Nonmicrobial nitrophenol degradation via peroxygenase activity of dehaloperoxidase-hemoglobin from amphitrite ornata

Article abstract of DOI:10.1021/acs.biochem.6b00143

The marine hemoglobin dehaloperoxidase (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of nitrophenols, an unprecedented nonmicrobial degradation pathway for nitrophenols by a hemoglobin. Using 4-nitrophenol (4-NP) as a representative substrate, the major monooxygenated product was 4-nitrocatechol (4-NC). Isotope labeling studies confirmed that the O atom incorporated was derived exclusively from H₂O₂, indicative of a peroxygenase mechanism for 4-NP oxidation. Accordingly, X-ray crystal structures of 4-NP (1.87 ?) and 4-NC (1.98 ?) bound to DHP revealed a binding site in close proximity to the heme cofactor. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. The 4-NC product was itself a peroxidase substrate for DHP, leading to the secondary products 5-nitrobenzene-triol and hydroxy-5-nitro-1,2-benzoquinone. DHP was able to react with 2,4-dinitrophenol (2,4-DNP) but was unreactive against 2,4,6-trinitrophenol (2,4,6-TNP). pH dependence studies demonstrated increased reactivity at lower pH for both 4-NP and 2,4-DNP, suggestive of a pH effect that precludes the reaction with 2,4,6-TNP at or near physiological conditions. Stopped-flow UV-visible spectroscopic studies strongly implicate a role for Compound I in the mechanism of 4-NP oxidation. The results demonstrate that there may be a much larger number of nonmicrobial enzymes that are underrepresented when it comes to understanding the degradation of persistent organic pollutants such as nitrophenols in the environment.

Full text of DOI:10.1021/acs.biochem.6b00143

Article
pubs.acs.org/biochemistry
Nonmicrobial Nitrophenol Degradation via Peroxygenase Activity of
Dehaloperoxidase-Hemoglobin from Amphitrite ornata
Nikolette L. McCombs, Jennifer D’Antonio, David A. Barrios, Leiah M. Carey, and Reza A. Ghiladi*
Department of Chemistry, North Carolina State University, Raleigh, North Carolina, 27695-8204
S
* Supporting Information
ABSTRACT: The marine hemoglobin dehaloperoxidase
(DHP) from Amphitrite ornata was found to catalyze the
H2O2-dependent oxidation of nitrophenols, an unprecedented
nonmicrobial degradation pathway for nitrophenols by a
hemoglobin. Using 4-nitrophenol (4-NP) as a representative
substrate, the major monooxygenated product was 4-nitro-
catechol (4-NC). Isotope labeling studies confirmed that the O
atom incorporated was derived exclusively from H2O2,
indicative of a peroxygenase mechanism for 4-NP oxidation.
Accordingly, X-ray crystal structures of 4-NP (1.87 Å) and 4-
NC (1.98 Å) bound to DHP revealed a binding site in close proximity to the heme cofactor. Peroxygenase activity could be
initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. The 4-NC
product was itself a peroxidase substrate for DHP, leading to the secondary products 5-nitrobenzene-triol and hydroxy-5-nitro-
1,2-benzoquinone. DHP was able to react with 2,4-dinitrophenol (2,4-DNP) but was unreactive against 2,4,6-trinitrophenol
(2,4,6-TNP). pH dependence studies demonstrated increased reactivity at lower pH for both 4-NP and 2,4-DNP, suggestive of a
pH effect that precludes the reaction with 2,4,6-TNP at or near physiological conditions. Stopped-flow UV−visible spectroscopic
studies strongly implicate a role for Compound I in the mechanism of 4-NP oxidation. The results demonstrate that there may be
a much larger number of nonmicrobial enzymes that are underrepresented when it comes to understanding the degradation of
persistent organic pollutants such as nitrophenols in the environment.
he biodegradation of xenobiotics in marine and aquatic
environments is mainly viewed as being principally a
their corresponding 2,6-dihaloquinones.9−13 More recently, it
has been shown that both known isoenzymes of DHP (A and
B) exhibit peroxygenase activity when haloindoles are
employed as substrates.14 These activities are believed to have
arisen from the evolutionary pressure to overcome high levels
of volatile brominated secondary metabolites (e.g., halophenols
and haloindoles) that are secreted as repellents by other
infaunal marine organisms that coinhabit the benthic
ecosystems within which A. ornata is found.11,15 Mechanistic
studies16−27 have shown that DHP appears to function via a
Poulos-Kraut type mechanism28 in which H2O2 reacts with a
ferric heme to form DHP Compound I,22,25 the iron(IV)-oxo
(ferryl) porphyrin π-cation radical species that is common to
both the peroxidase and P450 cycles. Given its activity against
naturally occurring halogenated phenols and indoles, the ability
for DHP to degrade similar compounds of anthropogenic
origin may shed light on how infaunal organisms may impact
POPs in previously unreported ways.
T
microbial function,1,2 with many recent studies focusing on the
biotransformations of such compounds by microbial enzyme
systems for potential applications in bioremediation.3−5 By
comparison, relatively little is known regarding how infaunal
systems impact the fate of persistent organic pollutants (POPs).
This lack of knowledge when compared to the microbial
systems may play a critical factor in (i) the successful modeling
of pollutants in the environment, (ii) fully understanding how
POP metabolites may arise, thus impacting the surrounding
ecosystem in an unforeseen way, and (iii) accurately estimating
the individual or combined effects of POPs and metabolites,
particularly with respect to their bioaccumulation, transport,
and fate through environments where human exposure is
substantial.6−8 Thus, there is a significant need to identify
systems of nonbacterial origin that may be involved in the
degradation or biotransformations of POPs in order to be able
to fully assess the impact of these chemicals, including their
metabolites, on the environment.
Here, we have explored if deactivated phenols, and in
particular nitrophenols [2-nitrophenol (2-NP), 3-nitrophenol
(3-NP), 4-nitrophenol (4-NP), 2,4-dinitrophenol (DNP), and
2,4,6-trinitrophenol (TNP)] could serve as potential substrates
Recently, the substrate scope of the catalytic globin
dehaloperoxidase from the infaunal marine worm Amphitrite
ornata has generated much interest given that this enzyme
exhibits activities commonly associated with the peroxidase and
P450 families of enzymes. This coelomic hemoglobin was
originally named dehaloperoxidase (DHP) due to its ability to
catalyze the oxidative dehalogenation of 2,4,6-trihalophenols to
Received: February 17, 2016
Revised: April 9, 2016
Published: April 12, 2016
© 2016 American Chemical Society
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Article
for DHP. Nitroaromatic compounds are commonly used in the
syntheses of industrial compounds including dyes, herbicides,
and explosives, and are extremely toxic to living organisms.29
The U.S. Environmental Protection Agency (EPA) has listed 4-
NP, which is exclusively anthropogenic in origin, as a priority
pollutant,30 and is thus the focus of the present report. We have
identified the reaction products of the DHP-catalyzed
degradation of 4-NP using a combination of HPLC and LC-
MS. Labeling studies demonstrate that DHP functions via a
peroxygenase mechanism with this substrate leading to 4-
nitrocatechol formation (4-NC), which itself was found to be a
substrate for DHP as well. The results reported here will be
discussed in the context of DHP as a multifunctional enzyme
capable of peroxygenase, peroxidase, and O2-transport activities,
and the structural and/or electronic properties of the protein,
heme cofactor and substrate that ultimately dictate which
activity is observed. More importantly, however, the results
demonstrate that there may be a much larger number of
enzymes than are currently known that are underrepresented
when it comes to understanding the degradation or
biotransformations of POPs, findings which may be highly
relevant across a number of areas including the environmental
impact assessments of chemicals and their metabolites,31−36 as
well as enzyme-based, toxicity-extrapolation, or other models of
terrestrial or marine ecosystems.37−41
pH 6 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 at the reaction pH. 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 4 min; 5:95 isocratic for 1 min; 5:95 to 95:5 linearly over 1
min, then isocratic for 7 min. Data analysis was performed
using the Empower software package (Waters Corp.).
Calibration curves for all nitrophenols were performed using
serial dilutions of commercially available analytes to determine
the amount of substrate conversion.
LC-MS Studies. Experiments were analyzed using a Thermo
Fisher Scientific Exactive Plus Orbitrap mass spectrometer with
heated-electrospray ionization (HESI) probe (Thermo Scien-
tific, San Jose, CA) equipped with a Thermo Hypersil Gold (50
× 2.1 mm) 1.9 μm particle size C18 column. Analyte separation
was performed using a linear gradient of binary solvents
(solvent A - H2O containing 0.1% formic acid: solvent B -
MeCN), with an elution profile (0.5 mL/min, A:B) of 99:1 to
0:100 over 20 min. Samples were analyzed using electrospray
ionization in negative mode to provide observation of the [M −
H]− species. Spectra were collected each second while scanning
in the range from 55−1000 m/z. Data analysis was performed
using Thermo Xcalibur software. Quantitation of the amount of
18O-labeled incorporated was performed using previously
established methods.43,44 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 (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 of enzyme, 10 μL of H2O2, and 12.5 μL of substrate
(in MeOH) and reacted for 5 min and then quenched with
catalase. Twenty microliter aliquots of undiluted reaction
mixtures were injected for LC-MS analysis.
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−visible spectroscopy was performed
on a Varian Cary 50 UV−visible spectrophotometer. Stock
solutions (2 mM) of all substrates were prepared in 100 mM
KPi at the desired pH, stored in the dark at −80 °C until
needed, and were periodically screened by 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 M−1 cm−1).42 Ferric samples of WT DHP B, DHP B
(Y28/38F), and WT DHP A were expressed and purified as
previously reported.17,18,22 Oxyferrous DHP was obtained by
the aerobic addition of excess ascorbic acid to a solution of
ferric DHP, followed by application of the enzyme over a PD-
10 desalting column.21 Enzyme concentration was determined
spectrophotometrically using εSoret = 116 400 M−1cm−1 for all
isoenzymes.18 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.
Substrate-Binding Studies. Adapted from previously
published protocols,45 50 mM stock solutions of nitrophenol
substrates were prepared in 100 mM KPi buffer at the
appropriate pH. The UV−visible spectrophotometer was
blanked with buffer only, and spectra were then acquired in
the presence of the nitrophenol substrate at the concentration
indicated in the figure legends while maintaining constant
enzyme (50 μM) concentration. 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 substrate concentration. A
nonlinear regression plot provided the reported apparent Kd
values.
Enzyme Assay Protocol. Reactions were performed in
triplicate at the specified pH in 100 mM KPi at 25 °C. Buffered
solutions (total reaction volume 250 μL) of DHP (10 μM final
concentration) and substrate (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), DMSO (10% v/v). For the studies with sodium formate, a
buffered solution (100 mM KPi; 500 μM sodium formate) at
Fluoride-binding studies were also performed per literature
protocol46 to determine the extent of substrate binding at pH 5
and 7. DHP (50 μM) and substrate (500 μM) in 100 mM KPi
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buffer at the appropriate pH were titrated with NaF (180 mM
stock solution prepared in buffer) to the concentration
indicated in the corresponding figure legend. The absorbance
at 605 nm was monitored, and the analysis was performed as
published.
nitrophenol (Table 1), but overall a less than 1.2-fold difference
was observed as the position of the nitro substituent was varied
(2-NO2 > 3-NO2 > 4-NO2). A trend of decreasing overall
reactivity was noted upon increasing substitution (4-nitro > 2,4-
dinitro > 2,4,6-trinitro). To determine if the pKa of the
substrate played a role in this observed trend, the reactions
were repeated with DHP B as a function of pH. No turnover
was observed for any substrate at pH 8, only 4-nitrophenol
(pKa 7.1552) showed activity at pH 7, and both 4-nitrophenol
and 2,4-dinitrophenol (pKa 4.0752) showed activity at pH 6 and
5. Picric acid (2,4,6-trinitrophenol; pKa 0.4252) was unreactive
under any condition examined, and no turnover of substrate
Stopped-Flow UV−Visible Studies. Optical spectra were
recorded using a Bio-Logic SFM-400 triple-mixing stopped flow
instrument coupled to a rapid scanning diode array UV−visible
spectrophotometer. The temperature was maintained at 20 °C
with a circulating water bath, and all solutions were prepared 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-
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 detailed in the figure legends are shown.
Data were baseline corrected using the Specfit autozero
function.
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 nitrophenol substrate (2.5−50
equiv). Control experiments were also performed as above in
the absence of hydrogen peroxide.
a
Table 1. Enzyme-Catalyzed Reactivity of Nitrophenols
enzyme
conversion (%)
Substrate Variation
DHP B Ferric
+ 2-nitrophenol
49.6 ( 0.8)
43.7 ( 1.3)
39.4 ( 0.7)
16.2 ( 1.6)
+ 3-nitrophenol
+ 4-nitrophenol
+ 2,4-dinitrophenol
+ 2,4,6-trinitrophenol
b
n.d.
Enzyme Variation
DHP B Oxyferrous
+ 4-nitrophenol
37.6 ( 1.7)
DHP B (Y28/38F) Ferric
+ 4-nitrophenol
+ 2,4-dinitrophenol
+ 2,4,6-trinitrophenol
DHP A Ferric
37.2 ( 1.0)
16.6 ( 0.6)
n.d.
Protein Crystallization and X-ray Diffraction Studies.
Non-His tagged DHP B was overexpressed and purified per
literature protocol27 with minor modifications as described in
the Supporting Information. DHP B crystals were obtained
through the hanging-drop vapor diffusion method. Prior to
hanging drops, DHP B was incubated for 1 h at 4 °C with the
desired substrate. Final incubated concentrations of DHP B,
4NP, and 4NC were 12 mg/mL, 8 mM, and 4 mM respectively.
The crystals were grown from mother liquor solutions of 30%
PEG 4000 and 0.2 M ammonium sulfate at pH 6.4, and were
equilibrated against identical reservoir solutions. Protein to
mother liquor ratios varied between 1:1, 1.33:1, 1.66:1, and 2:1.
At 4 °C crystals grew from each condition after 3 days. The
crystals were cryo-protected by briefly dipping them in
reservoir solution enhanced with 20% glycerol and then flash
frozen in liquid N2. Data were collected at 100 K on the SER-
CAT 22-ID beamline at the APS synchrotron facility, utilizing a
wavelength of 1.00 Å. All data were scaled and integrated using
HKL2000,47 molecular replacement was performed with
Phaser-MR48 from the PHENIX49 suite of programs using
3IXF27 as the search model, model building and manual
placement of waters utilized COOT50 and refinement was
carried out using phenix.refine.51
+ 4-nitrophenol
+ 2,4-dinitrophenol
+ 2,4,6-trinitrophenol
HRP
21.6 ( 0.1)
4.1 ( 1.3)
n. d.
+ 4-nitrophenol
hhMb
n. d.
n. d.
+ 4-nitrophenol
Mechanistic Probes
DHP B Ferric +4-nitrophenol
anaerobic
43.2 ( 0.4)
37.6 ( 0.6)
35.6 ( 0.2
37.8 ( 4.2)
38.3 ( 0.4)
n.d.
+ 500 μM mannitol
+ 500 mM formate
c
+ SOD
+ 10% DMSO
+ 4-Br-phenol
pH Effects with Ferric DHP B
pH 5
+ 4-nitrophenol
+ 2,4-dinitrophenol
+ 2,4,6-trinitrophenol
pH 7
44.9 ( 1.6)
24.6 ( 0.2)
n.d.
+ 4-nitrophenol
+ 2,4-dinitrophenol
+ 2,4,6-trinitrophenol
pH 8
34.0 ( 0.1)
n.d.
n.d.
RESULTS
■
DHP-Catalyzed Nitrophenol Reactivity with H2O2. The
hydrogen peroxide-dependent oxidation of nitrophenol as
catalyzed by WT DHP B when initiated from the ferric state
at pH 6 was monitored by HPLC. Reactions were initiated
upon addition of 500 μM H2O2 to a solution containing 10 μM
enzyme and 500 μM nitrophenol, incubated at 25 °C for 5 min,
and then quenched with catalase. Reactivity was greatest with 2-
+ 4-nitrophenol
+ 2,4-dinitrophenol
+ 2,4,6-trinitrophenol
n.d.
n.d.
n.d.
a
Reaction conditions: [nitrophenol] = [H2O2] = 500 μM, [enzyme] =
10 μM, 100 mM KPi buffer at pH 6 (unless indicated), 25 °C, 5 min.
b
c
n.d. = none detected. SOD = ∼2 U/μL.
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occurred when either DHP (nonenzymatic control) or peroxide
(nonoxidant control) were excluded from the reaction.
The reactivity with 4-nitrophenol 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 (peroxidase) and haloindole
(peroxygenase) oxidations.21,26 The mutant DHP B(Y28F/
Y38F), which forms Compound I rather than the Compound
ES species observed in WT DHP B,22 exhibited nearly the same
reactivity as the native enzyme. WT DHP B was also found to
be ∼1.8-fold more reactive than WT DHP A, which is
consistent with the ratio of the catalytic efficiencies (kcat/Km) of
these two enzymes for TCP oxidation (∼2.6) and haloindole
reactivity (∼2.4) that have demonstrated a greater activity for
isoform B over A.
In the presence of 500 μM 4-bromophenol, a known
inhibitor of TCP and haloindole oxidation,53 no oxygenation of
4-nitrophenol was observed. To determine if solvent accessible
radicals are playing a role in substrate turnover, reactions were
performed in the presence of radical scavengers (DMSO,
sodium formate, D-mannitol) and superoxide dismutase
(SOD); all showed nearly equivalent 4-nitrophenol conversion
when compared to the reaction run in their absence, suggestive
of a nonradical based mechanism. No changes in product
distribution were observed by HPLC (data not shown) in the
presence of these radical scavengers. Reactions were performed
anaerobically under N2 to probe if ambient molecular oxygen
from the air played a role in the oxygenation reaction. When
performed under such anaerobic conditions, no decrease in 4-
nitrophenol oxygenation was seen, consistent with previous
studies of WT DHP B and TCP that demonstrated an O2-
independent reaction pathway.18,21 Finally, studies performed
with horseradish peroxidase (HRP) or horse heart myoglobin
(Mb), the respective archetypes of the peroxidase and globin
superfamilies, showed no reactivity with 4-nitrophenol
substrate.
Identification of Reaction Products by HPLC. A
representative HPLC trace at 350 nm is shown in Figure 1
(top) for studies performed with 4-nitrophenol (4-NP).
Products were identified by their characteristic retention
times and electronic absorption spectra as compared to
authentic samples, by similarity to those of their respective
unsubstituted analogue when the substituted versions were not
commercially available, and/or by mass spectrometry (Figure 1,
bottom A−D).
The product that eluted at tR = 4.2 min exhibited an identical
retention time and UV−visible spectrum (Figure S1A,B), as
well as mass (m/z 154, [M − H+]−; Figure 1A), as an authentic
sample of 4-nitrocatechol (1a, 4-NC, Figure S1C,D), providing
unequivocal evidence for the identity of this species as being
formed from O atom incorporation into the 4-NP substrate.
The product that eluted at tR = 3.9 min exhibited a mass (m/z
170, [M − H+]−; Figure 1B), consistent with O atom
incorporation into 4-NC, suggestive of a nitrobenzene-triol
product. Three constitutional isomers are possible: 4-nitro-
benzene-1,2,3-triol (i.e., 4-nitropyrogallol, NPG), 5-nitroben-
zene-1,2,3-triol, or 5-nitrobenzene-1,2,4-triol. The UV−visible
spectrum (λmax = 346 nm; Figure S2B) of this product, while
consistent by Woodward-Fieser rules54−57 with its assignment
as a nitro-containing triol, rules out NPG (λmax = 380 nm58). As
such, the product was assigned as 5-nitrobenzene-triol (5-NT);
however the data cannot distinguish between the two
Figure 1. HPLC chromatograms for the reactions of (top) 4-
nitrophenol (1, 500 μM) or (middle) 4-nitrocatechol (1a, 500 μM)
with DHP B (10 μM) in the presence of H2O2 (500 μM) at 25 °C
(100 mM KPi, pH 6). The reactions were quenched upon addition of
catalase and subjected to HPLC analysis as described in the text.
(bottom) ESI-MS total ion chromatograms obtained for (A) 4-
nitrocatechol, 4-NC (1a); (B) 5-nitro-benzene-triol, 5-NT (1b); (C)
hydroxy-5-nitro-1,2-benzoquinone, H-5-NBQ (1c); and (D) the
substrate 4-nitrophenol, 4-NP (1).
remaining possible constitutional isomers, either as 5-nitro-
benzene-1,2,3-triol or 5-nitrobenzene-1,2,4-triol. When 4-NC
was employed as the substrate in the enzymatic reaction
(Figure 1 middle, and S2A,B), the 5-NT species was observed
with virtually identical retention time and UV−visible spectrum
(Figure S2C,D). This suggests that 5-NT formation in the
original nitrophenol reaction is derived from a secondary
reaction where an O atom is first incorporated into 4-NP to
yield 4-NC, followed by a second O atom incorporation to
yield 5-NT.
Regardless of whether 4-NP (Figure S3A,B) or 4-NC (Figure
S3C,D) was employed as substrate, both reactions also yielded
a product that eluted at tR = 1.9 min whose spectral features
(λmax = 380 nm) were consistent with the electronic structure
of a 1,2-benzoquinone.59 The mass of this species (m/z 168,
[M − H+]−; Figure 1C) was found to be 2 amu less than 5-NT,
and together with the absorption spectrum suggest that this
species is the 1,2-benzoquinone oxidation product of 5-NT. As
there are two possibilities for the structure of 5-NT, two
structures for the resulting oxidation product, defined here as
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H-5-NBQ, are also possible, either as 3-hydroxy-5-nitro-1,2-
benzoquinone or 4-hydroxy-5-nitro-1,2-benzoquinone.
H218O2 exhibited nearly the same results (m/z: 168, 52%; 170,
36%; 172, 11%) as with H218O and unlabeled H2O2 (Figure S5,
Table S1). The lack of influence on the label distribution as a
function of hydrogen peroxide again is indicative of a
peroxidase mechanism. As the double-labeled conditions
yielded ∼50% unlabeled product, we investigated if dioxygen
may have played a role in the label scrambling by repeating that
experiment under anaerobic conditions, however, we observed
identical results as the aerobic study (data not shown). Thus, as
the only other source of oxygen, we suggest that the 4-NC
substrate itself, present initially at 500 μM, is the source of the
unlabeled oxygen. As the results for H-5-NBQ (1c) formation
were in-line with those of 5-NT in that they were still indicative
of a peroxidase mechanism, the exact details of the label
scrambling were not further explored, but precedents for such
label scrambling via intermolecular reactions in model
complexes of catechol dioxygenases are known.60,61
Substrate Binding Studies. The electronic absorption
spectrum of 4-NP62 at pH 7 (Figure S6) exhibited maxima at
324 and 398 nm (the latter extending as far as ∼450 nm),
overlapping with the spectrum of ferric DHP B [408 (Soret),
508, 540 (sh), 635 nm], and thus precluding the use of the
Soret or Q bands for substrate binding studies. Rather, the
combination of the hypsochromic shift of the charge transfer
band to ∼615 (4-NP) or 625 nm (4-NC, 2,4-DNP) and its
hyperchromicity upon substrate binding was employed. Optical
spectra were recorded per literature protocol as a function of
substrate concentration (2.5−100 equiv; Figures S7−S9).45
Analysis by nonlinear regression plots provided a calculated
Isotopically Labeled Oxygen Studies. As the observed
reactivity was O2-independent (anaerobic study, Table 1),
studies employing labeled H218O2 and H218O (90% and 98% O
atom enriched, respectively) were performed with 4-nitro-
phenol and 4-nitrocatechol, and subsequently analyzed by LC-
MS to determine the source of the O atom incorporation. The
background-subtracted total ion chromatograms (TICs) for the
4-NP studies are shown in Figure 2. The isotopic distributions
for the monooxygenated product 4-NC were determined using
previously established methods.43,44 Products were identified by
the respective m/z of the (M − H+)− ion and retention time as
compared to available standards. In the presence of H218O and
unlabeled H2O2, no significant increase in mass was observed
(m/z: 154, 99%; 156, 1%; Figure 2A) when compared with the
absence of an 18O source (m/z: 154, 100%; 156, 0%; Figure
1A), thus ruling out solvent water as the source of the O atom
incorporated. The same experiment employing H218O2 and
unlabeled H2O showed a clear increase to higher mass for 4-
NC by 2 Da: m/z: 154, 2%; 156, 98% (Figure 2B). The results
showed a 98+% 18O enrichment (normalized), providing
unequivocal evidence that the oxygen atom was derived from
H2O2.
To determine the origin of the second O atom incorporated
into the dioxygenated products, 5-NT (1b) and H-5-NBQ
(1c), the above experiments were performed using 4-NC (1a)
as the starting substrate (Table S1). For 5-NT (1b) (unlabeled
reaction, m/z: 170, 100%; 172, 0%; Figure 1b), the presence of
H218O and unlabeled H2O2 led to a mass increase (m/z: 170,
9%; 172, 51%; 174, 40%) with a ∼ 1:1 ratio of single:double
labeled products (Figure S4). These results suggested that in
addition to the incorporation of a single 18O label from a
peroxidase cycle, that scrambling with the H218O solvent also
occurred. Whereas the same experiment employing H218O2 and
unlabeled H2O showed no significant increase to a higher mass
for 5-NT (m/z: 170, 95%; 172, 5%; 174, 0%), the double-
labeled experiment with H218O2 and H218O showed nearly the
same results (m/z: 170, 9%; 172, 47%; 174, 44%) as with
H218O and unlabeled H2O2. Thus, the data suggest that H218O2
does not play a role in the incorporation of the second O atom
into 5-NT, and together are strongly indicative of a peroxidase
mechanism for the secondary metabolite formation.
A
max value, which in turn was used to calculate α for the average
ΔA for each [substrate]. A second nonlinear regression plot
provided the apparent dissociation constants (Kd) for 4-NP, 4-
NC, and 2,4-DNP.
As shown in Table 2, binding for 2,4-DNP (pH 5, 48 μM;
pH 7, 105 μM) was ∼2.5-fold stronger than 4-NP (pH 5, 134
μM; pH 7, 262 μM) at both pH values. Moreover, a small pH-
dependence on binding was also noted: the Kd values for 4-NP
(pKa 7.1552) and 2,4-DNP (pKa 4.0752) both decreased ∼2-fold
upon lowering the pH from 7 to 5, suggesting that binding was
stronger to DHP when the substrates were in their neutral
phenol form. This increase in binding at pH 5 when compared
with pH 7 may also contribute to the increase in reactivity
observed at lower pH values (vide supra). For the product 4-
NC (pKa 6.6963), the Kd value increased ∼2-fold upon lowering
the pH, possibly suggesting stronger binding for the catecholate
For H-5-NBQ (1c) (unlabeled reaction, m/z: 168, 100%;
170, 0%; Figure 1c), the results were complicated by a
significant amount of label scrambling. Specifically, H218O/
H2O2 led to a moderate mass increase (m/z: 168, 58%; 170,
34%; 172, 8%), H2O/H218O2 showed only a minor increase to
higher mass (m/z: 168, 87%; 170, 9%; 172, 4%), and H218O/
Table 2. Kd Values for Substrate Binding to Ferric DHP B
F−
b
substrate
Kd (μM), pH 5 Kd (μM), pH 7 Kd (mM)
ref.
d
4-NP
134
79
7
262 23
40
18
31
90
30
1
3
9
1
a
a
a
a
d
d
d
4-NC
4
8
1
2,4-DNP
2,4,6-TNP
48
105 21
n.d.
c
n.d.
e
5.9 1.0
f
eg
,
4-BP
n.r.
1150
12
1
1
53, 46
46
dg
,
13
eg
,
2,4-DBP
n.r.
n.r.
n.r.
n.r.
172
24
9
46
eg
,
2,4,6,-TBP
1
46
a
b
Figure 2. ESI-MS total ion chromatograms obtained for the reaction
product 4-nitrocatechol (A: H218O, H216O2; B: H216O, H218O2).
Reaction conditions: [4-nitrophenol] = [H2O2] = 500 μM, [enzyme]
= 10 μM, 100 mM KPi (pH 7), 25 °C.
This work. Kd value of fluoride binding in the presence of 500 μM
c
nitrophenol substrate, n.d. = not determinable due to lack of spectral
changes, pH 5. pH 7. n.r. = not reported in reference. [halophenol]
= 1 mM.
d
e
f
g
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form. Structural elements of the DHP active site involving H-
bonding to 4-NP and 4-NC were elucidated by X-ray
crystallography and are discussed below (vide infra). Notably,
the Kd values for 4-NP and 4-NC were on a par with the ones
for the other known peroxygenase substrates, the 5-substituted
halogens (5-I-indole: 62 10 μM; 5-Br-indole: 150 10 μM;
5-Cl-indole: 317 23 μM), and significantly lower than that
for 4-bromophenol (Kd ≈ 1.15 mM53), a known inhibitor of
the enzyme.
ES,18 and subsequently mixed with 2.5, 10, or 25 equiv of 4-
nitrophenol. When reacted with 10 equiv. substrate (Figure 3),
the preformed Compound ES [UV−visible: 419 (Soret), 545,
585 nm] was reduced (kobs = 0.021 0.003 s−1) back to ferric
DHP B [407 (Soret), 507 nm], a decrease in the 4-nitrophenol
peak at 321 nm was noted, and an increase in the baseline was
noted at wavelengths shorter than 400 nm that can be
attributed to the absorption of the reaction products 4-NC, 5-
No Kd value for 2,4,6-TNP could be directly determined as
the UV−visible spectra did not exhibit any substrate-dependent
shifts (data not shown). We originally attributed this outcome
to either the unlikely binding of the phenolate form of 2,4,6-
TNP (pKa 0.4252) to the hydrophobic active site of DHP at
either pH 5 or 7, or simply due to the additional steric
hindrance present in the trisubstituted phenol. However, in
order to further probe 2,4,6-TNP binding to DHP, a
competition assay with the heme-fluoride binding equilibrium
was employed (Table 2).46 In this method, substrates that bind
within the DHP active site lead to a decreased binding affinity
F−
of fluoride to the heme (Kd = 4.5 0.4 mM). Interestingly,
F−
while 2,4,6-TNP was not observed to significantly affect Kd
(5.9 1.0 mM) at pH 7 (Figure S10A,B), the presence of this
F−
substrate at pH 5 led to an increase in Kd to 30 1 mM
(Figure S10C,D), a value that represents stronger substrate
binding than 4-NP (KdF− = 18 1 mM; Figure S11A,B), is on a
F−
par with 4-NC (Kd = 31 3 mM; Figure S11C,D), but still
weaker than 2,4-DNP (KdF− = 90 9 mM; Figure S11E,F). As
such, the lack of reactivity at this pH value likely represents
factors (e.g., substrate E°) unrelated to 2,4,6-TNP binding.
F−
Finally, a comparison of the Kd values at pH 7 reveals that
2,4,6-TNP binding is weaker than that of the bromophenols,
with substrate binding affinity in the order: 2,4,6-TNP < 4-BP <
2,4,6-TBP < 2,4-DBP.
Stopped-Flow UV−Visible Spectroscopic Studies with
4-NP. Single and double-mixing stopped-flow methods were
used to investigate the reaction of 4-nitrophenol with H2O2-
activated DHP, preformed either as Compound I [DHP
B(Y28F/Y38F)], Compound ES, Compound II, or with the
oxyferrous species itself as the starting state.
Compound I Reactivity. The 4-nitrophenol reactivity studies
performed with preformed Compound I using DHP B(Y28F/
Y38F) were identical at pH 6 (data not shown) and pH 7
(Figure S12): no activated enzyme (i.e., preformed Compound
I [406 (Soret), 528, 645 nm]22) was observed. Rather, ferric
DHP B(Y28F/Y38F) [406 (Soret), 507, 540 (sh) nm] was the
first spectrum recorded, suggesting that Compound I was
reduced by the substrate within the mixing time of the stopped-
flow apparatus. A conversion to a ferric-like species [411
(Soret), 503, 621 nm] was noted after 83 s. These spectral
features are markedly different from those observed for the
decay product of H2O2-activated DHP B (Y28F/Y38F) in the
absence of substrate [404 (Soret), 518, 542 (sh) nm], and they
do not match with the oxyferrous form. As such, they are
possibly indicative of substrate- and/or product-bound DHP B
(Y28F/Y38F), but this was not further explored. The rapid
reaction of Compound I with 4-nitrophenol is reminiscent of its
reactivity with 5-Br-indole, which was also found to reduce
Compound I to the ferric enzyme within the mixing time of the
stopped-flow apparatus via a peroxygenase mechanism.
Compound ES Reactivity. Ferric WT DHP B (10 μM) was
reacted with 10 mol equiv of H2O2 at pH 6, incubated for 220
ms to allow for the maximum accumulation of Compound
Figure 3. Kinetic data for the reaction of preformed Compound ES
with 4-nitrophenol. (A) Stopped-flow UV−visible 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 220 ms, with a 10-fold
excess of 4-nitrophenol at pH 6.0 (900 scans over 83 s); inset - the
single wavelength (419 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 two reaction components
derived from the SVD analysis: Compound ES (black) and ferric DHP
B in the presence of excess 4-nitrophenol (red). (C) Time
dependences of the relative concentrations for the two components
shown in the middle panel as determined from the fitting of the
spectra in the top panel.
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NT, and H-5-NBQ. The experimental values of kobs for
Compound ES reduction varied linearly with 4-NP in the range
of 2.5−10 molar equiv, yielding a biomolecular rate constant of
(2.73
0.06) × 102 M−1 s−1. Under these conditions,
Compound RH, the stable form of DHP that forms from H2O2-
activation in the absence of substrate, was not observed.
Notably, the conversion to the oxyferrous form of the enzyme
that is noted in similar assays that employ the physiological
substrates (i.e., trihalophenols and haloindoles) was not
observed.
When the above reaction was performed at pH 8, no changes
to the ferryl spectrum [UV−visible: 417 (Soret), 545, 585 nm]
were noted over the first 80 s (Figure S13). At longer
observation times up to 800 s, a slow conversion to the ferric
enzyme was observed (data not shown), with no significant
changes attributed to substrate loss or product formation. The
lack of reactivity observed in these stopped-flow experiments at
pH 8 correlates with the lack of reactivity noted in our HPLC
studies when performed at the same pH (Table 1).
Compound II Reactivity. As previously reported,19,24
oxyferrous DHP can be activated by H2O2 in the presence of
as little as 1 equiv of TCP21 or haloindole14 substrate, leading
to Compound II formation.21 Here, we investigated the
reactivity of preformed DHP Compound II with 4-NP using
sequential double-mixing stopped-flow methods: oxyferrous
DHP B containing 1 equiv of TCP was reacted with 10 mol
equiv of H2O2 at pH 6, incubated for 85 s to allow for the
maximum accumulation of Compound II [417 (Soret), 547,
585 nm],21 and subsequently mixed with an additional 10 equiv
of 4-NP (Figure 4). Notably, the Compound II spectrum
converted to the ferric spectrum [either as substrate or product-
bound: 405 (Soret), 503 nm] within 60 s, on a par with the
Compound II reactivity observed for TCP and indole.
Oxyferrous DHP Reactivity. In the absence of substrate,
previous studies have shown that a slight bleaching of the Soret
band and/or long time scale conversion to Compound RH
occurs upon the reaction of oxyferrous DHP with H2O2, and
only when TCP21,26 or indole14 substrates were present were
fast time scale reactive intermediates observable. This substrate-
dependent activation of DHP was investigated here with 4-NP
using stopped-flow methods. Upon rapid mixing of a solution
of oxyferrous DHP B preincubated with 10 equiv of 4-NP with
10 equiv of H2O2, substantial spectral changes were observed
(Figure 5). The oxyferrous form converted to a species whose
spectral features [417 (Soret), 545, 578, 590 (sh) nm; t = 8 s]
matched those of Compound II formed from our previous
identification of this species when employing indole as the
substrate.14 The Compound II species was found to convert at
longer times to the substrate or product-bound ferric enzyme
[405 (Soret), 500 nm] within 68 s, matching the reactivity
observed for preformed Compound II with 4-NP (vide supra).
The main observations from these stopped-flow studies with
4-NP were (i) Compound I was rapidly reduced by 4-NP
within the mixing time of the instrument to the ferric enzyme,
(ii) Compounds ES and II were reduced to the ferric enzyme
after ∼60−70 s, and (iii) 4-NP was found to activate oxyferrous
DHP with H2O2 via a Compound II intermediate.
Figure 4. Kinetic data for the reaction of preformed DHP B
Compound II with 4-nitrophenol. (A) stopped-flow UV−visible
spectra of the double-mixing reaction between preformed DHP B
Compound II (10 μM) and 10 equiv of 4-nitrophenol at pH 6.0 (900
scans over 83 s); inset - the single wavelength (419 nm) dependence
on time. DHP B Compound II was itself formed from an initial
reaction between oxyferrous DHP B preincubated with 1 equiv of
trichlorophenol and 10 equiv of H2O2 and reacted for 85 s prior to the
second mix with 4-nitrophenol. (B) experimentally obtained spectra
for Compound II derived from TCP (black, t = 2.5 ms), and its
reduction to ferric DHP B observed in the presence of 4-nitrophenol
(t = 60 s).
Compound I Reactivity. The 4-nitrocatechol reactivity
studies performed with preformed Compound I using DHP
B(Y28F/Y38F) yielded similar results to those observed for 4-
nitrophenol above (data not shown): no activated enzyme (i.e.,
preformed Compound I) was observed, but rather a ferric-like
DHP B(Y28F/Y38F) species [411 (Soret), 503, 621 nm] was
first identified, suggesting that Compound I was reduced by 4-
nitrocatechol within the mixing time of the stopped-flow
apparatus.
Compound ES Reactivity. Compound ES was preformed at
pH 6 as described above, and subsequently mixed with 2.5, 10,
or 25 equiv of 4-nitrocatechol. When reacted with 10 equiv.
substrate (Figure S14), the preformed Compound ES [UV−
visible: 417 (Soret), 544, 584 nm] was rapidly reduced to a
species that exhibited a ferric-like Soret absorption (410 nm)
within 200 ms, and underwent further reaction to yield ferric
DHP B [407 nm (Soret)] after 83 s. An increase in the baseline
attributed to the absorption of both the 4-NC substrate as well
as reaction products made assignment of the Q-band features
indeterminate.
Stopped-Flow UV−visible Spectroscopic Studies with
4-NC. Single and double-mixing stopped-flow methods were
also used to investigate the reaction of 4-nitrocatechol with
H2O2-activated DHP, preformed either as Compound ES (WT
DHP), Compound I [DHP B(Y28F/Y38F)], or Compound II,
or with the oxyferrous species itself as the starting state.
Compound II Reactivity. The reaction of preformed DHP
Compound II with 4-NC was investigated using sequential
double-mixing stopped-flow methods: oxyferrous DHP B
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and 4-nitrocatechol are found in Table S2, and selected
substrate−protein and heme-protein distances can be found in
Table S3. DHP B crystallized as a homodimer in the
asymmetric unit, consistent with each previously deposited
crystal structure. There is full occupancy of 4-NP in both
protomers, while 4-NC fully occupies subunit B however
exhibits partial occupancy of 0.5 in subunit A, with O2 present
in the absence of 4-NC. The orientation of each substrate is
identical in both of its corresponding subunits. The subsequent
structural analysis focuses solely on subunit B: both substrates
were found to bind in the distal pocket, oriented between the
heme β edge and Fe (Figures 6 and S17). Both molecules are
bound 4.2 Å above the heme with a tilt angle of 25° (in
reference to the heme normal) away from the β edge. The
aromatic rings are oriented for π-stacking interactions with F21.
The nitro groups are positioned internally, residing in the Xe1
binding site, with the OH groups situated near the pocket
entrance, stabilized by H-bonding interactions: the heme
propionate D and Y38 form a H-bonding network with 4-
NP, with distances of 2.6 and 2.7 Å, respectively, to the OH1 of
4-NP. As a tighter binder exhibited by the lower Kd, 4-NC
forms H-bonds with both of its OH groups: the heme
propionate D and Y38 form H-bonds with OH1, exhibiting
distances of 2.7 and 2.8 Å respectively. The OH2 interacts with
T56 at a distance of 2.6 Å, and also has a weaker H-bond with
Y38 at 3.2 Å. The hydrogen bonding interactions between OH1
of 4-NP or 4-NC and the protein/heme propionates aligns well
with the observation from Tables 1 and 2 that DHP prefers
protonated nitrophenolic substrates. This binding mode has
been observed previously for the p-halogenated phenols (Figure
6C,D). LSQ superposition of the subunit B distal pockets
Figure 5. Kinetic data for the reaction of oxyferrous DHP B with 4-
nitrophenol and hydrogen peroxide. (A) stopped-flow UV−visible
spectra of the single-mixing reaction between oxyferrous DHP B (10
μM) preincubated with 10 equiv. 4-nitrophenol and a 10-fold excess of
H2O2 at pH 6.0 (900 scans over 83 s); inset - the single wavelength
(418 nm) dependence on time. (B) experimentally obtained spectra
for oxyferrous DHP B (black, t = 2.5 ms), DHP B Compound II (blue,
t = 8 s), and its reduction to ferric DHP B observed in the presence of
4-nitrophenol (t = 68 s).
containing 1 equiv of TCP was reacted with 10 mol equiv of
H2O2 at pH 6, incubated for 85 s to allow for the maximum
accumulation of Compound II [419 (Soret), 545, 583 nm],21
and subsequently mixed with an additional 10 equiv of 4-NC
(Figure S15). The Compound II spectrum converted to the
ferric spectrum [either as substrate or product-bound: 405 nm
(Soret)] within 13 s, and remained relatively unchanged up to
83 s.
Oxyferrous DHP Reactivity. Upon rapid mixing of a solution
of oxyferrous DHP B preincubated with 10 equiv of 4-NC with
10 equiv of H2O2, it was observed (Figure S16) that the
oxyferrous form [418 (Soret), 543, 578 nm] converted to a
species whose spectral features [418 (Soret), 544, 584 nm; t = 3
s] matched those of Compound II formed from our previous
identification of this species when employing TCP as the
substrate.21 The Compound II species was found to convert at
longer times (t = 18 s) to the substrate or product-bound ferric
enzyme [405 nm (Soret)].
The main observation from these stopped-flow studies was
that the reactivity with 4-nitrocatechol mirrored that with 4-
nitrophenol in terms of reducing the activated enzyme back to
the ferric state, but appeared to be significantly faster overall.
X-ray Crystallographic Studies of DHP B Complexed
with 4-NP and 4-NC. Data collection and refinement statistics
for WT DHP B cocrystallized in complex with 4-nitrophenol
Figure 6. As viewed from the β edge, panels A and B provide atomic
distances for 4-NP and 4-NC, respectively. Panels C and D present the
distal superposition of 4-NP (5CHQ, green), 4-NC (5CHR, silver)
and 4BP (3LB2,53 purple) structures. As shown, the 4-NP and 4-NC
molecules superpose with little deviation, while 4BP is slightly
displaced with regard to the OH at the distal entrance. Panel C is
viewed from the β edge, and panel D presents the view from the distal
entrance, the γ edge.
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resulted in a 0.114 Å r.m.s.d, with the two substrates occupying
identical orientations.
to a nearby carbon atom on the substrate. By way of
comparison, 4-nitrophenol, and haloindoles by extension,
reside near the reactive heme center well-positioned for O
atom transfer directly from the ferryl intermediate, with no such
steric hindrance. Distal superposition of peroxidase and
peroxygenase substrate binding sites is provided in the
Supporting Information. Interestingly, TCP also possesses a
second binding site that closer resembles nitrophenol binding:
the Cl substituent of the C4 carbon is directed toward the
heme-Fe, H55 is found in the “open” (external) conformation
and the phenolic OH is within hydrogen bonding distance to
Y38, as seen with the nitrophenol substrates. This alternate
TCP binding site is situated at the γ edge of the heme, with the
molecule residing between both heme propionate arms. Thus,
when one considers all possible substrates (halophenols,
haloindoles, nitrophenols) for DHP, there is ample evidence
to suggest a common binding motif necessary for peroxygenase
activity, but such binding alone is not sufficient as to determine
peroxygenase vs peroxidase activity.
The peroxygenase substrate binding site appears to imply a
close interaction with the H2O2-activated form of DHP (as
necessitated by O atom transfer). As the principal reactive
species in heme peroxidases,80 peroxygenases,81 P450s,64,66 and
other hemoproteins,82,83 the Compound I, ES and II
intermediates were explored here for their mechanistic role(s)
in the DHP-catalyzed oxidation of 4-NP and 4-NC.
Compounds I and ES, either of which is formed from the
reaction of the ferric enzyme with hydrogen peroxide, both
contain an Fe(IV)-oxo species, yet differ in that Compound I
possesses a second oxidizing equivalent as a porphyrin π-cation
radical, whereas Compound ES has this radical residing on an
amino acid (i.e., a tyrosyl radical in DHP22) rather than on the
porphyrin ring.17,18 Compound II, formed either from the more
traditional 1e− reduction of Compounds I/ES or from the
reaction of oxyferrous DHP with 1 equiv H2O2 in the presence
of either trihalophenol21 or haloindole14 substrates, also
possesses an Fe(IV)-oxo species, but no additional oxidizing
equivalent residing on either the protein or the heme cofactor.
Using the DHP B(Y28F/Y38F) mutant to form Compound I
owing to the lack of tyrosines near the heme active site,22 its
reactions with both 4-nitrophenol and 4-nitrocatechol yielded
ferric DHP within the mixing time (<2.5 ms) of the stopped-
flow apparatus. By contrast, Compound ES was found to be at
least 2-orders of magnitude slower in its reactivity with these
two substrates, whereas preformed Compound II was found to
react with substrate significantly slower (seconds to tens of
seconds) than either Compounds I or ES. Thus, a greater than
104 magnitude difference in reactivity between the H2O2-
activated forms of DHP was observed from the presteady-state
kinetics with nitrophenol/nitrocatechol substrates. These
results agree with our previous observations with haloindole
substrates, where DHP Compound I (<2.5 ms), ES (∼150 ms),
and II (>30 s) also exhibited a ∼ 104-fold range in chemical
reactivity, and together strongly advocate for a two-electron
oxidized intermediate as the catalytic species mediating DHP
peroxygenase chemistry.
DISCUSSION
■
The activity studies presented here demonstrate that
dehaloperoxidase is able to catalyze the oxidation of nitro-
phenols as previously unreported substrates for DHP despite
their being of anthropogenic origin and not native to the
benthic ecosystems in which A. ornata is found. Isotope labeling
studies demonstrated that the oxygen atom incorporated into
the 4-nitrocatechol product was derived exclusively from
hydrogen peroxide, analogous to the “peroxide-shunt” of
cytochrome P450s and indoleamine 2,3-dioxygenase.43,64−66
Consistent 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 has only recently been reported for
dehaloperoxidase,14 but with haloindole substrates, as halophe-
nols proceed via a well-established peroxidase mechanism.67
Interestingly, the peroxygenase product, 4-nitrocatechol, itself
proved to be a substrate for DHP, yielding 5-nitrobenzene-triol
(5-NT) and hydroxy-5-nitro-1,2-benzoquinone (H-5-NBQ)
products in what labeling studies indicated was via a
peroxidase-based mechanism. Relevant to the studies here on
DHP, the microbial biodegradation of 4-nitrophenol has also
been shown to proceed via 4-nitrocatechol and 1,2,4-
benzenetriol intermediates in a number of systems that employ
hydroxylase/monooxygenase enzymes, including Arthrobacter
sp. strain JS443,68 Arthrobacter protophormiae RKJ100,69
Bacillus sphaericus JS905,70 Burkholderia cepacia RKJ200,71
Ralstonia sp. strain SJ98,72 Rhodococcus opacus AS2 and
Rhodococcus erythropolis AS3,73 and Serratia sp. strain DS001.74
In support of the observed peroxygenase activity, the crystal
structure of DHP B complexed with 4-NP showed the substrate
in close proximity to the heme Fe from which the inserted O
atom is derived. As there is no ferryl structure of DHP B,
analysis of a superposition of the 4-NP structure with a
surrogate oxyferrous DHP A structure (2QFN75) approximates
the reactive C3 carbon of 4-NP at ∼2.9 Å from the O bound to
the heme Fe, sufficiently positioned for O atom transfer to yield
the 4-nitrocatechol product. Interestingly, with the OH2 group
located 7.9 Å from the heme Fe, 4-NC resides in an orientation
that suggests rearrangement following initial 4-NP oxygenation.
The possibilities of molecular rotation inside the distal pocket
or product removal followed by rebinding as a substrate are
logical explanations for the crystallographic 4-NC binding site.
The orientation and proximity of both 4-NP and 4-NC
resemble that of the putative 5-haloindole binding site that was
determined by computational methods and supported by
resonance Raman studies,14 and suggest a common perox-
ygenase binding site for O atom transfer. By contrast, this
peroxygenase substrate site differs from the previously
determined peroxidase substrate binding sites: TBP76 and
TCP77 both bind deep in the distal cavity beneath F21 in close
proximity to L100 at the α edge of the heme, residing in the
Xe1 binding site78 with the hydroxyl group pointed toward the
heme Fe, and with H55 found in the “closed” (internal)
conformation (Figure S18).53,79 This is in contrast to the
nitrophenol structures where only the nitro group resides in the
Xe1 binding site. Moreover, the peroxidase substrates not only
reside near a heme edge, they are both positioned such that a
halogen substituent is facing the heme iron, thus sterically
hindering O atom transfer from an activated ferryl intermediate
Despite the broad range exhibited in their presteady state
kinetics, the results from the 4-nitrophenol end point assays
(Table 1) show that whether ferric DHP B(Y28F/Y38F)
(Compound I), WT ferric DHP B (Compound ES), or
oxyferrous DHP B (Compound II) were employed, virtually
identical substrate loss and product formation were observed.
One possible interpretation of these kinetic vs end point assay
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results is that DHP proceeds through a common pathway
regardless of the starting conditions. As all three reactive
intermediates (Compounds I, ES, and II) were reduced by
substrate to the ferric enzyme, we suggest that in addition to
the 2-electron O atom transfer chemistry associated with
Compound I, one-electron processes may play a role in the
initial turnover of DHP, for example, to reduce Compound II
to the ferric state, and that the subsequent activity (either
peroxygenase or peroxidase) proceeds as observed from the
ferric enzyme.
On the basis of the results obtained above, and through
modification of the previously established mechanisms for
peroxygenases,43,64−66 we propose the following catalytic cycle
for the in vitro hydrogen peroxide-dependent oxidation of
nitrophenol by ferric DHP from A. ornata (Scheme 1): ferric
DHP reacts with 1 equiv H2O2, forming either Compound I
(step i-a) or Compound ES (step i-b). The reaction of either of
these two ferryl species with nitrophenol leads to H atom
abstraction (steps ii-a or ii-b), followed by an oxygen rebound
step (step (iii), leading to regeneration of the ferric enzyme
with incorporation of the oxygen atom into 4-nitrophenol
yielding 4-nitrocatechol. The ferric enzyme is then available for
reactivation by a second equivalent of hydrogen peroxide,
thereby leading to additional 4-NC formation when reacting
with 4-NP as the substrate (via a second peroxygenase cycle),
or to 5-NT formation via a peroxidase cycle. While we propose
here that the initial step of substrate oxygenation can be viewed
as an H atom abstraction to yield a phenoxyl substrate radical
and an Fe(IV)−OH that together subsequently undergo
oxygen rebound to generate the nitrocatechol product, we
cannot rule out other possible mechanisms for O atom
insertion into an arene,84−86 such as epoxidation via an
electrophilic ferryl species, and additional studies, including
computational efforts, are currently underway.
Three additional comments can be made about the
nitrophenol peroxygenase mechanism in DHP: first, although
Scheme 1 shows the catalytic cycle initiating from the ferric
state, it has been demonstrated previously that DHP is able to
initiate both peroxidase and peroxygenase catalytic cycles from
the globin-active oxyferrous state: oxyferrous DHP reacts with
H2O2 in the presence of either trihalophenol21,26,87 or
haloindole14 substrate, thereby forming Compound II. Here,
we also observed Compound II formation when employing 4-
nitrophenol and 4-nitrocatechol as substrates and when starting
from oxyferrous DHP (step iv-a). Thus, it is likely that when
starting from the oxyferrous form, that Compound II is formed
initially, but undergoes a 1e− reduction (nonproductive; step iv-
b) to the ferric form, from which the catalytic cycle in Scheme 1
commences. Second, it is important to note under these
conditions that Compound RH, the stable form of DHP that
forms from H2O2-activation in the absence of substrate, was not
observed. Third, the conversion to the oxyferrous form of the
enzyme that is noted in similar assays that employ the
physiological substrates (i.e., trihalophenols and haloindoles)
was also not observed. Thus, the product-driven14,18 return of
ferric DHP to the globin-active oxyferrous form was not
observed here, an outcome that was not unexpected given that
nitrophenol is of anthropogenic origin and not a physiological
substrate. Although not the subject of the current study, this
latter point may be of consequence to the mechanism by which
nitrophenols are toxic to marine invertebrates such as A. ornata
in that they may lead to met-Hb formation.
Scheme 1. Proposed 4-Nitrophenol Peroxygenase Cycle for
Dehaloperoxidase B
As a consequence of the negative mesomeric and inductive
effects of the nitro substituent(s) leading to electron-deficient
aromatic systems,88,89 nitroaromatics can be more difficult to
degrade than their unsubstituted analogues. Microbial bio-
degradation of nitroaromatics has been shown to proceed via
both reductive and oxidative pathways,29,90 but for DHP it is
clearly an oxidative one. The ability for peroxidases to oxidize
substrates is dependent upon the ferryl/Fe(III) reduction
potential: for instance, 4-NP [E° = 1.23 V vs NHE, pH 7]91 is
not oxidized by Type VI-A horseradish peroxidase (HRP)
[E°CmpII/Fe(III) = 0.89
0.1 V], but is oxidized by fungal
peroxidases such as Coprinus cinereus peroxidase (CiP)
[E°CmpII/Fe(III) = 1.18 0.15 V] and Bjerkandera adusta versatile
peroxidase (VP) [E°CmpII/Fe(III) = 1.37 0.19 V].92 As a globin,
DHP possesses a much higher redox potential of +206
6
mV18,20 for the Fe3+/Fe2+ couple than those reported for
peroxidases (HRP, −266 mV;93 cytochrome c peroxidase, −182
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Biochemistry
Article
mV;94 myeloperoxidase, −5 mV95), and even for other globins
is at the high end of the range reported (sperm whale Mb, −43
mV;96 horse heart Mb, + 46 mV,97 horse heart Hb, + 152
mV;98 human Hb, + 158 mV99). Modulation of the heme redox
potential, and hence the reactivity of the enzyme, may be
achieved by a number of ways, including heme distortion,100
the hydrophobicity of the heme pocket,101,102 changes in heme
iron ligation103 and spin state,104 and/or modulation of the H-
bonding networks involving the distal105 and proximal20 ligands
or the heme propionates.106 The functional consequence of this
high redox potential is to minimize autoxidation to the ferric
state, thus ensuring that O2-binding is maintained, as only a
heme in the ferrous oxidation state can reversibly bind
molecular oxygen. However, such a high redox potential that
is a necessity for globin function may have another
consequence for DHP: in peroxidases, there is a strong
correlation between the redox potential of the Fe3+/Fe2+ couple
and the estimated redox potential for the ferryl/Fe(III)+
couple.92 On the basis of this correlation and the known
value for the Fe3+/Fe2+ couple in DHP, we have estimated that
the redox potential of the DHP ferryl/Fe(III) couple to be
∼1.58 V, which would make it one of the highest of any known
hemoprotein. Thus, it appears that the high reduction potential
of the ferryl species in DHP, which is a consequence of its
primary role as a globin, not only facilitates reactivity with
halogenated substrates, but also plays an important part in its
catalytic functions, i.e. peroxidase and peroxygenase activities,
against deactivated aromatics of anthropogenic origin such as
mono- and dinitrophenols, and nitrocatechols.
In summary, DHP catalyzes the degradation of nitrophenols
via a peroxygenase pathway that is highly reminiscent of
bacterial/fungal monooxygenases despite possessing neither
structural nor sequence homology to them. Such peroxygenase
activity now appears to be more widely applicable to DHP than
previously thought given that two classes of substrate,
nitrophenols and haloindoles, have been shown to undergo O
atom transfer. What makes DHP versatile as a biocatalyst may
stem from its globin origins: a relatively high redox potential
that imparts increased activity to the H2O2-activated enzyme
when compared to other peroxidases. DHP also possesses a
hydrophobic cavity that can accommodate small molecule
binding to support O atom transfer chemistry, a feature that
peroxidases generally lack. More importantly, the inclusion of
nitrophenols (2-NP, 4-NP, and 2,4-DNP) to the list of
pollutants that DHP can oxidize now increases its total of
EPA priority pollutants that are substrates to 7 (out of 126)
when including the known (halo)phenol substrates: phenol, 2-
chlorophenol, 2,4-dichlorophenol, and TCP. While it remains
to be seen if other priority pollutants are substrates for DHP
(e.g., 2,4-dimethylphenol, p-chloro-m-cresol, 4,6-dinitro-o-cre-
sol), it appears that multifunctional globins may be promising
platforms as promiscuous bioremediation catalysts, and other
marine globins from the benthic ecosystems from which A.
ornata originates should be explored for this potential.
Moreover, in addition to impacting our understanding of the
biotransformation pathways specific to persistent organic
pollutants, the consequences of these findings suggest the
need for expanding the breadth of organisms considered in
enzyme-decomposition ecosystem models or environmental
impact assessments of chemicals to include those beyond
microbial origin.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
chem.6b00143.
■
S
Protein purification for X-ray crystallographic studies;
HPLC data and UV−visible spectra of 1a−1c; mass
spectrometric analyses of isotope labeling studies with 4-
NC as substrate; UV−visible spectra of 4-NP at pH 5
and 7; optical spectra, titration curves, and fluoride-
binding competition assays of 4-NP, 4-NC, 2,4-DNP,
and 2,4,6-TNP; additional kinetic data and stopped-flow
studies of DHP B with 4-NP and 4-NC; X-ray data
collection and refinement statistics, and additional
structural analyses of DHP B cocrystallized with 4-NP
and 4-NC (PDF)
Accession Codes
The coordinates for the structures of 4-nitrophenol and 4-
nitrocatechol bound to DHP B have been deposited into the
protein data bank as PDB ID codes 5CHQ and 5CHR,
respectively.
AUTHOR INFORMATION
Corresponding Author
*Phone: (919) 513-0680. Fax: (919) 515-8920. E-mail: Reza_
Ghiladi@ncsu.edu.
Funding
■
This project was supported by NSF CAREER Award CHE-
1150709 (R.G.), the Army Research Office Grant 57861-LS
(R.G.), and the North Carolina State University Molecular
Biotechnology Training Program through an NIH T32
Biotechnology Traineeship grant (J.D.). 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. X-ray diffraction
data were collected at Southeast Regional Collaborative Access
Team (SER-CAT) 22-ID beamline at the Advanced Photon
Source, Argonne National Laboratory. Supporting institutions
may be found at www.ser-cat.org/members.html. Use of the
Advanced Photon Source was supported by the U.S. Depart-
ment of Energy, Office of Science, Office of Basic Energy
Sciences, under Contract No. W-31-109-Eng-38.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge Prof. Lukasz Lebioda (University of
South Carolina) for help in the refinement of the 4-NP and 4-
NC crystal structures.
ABBREVIATIONS
■
DHP, dehaloperoxidase; Hb, hemoglobin; Mb, myoglobin; 4-
NP, 4-nitrophenol; 4-NC, 4-nitrocatechol; 2,4-DNP, 2,4-
dinitrophenol; 2,4,6-TNP, 2,4,6-trinitrophenol or picric acid;
5-NT, 5-nitrobenzene-triol; H-5-NBQ, hydroxy-5-nitro-1,2-
benzoquinone; 4-BP, 4-bromophenol; 4-CP, 4-chlorophenol;
DBP, 2,4-dibromophenol; TBP, 2,4,6-tribromophenol; TCP,
2,4,6-trichlorophenol; POP, persistent organic pollutant;
Compound I, two-electron oxidized heme center when
compared to the ferric form, commonly as an FeIVO
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porphyrin π-cation radical; Compound II, one-electron
oxidized heme center when compared to the ferric form,
commonly as an FeIVO or FeIV−OH; Compound III,
oxyferrous [FeII−O2 or FeIII−(O2 )] state of the enzyme;
−
Compound ES, two-electron-oxidized state containing both a
ferryl center [FeIVO] and an amino acid (tryptophanyl or
tyrosyl) radical, analogous to Compound ES in cytochrome c
peroxidase; Compound RH, “Reversible Heme” state of
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