C. fumago chloroperoxidase is also a dehaloperoxidase: Oxidative dehalogenation of halophenols

Article abstract of DOI:10.1021/ja056213b

We have examined the H₂O₂-dependent oxidative dehalogenation of 2,4,6-trihalophenols and p-halophenols catalyzed by Caldariomyces fumago chloroperoxidase (CCPO). CCPO is significantly more robust than other peroxidases and can function under harsher reaction conditions, and so its ability to dehalogenate halophenols could lead to its use as a bioremediation catalyst for aromatic dehalogenation reactions. Optimal catalysis occurred under acidic conditions (100 mM potassium phosphate solution, pH 3.0). UV-visible absorption spectroscopy, high-performance liquid chromatography, and gas chromatography/mass spectrometry clearly identified the oxidized reaction product for CCPO-catalyzed dehalogenation of 2,4,6-trihalophenols as the corresponding 2,6-dihalo-1,4-benzoquinones. This reaction has previously been reported for two His-ligated heme-containing peroxidases (see Osborne, R. L.; Taylor, L. O.; Han, K. P.; Ely, B.; Dawson, J. H. Biochem. Biophys. Res. Commun. 2004, 324, 1194-1198), but this is the first example of a Cys-ligated heme-containing peroxidase functioning as a dehaloperoxidase. The relative catalytic efficiency (turnover number) of CCPO reported herein is comparable to that of horseradish peroxidase (Ferrari, R. P.; Laurenti, E.; Trotta, F. J. Biol. Inorg. Chem. 1965, 4, 232-237). The mechanism of dehalogenation has been probed using p-halophenols as substrates. Here the major product is a dimer with 1,4-benzoquinone as the minor product. An electron-transfer mechanism is proposed that accounts for the products formed from both the 2,4,6-trihalo- and p-halophenols. Finally, we note that this is the first case of a peroxidase known primarily for its halogenation ability being shown to also dehalogenate substrates. Copyright

Full text of DOI:10.1021/ja056213b

Published on Web 01/04/2006
C. fumago Chloroperoxidase is also a Dehaloperoxidase: Oxidative
Dehalogenation of Halophenols
Robert L. Osborne, Gregory M. Raner, Lowell P. Hager, and John H. Dawson*
Department of Chemistry and Biochemistry and School of Medicine, UniVersity of South Carolina, Columbia,
South Carolina 29208, Department of Chemistry and Biochemistry, UniVersity of North CarolinasGreensboro,
Greensboro, North Carolina 27402, Department of Biochemistry, UniVersity of Illinois at UrbanasChampaign,
Champaign, Illinois 61820
Received September 9, 2005; E-mail: dawson@mail.chem.sc.edu
Chloroperoxidase from Caldariomyces fumago (CCPO) is the
most extensively studied halogenating peroxidase, with multifunc-
tional catalytic activities typical of peroxidase as well as cytochrome
P450 enzymes. CCPO, like all peroxidases, follows a reaction cycle
2 2
that uses H O to generate the high-valent ferryl oxidant, Compound
In particular, phenols and their derivatives are commonly used in
a variety of industrial processes, and chlorinated phenol toxicity
has been proven both in vitro17 and in vivo. For example, exposure
to high levels of chlorophenols can cause damage to the liver and
the immune system. In addition, many chlorinated phenols, such
as 2,4,6-trichlorophenol (TCP), persist in the environment to such
an extent that they are included in the US EPA priority list of
18
1
I (Cpd I), which can then undergo two, one-electron reductions to
regenerate the ferric resting state with concomitant substrate
oxidation.1 Interestingly, CCPO has none of the three conserved
residues found near the heme unit in all other known peroxidases.
In addition to the lack of the conserved proximal His ligand, neither
the conserved distal His nor the Arg residues thought to facilitate
-4
19
dangerous pollutants. Interest in using biocatalysts in industrial
and bioremediation processes continues to increase as agencies, such
as the EPA, place more stringent restrictions on the use of reagents
that are potentially damaging to the environment.
3
O-O cleavage are found in CCPO. Previous studies implicate
CCPO catalyzes the oxidative dehalogenation of trihalophenols
Glu183 as the residue in CCPO analogous to the distal His in typical
(eq 1) and p-halophenols at appreciable rates and under reaction
5
peroxidases. Despite these differences in the proximal and distal
conditions often too harsh for other biocatalysts. As CCPO is signi-
ficantly more robust than other peroxidases, its ability to dehalo-
genate halophenols could lead to its use as a bioremediation catalyst
for aromatic dehalogenation reactions. Previous studies provide a well-
detailed analysis of the oxidative 4-dechlorination of polychlorinated
regions of the heme unit, the widely accepted enzymatically active
form of CCPO is Cpd I, as in all other known peroxidases.6
CCPO catalyzes some P450-type oxygen atom transfer reactions,
such as the epoxidation of olefins and the oxygenation of
sulfides.7
-10
Structurally, both CCPO and the cytochrome P450s
20
phenols by horseradish peroxidase (HRP), lignin peroxidase
contain protoporphyrin IX as the prosthetic group, and both are
proximally ligated to the protein by a deprotonated cysteine
thiolate.1 Cys-ligated heme enzymes are foremost among heme
proteins in their ability to catalyze the insertion of an oxygen atom,
LiP), and Amphitrite ornata dehaloperoxidase (DHP),22,23 all of
21
(
which are His-ligated heme proteins. This is the first example of a
Cys-ligated heme-containing peroxidase that can also function as a
dehaloperoxidase. It is also the first case of a peroxidase, known pri-
marily for its halogenation function, to also dehalogenate substrates.
-3
derived from either molecular oxygen or peroxide, into a variety
of organic substrates.1
,2,11
This is in contrast to most His-ligated
2 2
The H O -dependent oxidative dechlorination of TCP catalyzed
heme enzymes, which typically rely on electron-transfer-type
oxidations as opposed to direct oxygen atom insertion mechanisms
by CCPO at pH 3.0 was followed by electronic absorption (EA)
spectroscopy, high-performance liquid chromatography (HPLC),
12
due to limited substrate access to the heme iron.
2
4,25
and gas chromatography-mass spectrometry (GC-MS).
spectral changes observed for the CCPO/TCP/H reaction mixture
EA
Although CCPO demonstrates a broad range of catalytic activity,
its primary biological function appears to be the halogenation of
aliphatic substrates according to the reaction:
2 2
O
provided the first evidence for catalytic, oxidative dehalogenation
yielding 2,6-dichloro-1,4-benzoquinone (DCQ). The product was
characterized by its absorption maxima at λmax ) 272 and 342 nm,
-
+
-
-
-
AH + X + H + H O f AX + 2H O; X ) I , Br , Cl
2
2
2
20
21
as reported previously for HRP and LiP. Analysis of the product
by GC-MS (Figure 1) confirmed it to be DCQ, with a MS identical
to that of a DCQ standard (Aldrich). By monitoring the absorbance
at 272 nm versus time,25 the turnover number for the CCPO-
catalyzed reaction was found to be essentially identical to that
reported for the same reaction carried out by HRP.20 Formation of
DCQ was shown to occur only in the presence of CCPO.
-
-
-
-
where AH represents the substrate, and X can be Cl , Br , or I ,
-
4,13
but not F . The pathway for peroxidase-catalyzed halogenations
has been proposed to involve a ferric hypohalite intermediate known
as Cpd X.1 However, the mechanism of CCPO-catalyzed halo-
,2
14-16
genation of organic substrates is still under investigation.
Contrary to its primary biological function of chlorination, CCPO
can also dehalogenate trihalophenols (eq 1) and p-halophenols, a
feature that may have important environmental implications.
Because Cys-ligated heme enzymes are noted for their ability
to catalyze O-atom transfer reactions (vide supra), efforts were
initiated to establish whether the new quinone oxygen atom in DCQ
was derived from H
2
O
2
or H
O into the product quinone was found to be very rapid, with
complete exchange occurring in a matter of seconds. Thus, simple
2
O. However, the exchange rate for
18
H
2
18
O incorporation studies will not be possible to probe the
mechanism of oxidative dehalogenation.
Large-scale production of halogenated organic compounds has
resulted in contaminants that are often retained in the environment.
Mechanistic information suggesting that the CCPO-catalyzed
oxidative dehalogenation involves an electron-transfer process rather
1036
9
J. AM. CHEM. SOC. 2006, 128, 1036-1037
10.1021/ja056213b CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
In conclusion, we have presented the first evidence for catalytic,
oxidative dehalogenation by C. fumago chloroperoxidase. Not only
is the activity novel, but the rate of dechlorination is comparable
2
0
to that of the same reaction catalyzed by HRP. CCPO is
significantly more robust than other peroxidases and functions under
harsher reaction conditions compared to other biocatalysts. Expand-
ing the scope of reactivity achieved by CCPO may be beneficial
for industrial and biotechnological functions in the future. This
considerable extension of already known activities could lead to
the use of CCPO as a biocatalyst in the field of bioremediation
and a broader understanding of both how peroxidases and cyto-
chrome P450s react with halogenated organic substrates.
Acknowledgment. Financial support was provided by the Uni-
versity of South Carolina Environmental Research Initiative, the
NSF (MCB:964004), and the NIH (GM 26730) for additional
support. We also thank Dr. Masanori Sono and Dr. Mike Walla
for helpful discussions.
Figure 1. Mass spectrum of 2,6-dichloro-1,4-benzoquinone extracted from
the CCPO (0.1 µM), TCP (0.5 mM), and H2O2 (1 mM) reaction in 100
2
4
mM potassium phosphate solution, pH 3.0.
Scheme 1. Proposed Reaction Scheme of the CCPO/TCP/H2O2
Oxidative Dehalogenation Reaction
Supporting Information Available: EA, HPLC, and GC/MS data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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We have also found that CCPO catalyzes H
2 2
O -dependent
defluorination, debromination, and deiodination reactions (data not
shown). As with p-chlorophenol, two main products, p-benzo-
quinone (minor) and the halophenol dimer (major), are observed
for all p-halophenol-CCPO-catalyzed dehalogenation reactions.
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1996, 271, 4609-4612.
(
23) Osborne, R. L.; Taylor, L. O.; Han, K. P.; Ely, B.; Dawson, J. H. Biochem.
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24) EA spectral changes observed for the CCPO (0.1 µM), TCP (100 µM),
(
2 2
and H O (400 µM) reaction mixture in 100 mM potassium phosphate
solution, pH 3.0. Authentic 2,6-dichloro-1,4-benzoquinone gave nearly
identical spectral characteristics. After a 1 h incubation, 25 µL aliquots
of the reaction mixtures were injected onto a C18 HPLC column (150
2,4,6-tribromo- or 2,4,6-triiodophenol. These results show clearly
that CCPO not only possesses the ability to incorporate halogens
into aromatic substrates27 but can also remove those same groups
in an H O -dependent manner from the reaction products.
2 2
mm × 4.6 mm) under isocratic conditions (10% acetonitrile, 90% H O,
2
and 0.1% trifluoroacetic acid) with a flow rate of 1 mL/min. The reaction
products were extracted in ethyl acetate for GC analysis. A Restek RTX-
5
0 GC capillary column (30 m × 0.32 mm) i.d. × 0.25 µm d.f. was used.
Initial temperature of the column was set at 70 °C for 3.5 min, and from
there the temperature was increased at a rate of 15 °C/min up to a final
temperature of 280 °C and held for 7.5 min. MS detection was in the
positive ion mode. Authentic samples of 2,4,6-trichlorophenol and 2,6-
dichloro-1,4-benzoquinone gave identical retention times for both HPLC
and GC analyses.
CCPO is an easily obtainable protein (relatively low cost), and
we are interested in identifying the various factors that control this
novel activity, which could be applicable toward the biodegradation
of polychlorinated phenols and other noxious haloaromatic com-
pounds. The activity illustrated herein extends the already wide
range of reactions that can be carried out by CCPO. The types of
(25) See Supporting Information containing supplemental data.
26) The mass spectral molecular ion pattern is consistent with an isomer of a
(
8 2 2
chlorophenol dimer having a C12H O Cl composition.
reactions catalyzed by CCPO now include H
O
2 2
-dependent deha-
(27) Reddy, C. M.; Xu, L.; Drenzek, N. J.; Sturchio, N. C.; Heraty, L. J.;
Kimblin, C.; Butler, A. J. Am. Chem. Soc. 2002, 124, 14526.
logenation of mono- and trihalophenols, the exact opposite reaction
type to its primary biological function of chlorination.4
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