectrum of the unesterified product has a molecular ion at 70% of the total heme originally present, is again less polar m/z 719, in agreement with its identification as aphenylethyl on reverse phase HPLC thanheme itself. The Soret maximum heme adduct. The 500-MHz ’H NMR spectrum of the sample of the modified heme is at 404 nm. The molecular ion of the in pyridine confirms this inference and clearly identifies the modified ferric heme is found in the mass spectrum at m/z isolated material as meso-phenylethyl heme (Table 11). Three 629, as expected from addition of a methyl group to theheme. rather than four rneso-proton singlets are found in the 9-10- The electronic absorption spectrum of the dimethyl-esterified, ppm region of the NMRspectrum. The signals for the demetallated porphyrin consists 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 absorbance values, respectively, of100:5.2:2.7:2.4:1. These signals are observed for the corresponding methylene protons, values are very similar to those for the metal-free, dimethylpresumably because they are obscured by the water peak. esterified, phenylethyl adduct, which has peaks at 410, 508, These resonances are clearly visible in the spectrum of the 542,584, and 634 nm with relative absorbance values, respecdimethyl-esterified zinc complex taken in CDC13 (Table 111). tively, of 100:8.4:3.6:3.0:1. The ‘H NMR spectrum of the zincThe methylene adjacent to theporphyrin appears as a triplet complexed, dimethyl-esterified porphyrin confirms its iden( J = 8 Hz) at 5.424 ppm and that adjacent to thephenyl ring tity as one of the four isomers of meso-methylated protoporas a triplet ( J = 8 Hz) at 2.990. Assignment of these signals phyrin IX (Table 111). Singlets are found at 9.920, 9.808, and
14958
Horseradish Peroxidase meso-Heme Alkylation
TABLE111 'H NMR protonchemical shifts for the dimethylesterified zinc complexes of meso-alkylporphyrins The 'H NMR spectra of the zinc complexes were taken in deuteriochloroform. Peak positions are reported in parts/million relative to tetramethylsilane. Coupling constants are in hertz. Proton(s)
Methyl-
Ethyl
Phenylethyl
Meso
9.920 9.808 9.727
10.114 9.948 9.902
10.006 9.853 9.782
Internal vinyl"
8.234 8.141
8.222 8.116
External vinyl
6.327' 6.261' 6.200' 6.150'
-C&CHZCO:
4.315 4.345
Methyls
3.737 3.700 3.622 3.618 3.550 3.531
8.251 8.167 6.324' 6.280' 6.168' 6.128' 4.398 4.334 3.733 3.715 3.676 3.667 3.651 3.547
3.223 3.178
3.241 3.194
4.397
1.631d 5.223'
Meso group Methyl Methylene
~
6.317' 6.277" 6.157' 6.134' 4.344 4.294 3.717 3.699 3.682 3.628 3.620 3.513 3.215 3.156
5.424d 2.990d Phenvl 7.338' Each of these signals is a doublet of doublets, J = 11 and 18 Hz. This signal is a doublet, J = 17-18 Hz. e This signal is a doublet, J = 10-12 Hz. This signal is a triplet,J = 8 Hz. e This signal is a quartet, J = 8 Hz. 'This signal is a multiplet.
'
9.727ppm for three of the four meso-protons. The mesomethyl protons appear as an isolated singlet at 4.397 ppm. This methyl is clearly separated from the cluster of singlets between 3.74 and 3.53 ppm contributed by the methyl groups of the protoporphyrin IX skeleton and theesterified carboxyl groups. The othersignals in thespectrum are readily assigned to theprotons of the vinyl groups and thetwo propionic acid side chains of protoporphyrin IX. The positions of these protons fully support identification of the porphyrin as a meso-substituted protoporphyrin IX derivative. The regiochemistry of heme modification was established by nuclear Overhauser experiments. Irradiation of the phenylethyl protons adjacent to the porphyrin ring in the Zn2+complexed meso-phenylethyl adduct (ie. at 5.424 ppm) enhances the methyl resonances at 3.741 and 3.673 ppm. Likewise, irradiation of the corresponding methylene protons in the meso-ethyl adduct (5.223 ppm) enhances the methyl resonances at 3.735 and 3.667 ppm (Fig. 7). Finally, irradiation of the meso-methylprotons at 4.397 ppm in themethyl adduct enhances the methyl signals at 3.622 and 3.550 ppm. Small background nuclear Overhauser effects were observed in some instances due to intermolecular effects, but these were easily differentiated from the signals of interest. The positions of some of the porphyrin methyl signals deviate slightly from those listed inthe tables because pyridine was added to minimize the intermolecular effects. The key finding in all cases is that two methyl signals areenhanced by nuclear Overhauser effects 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
3 8
3 6
PPM
3 8
3 6
PPM
FIG.7. a, nuclear Overhauser signal enhancements observed when the alkyl methylene protons of the dimethyl ester of (Znz+)-mesoethylprotoporphyrin obtained with ethylhydrazine are irradiatedand the off-resonance decoupled spectrum is subtracted. b, methyl region of the 'H NMR spectrum of the same porphyrin. The instrumental conditions are given in the text.
1
100 Percent of HEME converted to meso adduct
FIG. 8. Correlation between loss of peroxidatic activity and formation of the 8-meso-alkyl-heme in reactions 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 nuclear Overhauser effects are those at positions 1 and 8. Correlation of Peroxidase Inactivation with Heme Modification-Complete inactivation of horseradish peroxidase by [l4C]pheny1hydrazinecorrelates with the binding of approximately 2 eq of phenylhydrazine/mol of enzyme (16). Since only a minor fraction of the heme groups are radiolabeled, the loss of activity is primarily caused by covalent binding of phenyl moieties to the protein rather than to the prosthetic group. In contrast, inactivation of the enzyme by phenylethylhydrazine closely parallels formation of the meso-phenylethyl heme adduct (Fig. 8). A similar correlation holds for ethylhydrazine, except that the phenylethyl adduct isolated from the fully inactivated enzyme accounts for more than 90% of the heme originally present, whereas the ethyl adduct only accounts for approximately 70% of the original heme (Fig. 8). The situation in the case of methylhydrazine is more complex in that the inactivation is reversible, but long term incubation of the enzyme with methylhydrazine converts approximately 60-70% of the heme into the meso-methyl adduct (not shown). Heme modification thus appears to be the critical event in the inactivation of horseradish peroxidase by alkylhydrazines. The presence of minor amounts of intact heme in the fully inactivated enzyme suggests that the protein is also sometimes modified, but protein modification is not, in contradistinction to phenylhydrazine, the major route of inactivation.
Alkylation Horseradish Peroxidasemeso-Heme
14959
with attachment of 2 eq of phenylhydrazine/mol of enzyme. The phenylhydrazine reaction also differs in that the mesoHorseradish peroxidase, as shown here, is inactivated by phenyl adduct accounts for only a minor part of the modified phenylethyl-, ethyl-, and methylhydrazine. As previously re- heme. The major product, the 8-hydroxymethyl derivative of ported for phenylhydrazine (16), inactivation depends on the heme (16), is not detectably formed with the alkylhydrazines. concentration of the hydrazine and requires catalytic turnover It is not clear why horseradish peroxidase is primarily inacof the enzyme (Figs. 1-3) (16). The reaction with phenylhy- tivated by phenylhydrazine by a reaction with the protein but drazine is too rapid for its kinetic properties to be determined by the alkylhydrazines by a reaction with the heme. by static spectroscopic methods (16), but clean pseudo-first Meso alkylation of the prosthetic heme group by the alkylorder kinetics are obtained for the reactions of the alkylhyhydrazines, by analogy with the phenylhydrazine reaction drazines with horseradish peroxidase (Figs. 1-3). The kinetic (16), involves addition of the alkyl radicals to the 6-mesodata (Table I) show that theethyl and phenylethyl analogues carbon of Compound I1 (Scheme 1).This mechanism is supbind reversibly to the enzyme with similar affinities, but ported, in the case of phenylhydrazine, by the demonstration methylhydrazine binds much more weakly.The high Kr value for methylhydrazine presumably reflects the fact that it is that phenyl radicals are formed (231, that phenyldiazene inactivates the enzyme only if Hz02is present, and that the less lipophilic thanthe ethyland phenylethyl analogues. Despite the similarities in their KT values, the phenylethyl ferrous state is not traversed during the catalytic cycle (16). If the analogy between the aryl- and alkylhydrazines holds, analogue inactivates the enzyme some 20 times more effecthe first stepin the reaction is oxidation of the alkylhydrazine tively than theethyl analogue. This difference in inactivation rates may include differences in the rates of oxidation of the (RNHNH,) to thediazene (RN=NH). Transfer of an electron two hydrazines to the diazenes, differences in the rates of from the diazene to Compound I, the first step in Scheme 1, produces Compound I1 and the diazenyl radical. Rapid elimradical release from the diazenes, and differences inthe partitioning between metabolite formation and enzyme inac- ination of nitrogen from the diazenyl radical then yields the tivation. An independent estimate of the partition ratios for alkyl radical which adds, as shown in Scheme 1, to the meso the phenylethyl and ethylanalogues shows that they differ by position of Compound 11. Electron redistribution followed by a factor of approximately 3 (11 for phenylethyl versus 32 for loss of the meso-proton finally produces the meso-alkylated ethyl) (Fig. 5). This difference accounts for some, but not all, heme. The alkyl and phenyl radicals differ in that abstraction of of the difference in the inactivation rates. a hydrogen from the 8-methyl group (bottom left methyl in Inactivation of horseradish peroxidase by the alkylhydrazines correlates closely with meso alkylation (see Scheme 1) Scheme 1)only competes with addition to the meso position of the prosthetic heme group (Fig. 8). Complete inactivation in the case of the phenyl radical. This difference presumably of the enzyme by the methyl, ethyl, andphenylethyl analogues derives from differences in the intrinsic reactivities of the coincides, respectively, with meso alkylation of approximately phenyl and alkyl radicals. The sp3-hybridizedethyl and phen60-70, 70, and 90% of the prosthetic heme groups. Inactiva- ylethyl radicals are less reactive thanthe sp2-hybridized tion by the alkylhydrazines is thus closely linked, particularly phenyl radical, although the methyl radical is more reactive in the latter instance, to alkylation of the prosthetic group. than the other alkyl radicals because it is not stabilized by Reactions with the apoprotein are required, however, to ex- inductive and hyperconjugative effects. The phenyl radical is plain the residual 10-30% of the activity loss that is not also less nucleophilic than the alkyl radicals. The higher associated with heme alkylation. This contrastssharply with intrinsic reactivity of the phenyl radical, which makes it a lower nucleophilicity, the finding that inactivation by phenylhydrazine correlates less discriminating reactant,andits which decreases its rate of reaction with the meso position, are probably responsible for its distinct reactivity profile. V v The reactions of methyl-, ethyl- and phenylethylhydrazine with horseradish peroxidase confirm and extend the proposal that substrates interact, as shown in Scheme 1, with the edge of the heme between the 6-meso-carbon and 8-methyl group rather than directly with the ferryl oxygen (16). No nitrogenor meso-alkylated derivatives are obtained except for the 6 meso adduct. The stringency of this regiospecificity, even for a moiety as small as the methyl radical, indicates that it is P b tightly enforced by the protein. These results contrast sharply with the finding that N-alkyl adducts arethe major products in the reactions of the alkylhydrazines with cytochrome P450 (1, 20). Nitrogen alkylation of the heme by the alkylhydrazines is thus expected if the iron and the nitrogens are v v V accessible to the reactive species. The absence of nitrogen alkylation in thereactions with horseradish peroxidase therefore suggests that the reaction is actively suppressed by the protein structure.Active regiochemicalcontrol of the reaction by the apoprotein is furthermore required by the finding that only one of the four essentially equivalent meso positions is alkylated. These results are consistent with electron transfer from the substrates to the heme edge associated with the 6 6 P SCHEME 1. Mechanism proposed for the inactivation of meso-carbon, but are more difficult to reconcile with direct horseradish peroxidase by alkylhydrazines. The fourth struc- reaction of the substrate with the ferryl oxygen. The formation of an intermediate with an absorption band ture in the sequence is an isoporphyrin. The final structure is that proposed for the heme adducts ( R = phenylethyl, ethyl, or methyl). at approximately 830 nm in the reactions of the alkylhydraDISCUSSION
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Alkylation meso-Heme Peroxidase Horseradish
zines with horseradish peroxidase is reminiscent of the forJ. Biol. Chem. 257, 6231-6241 5. Saito, S., and Itano, H. A. (1981) Proc. Natl. Acud. Sci. U. S. A. mation of a similar chromophoric species in the reaction of 78,5508-5512 the enzyme with cyclopropanone hydrate (21). The half-life 6. Ringe, D., Petsko, G. A., Kerr, D. E., and Ortiz de Montellano, of the intermediate obtained with the alkylhydrazines deP. R. (1984) Biochemistry 23, 2-4 pends on the structure of the alkylgroup but the intermediate, 7. Allison,W. S., Swain, L.C., Tracy, S. M., and Benitez, L.V. in most instances, is remarkably long-lived. The direct rela(1973) Arch. Biochem. Biophys. 155, 400-404 tionship between formation of the 830 nm chromophore and 8. Tank, S.-S., Trackman, P. C., and Kagan, H. M. (1983) J. Biol. Chem. 258,4331-4338 loss of catalytic activity clearly implicates the intermediate 9. Gibian, M. J., and Singh, K. (1986) Biochim. Biophys. Acta878, as a key species in the inactivation process. This conclusion 79-92 is supportedby the fact that reaction withalkylhydrazines the 10. Fitzpatrick, P. F., and Villafranca, J. J. (1986) J. Biol. Chem. converts the hemein high yield into meso-heme adducts. It is 261,4510-4518 also consistent with the observation, in the case of methyl- 11. Falk, M. C. (1983) Biochemistry 22, 3740-3745 hydrazine, that catalytic activity is recovered as the 830 nm 12. Lerner, H. R., Mayer, A. M., and Harel, E. (1974) Phytochemistry OX^.) 13, 397-401 chromophore disappears. It is likely that this chromophore, 13. Patek, D. R., and Hellerman, L. (1974) J. Biol. Chem. 249,2373as proposed for that observed with cyclopropanone hydrate, 2380 is due to the isoporphyrin (middle bottom structure in Scheme 14. Nagy, J., Kenney, W. C., and Singer, T. P. (1979) J. Biol. Chem. 1) expected from addition of the alkyl moiety to the meso254,2684-2688 carbon. The precise identity of the intermediate with the 830- 15. Hidaka, H., and Udenfriend, S. (1970) Arch. Biochem. Biophys. 140, 174-180 nm chromophore, the basis for its unexpected stability, and 16. Ator, M. A,, and Ortiz de Montellano, P. R. (1987) J. Biol. Chem. its role in the inactivation reaction are currently under inves262,1542-1551 tigation. 17. Mauk, M. R., and Girotti, A. W. (1974) Biochemistry 13, 1757Acknowledgments-We thank Dr. David Tew for obtaining the NMR spectrum and nuclear Overhauser data for the meso-methyl adduct. REFERENCES 1. Ortiz de Montellano, P. R., Augusto, O., Viola, F., and Kunze, K. L. (1983) J. Biol. Chem. 258,8623-8629 2. Jonen, H. G., Werringloer, J., Prough, R. A., and Estabrook, R. W. (1982) J. Biol. Chem. 257, 4404-4411 3. Ortiz de Montellano, P. R., and Kerr, D. E. (1983) J. Biol. Chem. 258, 10558-10563 4. Augusto, O., Kunze, K. L., and Ortiz de Montellano, P. R. (1982)
1763 18. Dean, R. T., DeFilippi, L. J., and Hultquist, D. E. (1976) Anal. Biochem. 76, 1-8 19. Furhop, J.-H., and Smith, K.M. (1975) in Porphyrins and Metalloporphyrins (Smith, K. M., ed) pp. 800-803, Elsevier/North-
Holland, New York) 20. Ortiz de Montellano, P. R., Kunze, K. L., and Beilan, H. S. (1983) J. Biol. Chem. 258,45-47 21. Wiseman, J. S., Nichols, J. S., and Kolpack, M. X. (1982) J. Biol. Chem. 257,6328-6332 22. Krueger, P. J. (1975) in The Chemistry of the Hydruzo, Azo, and Azoxv GFOLLDS (Patai.. S... ed). "DD. 153-224. John Wilev & Sons.
Ltd.,"New 9ork
23. Sinha, B. K. (1983) J. Biol. Chem. 258, 796-801
