Single turnover studies of oxidative halophenol dehalogenation by horseradish peroxidase reveal a mechanism involving two consecutive one electron steps: Toward a functional halophenol bioremediation catalyst

Article abstract of DOI:10.1016/j.jinorgbio.2012.09.017

Horseradish peroxidase (HRP) catalyzes the oxidative para-dechlorination of the environmental pollutant/carcinogen 2,4,6-trichlorophenol (2,4,6-TCP). A possible mechanism for this reaction is a direct oxygen atom transfer from HRP compound I (HRP I) to trichlorophenol to generate 2,6-dichloro 1,4-benzoquinone, a two-electron transfer process. An alternative mechanism involves two consecutive one-electron transfer steps in which HRP I is reduced to compound II (HRP II) and then to the ferric enzyme as first proposed by Wiese et al. [F.W. Wiese, H.C. Chang, R.V. Lloyd, J.P. Freeman, V.M. Samokyszyn, Arch. Environ. Contam. Toxicol. 34 (1998) 217-222]. To probe the mechanism of oxidative halophenol dehalogenation, the reactions between 2,4,6-TCP and HRP compounds I or II have been investigated under single turnover conditions (i.e., without excess H₂O₂) using rapid scan stopped-flow spectroscopy. Addition of 2,4,6-TCP to HRP I leads rapidly to HRP II and then more slowly to the ferric resting state, consistent with a mechanism involving two consecutive one-electron oxidations of the substrate via a phenoxy radical intermediate. HRP II can also directly dechlorinate 2,4,6-TCP as judged by rapid scan stopped-flow and mass spectrometry. This observation is particularly significant since HRP II can only carry out one-electron oxidations. A more detailed understanding of the mechanism of oxidative halophenol dehalogenation will facilitate the use of HRP as a halophenol bioremediation catalyst.

Full text of DOI:10.1016/j.jinorgbio.2012.09.017

Journal of Inorganic Biochemistry 117 (2012) 316–321
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Journal of Inorganic Biochemistry
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Single turnover studies of oxidative halophenol dehalogenation by horseradish
peroxidase reveal a mechanism involving two consecutive one electron steps:
☆
Toward a functional halophenol bioremediation catalyst
Suganya Sumithran , Masanori Sono , Gregory M. Raner , John H. Dawson a,c,⁎
a
a
b
a
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29073, USA
Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27402, USA
School of Medicine, University of South Carolina, Columbia, SC 29073, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 March 2012
Received in revised form 18 September 2012
Accepted 18 September 2012
Available online 25 September 2012
Horseradish peroxidase (HRP) catalyzes the oxidative para-dechlorination of the environmental pollutant/
carcinogen 2,4,6-trichlorophenol (2,4,6-TCP). A possible mechanism for this reaction is a direct oxygen
atom transfer from HRP compound I (HRP I) to trichlorophenol to generate 2,6-dichloro 1,4-benzoquinone,
a two-electron transfer process. An alternative mechanism involves two consecutive one-electron transfer
steps in which HRP I is reduced to compound II (HRP II) and then to the ferric enzyme as first proposed by
Wiese et al. [F.W. Wiese, H.C. Chang, R.V. Lloyd, J.P. Freeman, V.M. Samokyszyn, Arch. Environ. Contam.
Toxicol. 34 (1998) 217–222]. To probe the mechanism of oxidative halophenol dehalogenation, the reactions
between 2,4,6-TCP and HRP compounds I or II have been investigated under single turnover conditions
Keywords:
Horseradish peroxidase
Rapid kinetics
Chlorophenol dehalogenation
Phenoxy radical
Bioremediation
2 2
(i.e., without excess H O ) using rapid scan stopped-flow spectroscopy. Addition of 2,4,6-TCP to HRP I
leads rapidly to HRP II and then more slowly to the ferric resting state, consistent with a mechanism involving
two consecutive one-electron oxidations of the substrate via a phenoxy radical intermediate. HRP II can also
directly dechlorinate 2,4,6-TCP as judged by rapid scan stopped-flow and mass spectrometry. This observa-
tion is particularly significant since HRP II can only carry out one-electron oxidations. A more detailed
understanding of the mechanism of oxidative halophenol dehalogenation will facilitate the use of HRP as a
halophenol bioremediation catalyst.
Ferryl heme intermediates
©
2012 Elsevier Inc. All rights reserved.
1
. Introduction
fumago chloroperoxidase [33,34]. Among these peroxidases, HRP is an
especially promising candidate for use as a bioremediation catalyst be-
cause of its (a) stability resulting in easier storage and handling, (b) abil-
ity to oxidize a large number of chlorophenols, (c) flexibility to function
at wide ranges of temperature and pH, (d) and, most importantly, ready
availability and relatively low cost [13,14,35–41]. The oxidative
dehalogenation of chlorophenols is catalyzed by HRP in the presence
of H O . A better understanding of the mechanism of this process
2 2
would help in the design of a more efficient HRP-based bioremediation
method.
Chlorinated phenols are a major class of environmental pollutants
and are potential human carcinogens [1–4]. They are extensively used
as disinfectants, pesticides, herbicides and dyes; as a result they can
contaminate the soil and ground water, and eventually enter the
food chain. Removal of chlorophenols from industrial waste waters
is achieved by a variety of treatment processes, including (a) sequential
anaerobic and aerobic biodegradation [5–7], (b) adsorption by activated
carbon [8,9], (c) photo-degradation [10,11], (d) and, most recently,
enzymatic degradation [12–15].
Several peroxidase enzymes are being explored for the removal of
chlorophenols from industrial waste waters [15]. Detailed studies
have been performed regarding the oxidation of chlorophenols by
lactoperoxidase [16], lignin peroxidase (LiP) [17], horseradish perox-
idase (HRP) [18–20], dehaloperoxidase [21–32] and Caldariomyces
Heme peroxidases couple the oxidation of organic substrates to the
reduction of hydrogen peroxide to water by a mechanism involving
two ferryl heme intermediates. Peroxidase compound I (Cpd I), a ferryl
IV
(Fe =O) porphyrin π-cation radical [42,43] is formed first after addi-
2 2
tion of H O to the ferric enzyme. Reduction of Cpd I by a one-electron
oxidizable substrate yields the second ferryl porphyrin intermediate
known as compound II (Cpd II). Cpd II is then reduced by one more
electron to regenerate the ferric resting state [42,43]. Among the
chlorophenols that can be degraded by HRP, 2,4,6-trichlorophenol
☆
The review process of this manuscript was handled by the Guest Editor for this special
issue.
(
2,4,6-TCP) has received considerable attention due to its higher
⁎
Corresponding author at: Department of Chemistry and Biochemistry, University of
toxicity and its environmental release by diverse industries [4,33,34].
HRP catalyzes the oxidative dechlorination of 2,4,6-TCP generating
South Carolina, Columbia, SC 29073, USA. Tel.: +1 8037777234; fax: +1 8037779521.
E-mail address: jdawson@mailbox.sc.edu (J.H. Dawson).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.09.017

S. Sumithran et al. / Journal of Inorganic Biochemistry 117 (2012) 316–321
317
2
,4-dichloro-1,4-benzoquinone (Eq. (1)) [20]. A possible mechanism of
Stock solutions of (100 mM) 2,6-DCP, 2,3,6-TCP and 2,4,6-TCP were
made in a 50/50 ethanol/deionized-H O mixture by first dissolving the
halophenol in ethanol. The concentration of 2,4,6-TCP was confirmed
this reaction could involve a direct oxygen transfer from HRP Cpd I
to 2,4,6-TCP to generate 2,4-dichloro 1,4-benzoquinone in a single
two-electron oxo transfer process [44,45]. An alternative mechanism
involves two consecutive one-electron transfer steps with sequential
formation of both HRP compounds I (HRP I) and II (HRP II), as initially
proposed by Samokyszyn and co-workers [20]. The first substrate
intermediate in this oxidative dechlorination mechanism is a phenoxy
radical, which has been shown to react directly with DNA to form
covalent DNA adducts leading to the initiation of tumorigenesis [46].
The generation of these radicals in the body is explained by the
peroxidase- or myoglobin Cpd II-mediated oxidation of chlorophenols
which entered the body through food or water intake [46,47].
2
−
1
−1
spectrophotometrically using ε312=4.44 mM
cm
at pH 7.0 (20).
Potassium phosphate (100 mM) at pH 5.0 was used for all experiments.
UV-visible absorption spectra were recorded at 4 °C using a Cary
400 spectrophotometer interfaced to a Dell PC for data acquisition.
Rapid scan stopped-flow experiments were carried out at 4 °C on a
Hi-Tech Ltd. SF-61 DX2 instrument equipped with a diode array
detector. The dead time was determined to be 1.5 ms. In single and
double mixing experiments, numerous scans were recorded for differ-
ent run times to optimize the conditions. The kinetic data were fit to
various reaction models by the KinetAsyst program (Hi-Tech Ltd.) or
by the Specfit program from Spectrum Software Associates.
Either the single mixing or double mixing mode was used to
optimize the formation and determine the stability of HRP ferryl
intermediates, HRP I and HRP II. HRP (15 μM) in 100 mM potassium
OH
O
Cl
Cl
Cl
Cl
HRP
H O
2 2
phosphate solution, pH 5.0 was mixed with H O (15 μM in buffer)
+
H O + HCl
2
in the absence or presence of sodium ascorbate (7.5 μM), for the
formation of HRP I and HRP II, respectively. For the reactions of ferryl
intermediates with substrates, halophenol (15 μM, 7.5 μM or 3.75 μM)
in buffer was mixed with the preformed intermediates. Age times of
40 ms and 8 s were utilized for reactions with HRP I and II, respectively,
based on the conditions for their optimal formation. The run time was
varied depending on the time taken for the completion of the reaction.
A Restek RTX-5 GC capillary column (30 m×0.32 mm) was used
for product analysis. The initial temperature of the column was set
at 50 °C for 2 min and the temperature was subsequently increased
at a rate of 10 °C/min up to a final temperature of 300 °C and held
for 7.5 min. MS detection was in the electron impact ionization
mode, and a VG70S mass spectrometer scanning from 50 to 450 (m/z)
was used for product identification. Authentic samples of 2,6-DCP,
2,3,6-TCP, and 2,4,6-TCP gave identical corresponding retention times
for GC/MS analyses.
2
2
Cl
O
2
,6-dichloro-1,4-
benzoquinone
2,4,6-TCP
ð1Þ
With the use of rapid scanning stopped-flow absorption spectroscopy,
the reactivities of homogeneous short-lived ferryl HRP intermediates can
be investigated. Thus, the hypothesized two consecutive one-electron
transfer mechanism can be directly tested with single turnover experi-
ments using double mixing rapid-scan stopped-flow spectroscopy, in
which the ferryl intermediate (HRP I or HRP II) can be generated in the
first mix followed by reaction with chlorophenol in the second mix.
Using rapid-scan stopped-flow spectroscopy, we have previously
reported data with C. fumago chloroperoxidase that were consistent
with a mechanism of oxidative dehalogenation of chlorophenols involv-
ing two consecutive one-electron oxidations [34]. However, it was not
possible to carry out those studies under single turnover conditions
All kinetic rate constants were analyzed by using the non-linear
regression program (Sigma Plot) for absorbance vs. time plots to a
single exponential curve with three parameters mode. The values
reported here were based on three separate measurements and are
expressed as mean values± standard deviations.
2 2
since an excess of H O was needed to form chloroperoxidase com-
pound I. Although the turnover number (68 s ) for HRP-catalyzed
oxidation of 2,4,6-TCP in the presence of excess (>10 ×[HRP]) amounts
−
1
3
of H O
2 2
and the substrate at pH 5.4 has previously been determined by
3. Results and discussion
Ferrari and co-workers [21], no kinetic data on the direct reactions of
HRP I and HRP II with 2,4,6-TCP have been reported.
3.1. Oxidative halophenol dehalogenation
In the present study, we demonstrate the reaction steps involved in
the dehalogenation of 2,4,6-TCP and illustrate how HRP I and HRP II are
reduced by 2,4,6-TCP using single turnover experiments. It has previ-
ously been shown that the number and position of chlorine substituents
on the phenolic ring has an influence on the rate and the extent of
removal of halogens by HRP or by biodegradation [5,48]. Therefore,
we have also extended our studies to rapid-scan stopped-flow spectros-
copy and mass spectrometry analyses of the HRP-catalyzed degradation
of 2,3,6-trichlorophenol (2,3,6-TCP) and 2,6-dichlorophenol (2,6-DCP)
and report the rate constants for these reactions.
We have performed single turnover experiments employing rapid
scan stopped-flow spectroscopy to examine whether the mechanism
of oxidative dehalogenation catalyzed by HRP involves two consecutive
one-electron steps or a single two-electron oxidation. The present study
represents an important step forward in our understanding of the
mechanism of this reaction in that we are able to carry out the
stopped-flow investigation under single turnover conditions, meaning
that we can generate one equiv. of HRP I in the first mix and then
react it vs. the chlorophenol substrate in the second mix. First, we opti-
IV
mized the formation of HRP I (Fe =O/porphyrin π-cation radical) and
IV
HRP II (Fe =O) for use in double mixing rapid-scan stopped-flow
2
. Materials and Methods
spectroscopy experiments. To do this, HRP was reacted with an equimo-
lar amount of H
ascorbate, a two-electron donor substrate, to form HRP II) at pH 5.0
and 4 °C to generate the ferryl intermediate without any excess H
2 2
O to generate HRP I (plus one-half molar equiv. of
Horseradish peroxidase (HRP) (RZ=3.1), purchased from USB
United States Biochemical, Cleveland, OH), was dissolved in 100 mM
(
2 2
O .
potassium phosphate at pH 5.0. The HRP concentration was deter-
mined spectrophotometrically using the molar absorptivity of
This ensures that only a single turnover can be achieved. We chose pH
5.0 because the products are stable at acidic pH and can be isolated
for GC/MS analysis. Even though potassium phosphate is not a good
buffer at pH 5, it still helps maintain a constant pH value at 100 mM
(ionic strength: I=~0.1 M). Similar results were observed at pH 7.0.
Furthermore, despite the fact that an excess (>10) amount of
2,4,6-TCP over HRP would provide kinetic data that can be readily
−
1
−1
ε
403 =103 mM
cm
[49]. Reagent grade chemicals (Sigma,
ACROS, or Fisher) were used without further purification. Fresh
1
3
00 mM H
0% solution, and H
2
O
2
stock solutions in water were made from the commercial
concentrations were confirmed spectrophoto-
2 2
O
−
1
−1
metrically using ε240=39.4 M
cm [50].

318
S. Sumithran et al. / Journal of Inorganic Biochemistry 117 (2012) 316–321
analyzed as pseudo-first order reactions, the highest ratio used for the
reaction of HRP I with 2,4,6-TCP in this study was only 1:2. Unexpectedly,
kinetic traces for conversion of HRP I to HRP II were well fit to single
exponential reactions to the completion of the reaction even when the ra-
tios of [2,4,6-TCP] /[HRP] were near (2.0 and 1.0) or sub-stoichiometric
(0.5). Similarly, single exponential fits were also obtained for conversion
of HRP II to ferric HRP in the presence of 0.5, 1.0, 2.0, 2.5, 5.0 and 10 equiv.
of 2,4,6-TCP. These apparently unusual results will be discussed later in
terms of likely involvement of a 2,4,6-TCP radical in the reactions. All
the kinetic data (rate constants) that were obtained for three or more
separate runs in this study are summarized in Table 1 as mean± standard
deviation values.
3
2 2
.2. Reaction of ferric HRP with H O in the absence of organic substrate
To study the steps involved in the HRP-catalyzed oxidation of
Fig. 1. Rapid scan, stopped-flow spectra illustrating formation of the ferryl intermediates
and the regeneration of the ferric state after a single mixing of ferric HRP (15 μM) with
H O (15 μM) in 100 mM potassium phosphate solution at 4 °C and pH 5.0. The traces
halophenols and to probe the roles of high-valent intermediates in
the reaction mechanism, the rate constants for the spontaneous reac-
tion must first be measured. Rapid scan, stopped-flow spectroscopy
was thus used to initially optimize the formation and then establish
the stability of the HRP ferryl intermediates (HRP I and HRP II) at
2
2
represent (green dotted line) HRP I formed at 40 ms; (red solid line) HRP II formed at
00 s; (blue dashed line) ferric HRP regenerated at>45 min.
2
4
°C, pH 5.0. The absorption spectra of ferric HRP, HRP I and HRP II
mechanism is a direct two-electron oxidation with the O-atom transfer
from HRP I. The spontaneous (i.e., no added organic substrate) reduc-
tion of HRP I to ferric HRP, under the given experimental conditions
recorded at pH 5.0 (Fig. 1) [51] for this work were essentially identi-
cal to those previously reported at neutral pH value [52,53]. Reaction
of HRP with an equimolar amount of H
.75 μM) resulted in the homogenous formation of HRP I in 40 ms
as demonstrated by the characteristic absorption spectrum of HRP I
2
O
2
(final concentrations=
2 2
(HRP: H O =1:1, pH 5.0, 4 °C), clearly proceeded through the homo-
geneous formation of HRP II.
3
(
Soret peak at 400 nm). After 3 s, HRP I gradually converted to HRP
3.3. Reaction of HRP I with 2,4,6-TCP
II (kobs =0.027± 0.003 s− ), which has a Soret band at 419 nm and
distinct α (558 nm) and β (527 nm) transitions in the visible region.
The spectrum of HRP II remained unchanged for about 55 s and then
1
As just discussed, spontaneous conversion of preformed HRP I to
HRP II (monitored at 419 nm) occurred slowly with a rate constant
−
1
−1
decayed very slowly (kobs =0.0043± 0.0006 s ) to regenerate native
ferric HRP (Soret maximum at 403 nm). If the reaction of HRP I with a
reducing substrate proceeds via HRP II, then the reaction mechanism in-
volves two consecutive one-electron steps; if HRP I directly converts
back to the ferric state without involvement of HRP II, then the reaction
of 0.027± 0.003 s . The reaction of HRP I with 2,4,6-TCP is a much
faster process. When HRP I was mixed with 2 equiv. of 2,4,6-TCP,
HRP I was rapidly converted to HRP II in a first phase (kobs =238±
−
1
8 s ) (representative results shown in Fig. 2A). Note that, under
these conditions, ~50% of HRP I had been converted to HRP II within
the dead time (~1.5 ms) of the instrument. Then HRP II was slowly
−
1
reduced to the ferric state in the second phase (kobs =2.50± 0.20 s
Fig. 2B). The HRP I / 2,4,6-TCP reaction, followed at 419 nm, shows
single exponential behavior (Fig. 2A, inset); the rate of formation of
)
Table 1
(
Kinetics constants for the reactions of halophenols with HRP ferryl intermediates (HRP
I and HRP II).
−
1
HRP II (kobs =238± 8 s ) was ~8800 -fold faster with 2,4,6-TCP than
Substrate
[Substrate]/[HRP] at t=0 Rate constant (s−1)
HRP I to HRP IIa HRP II to Ferric HRPb
−1
without (kobs =0.027± 0.003 s ). The rate of subsequent conversion
−1
of HRP II to the ferric state (kobs =2.50± 0.20 s ) was also faster (by
~
580-fold) than in the absence of the substrate (kobs =0.0043±
No substrate
0.027± 0.003
56.6± 5.5
0.0043± 0.0006
−
1
2,4,6-TCP
0.5 (HRP I)
0.0006 s ) under these conditions where 1 equiv. of 2,4,6-TCP is
expected to remain unoxidized upon formation of HRP II.
0.5 (HRP II)
1.0 (HRP I)
1.0 (HRP II)
2.0 (HRP I)
2.0 (HRP II)
0.68± 0.09
0.96± 0.03
c
114± 12
238± 8
The mass spectrum of the dehalogenated product matches that of
the authentic sample of 2,6-dichloro-1,4-benzoquinone confirming
the product formation (m/z 176, data not shown). The data indicate
that in the 2,4,6-TCP dehalogenation mechanism, the first electron
is transferred from the substrate to HRP I to generate the phenoxy
radical and HRP II. Thus the oxidative dehalogenation of TCP to ben-
zoquinone product is mediated by both HRP I and HRP II as initially
hypothesized by Samokyszyn and co-workers [20] and subsequently
by Ferrari and co-workers [21].
1.15± 0.17
2.50± 0.20
c
1.56± 0.13
1.24± 0.09
1.37± 0.04
0.37± 0.02
c
2
,3,6-TCP
,6-DCP
2.0 (HRP I)
.0 (HRP II)
2.0 (HRP I)
.0 (HRP II)
30.1± 0.9
19.7± 0.7
d
2
c
2
2
0.26± 0.03
a
2 2
HRP I formed by mixing HRP (15 μM) with one equiv. of H O (15 μM). The
resulting HRP I (7.5 μM) was then mixed with buffer/halophenol (15 μM, 7.5 μM or
.75 μM ) in the second mix (age time 40 ms) (concentrations after mixing, [HRP]=
]=3.75 μM, [halophenol]=7.5 μM, 3.75 μM or 1.875 μM). The reaction was
3
2 2
[H O
monitored at 419 nm to follow the formation of HRP II.
3.4. Reaction of HRP I with other halophenols
b
2 2
HRP II formed by mixing HRP (15 μM) with one equiv. of H O (15 μM) and
one-half equiv. of sodium ascorbate (7.5 μM). The resulting HRP II (7.5 μM) was then
mixed with buffer/halophenol (15 μM, 7.5 μM or 3.75 μM) in the second mix (age
The spectral changes observed for the oxidation of 2,3,6-TCP and
,6-DCP by HRP I (data not shown) were the same as those seen for
2
time 8 s) (final concentrations, [HRP]=[H
halophenol]=7.5 μM, 3.75 μM or 1.875 μM). The reaction was monitored at 403 nm
to follow the regeneration of ferric HRP.
2 2
O ]=3.75 μM, [ascorbate]=1.875 μM,
[
2,4,6-TCP (Fig. 2). The HRP I to HRP II conversion rates are also
much higher (>103-fold) in the presence of these substrates than
the spontaneous reduction. HRP II is again seen as an intermediate
between HRP I and the ferric enzyme, consistent with a mechanism
involving two consecutive one-electron oxidation steps. However,
the rates for these substrates are not as high as in the 2,4,6-TCP
c
Second phase reaction following HRP I to HRP II conversion.
First (~60%) of two phases. When 2 molar equiv. of 2,3,6-TCP was reacted with
d
2
HRP II, the reaction trace was better fit to double-exponential decays (r =~0.999)
with fast (1.37 s−
1
−1
,
~60%) and slow (0.37 s
, ~40%) phases rather than to a
−
1
2
single-exponential decay (0.73 s , r =0.990).

S. Sumithran et al. / Journal of Inorganic Biochemistry 117 (2012) 316–321
319
spectrum (m/z 311, 313, 315, data not shown)] was formed, the structure
of which is under investigation.
3.5. Reaction of HRP II with 2,4,6-TCP, 2,3,6-TCP and 2,6-DCP
In the peroxidase reaction, the high-valent oxidant Cpd II can also
carry out one-electron oxidations. Therefore, the ability of HRP II to
dehalogenate halophenols was studied using double-mixing stopped-
flow experiments. In reactions of HRP I with the three chlorophenol
substrates described above, the HRP II formed in the first phase of
the reaction was reduced back to ferric HRP in the second phase. The
rate constants for the second phase reaction were much smaller
than those for the first phase by the factors of 94 (2,4,6-TCP), 24
(
2,3,6-TCP) and 54 (2,6-DCP) (Table 1). This reflects intrinsic differ-
ences between HRP I and HRP II for their substrate oxidation rates.
The somewhat lower concentrations of oxidizable substrate available
for HRP II relative to HRP I may also contribute a minor factor to the
observed differences.
Alternatively, HRP II can be generated by mixing ferric HRP
(
2 2
7.5 μM before mixing) with one equiv. of H O containing one-half
equiv. of the two-electron reductant sodium ascorbate (to reduce
the initially formed HRP I to HRP II). Complete HRP II formation
−
1
occurs in 2 s (kobs=~2.1 s ) resulting in its characteristic absorption
spectrum (Soret, β and α bands at 419 nm, 527 nm and 558 nm,
respectively), which is essentially unchanged over a period of~10 s.
The spontaneous reduction of HRP II to the ferric resting state is
−
1
~
6.2-fold slower (0.0043± 0.0006 s ) (Fig. 3 and Table 1) than that
−
1
of HRP I to HRP II (0.027± 0.003 s ) (Fig. 1A and Table 1). When
HRP II was preformed in the first mix and reacted with 0.5 equiv. of
Fig. 2. Conversion of HRP I to HRP II in 100 mM potassium phosphate solution at 4 °C,
pH=5.0. Ferric HRP was first mixed with one equiv. of H for the formation of HRP I
age time 40 ms), which was then mixed with TCP to observe the reaction course (final
concentrations, [HRP]=[H ]=3.75 μM, [TCP]=7.5 μM). Representative results
spectral changes and kinetic traces and their analysis) are displayed here; statistical
data based on three or more runs are summarized in Table 1. (A) First phase of the
reaction showing the conversion of HRP I to HRP II. Since the absorption spectrum of
HRP I can not be recorded under the conditions (see text, Section 2.3), its spectrum
2
,4,6-TCP in the second mix (Fig. 3), the rate of regeneration of the ferric
2 2
O
−
1
(
state (0.68± 0.09 s ) was >150-fold faster than the spontaneous rate.
2 2
O
Formation of the ferric state from HRP II in the presence of 2 equiv. of
(
−1
2
,4,6-TCP (k=1.56± 0.13 s ) and the other chlorophenol substrates
(
2,3,6-TCP, 2,6-DCP) has the same order (2,4,6-TCP>2,3,6-TCP≫
2,6-DCP⋙No substrate) with respect to the chloro substituent patterns
of the substrate as seen with HRP I (Table 1).
(dot-dashed line) taken from Fig. 1 is overlaid as reference. Inset, The same reaction
monitored at 419 nm shows, upon mixing with 2 mol equiv. of TCP, HRP I fully
GC/MS analysis confirmed that the products formed from the
reaction of HRP II with halophenol substrates are the same as seen
by Laurenti et al. in multiple turnover studies of the HRP/H O system
2 2
converting to HRP II with kobs=252 s− (dotted line), a single exponential kinetic fit
1
(solid line), spontaneous conversion without TCP (dashed line). (B) Second phase of
the reaction showing the conversion of HRP II to ferric HRP. Inset, The same reaction
monitored at 403 nm shows HRP II that was formed by mixing 2 mol equiv. of TCP
with HRP I, fully converting to ferric HRP in the subsequent reaction with kobs
=
−
1
2.71 s (dotted line), a single exponential kinetic fit (solid line), spontaneous conver-
sion without TCP (dashed line). In both panels A and B, the vertical arrows indicate the
directions of absorbance changes.
reaction (2,4,6-TCP≫2,3,6-TCP>2,6-DCP⋙No substrate) (Table 1).
Although the result in this study using the very limited number of
substrates may not be sufficient enough to draw a general conclusion,
the number of chlorine substituent for the substrates and whether a
chlorine atom is absent at 4 (para) position appear to have a significant
effect on the rate constants for reduction of HRP I and HRP II. This is
likely due to the fact that the slower substrates are harder to oxidize
due to the differences in chloro substitution pattern. Laurenti and
2 2
co-workers reported two products for the HRP/H O /2,6-DCP
reaction, 3,3′,5,5′-tetrachloro-4,4′-dihydroxybiphenyl and 3,3′,5,5′-
tetrachlorodiphenoquinone [18]. In the reaction of HRP I with 2
molar equiv. of 2,6-DCP in this study, the mass spectrum of the
reaction product (m/z 324, data not shown) is consistent with the
generation of 3,3′,5,5′-tetrachloro-4,4′-dihydroxybiphenyl; however, fur-
ther oxidation of this product to 3,3′,5,5′-tetrachlorodiphenoquinone was
not seen. This is because only one equiv. of HRP I was generated (single
Fig. 3. Conversion of HRP II to ferric HRP in 100 mM potassium phosphate solution at
°C, pH=5.0. Ferric HRP was first mixed with one equiv. of H and one-half equiv.
of sodium ascorbate for the formation of HRP II (age time 8 s), which was then mixed
with TCP to observe the reaction course (final concentrations, [HRP]=[H ]=
3.75 μM, [ascorbate]=1.875 μM, [TCP]=1.875 μM). The vertical arrows indicate the
directions of absorbance changes. Representative results (spectral changes and kinetic
traces and their analysis) are shown here; statistical data based on three or more runs
are summarized in Table 1. Inset, The same reaction monitored at 403 nm shows, upon
4
2 2
O
2 2
O
2 2
turnover with HRP:H O =1:1) consumption of which would leave no
remaining oxidant to carry out the further oxidation of 3,3′,5,5′-
tetrachloro-4,4′-dihydroxybiphenyl to the corresponding quinone. In
the case of 2,3,6-TCP oxidation, a different dimeric product [mass
mixing with 0.5 mol equiv. of TCP, HRP II fully converting to ferric HRP with kobs
=
−1
0.729 s
(dotted line), a single exponential kinetic fit (solid line), spontaneous
conversion without TCP (dashed line).

320
S. Sumithran et al. / Journal of Inorganic Biochemistry 117 (2012) 316–321
[
18]: 2,6-dichloro 1,4-benzoquinone quinone with TCP and 3,3′,5,5′-
sub-stoichiometric (0.5–2.0). The only exception was for the reaction
of 2,3,6-TCP with HRP II where two phases were observed (see
Table 1, footnote d).
tetrachloro-4,4′-dihydroxybiphenyl with 2,6-DCP. Further, these
chlorophenol substrates reacted faster with HRP I than HRP II,
which is usually the case with most peroxidase substrates [54].
These results clearly indicate that HRP II is directly capable of oxida-
tively dechlorinating 2,4,6-TCP and coupling 2,3,6-TCP and 2,6-DCP
to biphenyl products. Given the fact that HRP II can only catalyze
one-electron oxidations, the ability of this intermediate to catalyze
the two electron oxidation of 2,4,6-TCP can only be the result of a
mechanism involving two consecutive one-electron steps via a
phenoxy radical intermediate.
If a substrate is a one-electron donor, the reduction of HRP I to
HRP II in the presence of 1 molar equiv. of substrate would be
expected to completely consume the substrate at the end of the reac-
tion. A single exponential mode under such conditions would not be
expected. Yet the reaction proceeded as if the effective substrate con-
centration did not change during the entire reaction. Furthermore,
the reduction of HRP II to ferric HRP (either with initial 1 molar
equiv. of 2,4,6-TCP added to HRP I or 0.5 molar equiv. added to
HRP II) proceeded also in a single exponential fashion at rates that
are respectively 158 and 223-fold faster than the spontaneous con-
version (Table 1).
3
.6. Kinetics of the reactions of HRP I and HRP II with 2,4,6-TCP -
Determination of the 2nd order rate constants
A similar observation was reported in the reaction of lignin peroxi-
dase compound I (LiP I) with thioanisole (and p-methoxylthioanisole)
by Harvey and co-workers [55]. Using the rapid scan stopped-flow
spectroscopic technique, the authors found that even under single
turnover conditions (where stoichiometric amounts (0.75 μM) of
As mentioned above, kinetic traces for conversion of HRP I to HRP
II as well as HRP II to ferric HRP were best fit to single exponential
curves from the beginning [t=~0.75 and ~50 ms (i.e., the earliest
times of recording) after stopped-flow single mixing for HRP I and
double mixing for HRP II, respectively] to the completion of the
reaction even when the ratios of [2,4,6-TCP] /[HRP] were near or
2 2
pre-formed LiP I (ferric LiP+1 equiv. H O in the first mix) and the
substrate were mixed), LiP II that was quickly formed upon reduction
of LiP I by the substrate in the first phase, was converted to ferric LiP
in a single exponential fashion in the second phase. A thioanisole
radical(s) is presumably formed and used as substrate (reductant) in
the second-phase reaction [55]. The LiP II to ferric LiP conversion rates
under these conditions were ~20 times faster than the spontaneous
decay of LiP II to ferric enzyme (in the absence of substrate).
The above results with HRP can be explained in part by assuming
that (a) the initially formed phenoxy radical molecules are involved
as an electron donor for HRP I and HRP II or (b) the radicals rapidly
disproportionate to generate the two-electron oxidized quinone
product and more phenol, effectively preventing a rapid decrease of
the concentration of phenol. Fox and co-workers have reported that
the phenoxy radical of 2,4,5-trichlorophenol decays very rapidly
with a rate constant of (7.7± 1.7)×108
proportionation [56].
M
−1
s
−1
, presumably by dis-
It should be pointed out that although a single exponential decay
of HRP I to HRP II was still observed even in the presence of only
−
1
0
.5 molar equiv. of 2,4,6-TCP (kobs =56.6± 5.5 s ), the subsequent
second phase reaction for conversion of HRP II to ferric HRP occurred
at the same rate as for the spontaneous reaction. This is to be
expected since all of the substrate would have been consumed in
the first phase and shows that 2,4,6-TCP cannot provide more than
two electrons.
We have used the kinetic results for the conversion of HRP I to
HRP II and of HRP II to ferric HRP in the initial presence of various
molar equivalents (0.5–2.0) of 2,4,6-TCP (Table 1) to determine the
second order rate constants for the reactions of 2,4,6-TCP with HRP
I and HRP II. When the observed rate constants (kobs) for all of these
reactions are plotted against [2,4,6-TCP] /[HRP I] and [2,4,6-TCP]/
[
0
HRP II] values at t=0 (Fig. 4A and B, respectively, where subscript
is added to their concentrations at t=0), a good linear correlation
is seen for the HRP I reaction in which the rate constants are propor-
tional to [TCP] /[HRP I] (bottom X-axis scale) or [TCP] (top X-axis
0
0
0
1
scale) (Fig. 4A). For the HRP II case, a similar but slightly poorer
linear correlation is observed when the origin (X=Y=0) is included
(
Fig. 4B). Accordingly, the second order rate constants for the reaction
of HRP I with 2,4,6-TCP (from the slope in Fig. 4A), 2,3,6-TCP and
2
0 0
,6-DCP (based on the data for [chlorophenol] / [HRP I] =2.0 for
Fig. 4. Plots for observed pseudo-first rate constants vs. (A) [2,4,6-TCP]
B) [2,4,6-TCP] /[HRP II] (bottom abscissa scale) or [2,4,6-TCP] (top abscissa scale)
for reduction of (A) HRP I and (B) HRP II with 2,4,6-TCP, where the subscripts “0”
indicate the initial concentrations. [HRP I] =[HRP II] =3.75 μM. Spontaneous reduction
rates are plotted for [2,4,6-TCP] =0 μM for the both panels. Data derived from Table 1,
0 0
/[HRP I] or
1
(
0
0
0
Under non-pseudo-first-order reaction conditions ([2,4,6-TCP]/[HRP II]=0.5–2.0),
=k[S] [HRP I] ) need to be used to obtain the true second order rate
constants (k). However, the slopes in these plots, where the “apparent” pseudo-first
order rate constants were plotted vs. [S] for the conversion of HRP I to HRP II (or
HRP II to ferric HRP), would still give the same second order rate constants. Note that
[S] and [HRP I] are substrate and enzyme concentrations at t=0.
the initial rates (v
o
o
o
0
0
0
o
see Table footnotes for additional reaction conditions. Straight lines shown are linear
2
regression fits (r =0.9994 for A and 0.979 for B).
o
o

S. Sumithran et al. / Journal of Inorganic Biochemistry 117 (2012) 316–321
321
7
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−1 −1
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.6×10
M
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5
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[
[
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and HRP II are active catalysts for the oxidative dehalogenation of
halophenol substrates. It has been previously proposed that HRP cata-
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2 2
in the absence of excess H O leads rapidly to HRP II and then more
slowly to the ferric resting state. This constitutes direct and compelling
evidence for a mechanism involving two consecutive one-electron
oxidations of the substrate via a phenoxy radical intermediate. HRP II,
which can only carry out one-electron oxidations, also directly dechlori-
nates 2,4,6-TCP. In addition, the reactions with 2,3,6-TCP and 2,6-DCP,
in which the substrates are not dehalogenated, also appear to involve
one-electron processes. All evidence points to a mechanism involving
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Abbreviations
HRP
Horseradish peroxidase
HRP compound I
HRP compound II
Lignin peroxidase
LiP compound I
LiP compound II
Compound I
[
[
[
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HRP I
HRP II
LiP
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LiP I
14838–14839.
LiP II
Cpd I
Cpd II
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Compound II
2
2
2
,4,6-TCP 2,4,6-Trichlorophenol
,3,6-TCP 2,3,6-Trichlorophenol
,6-DCP 2,6-Dichlorophenol
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Acknowledgements
We thank Dr. David P. Ballou for helpful discussions and Dr. Mike
Walla, Director of Mass Spectrometer Services, Department of Chemistry
and Biochemistry, University of South Carolina, for the GC/MS analysis.
Financial support provided by the National Science Foundation (MCB
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