Peroxygenase and oxidase activities of dehaloperoxidase-hemoglobin from Amphitrite ornata

Article abstract of DOI:10.1021/ja500293c

The marine globin dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H₂O₂-dependent oxidation of monohaloindoles, a previously unknown class of substrate for DHP. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H₂O₂, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H₂O₂ also yielded 5,5′-Br₂-indigo above the expected reaction stoichiometry under aerobic conditions, and O₂-concentration studies demonstrated dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5′-Br₂-indigo, and together suggest a newly identified oxidase activity for DHP.

Full text of DOI:10.1021/ja500293c

Article
pubs.acs.org/JACS
Open Access on 05/02/2015
Peroxygenase and Oxidase Activities of Dehaloperoxidase-
Hemoglobin from Amphitrite ornata
David A. Barrios, Jennifer D’Antonio, Nikolette L. McCombs, Jing Zhao, Stefan Franzen,
Andreas C. Schmidt, Leslie A. Sombers, and Reza A. Ghiladi*
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, United States
S
* Supporting Information
ABSTRACT: The marine globin dehaloperoxidase-hemoglobin
(DHP) from Amphitrite ornata was found to catalyze the H2O2-
dependent oxidation of monohaloindoles, a previously unknown class
of substrate for DHP. Using 5-Br-indole as a representative substrate,
the major monooxygenated products were found to be 5-Br-2-
oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed
that the oxygen atom incorporated was derived exclusively from
H2O2, indicative of a previously unreported peroxygenase activity for
DHP. Peroxygenase activity could be initiated from either the ferric
or oxyferrous states with equivalent substrate conversion and product
distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme
to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-
active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded
5,5′-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, and O2-concentration studies demonstrated
dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen
in the catalytic generation of 5,5′-Br2-indigo, and together suggest a newly identified oxidase activity for DHP.
INTRODUCTION
■
A longstanding question in catalysis is how structure plays a
role in influencing chemical reactivity. This is readily apparent
in heme proteins where O2-binding, oxygenase, oxidase,
peroxygenase, and electron-transfer reactions all occur at active
sites that often contain a surprising number of similarities, both
structural and, by consequence, mechanistic. For example, the
reactive heme species known as Compound I contains an
Fe(IV)-oxo (ferryl) and porphyrin π-cation radical and has
been implicated in the mechanisms of cytochrome c oxidase,1
cytochrome P450 monooxygenase,2 human indoleamine 2,3-
dioxygenase,3 the fungal peroxygenase AaeAPO,4 horseradish
peroxidase,5 and prostaglandin endoperoxidase synthase (cyclo-
oxygenase)6 and has even been shown to form in myoglobin.7
Despite the common intermediate, the chemical reactivity of
each of the aforementioned systems remains exquisitely
controlled by the protein structure6 to ensure maximum
intended function with a minimum of unintended cross
reactivity. How nature achieves this control for the selectivity
of function continues to be of interest, yet remains a significant
challenge to fully understand.
Our chosen platform for the elaboration of the structural
features and other determinants that impart specific discrete
functions to heme proteins is the enzyme dehaloperoxidase
(DHP), the coelomic hemoglobin from the marine worm
Amphitrite ornata.8−10 DHP has also been shown to possess a
biologically relevant peroxidase activity that is believed to have
arisen from the evolutionary pressure to overcome high levels
Figure 1. Reaction of DHP with trihalogenated phenols and hydrogen
peroxide yields quinone products.
of volatile brominated secondary metabolites that are secreted
as repellents by other infaunal marine organisms that co-inhabit
the benthic ecosystems within which A. ornata is found.10,11
The oxidative dehalogenation of 2,4,6-trihalophenol yielding
2,6-dihaloquinone (Figure 1) has been investigated for both
known isoenzymes of DHP (A and B).12−23 The DHP
peroxidase cycle can be initiated from both the ferric and
ferrous (or oxyferrous) oxidation states.17,22 When starting
from the ferric state, DHP appears to function via a Poulos−
Kraut type mechanism24 in which H2O2 reacts with a ferric
heme to form DHP Compound I,18,21 the iron(IV)-oxo
porphyrin π-cation radical species that is formally two electrons
oxidized relative to the ferric resting state. Compound I rapidly
converts in DHP to an iron(IV)-oxo heme center with an
amino acid radical that has been termed Compound ES by
analogy with cytochrome c peroxidase.13−15,18,21 These
peroxide-activated forms of DHP return to the ferric resting
Received: January 10, 2014
Published: May 2, 2014
© 2014 American Chemical Society
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a
state upon oxidation of substrate in what is likely two
consecutive one-electron steps.20 Thus, although the activated
heme of dehaloperoxidase can theoretically support a direct
oxygen atom transfer, no peroxygenase activity has been
reported for DHP.
Table 1. Enzyme-Catalyzed Reactivity of Haloindoles
enzyme
conversion (%)
Substrate Variation
DHP B Ferric
+ indole
24.1 ( 2.3)
41.1 ( 2.0)
48.1 ( 2.3)
34.4 ( 0.6)
46.1 ( 1.7)
26.0 ( 1.3)
37.5 ( 1.6)
34.0 ( 2.0)
In an effort to identify whether DHP is capable of oxygen
atom transfer chemistry, we have expanded the substrate scope
of our reactivity studies to include other halometabolites that
are native to the marine environment of A. ornata. Given the
diversity of naturally occurring organobromine compounds
found in benthic ecosystems (e.g., mono-, di-, and tribromo-
phenols, mono- and dibromovinylphenols, bromopyr-
roles),25−27 we have limited the current study to brominated
indoles, as these have been isolated from several species of the
Hemichordata phylum that cohabitate with A. ornata in coastal
estuaries,28,29 and may represent another family of chemical
deterrents evolutionarily targeted by A. ornata and DHP.
Furthermore, similar marine-related indoles and their deriva-
tives continue to gain interest as antimicrobial and/or
anticancer agents (e.g., meridianins, fascaplysin)30−33 and are
emerging as a new class of compounds for further study.
Herein, we have investigated the ability for DHP to catalyze
the oxidation of haloindoles in the presence of H2O2. The
reaction products were identified in the presence and absence
of labeled peroxide, and the results provide unequivocal
evidence that DHP is capable of a previously unreported
peroxygenase activity that is similar to that observed for the
peroxide shunt pathways of both P450 monooxygenase and
indoleamine 2,3-dioxygenase.2,34−36 The peroxygenase activity
was observed to be initiated from either the ferric or oxyferrous
states, with the enzyme returning to the oxyferrous state upon
completion of its activity due to an unusual product-driven
oxidase reaction that ultimately forms indigo derivatives as
products. The fact that the peroxygenase activity is initiated
from, and returns to, the oxyferrous state is likely related to the
primary function of DHP of serving as an oxygen transport
protein, yet also highlights the plasticity of the DHP active site
for supporting multiple enzymatic functions.
+ 4-Br-indole
+ 5-Br-indole
+ 6-Br-indole
+ 7-Br-indole
+ 5-F-indole
+ 5-Cl-indole
+ 5-I-indole
+ tryptophan
b
n.d.
Enzyme Variation
DHP B Oxyferrous
+ 5-Br-indole
DHP B (Y28/38F) Ferric
+ 5-Br-indole
DHP A Ferric
+ 4-Br-indole
+ 5-Br-indole
HRP
44.9 ( 4.8)
62.2 ( 2.5)
15.7 ( 0.7)
20.3 ( 1.7)
+ 5-Br-indole
hhMb
n.d.
n.d.
+ 5-Br-indole
Mechanistic Probes
DHP B Ferric
anaerobic + 4-Br-indole
anaerobic + 5-Br-indole
DHP B Ferric (+ 5-Br-indole)
+ 500 μM mannitol
40.1 ( 0.9)
53.3 ( 0.3)
46.2 ( 1.6)
45.8 ( 1.1)
46.1 ( 0.7)
45.0 ( 4.4)
23.2 ( 5.1)
+ 500 mM formate
c
+ SOD
+ 10% DMSO
+ 500 μM 4-BP
pH Effects
DHP B Ferric (+ 5-Br-indole)
pH 5
pH 6
pH 7
pH 8
63.4 ( 0.1)
54.7 ( 1.9)
48.1 ( 2.2)
34.4 ( 2.4)
RESULTS
■
DHP-Catalyzed Haloindole Reactivity with H2O2. The
hydrogen peroxide-dependent oxidation of haloindoles as
catalyzed by ferric WT DHP B at pH 7 was monitored by
HPLC. Reactions were initiated upon addition of 500 μM
H2O2 to a solution containing 10 μM enzyme and 500 μM
indole, incubated at 25 °C for 5 min and then quenched with
catalase. Reactivity was greatest with 5-Br-indole (Table 1), but
overall a <1.5-fold difference in reactivity was observed as the
position of the bromine substituent was varied (5-Br ≈ 7-Br >
4-Br > 6-Br). Indole itself also showed reactivity, albeit 2-fold
lower than 5-Br-indole. Reactivity across the halogen series of
5-X-indoles was also <2-fold varied and was observed as
follows: Br > Cl ≈ I > F ≈ H. These results suggest that neither
the sterics nor the electronics of the substrate play a significant
role in its conversion. No reactivity was observed when either
DHP (non-enzymatic control) or peroxide (non-oxidant
control) were excluded from the reaction.
a
Reaction conditions: [haloindole] = [H2O2] = 500 μM, [enzyme] =
10 μM, 5% MeOH in 100 mM KPi buffer at pH 7 (unless indicated),
b
c
25 °C, 5 min. n.d. = none detected. SOD = ∼2 U/μL.
the Compound ES species observed in WT DHP B,18 was only
1.3-fold more reactive with 5-Br-indole, which is approximately
in line with the ratio of the catalytic efficiencies (kcat/Km) of
these two enzymes for TCP oxidation (∼1.6).18 WT DHP B
was also found to be ∼2.4-fold more reactive than WT DHP A,
which is nearly identical to the ratio observed (2.6-fold) for
TCP oxidation.14 In the presence of 500 μM 4-bromophenol, a
known inhibitor of TCP oxidation,37 a 2-fold attenuation in
reactivity was observed for 5-Br-indole.
Reactions performed in the presence of radical scavengers
(DMSO, sodium formate, D-mannitol) and superoxide
dismutase (SOD) all showed nearly equivalent 5-Br-indole
reactivity when compared to the reaction run in their absence.
No changes in product distribution were observed by HPLC
(vide infra) in the presence of these radical scavengers.
Reactions performed anaerobically under N2 showed no
decrease in reactivity, consistent with previous studies of WT
The reactivity with 5-Br-indole was virtually identical for
oxyferrous DHP B compared to the ferric enzyme, indicating
that the reaction can be initiated from either the globin active
(FeII−O2) or peroxidase-active (FeIII) states, a result that has
been observed previously for TCP oxidation.17,22 The mutant
DHP B(Y28F/Y38F), which forms Compound I rather than
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Figure 3. ESI-MS total ion chromatograms obtained for the reaction
products 5-Br-2-oxindole (A: H218O, H216O2; C: H216O, H218O2) and
5-Br-3-oxindolenine (B: H218O, H216O2; D: H216O, H218O2). Reaction
conditions: [haloindole] = [H2O2] = 500 μM, [enzyme] = 10 μM, 5%
MeOH in 100 mM KPi (pH 7), 25 °C.
Product Distribution as a Function of H2O2 Concen-
tration. The distribution of products for studies performed
with 5-Br-indole (500 μM) was monitored as a function of
H2O2 concentration (Figure 2B). With one exception, the
formation of all products was linear as a function of peroxide
concentration up to 100 μM ([indole] > [H2O2]), yet reached
a plateau above 500 μM ([indole] < [H2O2]). The ratio of the
products formed was nearly constant regardless of the H2O2
concentration employed (Table S1). The lack of further
product formation under conditions of excess peroxide is in line
with previous observations that DHP enters a peroxidase-
attenuated state, Compound RH,8,13−15,18 under conditions of
low [substrate]. As the exception, 5-Br-2,3-dioxindole formed
linearly throughout the tested H2O2 concentration range,
suggesting a non-enzymatic process under conditions of excess
peroxide. A similar H2O2 concentration-dependent product
distribution profile was observed for studies performed with
indole (Figure S2B, Table S1).
Isotopically Labeled Oxygen Studies. As the observed
reactivity was O2-independent (anaerobic study, Table 1),
studies employing labeled H218O2 and H218O (90% and 98%
oxygen atom enriched, respectively) were performed with 5-Br-
indole and subsequently analyzed by LC-MS to determine the
source of the oxygen atom incorporation. The background-
subtracted total ion chromatograms (TICs) are shown in
Figure 3. The isotopic distributions for the major products
observed, 5-Br-2-oxindole and 5-Br-3-oxindolenine, were
determined using previously established methods.36,38 Products
were identified by the respective m/z of the (M + H)+ ion and
retention time as compared to available standards. In the
absence of an 18O source, both 5-Br-2-oxindole (m/z: 212,
99.2%; 214:100%) and 5-Br-3-oxindolenine (m/z: 210, 96.9%;
212:100%) exhibited masses with the expected ca. 1:1 isotopic
distribution for bromine (Table S2). In the presence of H218O
and unlabeled H2O2, neither product exhibited an increase in
mass: 5-Br-2-oxindole (m/z: 212, 99.8%; 214, 100%; Figure
3A) and 5-Br-3-oxindolenine (m/z: 210, 100%; 212, 99.4%;
Figure 3B). The parent ions also exhibited their expected ∼1:1
relative abundance, and thus solvent water was ruled out as the
source of the oxygen atom incorporated. These same
experiments employing H218O2 and unlabeled H218O showed
a clear increase to higher mass for both products by 2 Da: 5-Br-
2-oxindole (m/z: 212, 6.1%; 214, 100%; 216, 75.8%; Figure
Figure 2. (A) HPLC chromatogram of the reaction of 5-Br-indole
(500 μM) with DHP B (10 μM) in the presence of H2O2 (500 μM) at
25 °C (5% MeOH in 100 mM KPi, pH 7). The reaction was quenched
upon addition of catalase and subjected to HPLC analysis as described
in the text. (B) Product distribution for the reaction described in panel
A under variable [H2O2].
DHP B and TCP that demonstrated an O2-independent
reaction pathway.14,17 Finally, studies performed with horse-
radish peroxidase (HRP) or horse heart myoglobin (Mb), the
respective archetypes of the peroxidase and globin super-
families, showed no reactivity with 5-Br-indole substrate.
Identification of Reaction Products by HPLC. A
representative HPLC trace at 242 nm is shown in Figure 2A
for studies performed with 5-Br-indole (1). Products were
identified by their characteristic retention time and electronic
absorption spectrum as compared to authentic samples (Figure
S1), by similarity to those of their respective unsubstituted
analogue when the halogenated versions were not commercially
available,36 and/or by mass spectrometry (vide infra). Two
major products were identified: 5-Br-2-oxindole (1c, 44%) and
5-Br-3-oxindolenine (1a, ∼47% as determined from mass
balance). Minor products were also noted: the doubly
oxygenated products 5-Br-2,3-dioxindole (1b, 2.9%) and 5-Br-
3-hydroxy-2-oxindole (1d, 3.7%), the pyrrole ring-opened
product N-(4-Br-2-formylphenyl)formamide (1e, 0.6%), and
the indigo derivative (E)-5,5′-dibromo-[2,2′-biindolinylidene]-
3,3′-dione (1f, ∼2%).
Control reactions with 5-Br-2-oxindole as the substrate under
the same reaction conditions showed no further chemistry,
suggesting no secondary oxidation products were derived from
this species. 5-Br-3-oxindole was not observed directly, with its
oxidation likely giving rise to the observed 5-Br-3-oxindolenine
and/or secondary oxidation products (vide infra). A similar
product distribution was observed for studies performed with
indole (Figure S2A).
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3C) and 5-Br-3-oxindolenine (m/z: 210, 4.7%; 212, 100%; 214,
89.5; Figure 3D). The results showed a 97+% 18O enrichment
(normalized), providing unequivocal evidence that the oxygen
atom was derived from H2O2. The minor dioxygenated
products were of too low concentration to be observed, and
their origins were not pursued further at the time.
between free DHP (no added indole substrate) and the various
indole substrates is larger than observed in previous work in
aqueous solution.
Neither indole nor bromoindoles (4-, 6-, or 7-Br) caused a
significant perturbation in the high-frequency region (Figure
S3) when compared to the DHP only sample. However, 5-Br-
indole exhibited an increase in the core size marker bands (ν3 =
1494 cm−1; ν2 = 1569 cm−1; ν10 = 1630 cm−1) attributed to
5cHS heme and a decrease in those for 6cHS heme (ν3 = 1477
cm−1; ν2 = 1563 cm−1). As such, the spectra for the 5-
substituted halogen series were investigated (Figure 4A). A
decrease in the modes assigned to the 6cHS heme population
and an increase in those for 5cHS heme that follows the
halogen series (I > Br > Cl > F) was observed.
In studies with indole (m/z: 134, 100%; Table S2), the
oxygen incorporated into the respective 2-oxindole product was
derived from H218O2 (m/z: 134, 10.1%; 136, 100%; ∼100%
normalized 18O enrichment) and not from H218O (m/z: 134,
100%; 136, 0.8%), consistent with the results from above for 5-
Br-indole. However, the results for the 3-oxindolenine product
showed some scrambling of the label. Experiments run in
H218O2 and unlabeled water exhibited 51.8% 18O enrichment
(normalized), whereas those run with H218O and unlabeled
water yielded minimal label incorporation (5.5%). Reactions
performed with both H218O2 and H218O showed 60.1% 18O
enrichment. The reasons for why the 18O enrichment for 5-Br-
3-oxindolenine was nearly quantitative versus ∼50% for 3-
oxindolenine for experiments performed under identical
reaction conditions and work-up times remain unclear.
Resonance Raman Studies. Resonance Raman spectra
were collected of ferric WT DHP B in the presence of 10 equiv
haloindoles in 10% MeOH/100 mM KPi (v/v) at pH 7.
Compared to aqueous buffer, there is a significant increase in
the 6-coordinated high spin (6cHS) heme Fe in 10% MeOH
buffer solution. The red spectrum in Figure 4A shows that the
heme Fe is nearly 100% 6cHS with H2O bound to the heme Fe,
while in aqueous solution there is a mixture of 5-coordinated
high spin (5cHS) and 6cHS metaquo heme as observed in
previous work.37 Presumably, this arises from a destabilization
of the distal histidine (His55) in the solvent exposed (or
“open”) conformation in the 10% MeOH buffer solution. The
fact that His55 appears to favor the internal or “closed”
conformation means that the contrast shown in Figure 4A
Similar observations for halophenols have been noted,37,41
where their binding to DHP has been shown to perturb the
relative population of 5cHS vs 6cHS high spin ferric heme.
Specifically, 4-halophenols, known inhibitors of DHP,37 bind in
the distal pocket, displacing the distal histidine (His55) into an
“open” or solvent exposed confirmation. This limits the
availability of His55 to stabilize an iron-bound water ligand
through hydrogen bonding, thus shifting the relative population
from primarily 6cHS heme to predominantly 5cHS heme, with
the magnitude of the shift correlating with the size of the
halogen atom.37,41 We envision a similar process occurring here
with 5-haloindoles, where their binding to the active site of
DHP results in the displacement of the distal histidine, loss of
the water ligand, and increase in the 5cHS heme population. As
shown in Figure 4B, the geometry optimized structure for 5-Br-
indole binding to DHP B, where the indole occupies the space
right above the heme iron (i.e., the monohalophenol inhibitor
site), is consistent with this hypothesis. By contrast, the
geometry optimized structure shown in Figure 4C shows that
the DHP B active site can accommodate a 6cHS heme when 7-
Br-indole is bound off of the α-edge of the heme (i.e., the
trihalophenol substrate site). These calculations show that 5-
bromo- and 7-bromoindole fit in the inhibitor and substrate
binding sites, respectively, consistent with the results from the
resonance Raman spectroscopic studies.
5-Haloindole Binding Studies. The electronic absorption
spectra of ferric DHP B in the presence of 50 equiv 5-Br-indole
[402 (Soret), 508, 540 (sh), 585, 635 nm] and alone [407
(Soret), 507, 540 (sh), 635 nm] were recorded in 100 mM KPi
(pH 7) containing 10% MeOH (v/v) (Figure S4). Using the
observed hypochromicity and hypsochromic shift of the Soret
band upon 5-Br-indole binding, optical difference spectra were
recorded per literature protocol as a function of substrate
concentration (2.5−100 equiv; Figure S5).42 Analysis by
nonlinear regression provided a calculated Amax of 0.51, which
was in turn used to calculate α for the average ΔA for each [5-
Br-indole]. A second nonlinear regression plot provided an
apparent dissociation constant (Kd) of 150 10 μM. Similarly,
the Kd values were experimentally determined for the other 5-
substituted halogens (5-Cl-indole: 317 23 μM; 5-I-indole: 62
10 μM). No significant changes in the optical difference
spectra were observed for 5-F-indole even at the highest
concentrations tested, and as such its binding affinity was
suggested to be weaker than 5-Cl-indole. Overall, a trend in Kd
was observed of increasing binding affinity as the size of the
halogen atom was increased: F < Cl < Br < I. For the latter
three indoles whose Kd values were experimentally determined,
all exhibited higher affinity for DHP than 4-bromophenol (Kd
∼ 1.2 mM),37 a known inhibitor of the enzyme.
Figure 4. (A) Resonance Raman spectra of 5-X-indole (500 μM; X =
F, Cl, Br, I) complexes of DHP B (50 μM) in 10% MeOH/100 mM
KPi (v/v) at pH 7. (B) Geometry optimized structure for 5-Br-indole.
The 5-bromoindole model was obtained by replacement of 4-
bromophenol in the PDB 3LB2 structure.37 In this model, the
bromine atom is located in the Xe binding site39 as observed in the 4-
bromophenol structure. (C) Geometry optimized structure for 7-Br-
indole. The native substrate 2,4,6-tribromophenol (2,4,6-TBP) in the
structure PDB 4HF640 was substituted with 7-bromoindole. Since
diatomic O2 was observed bound to the heme Fe in that structure, the
bound O2 was preserved in this model.
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Stopped-Flow Studies with Ferric DHP B. Double-
mixing stopped-flow UV−vis spectroscopic methods were used
to investigate the reaction of 5-Br-indole with H2O2-activated
DHP, preformed either as Compound ES (WT DHP) or
Compound I [DHP B(Y28F/Y38F)]. Ferric WT DHP B (10
μM) was reacted with 10 mol equiv of H2O2 at pH 7, incubated
for 350 ms to allow for the maximum accumulation of
Compound ES,14 and subsequently mixed with 10, 25, or 50
equiv of 5-Br-indole. When reacted with 10 equiv substrate
(Figure 5), the preformed Compound ES [UV−vis: 418
(Soret), 545, 583 nm] was rapidly reduced [kobs = (2.54
0.05) × 105 M−1 s−1] to a species whose spectral features we
attribute to substrate- (or possibly product-) bound ferric DHP
B [406 (Soret), 508, 540 (sh), and 633 nm] (see Figure S4 for
comparison). For substrate concentrations ≥25 equiv 5-Br-
indole, no Compound ES was observed. Rather, it was rapidly
reduced within the mixing time of the stopped-flow apparatus
(<2.5 ms), and only the ferric enzyme was observed initially.
For all substrate concentrations examined, the ferric enzyme
was found to convert to a species [418 (Soret), 544, 577, 590
(sh) nm] that is similar, but not identical, to Compound ES/II
(these two species being indistinguishable by optical spectros-
copy for DHP17,18) and is described as indole-derived ferryl
DHP (vide infra). This was likely attributable to the reaction of
the initially produced ferric enzyme with the excess H2O2
employed in this study. At longer observation times, a new
species [414 (Soret), 540, 577 nm; kobs = (1.39 0.08) × 10−2
s−1] was noted whose spectral features matched those of a
mixture of oxyferrous DHP B and the ferric enzyme.14,17 Under
these conditions of excess substrate, Compound RH, the stable
form of DHP that forms from H2O2 activation in the absence of
substrate, was not observed.
The 5-Br-indole reactivity studies performed with preformed
Compound I using DHP B(Y28F/Y38F) were qualitatively the
same as those reported above for preformed Compound ES
using WT DHP B (Figure S6) with the exception that no
activated enzyme (i.e., preformed Compound I [406 (Soret),
528, and 645 nm])18 was observed. Rather, substrate- (or
possibly product-) bound ferric DHP B(Y28F/Y38F) [401
(Soret), 508, 540 (sh) nm] was the first spectrum recorded,
and a conversion to oxyferrous DHP B(Y28F/Y38F) [415
(Soret), 542, 578 nm] was noted after 697 or 120 s for
reactions performed with 10 or 50 equiv 5-Br-indole,
respectively.
The main observations from these studies employing ferric
DHP B were (i) both Compound ES and Compound I were
rapidly reduced by 5-Br-indole to the ferric enzyme, and (ii)
ferric DHP was further reduced to oxyferrous DHP. Reduction
of ferric DHP to the oxyferrous form has been previously
observed with dichloroquinone,13,14,17 the product of trichlor-
ophenol oxidative dehalogenation by DHP, which suggested
that a reaction product related to the 5-Br-indole reactivity
described above may have led to a similar product-driven
reduction chemistry being observed.
Stopped-Flow Studies with Oxyferrous DHP B. As
previously reported, oxyferrous DHP is only activated toward
reactivity with H2O2 (forming Compound II)17 in the presence
of 1 equiv TCP substrate.17,22 In the absence of substrate, a
slight bleaching of the Soret band and/or long time scale
conversion to Compound RH have been noted, but no fast
time scale reactive intermediates were observed. Such substrate-
dependent activation of DHP was investigated here with indole
substrates using stopped-flow methods. Upon rapid mixing of a
Figure 5. Kinetic data obtained by optical spectroscopy for the
reaction of preformed Compound ES with 5-Br-indole. (A) Stopped-
flow UV−vis spectra of the double-mixing reaction of preformed DHP
B Compound ES (10 μM), itself formed in an initial mixing step from
ferric DHP reacted with a 10-fold excess of H2O2 in an aging line for
350 ms, with a 10-fold excess of 5-Br-indole at pH 7.0 (800 scans over
83 s). Inset: The single wavelength (418 nm) dependence on time
obtained from the raw spectra and its fit with a superposition of the
calculated spectral components. (B) Calculated spectra of the four
reaction components derived from the SVD analysis: Compound ES
(black), ferric DHP B in the presence of excess 5-Br-indole (blue),
ferryl DHP in the presence of 5-Br-indole (red), and a mixture of
oxyferrous and ferric DHP B (purple). (C) Time dependences of the
relative concentrations for the four components shown in the middle
panel as determined from the fitting of the spectra in the top panel.
solution of oxyferrous DHP B preincubated with 2.5 equiv of 5-
Br-indole with 5 equiv of H2O2, substantial spectral changes
were observed (Figure 6). The oxyferrous form converted to a
species whose spectral features [420 (Soret), 544, 584 nm; kobs
= (3.30 0.01) × 10−2 s−1] matched those of Compound II
formed from our previous identification of this species when
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Figure 7. Kinetic data obtained by optical spectroscopy for the
reaction of preformed DHP B Compound II with 5-Br-indole. (A)
Stopped-flow UV−vis spectra of the double-mixing reaction between
preformed DHP B Compound II (10 μM) and 25 equiv 5-Br-indole at
pH 8.0 (800 scans over 83 s). DHP B Compound II was itself formed
from an initial reaction between oxyferrous DHP B preincubated with
1 equiv trichlorophenol and 10 equiv H2O2 and reacted for 85 s prior
to the second mix with 5-Br-indole. (B) Experimentally obtained
spectra for Compound II derived from TCP (black, t = 2.5 ms), and
Compound II observed in the presence of 5-Br-indole (t = 30 s).
Overall, 5-Br-indole was found to activate oxyferrous DHP
toward reactivity with H2O2 via a Compound II intermediate.
To further investigate the reactivity of DHP B Compound II,
sequential double-mixing stopped-flow studies were employed
to monitor the reaction of preformed Compound II with 5-Br-
indole substrate. Oxyferrous DHP B containing 1 equiv TCP
was reacted with 10 mol equiv of H2O2 at pH 8, incubated for
85 s to allow for the maximum accumulation of Compound II
[420 (Soret), 545, 584 nm],17 and subsequently mixed with an
additional 10 equiv (Figure S7) or 25 equiv (Figure 7) of 5-Br-
indole. Conversion of the Compound II spectrum from the one
in the absence of indole to the one observed in the presence of
indole [420 (Soret), 545, 578, 584 (sh) nm] was noted in all
cases. The disappearance of indole-derived Compound II and
the formation of the ferric enzyme were linearly dependent on
the concentration of 5-Br-indole cosubstrate, yielding a
bimolecular rate constant of (8.15 0.26) × 102 M−1 s−1. At
concentrations of 5-Br-indole above 25 equiv, the ferric form of
the enzyme was further found to partially convert to oxyferrous
DHP (data not shown) as noted above for other experiments
with this substrate.
Figure 6. Kinetic data obtained by optical spectroscopy for the
reaction of oxyferrous DHP B with 5-Br-indole and hydrogen
peroxide. (A) Stopped-flow UV−vis spectra of the single-mixing
reaction between oxyferrous DHP B (10 μM) preincubated with 2.5
equiv 5-Br-indole and a 5-fold excess of H2O2 at pH 7.0 (800 scans
over 83 s). Inset: The single wavelength (418 nm) dependence on
time obtained from the raw spectra and its fit with a superposition of
the calculated spectral components. (B) Calculated spectra of the three
reaction components derived from the SVD analysis: oxyferrous DHP
B (black), a mixture of oxyferrous DHP with 5-Br-indole (blue), and
DHP B Compound II (red). (C) Time dependences of the relative
concentrations for the three components shown in the middle panel as
determined from the fitting of the spectra in the top panel.
employing TCP as the substrate.17 However, when higher
equivalents of indole were employed, the intermediate observed
had spectral features that were similar [420 (Soret), 545, 578,
584 (sh) nm] (data not shown; vide infra), but not identical, to
authentic TCP-derived Compound II. We surmise that the
presence of the indole in the active site led to these minor
spectral differences. The indole-derived Compound II species
was found to convert at longer times to the ferric enzyme.
Reactivity Studies of 5-Br-3-oxindole. The primary
products of 5-Br-indole reactivity, identified to be 5-bromo-2-
oxindole and 5-bromo-3-oxindolenine (vide supra), were
investigated as the putative reductants that led to the observed
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μM ferric DHP [407 (Soret), 508 nm] incubated with 250 μM
5-Br-3-acetoxyindole (λmax = 280 nm) exhibited no spectral
changes (data not shown).
When the above enzymatic reaction was repeated under
anaerobic conditions, the first spectrum obtained after addition
of LE in a glovebox and subsequent removal to the
spectrophotometer (∼3 min) in a sealed cuvette showed new
features [426 (Soret), 558 nm] that closely matched those of
ferrous DHP B,17 suggesting reduction of the ferric enzyme by
5-Br-3-oxindole (Figure 8C). 5,5′-Br2-indigo formation (λmax
=
659 nm) was also observed. No other significant changes in the
spectral features were observed at longer times. The
concentration of 5,5′-Br2-indigo was estimated to be ∼8 μM,
or nearly stoichiometric with respect to the enzyme
concentration, under anaerobic conditions after 10 min.
Overall, nearly 4-fold greater 5,5′-Br2-indigo formation was
observed under aerobic (31 μM) versus anaerobic (8 μM)
conditions when DHP was present, both of which were higher
than the non-enzymatic aerobic control (0.7 μM).
O2-Consumption Studies. The reaction of 5-Br-3-
oxindole (500 μM), formed in situ from the hydrolysis of 5-
bromo-3-acetoxyindole by liver esterase (LE, 45 U), with ferric
WT DHP B (10 μM) at pH 7 was monitored for O2-
consumption using a dissolved oxygen probe. Prior to the
addition of LE, the oxygen concentration was determined to be
17.49 0.20 mg/L (547 6 μM) at room temperature. After 5
min from the addition of LE, the [O2] was 10.12 0.84 mg/L
(316 26 μM) yielding a Δ[O2] of 7.37 0.86 mg/L. The
non-enzymatic control exhibited Δ[O2] of 1.71 0.51 mg/L
after 5 min.
Figure 8. (A) UV−vis spectra obtained under anaerobic conditions of
5-Br-3-oxindole after addition of liver esterase to a solution of 5-Br-3-
acetoxyindole (∼250 μM) in 5% MeOH in 100 mM KPi (pH 7)
scanned at times indicated (aerobic). Inset: Spectral region showing
minimal 5,5′-Br2-indigo formation. (B) UV−vis spectra obtained
under aerobic conditions of 5-Br-3-acetoxyindole (∼250 μM) and
ferric DHP B (10 μM) in 5% MeOH in 100 mM KPi (pH 7) prior to
(black spectrum) and 0−5 min after the addition of liver esterase. (C)
UV−vis spectra obtained under anaerobic conditions of 5-Br-3-
oxindole (∼250 μM) and ferric DHP B (10 μM) from 3 to 12 min
after the addition of liver esterase. Inset: Spectral region showing 5,5′-
Br2-indigo formation.
Stopped-Flow Studies with 5-Br-3-oxindole. Single-
mixing stopped-flow UV−vis spectroscopic methods were used
to investigate the reaction of 25 equiv 5-Br-3-oxindole with 10
μM ferric WT DHP B (Figure 9). Upon their rapid mixing,
oxyferrous DHP B [417 (Soret), 543, 578 nm] formation was
observed after 8 s, in line with the reactivity studies of 5-Br-3-
oxindole described above. At longer observation times, the
features related to oxyferrous DHP remained, whereas a new,
broad feature ∼600 nm, suggestive of 5,5′-Br2-indigo, was also
observed to form. When repeated with oxyferrous DHP B
(Figure S8), the spectral features of the enzyme were
unchanged as oxyferrous and 5,5′-Br2-indigo formed at the
same rate (kobs = 0.021 0.001 s−1) as was previously observed
when starting with the ferric enzyme. These results suggested
that the reactivity with 5-Br-3-oxindole yielding 5,5′-Br2-indigo
could be initiated from either the ferric or oxyferrous states.
Under these conditions, no Compound RH was observed.
Double-mixing stopped-flow methods were also used to
investigate the reaction of 5-Br-3-oxindole with preformed
Compound ES (Figure S9). Ferric WT DHP B (10 μM) was
reacted with 5 mol equiv of H2O2 at pH 7, incubated for 350
ms to allow for the maximum accumulation of Compound
ES,14 and subsequently mixed with 25 equiv of 5-Br-3-oxindole.
After 8 s, Compound ES [UV−vis: 417 (Soret), 545, 590 nm]
was converted to a species whose spectral features we attribute
to oxyferrous DHP B [417 (Soret), 543, 578 nm]. At longer
observation times, the spectral features of oxyferrous DHP
persisted, while significant 5,5′-Br2-indigo formation was also
noted after 83 s. No Compound RH was observed.
formation of oxyferrous DHP in the above stopped-flow
studies. 5-Br-2-oxindole, unreactive in the HPLC studies above
as a substrate, was unable to reduce ferric DHP B even at 100
equiv (data not shown). Thus, our attention focused on 5-
bromo-3-oxindole, the precursor of 5-bromo-3-oxindolenine, as
a potential reductant.
In the absence of DHP, UV−vis spectroscopic monitoring of
a 250 μM solution of 5-Br-3-oxindole (λmax = 383 nm), formed
in situ from the hydrolysis of 5-bromo-3-acetoxyindole by
porcine liver esterase (LE, 45 U), under aerobic conditions
showed only a trace of 5,5′-Br2-indigo formation (λmax = 289
and 640 nm; Figure 8A) after 5 min, likely attributable to the
known (non-enzymatic) oxidation of 3-oxindole and derivatives
by O2.43 In the presence of DHP, however, the first spectrum
obtained after addition of LE (∼3 s) showed new features [415
(Soret), 543, 577 nm] that closely resembled those of
oxyferrous DHP B with a minor component of ferric enzyme
present, indicative of DHP reduction by 5-Br-3-oxindole and
subsequent binding of O2 under aerobic conditions (Figure
8B). New spectral features at 289, 370, and 659 nm,
corresponding to the formation of 5,5′-Br2-indigo, were also
noted. Full formation of oxyferrous DHP B [418 (Soret), 543,
577 nm] was seen after 2 min, however the reaction proceeded
to generate 5,5′-Br2-indigo throughout the entire 5 min
observation period. Using the molar absorptivity coefficient of
indigo (22 140 M−1 cm−1)44 for 5,5′-Br2-indigo, we estimate
the latter’s concentration after 5 min to be ∼31 μM, or 45-fold
greater, when formed in the presence of DHP versus the
absence of enzyme. As a control, the absorption spectrum of 10
DISCUSSION
■
The activity studies presented herein demonstrated that
dehaloperoxidase was able to catalyze the conversion of
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equivalent resides on an amino acid (tyrosyl radical) rather than
on the porphyrin ring.13,14,18 Our results show that DHP
Compound I and Compound ES are both capable of reacting
with haloindoles, with both being rapidly reduced back to the
ferric state (<200 ms for Compound ES, <2.5 ms for
Compound I). By comparison, the reaction of preformed WT
DHP Compound ES with TCP substrate under similar
conditions also reforms the ferric enzyme but is ∼102−104-
fold slower (18.5 s).13,14
While no mechanistic insight was derived from the stopped-
flow studies for the reduction of the ferryl species given the
apparent time scale of these reactions, a secondary reaction was
noted. Namely, the reduction of the ferric enzyme and
formation of oxyferrous DHP were observed. Two lines of
evidence suggested a product-driven reduction of ferric DHP:
first, we have previously observed for ferric DHP that its
reaction with H2O2 and TCP ultimately regenerates the
oxyferrous enzyme and that separately the product of that
reaction, dichloroquinone, is capable of reducing ferric DHP to
the oxyferrous state.13,14,17 While the details of that reaction
remain poorly understood, we proposed that the unusually high
redox potential of DHP (206 6 mV)14,16 helped facilitate the
reduction of the enzyme by the reaction products after the
initial peroxidase activity was completed.8 Second, Mauk and
co-workers recently described the reduction of ferric IDO to
ferrous IDO by 3-oxindole that was itself generated as a
product during the oxidation of indole by ferric IDO and
H2O2.36 It was these two precedents of product-driven
reduction of a heme protein that provided the motivation to
explore if 5-Br-3-oxindole was able to reduce ferric DHP, which
was, in fact, observed to be the case.
On the basis of the results obtained above, and through
modification of the previously established mechanisms for
peroxygenases,2,34−36 we propose the following catalytic cycle
for the in vitro hydrogen peroxide-dependent oxidation of
haloindoles by ferric DHP from A. ornata (Scheme 1): ferric
DHP reacts with 1 equiv H2O2, forming either Compound I
(step i) or Compound ES (step iii). The reaction of either of
these two ferryl species with haloindole leads to the
regeneration of the ferric enzyme (steps ii and iv for
Compounds I and ES, respectively), with incorporation of
the oxygen atom into indole yielding either 5-Br-2-oxindole or
5-Br-3-oxindole. The latter subsequently reacts with the ferric
enzyme, leading to oxyferrous DHP formation (v) and the
observed 5-Br-3-oxindolenine product. The initial step of
substrate reactivity can likely be viewed as hydrogen atom
abstraction to yield an indolyl substrate radical48,49 and an
Fe(IV)−OH that subsequently undergoes oxygen rebound to
generate indoxyl/oxindole; however, we cannot rule out other
possible mechanisms for oxygen atom insertion into an arene,50
including indole epoxidation via an electrophilic ferryl species.
Alternatively, we have previously proposed that, although
counter to monofunctional peroxidase conventions, a bifunc-
tional peroxidase-hemoglobin such as DHP may be able to
initiate a peroxidase catalytic cycle from the globin-active
oxferrous, and not ferric, state.8 Oxyferrous DHP reacts with 1
equiv H2O2 in the presence of haloindole substrate forming
Compound II (step vi), analogous to this substrate-dependent
activation that has been observed previously with TCP.17 At
this point, two possibilities must be considered. The first is that
oxindole formation occurs upon transfer of the oxygen atom
from Compound II, regenerating ferrous DHP which can
rebind O2 under aerobic conditions (not shown). In this
Figure 9. Kinetic data obtained by optical spectroscopy for the
reaction of ferric DHP B with 5-Br-3-oxindole. (A) Stopped-flow UV−
vis spectra of the double-mixing reaction of ferric DHP B (10 μM)
with a 25-fold excess of 5-Br-3-oxindole at pH 7.0 (800 scans over 83
s). 5-Br-3-oxindole was itself formed from an initial reaction between
5-Br-3-acetoxyindole and liver esterase in an aging line prior to the
second mix with ferric DHP B. (B) Experimentally obtained spectra
for ferric DHP B (black, t = 2.5 ms), oxyferrous DHP B (blue, t = 8 s),
and a mixture of oxyferrous DHP B and 5,5′-Br2-indigo (red, t = 83 s).
haloindoles, a previously unreported class of substrate for DHP
that is likely physiologically relevant given their production by
infaunal organisms that coinhabit the benthic ecosystems within
which A. ornata resides.28,29 Activity was observed for all
monohaloindoles studied, regardless of the type of halogen or
its position on the six-membered ring; however, tryptophan
itself was not found to be a substrate. Isotope labeling studies
showed that the oxygen atom incorporated into the major
(monooxygenated) products was derived exclusively from
hydrogen peroxide, analogous to the “peroxide-shunt” of
cytochrome P450s and indoleamine 2,3-dioxygenase.2,34−36 In
line with a peroxygenase mechanism, substrate reactivity readily
proceeded under anaerobic conditions as well as in the
presence of superoxide dismutase and radical quenchers. Such
peroxygenase chemistry had been previously unreported for
dehaloperoxidase with any substrate.
Activation of most ferric heme proteins by H2O2 generally
leads to Compound I formation, the Fe(IV)-oxo porphyrin π-
cation radical species that is two electrons oxidized compared
to the resting enzyme and has been implicated as the principal
reactive species in peroxidases,45 P450s,34,35 and other
hemoproteins.46,47 By contrast, ferric WT DHP forms
Compound ES, a species that like Compound I is also two-
electrons oxidized above resting, yet whose second oxidizing
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Scheme 1. Proposed Peroxygenase Cycle for Ferric and Oxyferrous Dehaloperoxidase B
pathway, the two-electron chemistry (Fe2+/Compound II) is
occurring in a similar fashion as in the pathway initiated from
the ferric state (Fe3+/Compound I or ES). Our results,
however, suggest that this pathway does not occur as
Compound II reduction clearly yielded the ferric enzyme.
The second possibility is that an initial one-electron process
occurs where the enzyme is reduced from Compound II to the
ferric state by haloindole (step vii) and that the subsequent
reactivity proceeds as described above for a typical peroxidase.
Overall, however, it is clear that peroxygenase chemistry can be
initiated from the globin-active oxyferrous state and that the 3-
oxindole reactivity leads back to this state as well.
The oxygen atom transfer chemistry strongly suggests that
the haloindole substrate must be in close proximity to the active
ferryl intermediate to form the observed products. The results
of the haloindole resonance Raman study can be interpreted in
light of the two known halophenol binding sites. The addition
of 5-X-indole (X = I, Br, Cl) to DHP led to an observed
increase in the 5cHS heme population. This is reminiscent of
monohalophenol binding, which has been shown to bind in the
distal pocket above the heme, and forces the distal histidine to a
solvent exposed conformation.10,37,41,51,52 This ultimately leads
to the loss of the iron-bound water ligand and an increase in the
5cHS heme population. We suggest a similar binding location
for the 5-haloindoles, as denoted in Figure 4B. By contrast,
none of the other bromoindoles (4-, 6-, or 7-) led to changes in
the resonance Raman spectrum. We interpret this result to
suggest that these substrates bind elsewhere in the distal cavity,
possibly in the recently identified tribromophenol binding site
that is located in the pocket above the α-edge of the heme
(Figure 4C).40,53 Tribromophenol binding to DHP also does
not perturb the resonance Raman spectrum of the ferric
enzyme.14 Given that two binding sites may be present,
structural studies may play an important role in deducing
whether concerted or nonconcerted (radical) mechanisms form
the basis for the observed peroxygenase activity in DHP.
Indole oxidation chemistry has been reported previously:
HRP is reactive toward both indole and derivatives (including
5-Br-3-indole acetic acid) via a peroxidase mechanism,49,54 and
a recent report has shown that engineered myoglobin is able to
catalyze the oxidation of indole to form indigo blue.55 Under
the conditions examined here, however, neither of these native
monofunctional archetypes of the peroxidase and globin
families, respectively, were reactive toward 5-Br-indole, a result
that highlights the unique reactivity of the dehaloperoxidase-
hemoglobin system. Indoleamine 2,3-dioxygenase has also been
shown to catalyze the oxidation of indole via a peroxygenase
mechanism, however the product distribution is different.36
Notably, the major products of indole oxidation with IDO are
2- and 3-oxindole in a ∼3:1 ratio when [indole] > [H2O2], vs
an ∼1:1 ratio of 2-oxindole and 3-oxindolenine for DHP. We
surmise that the peroxidase activity of DHP led to the oxidation
of 3-oxindole to yield the observed 3-oxindolenine product.
Further, unlike IDO that was found to be reactive with both 2-
and 3-oxindole, DHP exhibited no reactivity toward 5-Br-2-
oxindole, a result that suggests that the minor dioxygented
products observed must be derived exclusively from the 5-Br-3-
oxindole pathway, and likely in a similar fashion that has been
reported for P450s.50,56 Other minor differences between these
two enzymes in the product distribution of the dioxygenated
products are also evident, but the secondary reactions that led
to them in DHP were not further explored.
The investigation of 5-Br-3-oxindole as the reducing agent
that led to oxyferrous DHP formation also revealed an
unexpected activity for DHP: not only did the aerobic reaction
of ferric DHP with 5-Br-3-oxindole (in situ generated)
immediately form oxyferrous DHP but also gave 5,5′-Br2-
indigo in an amount that exceeded the predicted reaction
stoichiometry. Under anaerobic conditions, the yield of 5,5′-
Br2-indigo was near stoichiometric with respect to the enzyme
concentration, showing that excess O2 was obligatory for the
catalytic generation of the indigo species by DHP. The non-
enzymatic, aerobic oxidation of 5-Br-3-oxindole only produced
a trace of 5,5′-Br2-indigo over the same time scale, showing that
DHP was also obligatory for the catalytic generation of the
indigo species. Moreover, O2 consumption studies showed a
significant decrease in dissolved oxygen concentration beyond
that exhibited by the background (non-enzymatic) reaction,
providing confirmatory evidence that the reaction was indeed
consuming molecular oxygen. Taken together, the require-
ments for both dioxygen and DHP in the catalytic formation of
5,5′-Br2-indigo from the respective oxindole demonstrated an
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HPLC to ensure that they had not degraded. Aliquots were stored on
ice during use. Solutions of H2O2 were prepared fresh daily and kept
on ice until needed. The concentration was determined by UV−vis
(ε240 = 46 mM−1 cm−1).57 Ferric and oxyferrous samples of WT DHP
B, DHP B (Y28/38F), and WT DHP A were expressed and purified as
previously reported.13,14,18 Enzyme concentration was determined
spectrophotometrically using εSoret = 116,400 mM−1 cm−1 for all
isoenzymes.14 Lyophilized horseradish peroxidase and horse heart
myoglobin were purchased from Sigma-Aldrich and stored at −20 °C
until utilized. Anaerobic studies were performed as described above in
an MBraun Lab Master 130 nitrogen-filled glovebox using argon
degassed solutions of buffer, peroxide, substrate and enzyme.
Enzyme Assay Protocol. Reactions were performed in triplicate
at pH 7 in 5% MeOH in 100 mM KPi at 25 °C. Buffered solutions
(total reaction volume 250 μL) of DHP (10 μM final concentration)
and (halo)indole (500 μM final concentration) were premixed, and
then the reaction was initiated upon addition of H2O2 (50, 100, 250,
500, or 1000 μM final concentrations). Experiments were performed
in the presence of D-mannitol (500 μM), superoxide dismutase (SOD)
(∼2 U/μL), and DMSO (10% v/v). For the studies with sodium
formate, a buffered solution (100 mM KPi; 500 mM sodium formate)
at pH 7 was utilized. After 5 min, reactions were quenched with excess
catalase. A 100 μL aliquot of reaction sample was diluted 10-fold with
900 μL of 100 mM KPi (pH 7). Diluted samples were analyzed using a
Waters 2796 Bioseparations Module coupled with a Waters 2996
Photodiode Array Detector and equipped with a Thermo-Scientific
ODS Hypersil (150 mm × 4.6 mm) 5 μm particle size C18 column.
Separation of observed analytes was performed using a linear gradient
of binary solvents (solvent A, H2O containing 1% trifluoroacetic acid;
solvent B, MeCN). Elution was performed using the following
conditions: (1.5 mL/min A:B) 95:5 to 5:95 linearly over 10 min; 5:95
isocratic for 2 min; 5:95 to 95:5 linearly over 1 min, and then isocratic
for 4 min. Data analysis was performed using the Empower software
package (Waters Corp.). Calibration curves for all indoles, and
available products were performed using serial dilutions of
commercially available analytes to determine the amount of substrate
conversion.
LC-MS Studies. Experiments were analyzed using a 6210 LC-TOF
mass spectrometer (Agilent Technologies, Santa Clara, CA). Analyte
separation was performed using the same conditions and column as
the HPLC studies with the exception of solvent A (water with 0.1%
formic acid). Samples were analyzed using electrospray ionization in
positive ion mode to provide observation of the [M + H]+ species.
Spectra were collected each second while scanning in the range from
100−1000 m/z. Data analysis was performed using Agilent software.
Quantitation of the amount of 18O-labeled incorporated was
performed using previously established methods.36,38 In the H218O2
studies, an aliquot of 2.15% (w/w) solution of 90% enriched H218O2
was diluted 3-fold to provide a peroxide solution at 20 mM
concentration. A final reaction volume (5% MeOH in 100 mM KPi,
pH 7) of 250 μL containing 10 μM enzyme, 50 equiv of substrate, and
50 equiv of labeled peroxide was allowed to react for 5 min before
quenching with catalase. For the H218O studies, stock solutions of the
reactants ([enzyme] = 120 μM; [H2O2] = 12.5 mM) in unlabeled
water were kept at sufficiently high concentrations to allow for the
98% enriched H218O to be diluted to ∼89% in the final reaction
mixture. Labeled water (207.5 μL) was charged with 21 μL enzyme, 10
μL H2O2, and 12.5 μL substrate (in MeOH) and reacted for 5 min and
then quenched with catalase. A 20 μL aliquot of undiluted reaction
mixtures was injected for LC-MS analysis.
apparent oxidase activity of dehaloperoxidase-hemoglobin. As
to the mechanism of the oxidase activity, based upon these
initial observations we propose that, in analogy with the
cytochrome P450 catalytic cycle, the one-electron reduction of
oxyferrous DHP by 5-Br-3-oxindole yields a ferric-peroxo
(Compound 0) intermediate that undergoes O−O bond
cleavage to yield Compound ES. The reaction of Compound
ES with 5-Br-3-oxindole, as supported by the stopped-flow
UV−vis spectroscopic studies, leads to the formation of
oxyferrous DHP, likely through a ferric intermediate that is
reduced (as above) to the ferrous enzyme and subsequently
binds dioxygen, thereby enabling the next round of the oxidase
catalytic cycle.
CONCLUSION
■
In summary, DHP has now been shown to possess
peroxygenase and oxidase activities in addition to its previously
identified O2-binding and peroxidase functions. What appears
to control for each activity is a complex set of parameters that
depends, in part, on the nature of the substrate as well as the
dual identity of DHP as both an enzyme and a globin: (i) the
relatively high redox potential and protein fold of DHP,
hallmarks of the globin family that impart reversible O2-
binding, also lead to its ease of reduction by 3-oxidole, thereby
leading to DHP oxidase activity; (ii) an active site pocket
capable of halophenol and haloindole binding, unnecessary for
an O2 transport globin, supports both peroxidase and
peroxygenase activities, respectively; (iii) a highly flexible distal
histidine ligand capable of both stabilizing bound O2 and
serving as a general acid/base catalyst; and (iv) a sterically
hindered substrate in 2,4,6-tribromophenol is a DHP
peroxidase substrate, whereas the unhindered (at the C2 and
C3 positions) haloindole is susceptible to DHP peroxygenase
activity, possibly due to the ability of the ferryl intermediate to
perform an electrophilic attack on the latter. Other factors, such
as the reactivity of Compound I vs Compound ES, the redox
potential of the substrate or its pKa, likely are important
determinants of enzymatic activity as well.
The diverse range of functions exhibited by DHP highlights
the versatility of this heme protein. The trade-off in being a
more versatile enzyme is that DHP exhibits catalytic efficiencies
below its monofunctional counterparts, albeit far greater than
what is noted as side reactivity in other systems. This increased
versatility at the expense of high catalytic efficiency is possibly
attributable to the nature of the DHP active site in that it lacks
the additional structural features normally observed in more
specialized systems that tune the protein toward one specific
discrete function. Thus, DHP provides a platform for probing
in detail how nature evolved structure−function relationships in
ancestral multifunctional systems of competent catalytic activity
from lower organisms to achieve the complexity necessary for
specialization of function and increased activity observed in the
monofunctional systems of higher organisms.
Stopped-Flow UV−vis Studies. Optical spectra were recorded
using a Bio-Logic SFM-400 triple-mixing stopped flow instrument
coupled to a rapid scanning diode array UV−vis spectrophotometer.
The temperature was maintained at 20 °C with a circulating water
bath, and all solutions were prepared in 5% MeOH in 100 mM KPi
(variable pH). Data were collected (900 scans total) over a three-time
domain regime (2.5, 25, and 250 ms; 300 scans each) using the Bio
Kinet32 software package (Bio-Logic). All data were evaluated using
the Specfit Global Analysis System software package (Spectrum
Software Associates) and fit to exponential functions as one-step/two-
EXPERIMENTAL SECTION
■
Materials and Methods. Isotopically labeled H218O2 (90% 18O-
enriched) and H218O (98% 18O-enriched) were purchased from Icon
Isotopes (Summit, NJ). Acetonitrile (MeCN) was HPLC grade and all
other chemicals were purchased from VWR, Sigma-Aldrich, or Fisher
Scientific and used without further purification. UV−vis spectroscopy
was performed on a Cary 50 UV−vis spectrophotometer. Stock
solutions (10 mM) of all indoles were prepared in MeOH, stored in
the dark at −20 °C until needed, and were periodically screened by
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Journal of the American Chemical Society
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species, two-step/three species or three-step/four species irreversible
mechanisms where applicable. For data that did not properly fit these
models, experimentally obtained spectra at selected time points shown
in the figure legends are shown. Data were baseline corrected using the
Specfit autozero function.
5% MeOH in 100 mM KPi at pH 7 (<1 min) in a quartz cuvette.
Reactions were initiated by the addition of porcine liver esterase (LE,
45 U), and spectra collected at the times indicated. Anaerobic studies
were initiated in the glovebox, the quartz cuvette was sealed from the
atmosphere, and the spectra were obtained on the benchtop.
O2-Consumption Studies. All experiments were performed in
triplicate with a Fisher Scientific AR 60 dissolved oxygen Accumet
BOD probe that was standardized in dH2O prior to use. Solutions of
5-Br-3-acetoxyindole in methanol were freshly prepared prior to each
experiment. Ten mg of liver esterase was dissolved in 500 mL of 100
mM KPi buffer and kept on ice. A 5 mL solution was made containing
10 μM ferric WT DHP B and 500 μM 5-Br-3-acetoxyindole in 5%
MeOH in 100 mM KPi buffer (pH 7), and the meter was stabilized in
the solution. A 50 μL aliquot of porcine liver esterase (∼1 U/μL) was
added to the 5 mL solution, and the final O2 concentration was
measured after 6 min.
Experiments were performed in single-mixing mode where enzyme
at a final concentration of 10 μM was reacted with 2.5−25 equiv of
H2O2. For substrate preincubation studies, the enzyme solution also
contained substrate (2.5−50 equiv). Double mixing experiments were
performed using an aging line prior to the second mixing step to
observe Compound ES/Compound I reactivity with 5- or 7-Br-indole
substrate (2.5−50 equiv). For studies with 5-Br-3-oxindole, a solution
of 5-Br-3-acetoxyindole in 100 mM potassium phosphate buffer was
prepared from a 10 mM stock solution in pure methanol for final
concentrations of 100, 250, or 500 μM. The 5-Br-3-acetoxyindole
solutions were loaded into the stopped-flow syringe, and ∼1 mg (20
U) of liver esterase was directly added to the syringe and mixed
thoroughly, and then the solution was loaded into the apparatus.
Control experiments were also performed as above in the absence of
hydrogen peroxide.
ASSOCIATED CONTENT
■
S
* Supporting Information
UV−vis spectra of 1a−1f and 2a−2f, reactivity studies of DHP
with indole and product distribution tables, resonance Raman
spectra of indole and 4-, 5-, 6-, and 7-Br-indole with DHP B,
optical difference spectra and titration curves of 5-X-indole
binding to DHP B, additional kinetic data and stopped-flow
studies of DHP B with 5-Br-indole and 5-Br-3-oxindole, mass
spectrometric analyses of isotope labeling studies, and the
charge set and templates for the NAMD topology file. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Resonance Raman Studies. Samples were prepared with final
concentrations of 50 μM WT DHP B and 500 μM substrate in 100
mM KPi (pH 7) containing 10% MeOH (v/v) and then transferred to
a 5 mm diameter glass NMR tube. Spectra were obtained by Soret
band excitation using a Coherent Mira 900 titanium sapphire
(Ti:sapphire) laser. The Ti:sapphire laser was pumped using a
Coherent Verdi 10 frequency doubled diode pumped Nd:vanadate
(Nd:VO4) laser producing 10 W at 532 nm. The beam generated was
sent through a Coherent 5-050 doubler to generate a normal working
range of 400−430 nm for Soret band excitation of both DHP only and
the DHP/indole complexes. The beam was collimated and
cylindrically focused to a vertical line of ∼0.5 mm on the sample.
Laser power at the sample was 60 mW. Scattered light was collected
with a Spex 1877 triple spectrometer equipped with a liquid nitrogen-
cooled CCD detector controlled by Spectramax software.
AUTHOR INFORMATION
■
Corresponding Author
Reza_Ghiladi@NCSU.edu
Geometry Optimization Studies. The native substrate 2,4,6-
tribromophenol (2,4,6-TBP) in the structure PDB 4HF640 was
substituted with 7-bromoindole. Since diatomic O2 was observed
bound to the heme Fe in that structure, the bound O2 was preserved in
this model. The 5-bromoindole model was obtained by replacement of
4-bromophenol in the PDB 3LB2 structure.37 In this model, the
bromine atom is located in the Xe binding site39 as observed in the 4-
bromophenol structure. The parameters for the 5-bromo and 7-
bromoindole molecules were obtained by modification of the tyrosine
amino acid from the CHARMM force field implemented in the
NAMD code58,59 with a charge set calculated using electrostatic
potential fitting60 implemented in the density functional theory (DFT)
code DMol3.61,62 The DFT calculations were conducted using a
double numerical basis set with polarization functions and the PBE
functional.63,64 The charge set and templates for the NAMD topology
file are provided in the Supporting Information. The bond stretching,
bending, and torsional parameters for the C−Br bond and associated
angles and dihedrals were obtained from the structure and an estimate
of the relative bond stretching force constant. Visual molecular
dynamics (VMD) was used for visualization and generation of the
figures.65
Haloindole-Binding Studies. Adapted from previously published
protocols,42 stock solutions of 2 mM 5-X-indole (X = F, Cl, Br and I)
in MeOH were prepared. The UV−vis spectrophotometer was
blanked with 10 μM ferric WT DHP B in 100 mM KPi (pH 7)
containing 10% MeOH. Spectra were then acquired in the presence of
2.5, 5, 10, 25, 50, and 100 equiv of the indole substrates while
maintaining both constant enzyme and MeOH concentrations.
Analysis by nonlinear regression using the GraFit software package
(Erithacus Software Ltd.) of the experiments performed in triplicate
provided a calculated Amax, which was in turn used to calculate α for
the average ΔA for each indole concentration. A nonlinear regression
plot provided the reported apparent Kd values.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was supported by a National Science Foundation
CAREER Award (CHE-1150709 to R.G.), the North Carolina
State University Molecular Biotechnology Training Program
through an NIH T32 Biotechnology Traineeship grant (J.D.),
and the Army Research Office Grant 57861-LS (R.G.). EPR
instrumentation employed in this work has been supported by
NIH S10RR023614, NSF CHE-0840501, and NCBC 2009-
IDG-1015. Mass spectra were obtained at the Mass
Spectrometry Facility for Biotechnology at North Carolina
State University. Partial funding for the facility was obtained
from the North Carolina Biotechnology Center and the
National Science Foundation.
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