Conversion of pentahalogenated phenols by microperoxidase-8/H2O2 to benzoquinone-type products

Article abstract of DOI:10.1021/tx980037l

This study reports the microperoxidase-8 (MP8)/H₂O₂-catalyzed dehalogenation of pentafluorophenol and pentachlorophenol, compounds whose toxic effects and persistence in the environment are well documented. The primary products of this dehalogenation reaction appear to be the corresponding tetrahalo-p-benzoquinones. Under the conditions used, the fluorinated phenol and its intermediate products are more susceptible to degradation than the corresponding chlorinated analogue and its products. The main degradation products of tetrachloro-p-benzoquinone and tetrafluoro-p- benzoquinone were identified as trichlorohydroxyp-benzoquinone and trifluorohydroxy-p-benzoquinone, respectively. This secondary conversion of tetrafluoro-p-benzoquinone and tetrachloro-p-benzoquinone was not mediated by MP8, but was driven by H₂O₂. Evidence is presented for a mechanism where H₂O₂ molecules and not hydroxide anions are the reactive nucleophilic species attacking the tetrahalo-p-benzoquinones. In addition to the formation of the trihalohydroxy-p-benzoquinones, the formation of adducts of the tetrahalo-p-benzoquinone products with ethanol, present in the incubation medium, was observed. The adduct from the reaction of tetrachloro-p- benzoquinone with ethanol was isolated and identified as trichloroethoxyquinone. Thus, the present paper describes a system in which the formation of tetrahalo-p-benzoquinone-type products by an oxidative heme- based catalyst could be unequivocally demonstrated.

Full text of DOI:10.1021/tx980037l

Chem. Res. Toxicol. 1998, 11, 1319-1325
1319
Con ver sion of P en ta h a logen a ted P h en ols by
Micr op er oxid a se-8/H2O2 to Ben zoqu in on e-Typ e P r od u cts
Ahmed M. Osman,*,† Maarten A. Posthumus,‡ Cees Veeger,†
Peter J . van Bladeren,§ Colja Laane,† and Ivonne M. C. M. Rietjens†
Department of Biomolecular Sciences, Laboratory of Biochemistry,
Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands,
Department of Biomolecular Sciences, Laboratory of Organic Chemistry, Agricultural University,
Dreijenplein 8, 6703 HB Wageningen, The Netherlands, and TNO Nutrition and Food Research
Institute, P.O. Box 360, 3700 AJ Zeist, The Netherlands
Received February 26, 1998
This study reports the microperoxidase-8 (MP8)/H2O2-catalyzed dehalogenation of pentafluo-
rophenol and pentachlorophenol, compounds whose toxic effects and persistence in the
environment are well documented. The primary products of this dehalogenation reaction
appear to be the corresponding tetrahalo-p-benzoquinones. Under the conditions used, the
fluorinated phenol and its intermediate products are more susceptible to degradation than
the corresponding chlorinated analogue and its products. The main degradation products of
tetrachloro-p-benzoquinone and tetrafluoro-p-benzoquinone were identified as trichlorohydroxy-
p-benzoquinone and trifluorohydroxy-p-benzoquinone, respectively. This secondary conversion
of tetrafluoro-p-benzoquinone and tetrachloro-p-benzoquinone was not mediated by MP8, but
was driven by H2O2. Evidence is presented for a mechanism where H2O2 molecules and not
hydroxide anions are the reactive nucleophilic species attacking the tetrahalo-p-benzoquinones.
In addition to the formation of the trihalohydroxy-p-benzoquinones, the formation of adducts
of the tetrahalo-p-benzoquinone products with ethanol, present in the incubation medium, was
observed. The adduct from the reaction of tetrachloro-p-benzoquinone with ethanol was isolated
and identified as trichloroethoxyquinone. Thus, the present paper describes a system in which
the formation of tetrahalo-p-benzoquinone-type products by an oxidative heme-based catalyst
could be unequivocally demonstrated.
halogenated phenols, resulting in the elimination of the
halogen as anion and the formation of p-benzoquinone
as the only dehalogenated product of the reaction. By
using H218O2 and H218O, it was demonstrated that the
oxygen atom inserted into the product, p-benzoquinone,
derived from the medium (water) and not from hydrogen
peroxide.
In tr od u ction
Microperoxidase-8 (MP8)1 is a heme octapeptide de-
rived from the enzymatic digestion of horse heart cyto-
chrome c. Microperoxidases are often used as model
systems for redox catalytic heme proteins such as per-
oxidases and cytochrome P450 enzymes (1). Recent
investigations suggested that MP8 reacts with hydrogen
peroxide to form intermediate compounds analogous to
compounds I and II of horseradish peroxidase (2-4).
The elucidation of the degradation pathways of aro-
matic polyhalogenated compounds is of particular im-
portance because of their persistence in the environment
with potential deleterious effects to both plants and
animals. Although peroxidase-catalyzed oxidation of
chlorinated phenols to the corresponding benzoquinones
was previously shown (5-8), neither the secondary
degradation of the latter products nor the oxidation of
pentafluorophenol has, to our knowledge, been reported.
Previous work (8) showed that the MP8/H2O2 system is
capable of catalyzing the dehalogenation of mono-p-
The present study was carried out to investigate
whether MP8 in the presence of H2O2 could also catalyze
the dehalogenation of polyhalogenated phenols and give
rise to reactive tetrahalo-p-benzoquinones. Direct proof
of the formation of these reactive tetrahalo-p-benzoquino-
nes would be of importance since their formation in
oxidative (P450-catalyzed) dehalogenation reactions has
always been derived from indirect evidence, namely, the
detection of the reduced tetrahalohydroquinone ana-
logues upon the addition of ascorbic acid or NADPH into
the reaction mixtures (9, 10). The results presented in
this paper show that MP8 could indeed convert penta-
halophenols to tetrahalo-p-benzoquinones as the primary
metabolites. The absence of a large protein moiety in
the case of MP8 as compared to cytochromes P450
apparently eliminates the swift protein binding, provid-
ing the possibility for direct detection of the reactive
tetrahalo-p-benzoquinones.
* Address correspondence to this author at the Department of
Biomolecular Sciences, Laboratory of Biochemistry, Agricultural Uni-
versity, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
Phone: 31-317-482868. Fax: 31-317-484801.
† Laboratory of Biochemistry, Agricultural University.
‡ Laboratory of Organic Chemistry, Agricultural University.
§ TNO Nutrition and Food Research Institute.
Ma ter ia ls a n d Meth od s
1
Abbreviations: MP8, microperoxidase-8; HPLC, high-performance
liquid chromatography; GC-MS, gas chromatography/mass spectro-
metry.
Ch em ica ls. MP8 was prepared by limited proteolytic diges-
tion of horse heart cytochrome c as described in the literature
10.1021/tx980037l CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/13/1998

1320 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Osman et al.
(11), and its concentration was determined either by the
chromogen method (11) or by measuring the absorbance at 397
nm (ꢀ397 nm ) 1.5 × 105 M-1 cm-1) in 0.1 M potassium phosphate
(pH 7.0) (3). The purity of this preparation of MP8 was judged
from HPLC analysis to be over 95%. Pentachlorophenol,
tetrafluorophenol, tetrafluorohydroquinone, and protoporphyrin
IX were obtained from Aldrich (Steinheim, Germany). Tetra-
chlorophenol and tetrachloro-p-benzoquinone (chloranil) were
purchased from J anssen (Beerse, Belgium). Pentafluorophenol
was from Sigma (St. Louis, MO), and tetrafluoro-p-benzoquinone
was from Fluorochem (Derbyshire, U.K.). L-Ascorbic acid and
hydrogen peroxide (v/v, 30%) were from Merck (Darmstadt,
Germany). Hydrogen peroxide was diluted before use to a stock
solution of 50 mM in demineralized water. The concentrations
of the stock solutions of H2O2 were determined spectrophoto-
dard incubation condition) to obtain more product formation.
After 1 min of incubation, the samples were put into liquid
nitrogen. The product, trichlorohydroxy-p-benzoquinone, formed
in these incubations was isolated by HPLC and was identified
by MS.
Isola tion of Tr ich lor oeth oxy-p-ben zoqu in on e. To isolate
trichloroethoxy-p-benzoquinone, 6.2 mg of tetrachloro-p-benzo-
quinone was dissolved in 5 mL of absolute ethanol. Of this
solution, 100 µL was taken and added to a mixture of 350 µL of
0.1 M Tris-HCl (pH 7.0) and 250 µL of absolute ethanol. The
resulting solution was well mixed and incubated at 37 °C for 2
min. This sample was frozen in liquid nitrogen until analyzed
by HPLC.
High -P er for m a n ce Liqu id Ch r om a togr a p h y (HP LC). A
volume of 10-50 µL of each of the reaction mixtures (see
incubation conditions) was loaded onto a Lichrosphere RP8
column (150 × 4.6 mm). Elution was performed at 1 mL/min,
starting with 100% solvent A (1% acetic acid in water) and then
maintaining 100% of this solvent for 1 min, followed by a linear
gradient to obtain 100% methanol in 25 min. For the isolation
of trichloroethoxy-p-benzoquinone from tetrachloro-p-benzo-
quinone, the gradient was increased linearly to obtain 94%
methanol in 19 min and was maintained at 94% methanol for
1 min, followed by a linear increase of the gradient to 100%
methanol in 25 min. UV detection and measurement of UV
absorption spectra of the eluted compounds were performed with
a Waters 996 diode array detector.
An a lysis by Ga s Ch r om a togr a p h y-Ma ss Sp ectr om etr y
(GS-MS). For fluorinated derivatives, the gas chromatograph
(Hewlett-Packard 5890) was equipped with a 30 m × 0.25 mm
ID capillary DB 17 column (J and W Scientific, Folsom, CA).
The carrier gas was helium at a flow rate of about 1 mL/min. A
temperature gradient from 70 to 200 °C for 13 min was applied.
The column was connected to a Hewlett-Packard 5970B mass
spectrometer.
metrically (ꢀ240
) 39.4 M-1 cm-1) (12); 91% enriched labeled
nm
H218O2 (v/v, 2%) was purchased from Icon (Sunnit, NJ ).
Trichlorohydroxy-p-benzoquinone was prepared as described
in the literature (13) by dissolving solid chloranil (30 mg) in an
ice-cold 2 M sodium hydroxide solution. This solution was
acidified, keeping it in ice, with concentrated hydrochloric acid.
The purity of this synthesized compound was judged from HPLC
analysis which gave only one peak, and the compound was
identified by mass spectrometry (MS) (data not shown). The
recovery was quantitative. Trifluorohydroxy-p-benzoquinone
was synthesized from tetrafluoro-p-benzoquinone following the
same procedure described above for the synthesis of trichloro-
hydroxy-p-benzoquinone. The purity and the identity of this
synthesized trifluorohydroxy-p-benzoquinone were judged from
both HPLC and GC-MS analysis (see Results).
In cu ba tion Con d ition s. A typical reaction mixture con-
sisted of the halophenol (0.25-2 mM final concentration) and
MP8 [7.5 µΜ final concentration in 0.1 M Tris-HCl (pH 7.0)
containing 50% ethanol, or in some cases, when indicated, in
0.1 M potassium phosphate (pH 7.6)]. Sometimes, as indicated,
ascorbic acid (0.5-3 mM final concentration) was added to the
reaction mixture. Reaction mixtures were preincubated at 37
°C for 2 min. The reaction was started by the addition of
hydrogen peroxide (2.5 mM final concentration). After 1 min
of incubation, the reaction was stopped by freezing the samples
in liquid nitrogen. Furthermore, since iron in the presence of
H2O2 could lead to Fenton-type chemistry, the following incuba-
tions were carried out as control experiments: 2 mM pentachlo-
rophenol was incubated either with 10 µM protoporphyrin IX
or with 100 µM ferrous salts (FeCl2‚4H2O or FeSO4‚7H2O) in a
reaction mixture of 1 mL in 0.1 M Tris-HCl (pH 7.0) containing
50% ethanol. Following the addition of 2.5 mM hydrogen
peroxide (final concentration) to the reaction mixture, the
reaction was incubated for 1 min at 37 °C. The reaction was
terminated by freezing the samples in liquid nitrogen.
For chlorinated derivatives, GC was not successful, and the
solutions in dichloromethane were introduced via a direct
insertion probe into the ion source of a Finnigan MAT 95 mass
spectrometer operated in the 70 eV, EI (electron impact)
ionization mode at resolution RP ) 1000 for the mass spectrum
determination and RP ) 7000 for the accurate mass measure-
ments, respectively.
Molecu la r Or bita l Ca lcu la tion s. Molecular orbital calcu-
lations were carried out on a Silicon Graphics Indigo2 using
Spartan version 5.0 (Wave Function Inc., Irvine, CA). The
semiempirical molecular orbital method was used, applying the
PM3 Hamiltonian. Geometries were optimized for all bond
lengths, bond angles, and torsion angles.
Resu lts
Effect of H2O2 on th e Non ca ta lyzed Con ver sion of
Tet r a ch lor o-p-ben zoq u in on e a n d Tet r a flu or o-p-ben zo-
qu in on e to th e Cor r esp on d in g Tr ih a loh yd r oxy-p-ben zo-
qu in on es. To examine the effect of H2O2 on the noncatalyzed
conversion of the tetrahalo-p-benzoquinones to the correspond-
ing trihalohydroxy-p-benzoquinones, various concentrations of
hydrogen peroxide (0-2.5 mM) were incubated with 1 mM either
tetrachloro-p-benzoquinone or tetrafluoro-p-benzoquinone in 0.1
M Tris-HCl pH 7.0, containing 50% ethanol for 1 min at 37 °C.
After 1 min of incubation, the samples were put in liquid
nitrogen until analysis by HPLC.
In cu ba tion of P en ta ch lor op h en ol w ith MP 8/H2O2.
Upon analyzing various conditions, it was found that the
reaction products were relatively stable in 0.1 M Tris-
HCl (pH 7.0) containing 50% ethanol which was conse-
quently employed.
Figure 1A presents the results from HPLC analysis of
the incubation of pentachlorophenol with MP8 in the
presence of hydrogen peroxide. After 1 min of incubation,
about 14% of the substrate was converted to products.
No further degradation of the substrate was observed
when the time of incubation was increased to 5 min, due
to the inactivation of MP8 (14). No product formation
was observed in control incubations in which either MP8
or H2O2 was omitted from the reaction mixtures. In the
presence of MP8 and H2O2, three major products were
found (Figure 1A). The peak with the retention time of
13.3 min, which is the dominant product, was identified
as trichlorohydroxy-p-benzoquinone. This product was
identified by comparing its retention time and the UV
spectrum with those of an authentic trichlorohydroxy-p-
Isola t ion of Tr ich lor oh yd r oxy-p -b en zoq u in on e Ob -
ta in ed by In cu ba tin g La beled H218O2 a n d Un la beled H2O2
with Tetr ach lor o-p-ben zoqu in on e. To understand the source
of the oxygen atom incorporated into the trichlorohydroxy-p-
benzoquinone, formed from the reaction of H2O2 with the
tetrachloro-p-benzoquinone, 2 mM tetrachloro-p-benzoquinone
was incubated with 5 mM labeled H218O2 in a final volume of 1
mL of Tris-HCl (pH 7.0) containing 50% ethanol. A control
experiment with normal H2O2 was also performed. In these
incubations, the concentrations of both labeled and unlabeled
hydrogen peroxide were increased 2-fold (relative to the stan-

MP8-Catalyzed Oxidation of Pentahalophenols
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1321
F igu r e 1. (A) HPLC elution profile of the incubation mixture of 7.5 µM MP8 and 2.5 mM H2O2 with 2 mM pentachlorophenol in 0.1
M Tris-HCl (pH 7.0) containing 50% ethanol. (B) HPLC profile of a sample of tetrachloro-p-benzoquinone dissolved in 0.1 M Tris-
HCl (pH 7.0) containing 50% ethanol and incubated for 2 min at 37 °C.
F igu r e 2. Mass spectrum (MS) of the product with a retention time of 20.1 min (Figure 1A), identified as trichloroethoxyquinone.
benzoquinone standard. This product gives the reaction
mixture a characteristic purple color at neutral pH, that
could be attributed to the anionic form of trichlorohy-
droxy-p-benzoquinone (pKa ) 1.09) (15). The protonated
form of this product, obtained by dissolving it in 1 N HCl,
as well as its reduced form obtained by incubating with
ascorbic acid, resulted in the disappearance of the purple
color. The peak with the retention time of 19 min was
identified as tetrachloro-p-benzoquinone by comparing its
retention time and UV spectrum with tetrachloro-p-
benzoquinone. The peak at retention time 20.1 min is
derived spontaneously from tetrachloro-p-benzoquinone.
Dissolving tetrachloro-p-benzoquinone in the same buffer
used for the enzymatic reaction and incubating for 2 min
at 37 °C, omitting MP8 and H2O2, resulted in the
formation of the metabolite with retention time 20.1 min
(Figure 1B). This product was isolated by HPLC and
analyzed by mass spectrometry (Figure 2). The mass
spectrum shows a trichloro compound of molecular
weight 254, losing masses 28 and 18 consecutively to form
fragments at 226 and 198, respectively. Accurate mass
measurement at a resolution of 7000 for the molecular
ion peak m/z 254 was 253.9308 amu, in agreement with
the elemental composition C8H5Cl3O3 (calculated mass
) 253.9304). Therefore, the product can be identified as
trichloroethoxy-p-benzoquinone. The slight deviation of
the isotopic pattern of the peak clusters at m/z 226 and
254 from the theoretical pattern is due to the presence
of a small amount of the reduced form of this compound
(trichloroethoxyhydroquinone MW ) 256). During freeze-
drying, the reduction of this adduct by methanol oc-
curred. We showed previously (16) that alcohols could
act as reductants for activated benzoquinone-type prod-
ucts. No detectable amount of this adduct (trichloro-
ethoxyquinone) was observed when a solution of tetra-
chloro-p-benzoquinone was dissolved in pure ethanol and
incubated for 2 min at 37 °C. Moreover, the rate of
formation of this adduct increased 9-fold when tetra-
chloro-p-benzoquinone was incubated in 0.1 M Tris-HCl
(pH 8.0) containing 50% ethanol. This indicates that the
aqueous buffered solution favored the interaction of
ethanol with chloranil. Together, the results presented
in Figures 1 and 2 demonstrate that tetrachloro-p-
benzoquinone is the primary product resulting from the
MP8-mediated dehalogenation of pentachlorophenol.
In cu b a t ion s of P en t a flu or op h en ol w it h MP 8/
H2O2. Figure 3A shows the HPLC chromatogram of the
reaction mixture obtained from the incubation of pen-
tafluorophenol with MP8 and hydrogen peroxide. The
HPLC profile is analogous to that described above for
pentachlorophenol. Pentafluorophenol was more readily
converted than the chlorinated analogue. After 1 min
of incubation, about 75% of the pentafluorophenol was
converted as compared to 14% for pentachlorophenol.
However, although readily observable, the product, tet-
rafluoro-p-benzoquinone (the minor peak with retention
time 12 min in Figure 3A), was less stable as compared
to tetrachloro-p-benzoquinone. One of the major products
was identified as trifluorohydroxy-p-benzoquinone by
comparing its retention time (8 min) and UV spectrum
to an authentic reference compound synthesized from
tetrafluoro-p-benzoquinone. As shown in Figure 3B, a
minor product with a retention time of 2.3 min was also
present in the preparation. This minor product increased

1322 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Osman et al.
F igu r e 3. (A) HPLC elution profile of the incubation mixture of 7.5 µM MP8 and 2.5 mM H2O2 with 2 mM pentafluorophenol in 0.1
M Tris-HCl (pH 7.0) containing 50% ethanol. The pKa of trifluorohydroxy-p-benzoquinone is 1.73, and the two pKa’s for 2,5-difluoro-
3,6-dihydroxy-p-benzoquinone are pKa1 ) 1.4 and pKa2 ) 3.3 (15). The peak with retention time 10.4 min is a degradation product
of trifluoroethoxy-p-benzoquinone. (B) HPLC chromatogram of the preparation of trifluorohydroxy-p-benzoquinone synthesized from
tetrafluoro-p-benzoquinone.
(Figure 4B) shows a molecular ion peak at m/z 176, which
is 2 amu lower than the molecular weight of trifluoro-
hydroxy-p-benzoquinone, m/z 178 (Figure 4C). The mass
spectrum of the degradation product (Figure 4B) exhibits
spectral fragmentation similar to that of the parent
compound, trifluorohydroxy-p-quinone (Figure 4C). How-
ever, the fragment m/z 62 (C2F2) is absent in the mass
spectrum of the degradation product. This is consistent
with the replacement of one of the neighboring fluorine
atoms by a hydroxyl group. The product was, therefore,
identified as 2,5-difluoro-3,6-dihydroxy-p-benzoquinone.
Two other products (retention times of 16.2 and 10.4 min)
were also found in the HPLC chromatogram of the
incubation reaction mixture (Figure 3A). The peak with
the retention time of 16.2 min appears to be the ethoxy
adduct of tetrafluoro-p-benzoquinone, since from fluor-
anil, dissolved in 0.1 M Tris-HCl (pH 7.0) containing 50%
ethanol, this peak was formed, as in the case of the
chlorinated compound (data not shown). Thus, it is
inferred that this product is the corresponding trifluo-
roethoxy-p-benzoquinone. Attempts to isolate this prod-
uct failed. The product with the retention time of 10.4
min, shown in Figure 3A, is a degradation product from
trifluoroethoxy-p-benzoquinone. Thus, it is concluded
that tetrafluoro-p-benzoquinone is the primary product
formed from the MP8/H2O2-mediated dehalogenation of
pentafluorophenol.
Effect of H2O2 on th e Non ca ta lyzed Con ver sion
of Tetr a h a lo-p-ben zoqu in on es to th e Cor r esp on d -
in g Tr ih a loh yd r oxy-p-ben zoqu in on es. As indicated
in Figure 1B, tetrachloro-p-benzoquinone does not give
rise to spontaneous formation of trichlorohydroxy-p-
benzoquinone in the absence of MP8 and H2O2. Thus,
F igu r e 4. (A) Gas chromatographic analysis of the preparation
of trifluorohydroxy-p-benzoquinone, freeze-dried overnight and
the mechanism in which a hydroxide ion replaces the
chlorine under alkaline conditions (13) does not occur
significantly under our experimental conditions. When
tetrachloro-p-benzoquinone was incubated with H2O2 in
the presence and in the absence of MP8, in both cases
the hydroxy compound was formed, showing that its
formation depends only on H2O2. The data in Figure 5A
show that the formation of trichlorohydroxy-p-benzo-
quinone from tetrachloro-p-benzoquinone increases with
increasing concentration of H2O2 and is accompanied by
a parallel decrease of the formation of the trichloroethoxy
adduct. At low concentrations of hydrogen peroxide, the
dissolved in ethyl acetate. (B) Mass spectrum of the peak with
retention time 12.1 min, identified as 2,5-difluoro-3,6-dihydroxy-
p-benzoquinone. (C) Mass spectrum of the peak with retention
time 9.2 min, identified as trifluorohyroxy-p-benzoquinone.
with time, accompanied by a parallel decrease of the
concentration of trifluorohydroxy-p-benzoquinone. This
indicates that this minor product is spontaneously gener-
ated from trifluorohydroxy-p-benzoquinone. Figure 4A
shows the results obtained from the GC analysis of this
mixture. The peak with the retention time of 12.1 min
is the degradation product of trifluorohydroxy-p-benzo-
quinone. The mass spectrum of this degradation product

MP8-Catalyzed Oxidation of Pentahalophenols
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1323
F igu r e 5. Effect of H2O2 on the spontaneous conversion of (A)
tetrachloro-p-benzoquinone to trichlorohydroxy-p-benzoquinone
and (B) tetrafluoro-p-benzoquinone to trifluorohydroxy-p-ben-
zoquinone. Concentrations of hydrogen peroxide (0-2.5 mM)
were incubated with 1 mM either tetrachloro-p-benzoquinone
or tetrafluoro-p-benzoquinone in 0.1 M Tris-HCl (pH 7.0)
containing 50% ethanol for 1 min at 37 °C. (O) Tetrahalo-p-
benzoquinone; (4) trihaloethoxyquinone; (b) trihalohydroxy-p-
benzoquinone. The maximum peak area of each product at the
specified wavelength was taken as 100%.
formation of the trichloroethoxy adduct prevails over the
formation of trichlorohydroxy-p-benzoquinone. In con-
trast, at concentrations of H2O2 above 1 mM, the forma-
tion of trichlorohydroxy-p-benzoquinone dominates over
trichloroethoxy formation. Similar results were obtained
in the case of the conversion of tetrafluoro-p-benzo-
quinone (Figure 5B). Hydrogen peroxide was shown also
to stimulate the formation of trifluorohydroxy-p-benzo-
quinone from tetrafluoro-p-benzoquinone at the expense
of the formation of trifluoroethoxy-p-quinone.
Or igin of th e Oxygen in Tr ich lor oh yd r oxy-p-
ben zoqu in on e F or m ed fr om Tetr a ch lor o-p-ben zo-
qu in on e w ith H2O2. Tetrachloro-p-benzoquinone was
incubated with labeled H218O2 or with unlabeled H2O2,
and, as expected, the major product formed was trichlo-
rohydroxy-p-benzoquinone. The mass spectra of the
molecular ion region of trichlorohydroxy-p-benzoquinone,
obtained with unlabeled and labeled H218O2 (Figure
6A,B), indicate the shift of the molecular ion isotope
cluster peaks of the unlabeled compound (m/z 226, 228,
230, and 232) with 2 mass units, as can be expected for
the incorporation of 18O. The small peak at m/z 226 in
the labeled experiment originates from the unlabeled
trichlorohydroxy-p-benzoquinone formed as a result of
using not fully (91%) labeled H218O2.
F igu r e 6. Mass spectra of the molecular ion region of trichlo-
rohydroxy-p-benzoquinone obtained from the incubations of
tetrachlorohydroxy-p-benzoquinone with (A) unlabeled H2O2 or
with (B) labeled H218O2.
liberated from the heme cofactor upon inactivation, could
catalyze the oxidative dehalogenation of the pentahalo-
genated phenols. This implies that Fenton-type chem-
istry is not involved in the MP8-catalyzed oxidation of
the pentahalogenated phenols. This conclusion is in
agreement with a previous report where the superoxide
anion and hydroxyl radical scavengers were shown to
have no effect on the MP8/H2O2-mediated hydroxylation
of aniline (17).
Discu ssion
The results presented in this paper show that MP8, in
the presence of hydrogen peroxide, can convert penta-
halophenols to tetrahalo-p-benzoquinones as the primary
products. The incubation of the pentahalophenols with
MP8/H2O2 resulted in the formation of three principal
products identified as trihalohydroxy-p-benzoquinone,
tetrahalo-p-benzoquinone, and trihaloethoxy-p-benzo-
quinone. The active species of MP8 formed upon reaction
with H2O2 could be analogous to compounds I and II of
horseradish peroxidase (2-4). Previous studies (8) showed
that ascorbic acid inhibited the MP8/H2O2-catalyzed
dehalogenation of mono-p-halogenated phenols whereas
ascorbic acid had no effect on the microsomal P450-
mediated dehalogenation of the same substrates. Based
on this observation as well as on studies of 18O-labeled
H218O and H218O2, which showed that the oxygen atom
inserted into the product, p-benzoquinone, originates
from water, a peroxidase type of reaction rather than a
P450 type of mechanism was suggested for this MP8/
H2O2-mediated oxidative dehalogenation (8). It is im-
portant to stress that the results reported in the present
study argue against the involvement of Fenton-type
chemistry in this MP8/H2O2-mediated dehalogenation
reaction.
The result of these experiments indicates that H2O2 is
the source of the oxygen atom inserted into the trichlo-
rohydroxy-p-benzoquinone.
Effect of Ascor bic Acid a n d F en ton -Typ e Rea c-
tion Ca ta lysts on th e Degr a d a tion of th e P en ta h a -
logen a ted P h en ols. The formation of all products
reported in Figures 1A and 3A was completely inhibited
by the addition of 3 mM ascorbic acid to the reaction
mixture (data not shown). This result is in agreement
with previous findings (8), where also MP8-mediated
oxidative dehalogenation of mono-p-halogenated phenols
was completely suppressed by ascorbic acid. Finally, the
possibility that Fenton-type chemistry might be involved
in the MP8/H2O2-mediated oxidation of the pentahalo-
genated phenols was investigated. The incubation of 2
mM pentachlorophenol with 10 µM protoporphyrin IX or
with 100 µM ferrous salts both in the presence and in
the absence of H2O2 under experimental conditions
similar to those used for the MP8-catalyzed reaction did
not result in product formation. This result excludes the
possibility that traces of iron cations, which might be
A scheme outlining the mechanism of the MP8/H2O2-
driven oxidative dehalogenation reaction is presented in

1324 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Osman et al.
F igu r e 7. (A) Mechanism of the MP8-catalyzed dehalogenation of pentafluorophenol and pentachlorophenol to the corresponding
tetrahalo-p-benzoquinones (8). The active species of MP8 formed upon addition of H2O2 could catalyze two consecutive one-electron
oxidations of the pentahalogenated phenols, leading to the formation of carbocation derivatives which are attacked by OH- to give
the tetrahalo-p-benzoquinones (the active species of MP8 formed upon reaction with hydrogen peroxide are supposed to be high-
valent iron-oxo intermediates that could be analogous to compounds I and II of horseradish peroxidase; see refs 2-4). (B) Mechanism
of the spontaneous conversions of tetrachloro-p-benzoquinone and tetrafluoro-p-benzoquinone to trichlorohydroxy-p-benzoquinone
and trifluorohydroxy-p-benzoquinone, respectively. The tetrahalo-p-benzoquinones could undergo the following nucleophilic reac-
tions: nucleophilic attack by the solvent molecules, i.e., EtO- (pathway a) or OH- (pathway b). The reaction with water is insignificant
under our experimental conditions. Nucleophilic attack by H2O2 molecules (pathway c). All these reactions lead to the displacement
of the halogen (X ) F- or Cl-) as an anion and the formation of the corresponding trihalohydroxy-p-benzoquinones and ethoxy
adducts as indicated.
Figure 7A. The active high-valent iron-oxo intermedi-
ates of MP8 could catalyze two consecutive one-electron
oxidations of the pentahalogenated phenols. This would
lead to the formation of carbocation intermediates, which
are then subjected to nucleophilic attack by a hydroxide
ion, giving rise to the formation of the tetrahalo-p-
benzoquinones and the elimination of the halogen at the
para position as an anion. The solvent consists of water
and ethanol; however, the reaction of an ethoxide anion
does not lead to a product, since an adduct is formed
which has no acidic proton as in the case of the hydroxy-
lated adduct and, as a result, cannot give rise to elimina-
tion of the halogen anion and the formation of the
corresponding quinone (Figure 7A). This mechanism is
consistent with the higher rate of conversion observed
in the case of pentafluorophenol when compared with the
rate of conversion of pentachlorophenol, since pentafluo-
rophenol is oxidized more easily (18). This conclusion is
also consistent with the data obtained from molecular
orbital calculations, where the energy of the highest
occupied molecular orbital, E(HOMO), of pentafluorophe-
nol, computed to be -3.87 eV, is higher than the
E(HOMO) of pentachlorophenol (-4.02 eV). This pro-
posed mechanism is also in agreement with the previ-
ously suggested sequential one-electron oxidations of
pentachlorophenol and 2,4,6-trichlorophenol by horserad-
ish peroxidase (6, 7) and by lignin peroxidase (5). It was
even demonstrated that HRP-catalyzed oxidation of
pentachlorophenol results in the formation of ESR-
detectable pentachlorophenoxyl intermediates (6). Al-
though steric restrictions and electrostatic repulsions
beween the halogen substituents do not favor interactions
between the phenoxyl radicals, we cannot yet exclude
disproportionation between the radical intermediates as
an alternative pathway to regenerate the parent phenol
and form the cation (Figure 7A).
Once formed, the tetrahalo-p-benzoquinones react with
solvent molecules and with hydrogen peroxide present
in the incubation mixture, giving rise to the correspond-
ing trihaloethoxy-p-benzoquinones and the corresponding
trihalohydroxy-p-benzoquinones (Figure 7B). The fact
that adducts are formed from tetrachloro-p-benzoquinone
with ethanol in aqueous buffered solutions could explain
the observations reported in the literature on the insta-
bility of tetrachloro-p-benzoquinone in solutions of 50%
aqueous ethanol buffered at different pH values (19, 20).
Because the nucleophilic reactivity of the ethoxide ion is
greater than that of the hydroxide ion (21, 22), the
ethoxide and not the hydroxide adduct is formed. The
lack of detection of trichlorohydroxy-p-benzoquinone
reported for the peroxidase-mediated oxidation of pen-
tachlorophenol in previous studies (5, 6) could be at-

MP8-Catalyzed Oxidation of Pentahalophenols
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1325
(6) Samokyszyn, V. M., Freeman, J . P., Maddipati, K. R., and Lloyd,
R. V. (1995) Peroxidase-catalyzed oxidation of pentachlorophenol.
Chem. Res. Toxicol. 8, 349-355.
tributed to the relatively lower concentrations of the
pentahalogenated phenol and H2O2 used in these studies.
While ethoxide ions derived from ethanol molecules
react with the tetrahalo-p-benzoquinones, leading to the
formation of the trihaloethoxy-p-benzoquinones (pathway
a, Figure 7B), the results presented in this study clearly
show that the formation of the trihalohydroxy-p-benzo-
quinones did not result from the expected reaction of
hydroxide ions (pathway b, Figure 7B) as was suggested
to be the case under alkaline conditions (13). Instead,
hydrogen peroxide acts as the nucleophile in the conver-
sion of tetrahalo-p-benzoquinones to the corresponding
trihalohydroxy-p-benzoquinones and provides the oxygen
atom incorporated into the trichlorohydroxy-p-benzo-
quinone (pathway c, Figure 7B). The neutral H2O2
molecule is known to be a good nucleophile that has a
higher reactivity than water (23).
The results presented here imply that plants and
animals could degrade polyhalophenols to reactive ha-
logenated benzoquinones through a peroxidase pathway.
The likelihood that mammalian peroxidases might con-
vert the polyhalophenols to reactive intermediates has
important implications with respect to the cytotoxic
effects of these products in vivo, since it provides an
additional bioactivation pathway comparable to the cy-
tochrome P-450 catalyzed bioactivation of polyhalophe-
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Ack n ow led gm en t. This study was supported by
TMR large-scale NMR facility Grant ERB-CHGE-
CT940061, by a grant for the Research School Environ-
mental Chemistry and Toxicology (M and T) and TNO
Nutrition and Food Research Institution, Zeist, The
Netherlands, and by EU Copernicus Grant ERBIC 15
CT961004. We thank Mr. Martin Bouwmans for having
drawn some of the figures.
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media. Proc. Natl. Acad. Sci., U.S.A. 94, 4295-4299.
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