Chem. Res. Toxicol. 1994, 7, 843-849
843
Inactivation of Horseradish Peroxidase by 3,5-Dicarbethoxy-2,6-dimethyl-4-ethyl1,4-dihydropyridine Katsumi Sugiyama,? Amina Woods,$ Robert J. Cotter,* Robert J. Highet,B John F. Darbyshire," Yoichi Osawa,* and James R. Gillette Laboratory of Chemical Pharmacology and Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1760, and Middle Atlantic Mass Spectrometry Laboratory, Johns Hopkins School of Medicine, Baltimore, Maryland 21205 Received June 23, 1994@ I n the presence of H202, horseradish peroxidase (HRP) catalyzes the one-electron oxidation of a porphyrinogenic agent, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-l,4-dihydropyridine (DDEP), leading to formation of a n ethyl radical, inactivation of the enzyme, and formation of a n altered heme product. The loss of heme during the inactivation of HRP was dependent on the duration of exposure to DDEP as well as the concentration of H202 and DDEP. The pseudo first order rate constant for the oxidation of DDEP by compound I of HRP at p H 7.4was 0.07min-', and t h e maximal extent of heme loss was 35%. The altered heme product, which was isolated by reverse phase HPLC, was characterized by the use of mass and l H NMR spectrometry as a substitution product of a CzH40H moiety for a meso proton of the prosthetic heme [meso(hydroxyethy1)hemel. The source of the oxygen in the C2H40H moiety appeared not to be H202 or H2O as l80was not incorporated in the heme adduct when H P 0 2 or H2180 was used. The DDEP-mediated reaction did not form the expected 6-meso-ethylheme adduct analogous to the ethyl radical-mediated inactivation of HRP by ethylhydrazine (EH) [Ator e t al. (1987) J . B i d . Chem. 262, 14954- 149601. However, we have found that meso-(hydroxyethy1)heme was formed in the EH-mediated reaction, albeit in apparently lower amounts t h a n d-mesoethylheme. Perhaps the proximity of the heme to the ethyl radical may play a role in determining the nature of the heme products formed.
Introduction Horseradish peroxidase [EC 1.11.1.7, donor hydrogen peroxide (H202)oxidoreductase, HRPll is a hemoprotein that catalyzes the oxidation of various substrates in the presence of H202 (1,2). The catalytic turnover of HRP involves H202-mediated oxidation of the prosthetic heme group to an intermediate species referred to as compound I, which is a ferryl porphyrin radical cation species (1, 2). A one-electron reduction of compound I yields compound 11, which is thought to be a ferryl heme species with a n oxene ligand (Few=O) (3). One-electron oxidation of substrates containing hetero atoms by compound I of HRP frequently results in the formation of alkyl radicals, which may alter the prosthetic heme group and modify or terminate the catalytic function of the enzyme (4-7). Possible reactions of radicals with the prosthetic heme group include addition
* Correspondence should be addressed to this author at the Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8N-110, Bethesda, MD 20892-1760; Phone: (301) 496-4841; FAX: (301) 4020171. + Present address: Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Building 10, Room 10B-09, Bethesda, MD 20892. t Johns Hopkins School of Medicine. Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute. Present address: Department of Medicinal Chemistry, BG-20, University of Washington, Seattle, WA 98195. Abstract published in Advance ACS Abstracts, October 15, 1994. Abbreviations: P450, cytochrome P450; HRP, horseradish peroxidase; DDEP, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-l,4-dihydrop~dine; DP, 3,5-dicarbethoxy-2,6-dimethylpyridine; EDP, 3,5-dicarbethoxy2,6-dimethyl-4-ethylpyridine; EH, ethylhydrazine; DF, deferoxamine mesylate; IOB, iodosobenzene; ABTS, 2,2'-azidobis[3-ethylbenzothiazolinesulfonic acid]. @
0893-228d94/2707-0843$04.50/0
to the iron atom, pyrrole nitrogens, pyrrole carbons, vinyl carbons, and meso carbons or abstraction of a methyl hydrogen (8). The nature and regiospecificity of the reaction are determined by the steric and electronic properties of the radicals, the structure of the active site of the enzyme, and the mechanism by which radicals are generated. For example, phenylhydrazine inactivates HRP by the formation of 6-meso-phenyl and 8-(hydroxymethyl) derivatives as well as the covalent alteration of the apoprotein (91, whereas the reaction of ethylhydrazine (EH) with HRP has been shown to lead to the formation of the 6-meso-ethylheme adduct, most likely by way of the generation of a n ethyl radical leading to the formation of a B-meso-isopophyrin intermediate (10). Cytochrome P450s (P450) are also known to metabolize the porphyrinogenic agent 3,5-dicarbethoxy-2,6-dimethyl4-ethyl-1,4-dihydropyridine (DDEP) to a n ethyl radical, leading to the inactivation of the enzymes. While the inactivation of P450 2 C l l and 2C6 appeared to be due to the formation of N-ethylheme (11, 121, the exact mechanism of heme alteration in the inactivation of P450 3A1 is unknown (12). We have found that HRP is also inactivated by DDEP. We sought to determine if DDEP could lead to the formation of N-ethylheme or covalent modification of apoprotein, analogous to the inactivation of P450, or the formation of a 6-meso-ethylheme adduct, analogous to the reaction with EH and HRP. Unexpectedly, we found the 6-meso-ethylheme is not formed from the reaction with DDEP; instead, a CzH40H mesosubstituting product of heme was formed. The present report describes the characterization of this heme adduct and the kinetics of its formation.
0 1994 American Chemical Society
844 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
Experimental Section HRP (type VI-A) and deferoxamine mesylate (DF) were obtained from Sigma (St. Louis, MO). Bovine liver catalase was purchased from Calbiochem (San Diego, CAI. Potassium ferricyanide and hydrogen peroxide (30%) were obtained from Fisher (Pittsburgh, PA). Ethylhydrazine (EH) oxalate was purchased from Fluka (Ronkonkoma, NY).Hydrogen peroxide H21802 (2% soln, 90 atom %) and H21BO (95 atom %) were obtained from ICON (Summit, NJ), and Sep-Pak cartridges were purchased from Millipore (Milford, MA). 2,2'-hidobis[3-ethylbenzothiazolinesulfonic acid] (ABTS) was purchased from Research Organics (Cleveland, OH). Pyridine-ds (&, 99.94%) and Methanol-& (d4,99.96%)were purchased from Merck (Rahway, NJ) and Cambridge Isotope Laboratories (Woburn, MA), respectively. Stannous chloride was obtained from Aldrich (Milwaukee, WI). Absorption spectra were obtained with a SLM Aminco DW-2000 UV-vis spectrometer (Urbana, IL). High pressure liquid chromatography was performed using a HewlettPackard 1050 system equipped with a 1040M diode-array detector (Hewlett-Packard, Rockville, MD). The concentration of HRP was determined by using E403 = 102 cm-' mM-l (13). All experiments were performed using potassium phosphate buffer pretreated with Chelex 100 (Bio-Rad, Richmond, CAI. Caution: Contact with EH is harmful and may cause irreversible effects. Synthesis. The synthesis and chemical characterization of DDEP have been described previously (14).
Inactivation of HRP by DDEP or EH. (i) Inactivation of HRP by DDEP. Activity of HRP was determined with the use of the ABTS assay (15). To 4 nmol of HRP in 0.1 M potassium phosphate (pH 7.0) was added 25 nmol of iodosobenzene. Catalase (306 units) was added in one experiment. Then 250 nmol of DDEP was added. The total volume was 0.5 mL. The reactions were allowed to proceed a t 37 "C for 20 min and terminated by freezing. (ii) The HzOz Concentration-Dependent Loss of the Heme of HRP. To 4 nmol of HRP in a 0.1 M potassium phosphate buffer (pH 7.0) were added 0-500 nmol of HzOz and 1.5 pmol of DDEP. The total volume was 0.5 mL. Reactions were allowed to proceed for 1h at 37 "C and were terminated by the addition of 25 pL of 1.9 mM DF. Aliquots (100 pL) of the reaction mixtures were injected into a HPLC Vydac C4 column, 0.46 x 25 cm (Vydac, Hesperia, CA) equilibrated with 1OO:O.l H20fl'FA. A linear gradient was run to 35:65:0.1 CH3CN/HZOI"FA (v/v) a t a flow rate of 1m u m i n over 45 min. The loss of heme and the amount of altered heme were monitored by HPLC profiles at 400 nm. Since the extinction coefficients of the altered heme products are unknown, the percentage of the altered heme products formed was defined as the ratio of the peak areas of the altered heme product over that of the initial amount of heme. (iii) Time-Dependent Loss of the Heme of J3RP. To 4 nmol of HRP in 25 mM potassium phosphate (pH 7.0) was added 5 nmol of H2Oz. Spectroscopic measurements confirmed that only compound I was formed at pH 7.0. The complex was stable prior to the addition of substrate. After 1min the residual H2Oz was destroyed by the addition of catalase (306 units). Then 250 nmol of DDEP was added. The total volume was 0.5 mL. The reaction was allowed to proceed a t 37 "C and then terminated by the addition of 25 p L of 1.9 mM DF a t selected times. Similar experiments were also performed where 125 nmol of EH was substituted for DDEP. (iv) Effect of the Concentration of DDEP or EH on Heme Loss. To 8 nmol of HRP in 50 mM potassium phosphate buffer (pH 7.4) was added 10 nmol of HzOz. Residual H2Oz was destroyed by the addition of catalase (611 units). Then, reactions were started by the addition of either 0-1 pmol of DDEP or 0-0.5 pmol of EH. The total volume was 1mL. The mixtures were incubated for 0-15 min a t 37 "C, and the reactions were terminated by the addition of 5OpL of 1.9 mM DF. The maximal extent of heme loss (the ratio of the amount of heme loss to the initial amount of heme) was determined from corresponding
Sugiyama et al.
Scheme 1. Kinetic Scheme of H e m e Loss in the Inactivation of Compound I by DDEP kl2
Compound I +
s
k23
CompoundlS
Hemeloss
__t
t E
E HRP
+ M
values for 1 mM of DDEP or EH after 90 min of incubation, when all of compound I was consumed.
Kinetics of Heme Loss in the Osidation of DDEP or EH Catalyzed by Compound I. A kinetic scheme for the loss of heme in the oxidation of DDEP or EH by compound I is presented in Scheme 1. The substrate (S) combines reversibly with compound I to form an enzyme-substrate complex (compound IS), which in turn leads to either alteration of heme or formation of product, M, and native HRP. We have assumed that the formation of intermediate, compound IS, rapidly approaches steady state,but that the concentration of compound I, heme loss, and M are not in steady state. Then, we may write the following equations:
d(compound I)/dt = (compound IS)k,, (compound 1)(S)kl2 t 0 d(compound IS)/dt = (compound I)(S)k12 (compound IS)(k,, k23
+
d(heme loss)/dt = (compound IS)k2,
+
k24)
f
0
=0
d(M)/dt = (compound IS)kZ4f 0 Solving for the Laplace transforms for compound I, heme loss, and M, and then for the inverse Laplace transform equations gives:
(heme loss) = (compound I)[k23/(k23
+ k2,)1(1 - e-At) (1)
From eq 1, a factor A is obtained with known values of P, k23/ (k23
+ k24):
A = -{ln[l - (heme loss)/(P)(compound I)l}/t
(2)
P is the maximal extent of heme loss (the ratio of the amount of heme loss to the initial amount of heme) when all of the compound I is consumed. The amount of compound I was assumed to equal the amount of HRP added. Isolation of the Altered Heme Product Generated in the Reaction of HRP with DDEP or EH. Mixtures containing 2.5 pmol of HRP, 2.5 pmol of HzOz, and 62.5 pmol of DDEP in 100 mL of 0.1 M potassium phosphate (pH 7.4) were incubated for 1.5 h a t 37 "C, and the reaction mixtures were filtered. Altered heme products generated in the reaction of HRP with EH were prepared in a similar manner. The filtrates of the incubation mixtures were directly chromatographed on a 21.5 x 250-mm Bio-Rad Hi-Pore RP-304 HPLC column (Richmond, CA) at a flow rate of 20 m u m i n using a 50 min linear gradient from 75:25:0.1 CH3CN/H20/TFA to 64:36:0.1 CH3CN/HzO/TFA (v/v). The retention time of the altered heme product generated by DDEP was 52 min, and those of the products formed from EH were 48 and 52 min. A corresponding fraction of the altered heme product was loaded on a Sep-Pak
Alteration of the Prosthetic Heme of HRP
Chem. Res. Toxicol., Vol. 7, No. 6,1994 846
Table 1. Inactivation of Horseradish Peroxidase by D D E P heme loss, activity loss, system +IOB +IOB
+ DDEP + catalase + DDEP
%b
%
22.2 f 1.9
14.4 15.2
activity/ heme lossc 0.65
Experimental design is described under Experimental Section. *Results represent mean & SD of duplicate measurements measured by HPLC as described in the Experimental Section. Values were corrected for a contribution of heme from catalase, which was 1%of total amount of heme. cartridge and eluted with methanol. A similar experiment using Hz180z was performed for the study of l80incorporation. For O , (1pmol) was the study of l80incorporation from H Z ~ ~HRP treated with HzOz (1pmol) and DDEP (13pmol) in 2 mL of 0.1 M potassium phosphate (pH 7.4) made with Hzl80 (final 50 atom %). Samples for NMR studies were prepared using the above procedure starting with a 10 pmol of HRP, 10 pmol of HzOz, and 250 pmol of DDEP in 400 mL of 0.1 M potassium phosphate (pH 7.4). Tandem Time-of-Flight Mass Spectrometry. Mass spectra were obtained on a tandem time of flight mass spectrometer built in house and described previously (16).This instrument consists of two reflectron mass analyzers in tandem separated by a collision chamber for inducing fragmentation. However, in this work mass spectra were recorded in the “double reflectron” mode in order to obtain sufficient mass resolution to resolve completely the isotopic contributions to the molecular ion peak. Samples were diluted in a solution of methanoUO.l% trifluoroacetic acid in HzO ( l : l ) , mixed with a solution of caffeic acid in water, and deposited on the probe tip of the mass spectrometer. The sample was ionized using a PTI PL2300 (Ontario, Canada) pulsed nitrogen laser, and spectra were recorded by a Tekronix TDS 540 (Beaverton, OR) digital oscilloscope, downloaded to a 486 PC, and averaged using Tofware (ILYS Software, Pittsburgh, PA). NMR Spectrometry. NMR spectra of the heme products in pyridine-ds solution were obtained on a Varian XL300 spectrometer following reduction by stannous chloride ( 17); 0.20.3 mg of sample was added to 2-3 mg of SnClz in a volume of 0.5 mL. Typically, 256 free induction decays with an accumulation time of 4 s were collected for 1D spectra.
E *
6.
0 0
(I
l-
a w 0
z a
m a
s:m a
C. 1
2
h
20
10
30
40
!
TIME Imin)
Figure 1. Reverse phase HPLC of HRP (8 pM) treated with (A) HzOz (0.25 mM), (B) HzOz (10 pM), catalase (611 units), and DDEP (3 mM), and (C) HzOz (10 pM), catalase (611 units), and EH (0.125 mM). For samples in panels B and C, catalase was added 1 min after addition of HzOz. Incubations were carried out at 37 “C for 1h in a total volume of 1.0 mL. A 1OOpL aliquot was injected and analyzed as described in the Experimental Section (ii).Peaks at 26.5 and 28 min correspond to DDEP and heme, respectively. loo
OI
E
80
;x
r0
I\
‘-0 ..........
0.--......
”-.....o
-.*-
Results DDEP caused a loss of peroxidase activity (14%) of HRP (Table 1). In studies on peroxidase activity, iodosobenzene was used as a n oxidant rather than H z 0 ~ in order to avoid the possible involvement of the Fenton reaction, but similar losses in heme were observed with HzOz. The loss of activity accounted for about 65% of heme loss, implying that HRP with an altered prosthetic heme still possessed catalytic activity. HPLC analysis revealed a concomitant loss of heme (Table 1)as well as formation of a n altered heme product (Figure 1). HPLC analysis showed different heme products of HRP by HzOz, DDEP, and EH (Figure 1). Treatment of HRP by HzOz alone produced a polar heme product 1 ( t 18.5 ~ min). By contrast, two altered heme products were observed in the EH-mediated reaction. One was a 6-meso-ethylheme (tR 34 m i d , 2, and the other one was a more lipophilic product, 3 ( t 36 ~ min). However, only product 3 was formed in the DDEP-mediated reaction. Kinetics. The loss of heme of HRP during the metabolism of DDEP was dependent on the amount of HzOz (Figure 2). About 15%of heme was lost with HzOz alone whereas about 30% of heme was lost in the presence of DDEP and HzOz. The prosthetic heme of HRP was not completely lost even with large quantities of H202. The formation of the altered heme product was
50
k
0
1
I
I
I
30
60
90
120
‘ 0
[H,O*MHRPl
Figure 2. The loss of heme and the formation of the altered heme product 3 as a function of HzOz to enzyme ratio in the inactivation of HRP for 1 h. A loss of heme with HzOz in the absence of DDEP (open circles) and in the presence of DDEP (closed circles). The formation of altered heme product in the presence of DDEP (triangles).
also dependent on HzOz concentration; maximum formation occurred with a ratio of HzOz to HRP of about 30. Time courses of heme loss and the formation of altered heme products during the oxidation of DDEP or EH by compound I are presented in Figure 3. The loss of heme of HRP during the oxidation of DDEP was time-dependent and paralleled the formation of the altered heme product (Figure 3A). Since the mechanism of DDEPmediated heme loss of HRP is thought to be similar to that of EH, i.e., the generation of ethyl radicals, the corresponding time course of the loss of heme and the formation of two altered heme products during the oxidation of EH catalyzed by compound I were compared (Figure 3B). The losses of the heme of HRP by DDEP
Sugiyama et al.
846 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
r
251A
t
0
(kz3 + kz4)/Km
Substrate
25
P
(min -1.mM -1) DDEP
2o
0.07 0.37
EH
0.35 0.81
15
0)
E
Po
I
10
.-C E
30
0
60
90
-
120
4:
Time (min)
E
601I d
8
20
m a,
I
m -0
40
I
B
40
cll
0.25
0
ca,
.g
0.5
0.75
1
E
22
[SI (mM) Figure 4. Effect of the concentration of DDEP or EH on heme loss a t pH 7.4: EH (open circles), DDEP (closed circles). A = -{ln[l - (heme loss)/(P)(compound I)]}/t. Kinetic parameters are shown in the inset. P is the maximal extent of heme loss given by (heme loss)/(heme,,iti,l) of a 90 min incubation.
0
30
0
60
90
120
--I
Time (min)
Figure 3. Time-dependent loss of heme and the formation of altered heme products, 2 and 3, in the oxidation of DDEP and EH by compound I. (A) Reactions were started with 4 nmol of compound I and 250 nmol of DDEP. A loss of heme (open circles) and the formation of altered heme product 3 (closed circles). (B) Reactions were started with 4 m o l of compound I and 125 nmol of EH. A loss of heme (open circles) and the formation of d-meso-
l i
ethylheme (the altered heme product 2) (triangles) and the altered heme product 3 (closed circles). The exact amounts of product formed are unknown since the extinction coefficients have not been determined.
Table 2. Molecular Ions of Altered Heme Products, 2 and 3, Generated in DDEP- and EH-Mediated Inactivation of Horseradish Peroxidase system heme HRP DDEP HzOz HRP DDEP H2l80z HRP DDEP HzOz HzlsO HRP EH HzOz HRP EH HzOz
+ + + + +
+ + +
+ +
+
obsd molecular ion mlz
predicted molecular formula
660.3" 660.2'" 660.2"
C34H32FeN404 C36H36FeN406 C36H36FeN405e C36H36FeN405C
616.5 660.2 660.2 660.2
644.3b 660.1"
C36H36FeN404 C36H36FeN405
644.6 660.2
theoretical value
300
400
500
600 Wa,eIe"g'" inn,,
700
800
900
Figure 5. Visible spectral analysis of heme (- - -) and an altered heme product 3 (-) generated in the DDEP-mediated inactivation of HRP (in methanol).
a Altered heme 3 corresponding to a retention time of 36 min on HPLC. b Altered heme 2 corresponding to a retention time of 34 min on HPLC. Predicted molecular formula for compound that did not incorporate I80.
Characterization of the Altered Heme Products. (a) Visible Spectral Analyses. The altered h e m e product 3 h a d absorption m a x i m a of 403 n m (100)a n d 505 n m (8.5)in methanol (Figure 5). (b)MS Analyses. Tandem time-of-flight m a s s spectra
a n d EH were also concentration-dependent (Figure 4). Kinetic p a r a m e t e r s for h e m e loss associated with oxidation of D D E P or EH catalyzed by compound I are also presented in Figure 4. When all of compound I w a s reduced t o the ferric state, 35% of the prosthetic h e m e w a s lost by D D E P . T h e second order rate c o n s t a n t for the disappearance of compound I d u r i n g t h e oxidation of DDEP, ( A 2 3 k24)/Km,w a s 0.07 min-l mM-l. EH w a s found t o be a more efficient inactivator of H R P than DDEP; the extent of h e m e loss, P, a n d t h e rate constant, (k23 k24)/&, with EH were 0.8 a n d 0.37 min-' mM-l, respectively.
of the altered h e m e products 2 a n d 3 are presented in Figure 6. Molecular ions a n d theoretical values of corresponding molecular formulas of altered h e m e products are presented in Table 2. The m a s s spectrum of the altered h e m e product 2 exhibited a molecular ion at m l z 644.3, which agreed with a theoretical value of 644.6 for a product corresponding t o the net addition of C2H4 t o heme. T h e m a s s spectra of the altered h e m e product 3 formed with either D D E P or EH as substrates exhibited a molecular ion at m / z 660.3 a n d 660.1, respectively, which agreed with a theoretical value of 660.2 for a product corresponding t o the net addition of Cz&O t o heme. These results confirmed that the altered h e m e
+
+
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 847
Alteration of the Prosthetic Heme of HRP
Scheme 2. DDEP Metabolic Pathway Associated with Inactivation of HRP
'fi
HRP.
1
HSC R
/ Ryg C2H5
H202
CH, p
H
DDEP R = C02C2HS
I **
EDP
Inactlvatlon
p~
DP
660.3
> t UJ
t I-
z
Lu
I
I-
4w
644 3
\
a
10
8
6
4
2
0
PPM I
640
1
650 Mi2
I
I
660
Figure 6. Tandem time-of-flight mass spectra. (A) The altered heme product 3.(B)The altered heme product 2.
product generated in the DDEP-mediated heme loss is one of the two heme products formed with EH-mediated reaction. Product 3 isolated from reaction mixtures containing Hz1802or Hz180 was analyzed t o identify the source of the oxygen. Hz180z or HZl80 did not alter the molecular mass of 3,which implies that the oxygen comes from 0 2 (Table 2). ( c )NMR Analyses. The 'H NMR of the altered heme product 3 showed only three meso proton signals, which were similar to those of 8-meso-ethylheme, 2, generated in EH-mediated inactivation of HRP (Figure 7, Table 3). All signals corresponding to two vinyl substituents, four methyl groups, and propionic methylene groups were intact. Also, no signals corresponding to a n N-ethyl group were observed. The evidence showed that the CzH50 group is attached to a meso carbon of heme. While lH NMR of 8-meso-ethylheme, 2, clearly exhibited signals of ethyl and methyl protons a t 4.8 and 0.5 ppm, respectively, with a n interaction to each other in the COSY spectrum (Figure 7, Table 3), these signals are absent for the (hydroxyethyl)heme, 3. The possible structures of a substituent group a t a meso position, therefore, were -0CHZCH3, -CH(OH)CHs, -CHzOCH3, and -CHZCHzOH. The first two candidates were eliminated on the ground that no methyl proton signals coupling to the adjacent methylene protons were observed
Figure 7. 300-MHz 'H NMR spectrum of (A) the altered heme product 3 and (B)the altered heme product 2 (d-meso-ethylheme). Impurity, pyridine, and water peaks are marked X, F'yr, and W, respectively.
in the COSY spectrum. Since there was no additional methoxyl proton signal, the third candidate was also eliminated. The probable adduct is, therefore, a -CHzCHzOH moiety. In support of this view the meso substituent group of the altered heme product 3 gave four proton signals with two pairs of coupling between 3 and 4 ppm. Two pairs of four proton signals coupled to each other indicated that four methylene protons may be held in a rigid conformation by hydrogen bonding. Similar effects of intramolecular hydrogen bonding on coupling of methylene protons have been observed previously (18). The unusual upfield chemical shift exhibited by 2-ethyl protons suggests that they are shielded from the ring current of heme.
Discussion HRP catalyzes the oxidation of DDEP by HzOz to yield 3,5-dicarbethoxy-2,6-dimethylpyridine(DP) and 3,5di~arbethoxy-2~6-dimethyl-4-ethylpyridine (EDP) (19) (Scheme 2). A one-electron oxidation of DDEP by HRP and HzO2 also results in the formation of DP and the inactivation of HRP (19). We have shown that a new altered heme product was formed during the loss of the prosthetic heme of HRP by DDEP in the presence of HzOz. The altered heme product was characterized by mass and lH NMR spectrometry as a substitution product of a CzH40H moiety for a
848 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
Sugiyama et al.
Table 3. NMR Data for the Altered Heme Product 3 and 6-meeo-Ethvlhemea altered heme product 3 8-meso-ethylheme, 2 meso -CH=CH2 4 2 -CH=CHz trans 4 trans 2 cis 4 cis 2 -CHzCH2COzH -CH&H2COzH methyls
meso-alkyl substituent
chemical shift 10.15 10.10 9.94
COSY interactions
chemical shift 9.98 9.93 9.84
COSY interactions
8.41 (dd, J = 18, 12) 8.32 (dd, J = 18, 11)
6.23,5.90 6.23, 5.97
8.35 8.19
6.24, 5.91 6.11, 5.99
6.23 (d, J = 18) 6.23 (d, J = 18) 5.90 (d, J = 12) 5.97 (d, J = 11) 4.42 (m) 3.46 (m) 3.83 3.68 3.54 3.41 4.09 (m) 3.97 (m) 3.73 (t) 3.31 (m)
8.41,5.90 8.32, 5.97 8.41,6.23 8.32,6.23 3.46 4.42
6.24 6.11 5.91 5.99 4.37 3.44, 3.34 3.69 3.55 3.51 3.38 4.69 (q, J = 7) 0.46 (t, J = 7)
8.35, 5.91 8.19 8.35, 6.24 8.19 3.44, 3.34 4.37
3.31 3.73 3.97 4.09
0.46 4.69
a Chemical shift values are ppm relative to tetramethylsilane, and coupling constants are given in Hz;resonances are singlets except as noted; d = doublet, t = triplet, q = quartet, m = multiplet.
Scheme 3. Hypothetical Mechanism of the Formation of b-meso-(Hydroxyethy1)hemeAdduct by DDEP-Mediated Inactivation of HRP
-
HOH2CH2
H02C
meso proton of the prosthetic heme. Although the exact meso site that was altered was not determined, the formation of only d-meso-alkylheme by treatment of HRP with methyl-, ethyl-, n-butyl-, and phenethylhydrazines (6)suggests that the altered heme product 3 may also be a b-meso-alkylhemeadduct (Figure 8). While the ethyl radical-mediated inactivation of HRP by EH leads mainly to the formation of d-meso-ethylheme, the new altered heme product 3 contains a n additional oxygen atom. A hypothetical reaction pathway for the formation of the altered heme product, 3, is proposed in Scheme 3. Ethyl radicals generated by one-electron oxidation of DDEP may be rapidly trapped by oxygen molecules to form ethylperoxy radicals, which may undergo isomerization by a n intramolecular 1,4H-atom shift to form ethyl hydroperoxide radicals (20). The ethyl hydroperoxide radicals may then react with heme to form (hy-
HO)
droxyethy1)heme. Perhaps the proximity of generation of the ethyl radical relative to the location of the heme determines the reaction pathway of the ethyl radical. This hypothetical mechanism for the formation of (hydroxyethy1)heme was derived from the following observations. Since only three of the four meso protons were detected in the NMR spectrum of the altered heme adduct 3,it is conceivable that the initial step of heme loss is the addition of a radical to a meso position. However, if a radical is a n ethyl radical, it is expected to form a n isoporphyrin intermediate (IO). Though the reported half-life of ethylisoporphyrin is 450 min (IO), a characteristic absorption of isoporphyrin around 835 nm was not detected with DDEP at pH 7.0 during the reaction. The absence of b-meso-ethylheme in DDEPmediated heme loss also suggests that the radical species is not a n ethyl radical; instead it implies that a n oxygen
H3ca Chem. Res. Toxicol., Vol. 7, No. 6,1994 849
Alteration of the Prosthetic Heme of HRP
'
/ Fe'\ /
HOH2C' C H2
H3C
\
H2
\
I
C02H
I
C02H
Figure 8. A proposed structure of 6-meso-(hydroxyethy1)heme (the altered heme product 3)generated in the DDEP-mediated inactivation of HRP.
is incorporated into an ethyl radical prior to its addition to a meso position. The source of oxygen remains unknown. Though DDEP contains oxygen atoms, EH does not. Therefore, the oxygen in (hydroxyethy1)heme most probably does not come from the substrate. Also, it is unlikely that a n oxygen atom comes from protein by the cleavage of a covalent bond between carbon and oxygen of tyrosine, serine, or threonine. Since lS0in H21sO~or H2180was not incorporated into (hydroxyethy1)heme, the source is probably molecular oxygen. Thus, a radical reaction involving 0 2 and not a cationic mechanism involving water seems more likely for the formation of (hydroxyethy1)heme as depicted in Scheme 3.
Acknowledgment. We thank Dr. Timothy Cornish for his technical help in mass spectrometry.
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