The multifunctional globin dehaloperoxidase strikes again: Simultaneous peroxidase and peroxygenase mechanisms in the oxidation of EPA pollutants

Article abstract of DOI:10.1016/j.abb.2019.108079

The multifunctional catalytic hemoglobin dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata was found to catalyze the H₂O₂-dependent oxidation of EPA Priority Pollutants (4-Me-o-cresol, 4-Cl-m-cresol and pentachlorophenol) and EPA Toxic Substances Control Act compounds (o-, m-, p-cresol and 4-Cl-o-cresol). Biochemical assays (HPLC/LC-MS) indicated formation of multiple oxidation products, including the corresponding catechol, 2-methylbenzoquinone (2-MeBq), and oligomers with varying degrees of oxidation and/or dehalogenation. Using 4-Br-o-cresol as a representative substrate, labeling studies with ¹⁸O confirmed that the O-atom incorporated into the catechol was derived exclusively from H₂O₂, whereas the O-atom incorporated into 2-MeBq was from H₂O, consistent with this single substrate being oxidized by both peroxygenase and peroxidase mechanisms, respectively. Stopped-flow UV–visible spectroscopic studies strongly implicate a role for Compound I in the peroxygenase mechanism leading to catechol formation, and for Compounds I and ES in the peroxidase mechanism that yields the 2-MeBq product. The X-ray crystal structures of DHP bound with 4-F-o-cresol (1.42 ?; PDB 6ONG), 4-Cl-o-cresol (1.50 ?; PDB 6ONK), 4-Br-o-cresol (1.70 ?; PDB 6ONX), 4-NO₂-o-cresol (1.80 ?; PDB 6ONZ), o-cresol (1.60 ?; PDB 6OO1), p-cresol (2.10 ?; PDB 6OO6), 4-Me-o-cresol (1.35 ?; PDB 6ONR) and pentachlorophenol (1.80 ?; PDB 6OO8) revealed substrate binding sites in the distal pocket in close proximity to the heme cofactor, consistent with both oxidation mechanisms. The findings establish cresols as a new class of substrate for DHP, demonstrate that multiple oxidation mechanisms may exist for a given substrate, and provide further evidence that different substituents can serve as functional switches between the different activities performed by dehaloperoxidase. More broadly, the results demonstrate the complexities of marine pollution where both microbial and non-microbial systems may play significant roles in the biotransformations of EPA-classified pollutants, and further reinforces that heterocyclic compounds of anthropogenic origin should be considered as environmental stressors of infaunal organisms.

Full text of DOI:10.1016/j.abb.2019.108079

Journal Pre-proof
The multifunctional globin dehaloperoxidase strikes again: Simultaneous peroxidase
and peroxygenase mechanisms in the oxidation of EPA pollutants
Talita Malewschik, Vesna de Serrano, Ashlyn H. McGuire, Reza A. Ghiladi
PII:
S0003-9861(19)30371-6
https://doi.org/10.1016/j.abb.2019.108079
YABBI 108079
DOI:
Reference:
To appear in: Archives of Biochemistry and Biophysics
Received Date: 16 May 2019
Revised Date: 19 August 2019
Accepted Date: 20 August 2019
Please cite this article as: T. Malewschik, V. de Serrano, A.H. McGuire, R.A. Ghiladi, The multifunctional
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The Multifunctional Globin Dehaloperoxidase
Strikes Again: Simultaneous Peroxidase and
Peroxygenase Mechanisms in the Oxidation of EPA
Pollutants
Talita Malewschik, Vesna de Serrano, Ashlyn H. McGuire and Reza A. Ghiladi*
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204
Manuscript Correspondence:
Prof. Reza A. Ghiladi
Department of Chemistry
North Carolina State University
Raleigh, NC 27695
(
(
919) 513-0680 (phone)
919) 515-8920 (fax)
E-mail: Reza_Ghiladi@ncsu.edu
1

ABBREVIATIONS
Asymmetric Unit (ASU); 2-MeBq, 2-methyl-1,4-benzoquinone; 4-Br-C, 4-bromo-o-cresol; 4-
BP, 4-bromophenol; 4-Cl-C, 4-chloro-o-cresol; 4-F-C, 4-fluoro-o-cresol; 3-MC, 3-
methylcatechol; 4-MC, 4-methylcatechol; 4-Me-C, 4-methyl-o-cresol; 4-NO -C, 4-nitro-o-
2
cresol; 4-NP, 4-nitrophenol; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; Compound I, two-
IV
electron oxidized heme cofactor compared to the ferric form, commonly as an Fe =O
porphyrin π-cation radical; Compound II, one-electron oxidized heme cofactor when compared
IV
IV
to the ferric form, commonly as an Fe =O or Fe –OH; Compound ES, two-electron-oxidized
IV
state containing both a ferryl center [Fe =O] and an amino acid (tryptophanyl or tyrosyl)
radical, analogous to Compound ES in cytochrome c peroxidase; Compound RH, ‘Reversible
Heme’ state of dehaloperoxidase, formed from the decay of Compound ES in the absence of co-
substrate; DHP, dehaloperoxidase; EI, electron ionization; EPA, Environmental Protection
Agency; ESI, electrospray ionization; Hb, hemoglobin; HRP, horseradish peroxidase; HSM,
horse skeletal muscle; Mb, myoglobin; PCP, pentachlorophenol; TBP, 2,4,6-tribromophenol;
TCP, 2,4,6-trichlorophenol; TFA, trifluoroacetic acid; WT, wild-type.
2

HIGHLIGHTS
•
•
•
•
•
Cresols are a new class of substrates for dehaloperoxidase (DHP)
Substrate oxidation yields monomeric and oligomeric products with dehalogenation
Crystallographic studies reveal substrate binding in the distal heme pocket
Labeling studies show concurrent peroxidase and peroxygenase oxidation mechanisms
DHP oxidizes 10 of the 11 phenolic compounds on the EPA Priority Pollutants List
3

ABSTRACT The multifunctional catalytic hemoglobin dehaloperoxidase (DHP) from the
terebellid polychaete Amphitrite ornata was found to catalyze the H O -dependent oxidation of
2
2
EPA Priority Pollutants (4-Me-o-cresol, 4-Cl-m-cresol and pentachlorophenol) and EPA Toxic
Substances Control Act compounds (o-, m-, p-cresol and 4-Cl-o-cresol). Biochemical assays
(HPLC/LC-MS) indicated formation of multiple oxidation products, including the corresponding
catechol, 2-methylbenzoquinone (2-MeBq), and oligomers with varying degrees of oxidation
and/or dehalogenation. Using 4-Br-o-cresol as a representative substrate, labeling studies with
1
8
O confirmed that the O-atom incorporated into the catechol was derived exclusively from
, whereas the O-atom incorporated into 2-MeBq was from H O, consistent with this single
H
2
O
2
2
substrate being oxidized by both peroxygenase and peroxidase mechanisms, respectively.
Stopped-flow UV−visible spectroscopic studies strongly implicate a role for Compound I in the
peroxygenase mechanism leading to catechol formation, and for Compounds I and ES in the
peroxidase mechanism that yields the 2-MeBq product. The X-ray crystal structures of DHP
bound with 4-F-o-cresol (1.42 Å; PDB 6ONG), 4-Cl-o-cresol (1.50 Å; PDB 6ONK), 4-Br-o-
2
cresol (1.70 Å; PDB 6ONX), 4-NO -o-cresol (1.80 Å; PDB 6ONZ), o-cresol (1.60 Å; PDB
6
OO1), p-cresol (2.10 Å; PDB 6OO6), 4-Me-o-cresol (1.35 Å; PDB 6ONR) and
pentachlorophenol (1.80 Å; PDB 6OO8) revealed substrate binding sites in the distal pocket in
close proximity to the heme cofactor, consistent with both oxidation mechanisms. The findings
establish cresols as a new class of substrate for DHP, demonstrate that multiple oxidation
mechanisms may exist for a given substrate, and provide further evidence that different
substituents can serve as functional switches between the different activities performed by
dehaloperoxidase. More broadly, the results demonstrate the complexities of marine pollution
where both microbial and non-microbial systems may play significant roles in the
4

biotransformations of EPA-classified pollutants, and further reinforces that heterocyclic
compounds of anthropogenic origin should be considered as environmental stressors of infaunal
organisms.
KEYWORDS: cresols; dehaloperoxidase; hemoglobin; oxidation; peroxidase; peroxygenase
5

GRAPHICAL ABSTRACT
6

INTRODUCTION
A number of phenolic compounds have been classified as persistent organic pollutants
(
POPs) by the EPA and World Health Organization owing to their resistance to biodegradation
1, 2]. As a consequence, bioaccumulation of POPs may cause severe and often long-lasting
[
effects on the environment, spanning atmospheric, terrestrial, and marine systems [3, 4]. Of
similar concern, once released into the environment from anthropogenic sources, some phenolic
compounds can undergo biotransformations owing to their similarity to naturally-produced
compounds, forming secondary metabolites that can be even more toxic than the parent
compound [5]. Relevant to the present study, examples of phenolic POPs include i) cresols
(methylphenols), which are released into the environment mainly by industrial effluent or
through the combustion of petroleum, coal, wood, and tobacco [6], and ii) pentachlorophenol, a
common water contaminant that is primarily employed as a wood preservative and pesticide [7].
Due to their potential for significant environmental toxicity, adverse health effects after acute or
chronic exposure, and long-term persistence leading to bioaccumulation, a range of phenol
derivatives, including halophenols, nitrophenols, and cresols, have elevated threat levels and are
either classified by the EPA as Priority Pollutants [1] or included in the Toxic Substances
Control Act [2].
In light of their environmental toxicity, phenol biodegradation pathways have been the
subject of numerous investigations. Microorganisms (e.g., bacteria and fungi) employ cresol
isomers (
methylcatechol and
fission that leads to smaller products [8-11]. Additionally,
chloroperoxidase [12], manganese peroxidase [13], and polyphenol oxidase [14], and in the case
o
-,
m
-,
p
-cresol) as carbon sources, with initial oxidation of
-cresol to 3-methylcatechol and 2-methylhydroquinone, followed by ring
-cresol is oxidized by
p-cresol to 4-
o
p
7

of horseradish peroxidase (HRP) and ascorbate peroxidase (APX), common oxidation products
include 2-dihydroxy-5,5-dimethylbiphenyl and tetrahydrodibenzofurane (Pummerer’s quinone)
[15, 16]. Enzymes are also employed in the degradation of pentachlorophenol (PCP), including
HRP and LiP [16], and PCP-degrading bacteria, such as Sphingobium chlorophenolicum and
Flavobacterium sp., employ multiple enzymes for full mineralization of the chlorinated substrate
[17, 18]. Despite the insights afforded by these studies, the majority of them have focused on
plant or microbial pathways, and comparatively fewer studies have addressed how infaunal
organisms impact the fate of, or are themselves impacted by, persistent organic pollutants [19-
2
2].
One such infaunal system that is potentially involved in the degradation of POPs and related
xenobiotics is the multifunctional catalytic hemoglobin dehaloperoxidase (DHP) from Amphitrite
ornata [23-29]. As a sediment-dwelling marine worm [30], A. ornata tolerates a diverse array of
biogenically-produced organobromine compounds (secreted by other marine organisms as
defense mechanisms [31]) by employing its hemoglobin as a detoxification enzyme. Named
dehaloperoxidase [27, 32], this O
of compounds, including mono, di- and trihalophenols [32], haloindoles [35], pyrroles [36],
halo)guaiacols [23] and nitrophenols [25], and also strongly binds azoles [24]. Substrate
2
-transport protein [33, 34] is capable of oxidizing a wide array
(
oxidation/degradation in DHP takes place via one of four canonical heme-based mechanisms:
peroxidase [37-40], peroxygenase [25, 35, 36], oxidase [35], and oxygenase [41], with no
crossover between these activities for a given class of substrate, i.e., halophenols, nitrocatechol,
and haloguaiacols are oxidized solely via a peroxidase-based mechanism, and haloindoles,
pyrrole, and nitrophenols are exclusively oxidized via a peroxygenase activity. To date, it is still
not understood how DHP (as a multifunctional enzyme) controls for this activity differentiation
8

given that all activities occur at a single site employing reactive intermediates (i.e., Compounds I
42, 43], ES [37, 38], and II [44]) that are commonly invoked in these four mechanisms [45-47].
[
Moreover, no mechanistic studies have explored the oxidation of cresols or pentachlorophenol
by DHP as a model for infaunal organisms.
To address these questions, and in light of the need for further investigating biological
systems of nonmicrobial origin in assessing the environmental fate and/or impact of phenolic
compounds from the EPA Priority Pollutants and Toxic Substances Control Act lists, here we
present structural, spectroscopic, and mechanistic studies describing the reactivity of cresols and
pentachlorophenol with the multifunctional globin dehaloperoxidase, and establish cresols as a
new class of substrate for DHP. We will show that unlike phenols/guaiacols (peroxidase, red
box) and nitrophenols (peroxygenase, blue box), DHP is able to oxidize a given cresol substrate
by both peroxidase and peroxygenase mechanisms (Figure 1, purple box), and the ability to
‘tune’ DHP activity through the selection of the substrate (e.g., substituent effects [23, 48, 49])
will be discussed. The results here will demonstrate that DHP is a unique example of a
multifunctional protein that challenges many of the assumptions behind the protein structure-
function correlation that has been built from decades of study of monofunctional proteins, and
provides us with a system for exploring how the substrate itself can have a significant influence
in tuning the chemical reactivity exhibited by an enzyme.
Phenol/Guaiacol
Cresol
Nitrophenol
R = H, OCH3
Peroxidase
+
Peroxidase
Peroxygenase
Peroxygenase
Figure 1. Substituent effects modulate different oxidation mechanisms in DHP: peroxidase (red)
and peroxygenase (blue), with cresols being oxidized by both mechanisms (purple).
9

EXPERIMENTAL
Materials. Ferric WT DHP B was expressed and purified as previously reported [38, 50].
Oxyferrous DHP B was prepared by the aerobic addition of excess ascorbic acid to ferric DHP
B, followed by desalting (PD-10 column) [44]. DHP concentration was determined
-1
-1
spectrophotometrically using εsoret = 116,400 M cm [38]. Horse skeletal muscle (HSM)
−
1
−1
myoglobin (εsoret = 188,000 M cm [51]), horseradish peroxidase (HRP) (εsoret = 102,000
−
1
−1
M cm [52]) and mushroom tyrosinase from Agaricus bisporus (7164 U/mg; hydrated using
1
00 mM KP at pH 7) were purchased from Sigma-Aldrich and used as received. Substrate stock
i
solutions (10 mM) were prepared in methanol and stored at -80 °C. Solutions of H
2 2
O were
prepared fresh daily in 100 mM KP (pH 7) and kept on ice until needed. Isotopically labeled
i
18
18
18
18
H
2
O
2
(90% O-enriched) and H
2
O (98% O-enriched) were purchased from Icon Isotopes
(Summit, NJ). Acetonitrile (MeCN) was HPLC grade, and all other reagent-grade chemicals
were purchased from VWR, Sigma-Aldrich or Fisher Scientific and used without further
purification.
HPLC Reactivity Studies. Enzyme assays (250
µL total volume) were performed in triplicate
in 100 mM KP at pH 7 (unless otherwise indicated) containing 5% MeOH at room temperature.
i
A typical reaction was initiated by the addition of 500
enzyme and 500 M substrate, and quenched after 5 minutes with an excess of catalase. Enzyme
variants included ferric WT DHP B, ferric DHP B (Y28F/Y38F) [42], oxyferrous WT DHP B,
HSM and HRP. Mechanistic probes were added prior to the addition of H O , including 500
µM H O to a solution containing 10 µM
2 2
µ
µM
2
2
4
-bromophenol (MeOH), 500
DMPO; MeOH) or 10% v/v DMSO, and adjustments were made to ensure a final 5% MeOH
concentration for all reactions except DMPO (10% MeOH final). A 200 L aliquot of the
µM D-mannitol (KP ), 100 mM 5,5-dimethyl-1-pyrroline N-oxide
i
(
µ
1
0

reaction mixture was diluted 4-fold with 600 µL 100 mM KP at the reaction pH, and the diluted
i
samples were analyzed using a Waters e2695 Separations Module coupled to a Waters 2998
photodiode array detector and equipped with a Thermo Fisher Scientific ODS Hypersil (150 mm
×
4.6 mm) 5 µM particle size C18 column. Separation was performed using a linear gradient of
binary solvents (solvent A, water + 0.1% TFA; solvent B, acetonitrile + 0.1% TFA). The elution
consisted of the following conditions: (1.5 mL/min A:B) 95:5 to 5:95 linearly over 12 min, 5:95
isocratic for 2 min, 5:95 to 95:5 over 1 min, then isocratic for 3 min. Data analysis was
performed using the Waters Empower software package.
Product Determination by LC-MS. The reactions were performed and quenched as described
above with the exception that 5 mM KP
reaction (20 L injection aliquot) was carried out using a Thermo Fisher Scientific Exactive Plus
Orbitrap mass spectrometer employing a heated electrospray ionization (HESI) probe and
equipped with a Thermo Hypersil Gold (50 x 2.1 mm, particle size 1.9 m) C column. The flow
rate was set to 250 L/min (solvent A, water + 0.1% formic acid; solvent B, acetonitrile + 0.1%
formic acid) and the mass spectrometer was operated in both negative and positive ion modes to
i
(pH 7) buffer was employed. Analysis of the undiluted
µ
µ
4
µ
-
+
yield the [M-H] and [M+H] species, respectively. Spectra were collected while scanning from
1
8
1
00-1500
m/z
and data analysis was performed using Thermo Xcalibur software. For the
O
labeling studies, the reactions were performed for 10 minutes in the presence of DHP, substrate,
18
and H O , where unlabeled H O was replaced with H O , and/or the KP buffer was replaced
2
2
2
2
2
2
i
18
18
with H O to ensure >90% of labeled O was present.
2
Tyrosinase-Catalyzed Cresol Hydroxylation. The tyrosinase-catalyzed hydroxylations of 4-
-cresol (R = F, Cl, Br, NO ), 4-Cl- -cresol, -cresol and -cresol were performed per
literature protocol with modifications [53-55]. Briefly, to a 1.5 dram glass vial was added neat
R-o
m
m
p
2
1
1

cresol substrate (0.05 mmol), 1.5 equiv ascorbic acid (solid), 1.665 mL 100 mM KP
) and 335 L tyrosinase (7880 U/mL). The reaction was stirred open to air for 24 hours at room
temperature, quenched with 1 M HCl (1 mL), and subsequently analyzed by HPLC and LC-MS
negative ion mode) (vide supra for instrument and method information) prior to workup. The
i
buffer (pH
7
µ
(
reaction mixture was then extracted with ethyl acetate (EtOAc; 2 x 2 mL), and the combined
organic extracts were dried over sodium sulfate, filtered, and concentrated in vacuo to yield oils
that ranged from colorless to dark brown. The crude product was redissolved in 150
µ
L EtOAc,
L of
followed by the addition of 100 L pyridine, 100 L hexamethyldisilazane and 50 µ
µ
µ
trimethylsilyl chloride (HMDS-TMCS, 2:1 v/v) [55]. This mixture was vigorously stirred for 30
minutes, allowed to stand for 5 minutes, and then centrifuged for 5 minutes at 10,000 rpm. The
supernatant was analyzed on an Agilent 6890 Series GC System coupled to an Agilent 5973
Mass Selective Detector using a DB-5MS column (0.25 mm i.d. x 30 m, 0.25
µm film
thickness). The method employed was as follows: isothermal at 100 °C for 2 minutes, a gradient
of 10 °C/min to 280 °C, and maintained at 280 °C for 10 minutes [53]. The flow rate (He) was
1
5 mL/min, and the injection volume was 1
µL. Mass spectra were recorded with an electron
beam of 70 eV, scanning from 50-550 m/z
.
Binding studies. The substrate dissociation constants (Kd values) were determined in
triplicate for ferric WT DHP B using a Cary 50 UV-vis spectrophotometer as per previously
published protocols [35, 56]. Against a reference spectrum of 50
KP (pH 7) containing 5% MeOH, difference spectra were obtained for the addition of 0.5-150
equiv substrate to 50 M DHP B while maintaining constant enzyme, buffer, and MeOH
µM ferric DHP B in 100 mM
i
µ
concentrations. Analysis of the visible region (450-700 nm) was performed using the ligand
binding function in Grafit (Erithacus Software Ltd.).
1
2

Crystallization and Data Collection. Crystallization and data collection were performed
using non-His-tagged ferric DHP B that was overexpressed and purified as per literature
precedent [23, 48-50]. The protein was crystallized from the ferric form using the hanging-drop
vapor diffusion method. Crystals grew from 30-32% MPEG 2000, 0.2 M ammonium sulfate and
0
.02 M sodium cacodylate (pH 6.5) by mixing protein at 10 mg/mL in 20 mM sodium cacodylate
(pH 6.5) and crystallizing solution at 1:1, 1.5:1 or 2:1 of protein:reservoir solution. Crystals
appeared after 3 days of incubation at 4 °C and were harvested after a growth period of one to
two weeks. All the substrate-bound forms were obtained by overnight soaking of crystals in
solutions containing 34% MPEG 2000, 0.2 M ammonium sulfate, and supplemented with
varying concentrations of substrate (1–100 mM) dissolved in DMSO, with a final concentration
of 5% DMSO in the soaking solution. The crystals were subsequently cryoprotected by dipping
in soaking solution supplemented with 25% ethylene glycol, and flash cooled in liquid nitrogen
before data collection. X-ray diffraction data were collected remotely at the SER-CAT ID22 and
BM22 beamlines at the Advanced Photon Source, Argonne National Laboratories. The data sets
were collected at 100 K, using a wavelength of 1.0 Å, employing a shutterless Rayonix
MX300HS detector. The data were integrated and scaled with either iMosflm from CCP4 suite
or HKL2000 program suite [57], molecular replacement was done with Phaser-MR [58] using
3
IXF [50] coordinates as a search model, manual model building was performed in COOT [59],
whereas refinement was carried out using REFMAC5 [60] in the CCP4 suite [61] and
phenix.refine in the PHENIX suite of programs [62, 63]. The final model was validated using
COOT, MolProbity [64] and PDB_REDO [65].
Stopped-Flow UV−Visible Spectroscopic Studies. Optical spectra were recorded using a
Bio-Logic SFM-400 triple-mixing stopped-flow instrument coupled to a rapid scanning (1.5 ms)
1
3

2 2
diode array UV−vis spectrophotometer. The protein and H O solutions were prepared in 100
mM KP (pH 7), and the substrates solutions were made in buffer containing 5% MeOH. Double-
i
mixing experiments were performed using an aging line prior to the second mixing step to
observe Compound I/ES/II reactivity with 10 equiv substrate, as follows: (i) Compound I was
pre-formed from the reaction of ferric DHP B (Y28F/Y38F) [42] with 10 equiv H O in an aging
2
2
line for 85 ms prior to mixing with the substrate; (ii) Compound ES was pre-formed by reaction
of ferric WT DHP B with 10 equiv of H in an aging line for 400 ms prior to mixing with the
substrate [38]; (iii) Compound II was pre-formed from oxyferrous DHP B that was preincubated
with 1 equiv TCP and then reacted with 10 equiv of H in an aging line for 2.75 s prior to
2
O
2
2
O
2
mixing with the substrate [23, 44]. Data were collected (900 scans total) over a three-time
domain (1.5, 15, and 150 ms) observation period using the Bio-Kine 32 software package (Bio-
Logic). All data were evaluated using the Specfit Global Analysis System software package
(Spectrum Software Associates) and fit with SVD analysis as either one-step, two-species or
two-step, three-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.
RESULTS AND DISCUSSION
DHP-Catalyzed Substrate Reactivity with H O . The hydrogen peroxide-dependent reaction
2
2
of ferric WT DHP B with the EPA Priority Pollutants and related substrates was monitored by
HPLC, and the corresponding substrate conversion percentages (based upon substrate loss) are
reported in Table 1. Reactions were initiated upon the addition of 500
2 2
µM H O to a solution
containing 10 M ferric WT DHP B and 500 M substrate, incubated at 25 °C for 5 min, and
µ
µ
1
4

then quenched with catalase (as DHP does not possess any catalase activity). At pH 7, the
substrate conversion ranged from a low value of 11% for pentachlorophenol to a high of ~67%
for
attributable to protein-dependent pH effects given that the substrate pKa values are below ~5 or
above ~9.5 (pKa values: -cresol, 10.29 [66]; -cresol, 10.26 [66]; -cresol, 10.09 [66]; 4-Me-
cresol, 10.60 [67]; 4-Cl- -cresol, 9.71 [67]; 4-Cl- -cresol, 9.55 [67]; pentachlorophenol, 4.70
68]; 2,4-dinitro- -cresol, 4.31 [69]). The order of oxidation of the cresol isomers was found as
-cresol ~ -cresol < -cresol, which mirrors their binding affinities (vide infra). An inverse
relationship was observed between halogen size and enzyme reactivity for the 4-X- -cresols
p-cresol. When repeated at pH 8, only modest changes (< 2-fold) were noted, likely
o
m
p
o-
o
m
[
o
o
m
p
o
halogen series (X = F, Cl, Br), with a 2-fold difference (F > Cl > Br) between the highest and
lowest conversions. While an electronic effect cannot be ruled out, we note a significant
difference in the substrate orientation within the active site for 4-F-o-cresol (51.8%) vs. 4-Cl-o-
cresol (35.2%) and 4-Br- -cresol (27.5%) as shown by X-ray crystallography (vide infra, Figure
o
S10). Finally, DHP B was also found to catalyze the oxidation of pentachlorophenol (11%),
which now adds the fully substituted analog to the halophenol series 4-chlorophenol (inhibitor)
[
32, 70], 2,4-dichlorophenol (substrate [32, 71]) and 2,4,6-trichlorophenol (peroxidase substrate
32, 38, 72]). Finally, no reactivity was observed for 2,4-dinitro- -cresol under any conditions
[
o
examined, even when performed at pH 5 (data not shown), which is near its reported pKa of 4.31.
As DHP is able to catalyze the oxidation of 2,4-dinitrophenol (pKa 4.07 [66]) at pH 5 and 6 [25],
the lack of reactivity here with 2,4-dinitro-o-cresol is possible due to binding or electronic
factors, and not pH-related. As expected, neither substrate turnover nor product formation were
observed in the absence of H
not shown).
2 2
O (non-oxidant control) or enzyme (non-enzymatic control) (data
1
5

Table 1. DHP-Catalyzed Substrate Oxidation Studies.
Substrate
Conversion (%)
DHP B WT Ferric
pH 7
pH 8
+
+
+
+
+
+
+
+
+
+
+
o
p
-cresol
-cresol
31.6 (± 6.2)
67.3 (± 3.6)
33.8 (± 2.2)
65.3 (± 6.7)
51.8 (± 2.2)
35.2 (± 3.7)
27.5 (± 3.5)
30.3 (± 3.0)
n.d.
35.2 (± 2.9)
75.9 (± 3.5)
35.7 (± 3.0)
81.4 (± 11.5)
53.7 (± 2.0)
46.8 (± 5.7)
43.8 (± 4.3)
11.8 (± 2.3)
n.d.
m
-cresol
4-Me- -cresol
4-F- -cresol
4-Cl- -cresol
-cresol
-cresol
-cresol
-cresol
pentachlorophenol
o
o
o
4-Br-
o
2
4-NO -o
2,4-dinitro-
o
4-Cl-
m
35.4 (± 1.9)
11.0 (± 2.1)
23.1 (± 1.6)
8.9 (± 1.3)
Reaction conditions: [ferric DHP B] = 10
2 2
µM, [substrate] = [H O ] = 500 µM, 5% MeOH/100
mM KP (v/v) at pH 7 or 8, 25 °C, 5 min; n.d. = no reactivity detected.
i
As the native substrate scope of dehaloperoxidase is believed to be organobromine
compounds [32], and as DHP B has been shown to oxidize brominated analogs of phenols [38],
indoles [35], and guaiacols [23], mechanistic investigations were performed here with 4-Br-o-
cresol (4-Br-C) as a representative substrate for the cresol family, the results of which are
summarized in Table 2.
(
i) pH dependence: a ~3-fold increase in substrate oxidation was observed as the reaction pH
was increased from 5 (16.0%) to 8 (43.8%), with only a minor further increase at pH 9 (46.2%).
This suggests that maximal activity occurs as the pH Ka of the DHP metaquo acid–alkaline
≥
p
transition of 8.1 [73]. This is the opposite behavior as has been previously shown for DHP
peroxygenase substrates 4-nitrophenol [25], pyrrole [36], and 5-Br-indole [35], where activity
increased as the pH decreased.
(ii) Enzyme variations: the oxidation of 4-Br-o-cresol as catalyzed by oxyferrous DHP B
(21.0%) was slightly attenuated compared to that of ferric DHP B (27.5%), but still demonstrated
1
6

II
that the substrate oxidation reaction can be initiated from either the globin-active (Fe -O
2
) or the
III
peroxidase active (Fe ) oxidation states, a result consistent with literature precedent [23, 25, 35,
3
6, 44, 74, 75]. Studies performed with DHP B (Y28F/Y38F), a mutant that yields Compound I
as the initially-observed reactive intermediate rather than the Compound ES species observed in
WT DHP B [42], showed only slightly lower substrate conversion (21.9%) compared to WT
DHP. Whereas the canonical horseradish peroxidase (HRP) yielded virtually complete
conversion (98.8%) of 4-Br-o-cresol, horse skeletal muscle myoglobin showed similar
conversion (33.1%) compared to WT DHP B; in both cases, the product distributions were
different from that observed for DHP (data not shown). Finally, DHP isoenzyme A, which
differs from DHP B by only five amino acid substitutions [49, 50], showed a higher conversion
of 49.7% than the DHP B analog; the higher activity of isoenzyme A over that of B has only
been observed previously with pyrrole (a peroxygenase substrate) [36].
(
iii) Mechanistic studies: The addition of 4-bromophenol (Kd = 1.15 mM [70]), a known
peroxidase and peroxygenase inhibitor [76, 77], decreased substrate conversion by 1.7-fold,
suggesting that 4-bromophenol has an inhibitory effect on 4-Br- -cresol oxidation. When the
o
reaction was performed in the presence of the known radical scavengers D-mannitol (26.1%) and
DMPO (21.1%), no significant differences in substrate conversion or product distribution were
noted, a result that suggests that solvent accessible radicals do not play a role in the oxidation of
4
-Br-
o
-cresol. As a comparison, 4-Br-
o
-guaiacol, a peroxidase substrate, was similarly
-guaiacol and 4-NO2-
unaffected by DMPO, whereas this radical scavenger did inhibit 5-Br-
o
o-
guaiacol conversion, suggesting that these peroxidase substrates do proceed through diffusible
radicals [23]. Finally, in the presence of DMSO, a slight increase (to 34.5%) in reactivity without
change in product distribution was shown, which we attributed to the organic solvent facilitating
1
7

access of the substrate to the hydrophobic binding pocket of DHP [78].
Taken together, the results obtained from the above enzymatic assays showed that
dehaloperoxidase was able to catalyze the conversion of the EPA pollutants under physiological
conditions, and establishes cresols as yet another class of phenolic substrate for DHP. These
studies do not, however, provide definitive evidence for oxidation via either a peroxidase or
peroxygenase-based mechanism, necessitating labeling studies (vide infra).
Table 2. DHP-catalyzed oxidation of 4-Br-o-cresol as a function of enzyme variant, pH, and
mechanistic probes.
Condition
Conversion (%)
pH Studies
pH 5.0
pH 6.0
pH 7.0
16.0 (± 1.1)
19.1 (± 1.0)
27.5 (± 3.5)
n. d.
-
-
2 2
H O
enzyme
n. d.
pH 8.0
pH 9.0
43.8 (± 4.3)
46.2 (± 6.6)
Enzyme Variation
DHP B Oxyferrous
DHP A Ferric
DHP B (Y28F/Y38F)
HRP
21.0 (± 3.2)
49.7 (± 1.4)
21.9 (± 1.5)
98.8 (± 0.2)
33.1 (± 3.2)
HSM Mb
Mechanistic Probes
5
1
1
5
00
µ
M D-mannitol
26.1 (± 1.3)
34.5 (± 2.3)
21.1 (± 5.0)
0% DMSO
00 mM DMPO
00
a
µ
M 4-bromophenol 15.0 (± 2.8)
M, [4-Br-o-cresol] = [H O ] = 500 µM, 5% MeOH/100 mM
2 2
Reaction conditions: [DHP] = 10
µ
a
KP (v/v), pH 7, 25 °C, 5 min; n.d. = no reactivity detected. = reaction performed in 10%
i
MeOH.
Identification of Reaction Products by HPLC and LC-MS. A representative chromatogram
for each DHP B-catalyzed substrate oxidation reaction in the presence of H O is found in Figure
2
2
2
, and the reaction mixtures were further analyzed by LC-MS. Under the conditions employed,
1
8

multiple products were observed, and although identification of the exact chemical structures
was not pursued, the products consisted of oligomers (up to n=6) with varying degrees of
oxidation; a list of the retention times, masses and chemical formulae obtained for the products
(up to n = 3) can be found in Tables S1-S4. For the 4-X-cresol series (X = F, Cl, Br),
dehalogenation products were also observed, consistent with previous studies employing 2,4,6-
trihalophenols (yielding the corresponding 2,6-dihaloquinones [38]) and 4-X-guaiacols (forming
oligomeric products) that also underwent oxidative dehalogenation chemistry [23].
*
pentachlorophenol
*
4
4
-Cl-m-cresol
▲
*
-NO -o-cresol
▲
2
*
▲
4
4
-Br-o-cresol
-Cl-o-cresol
▲
▲
*
*
4
4
-F-o-cresol
*
-Me-o-cresol
▲
*
m-cresol
p-cresol
*
▲
*
o-cresol
3
4
5
6
7
Retention Time (min)
8
9
10
Figure 2. HPLC chromatograms for the reaction of DHP B with the EPA priority pollutants and
cresol derivatives in the presence of H O . Reaction conditions: 10 M DHP B, 500
(v/v), pH 7, 25 °C. The reaction was quenched
after 5 minutes with an excess of catalase. The 2-MeBq product is highlighted by the triangle
with a ~ 4.6 min. The asterisks denote the substrate. Reactions were monitored at 260 nm
except pentachlorophenol (280 nm), and 4-X- -cresol (X = F, Cl, Br; 265 nm).
µ
µM
2
2
2 2 i
substrate, 500 µM H O , 5% MeOH/100 mM KP
t
R
o
For seven of the eleven substrates [o-cresol, m-cresol, 4-Cl-m-cresol and 4-R-o-cresol (R = F,
Cl, Br, NO )] studied, a common oxidation product was observed. This product exhibited
2
identical spectroscopic features (
λ
max = 249 nm), retention time (
t
R
= 4.6 min), and mass (m/z
-
+
1
21.0, [M-H] ; 123.0, [M+H] ) that matched a commercial sample of 2-methyl-1,4-
1
9

benzoquinone (2-MeBq; Figure S1). On the basis of a calibration curve (Figure S2), the amount
of 2-MeBq was determined for each reaction (Table S5), and it was found that 2-MeBq
formation varied up to 150-fold (0.2 – 28.9
µM) at pH 7, but only ~5-fold at pH 8 (5.2 – 27.8
µM, pH 8). The formation of the 2-MeBq indicates dehalogenation (-HX, X = F, Cl, Br) and loss
of nitrite (HNO ), and although no attempts were made to identify the leaving groups, stopped-
2
flow UV/visible spectroscopic studies suggest that the halogen ions could serve as ligands to the
heme-Fe, forming Fe-X adducts (vide infra). No 2-MeBq product was observed in the reactions
with
unaffected when 100 mM DMPO was included in the 4-Br-
formation has precedent for the DHP-catalyzed oxidative dehalogenation of 2,4,6-trihalophenol
X = F, Cl, Br) yielding the corresponding 2,6-dihaloquinone [32], and 4-X- -guaiacol (X = F,
p
-cresol, 4-Me-o-cresol and pentachlorophenol, and the yield of 2-MeBq product was
o
-cresol reaction. Quinone product
(
o
Cl, Br) yielding 2-methoxybenzoquinone (2-MeOBQ) [23], with both reactions proceeding via a
peroxidase-based mechanism. Formation of these quinone products has been previously linked to
a product-driven reduction [23, 38] owing to the unusually high redox potential of DHP [79],
thereby providing a resolution to the ‘dehaloperoxidase paradox’ [80] wherein the product of its
III
peroxidase activity (with an Fe resting state) reduces DHP back to the ferrous oxidation state,
thereby rescuing its oxygen-transport function.
Identification of a Catechol Reaction Product. With the exception of pentachlorophenol,
each substrate oxidation reaction exhibited a product that corresponded to the incorporation of an
O-atom into the substrate (monomer + 1 O; Tables S1-S3). To determine if these products could
be specifically ascribed to catechol formation, authentic samples of the corresponding catechol,
either from commercial sources or prepared enzymatically (see Supporting Information), were
employed as follows:
2
0

R
(i) 4-methylcatechol (4-MC) was found to have the same characteristic retention time (t =
-
4
.4 min), UV-visible spectrum (λmax = 225 nm) and mass (m/z [M-H] = 123) as one of the
oxidation products of
p
-cresol in the HPLC chromatogram (Figure S3), confirming the formation
-cresol.
ii) 3-methylcatechol (3-MC) showed a retention time of 4.7 min (λmax = 225 nm), however,
of 4-MC during the DHP-catalyzed oxidation of
p
(
when compared to the
o-cresol chromatogram, no peak with the same retention time and
spectroscopic profile was observed (data not shown), despite the presence of the corresponding
-
mass (m/z [M-H] = 123; Table S1) being observed by LC-MS for the oxidation of
o-cresol. This
suggests that the 3-methylcatechol product is present at very small concentrations in the reaction
mixture.
(
iii) As they were not commercially available, the catechol derivatives for 4-R-
o-cresol (R =
F, Cl, Br, NO ), 4-Cl- -cresol, and -cresol were synthesized enzymatically employing
m
m
2
tyrosinase following literature protocols [53-55], and their characterization by GC-MS, LC-MS
and HPLC can be found in the Supporting Information (Figures S4-S6). As shown in Figures S5
and S6, catechol products were identified by their retention time and UV-visible spectrum in the
2
DHP-catalyzed oxidation of 4-F-o-cresol, 4-NO -o-cresol, 4-Cl-m-cresol and m-cresol.
Altogether, O-atom incorporation for five cresol substrates was confirmed from authentic
samples that were either obtained commercially or enzymatically, with hydroxylation of the
remaining cresol substrates (excluding pentachlorophenol) indicated by the LC-MS data. These
results suggest the possibility of peroxygenase activity with the cresol substrates, an activity that
has been observed for DHP with pyrrole [36], 5-Br-indole [35], and nitrophenol [25] substrates,
but not with halophenol nor haloguaiacol substrates, these latter two being oxidized via a
peroxidase activity.
2
1

Labeled Oxygen Studies. In order to determine the origin of the O-atom that was
incorporated in the para (2-MeBq) and ortho (catechol) positions of the oxidation products,
labeling studies with 4-Br-
o
-cresol (as a representative substrate) were conducted employing
18
18
H O and H O (98% and 90% enriched, respectively), and the results were subsequently
2
2
2
+
-
analyzed by LC-MS in positive and negative modes to yield the [M+H] and [M-H] species,
respectively. The background-subtracted total ion chromatograms (TICs) are shown in Figures 3
and 4. The data were obtained in both ionization modes as the catechol (5-bromo-3-methyl-1,2-
benzenediol) and 2-methyl-1,4-benzoquinone (2-MeBq) products could only be observed in
negative and positive ion modes, respectively.
1
8
2
-Me-1,4-benzoquinone (2-MeBq): In the presence of unlabeled H O and labeled H O , no
2 2 2
increase in mass for 2-MeBq was observed [m/z: 123.08, 100% (Figure 3C)] when compared
1
8
18
with the results obtained in the absence of an O source (Figure 3A). The presence of H O and
2
unlabeled H O , however, resulted in an increase of 2 and 4 Da in the 2-MeBq mass [m/z
:
2
2
1
23.08, 10.9%; 125.05, 38.1%; 127.05, 51.0% (Figure 3B)], suggesting incorporation of labeled
oxygen from water, with further scrambling from solvent occurring to account for the second
1
8
labeled oxygen in 2-MeBq. The results obtained for the reaction employing both labeled H
2
O
18
and H O [m/z 123.05, 16.2%; 125.05, 36.7%; 127.05, 47.1% (Figure 3D)] were virtually
2
2
18
identical to those of the H O/H O reaction, providing strong evidence for O-atom
2
2
2
incorporation derived exclusively from water (and not from hydrogen peroxide), and consistent
with a peroxidase mechanism for cresol oxidation by DHP yielding 2-MeBq. Additionally, the
1
8
oligomeric products were observed to contain O only in reactions that contained labeled water
data not shown).
(
2
2

Figure 3. ESI-MS total ion chromatograms obtained in positive ion mode for the 2-
methylbenzoquinone (2-MeBq) product observed in the reaction of 4-BrC with DHP B: (A)
18
18
2
18
18
H O/H O , (B) H O/H O , (C) H O/H O , and (D) H O/H O . Reactions conditions: 10
2
2
2
2
2
2
2
2
2
2
2
µM DHP B, 500
µ
M 4-Br-
o
-cresol, 500
µ
M H O , 5% MeOH / 5 mM KP (v/v), pH 7, 25 °C, 10
2
2
i
minutes.
1
8
5
-Br-3-Me-1,2-benzenediol: In the presence of labeled H O and unlabeled H O , no
2 2 2
7
9
81
increase in mass for the catechol [m/z: 200.96 ( Br), 48.3%; 202.96 ( Br), 51.6% (Figure 4B)]
1
8
was observed when compared with the results obtained in the absence of an O source (Figure
18
4
A). However, in the presence of unlabeled H O and labeled H O there was an increase of 2
2 2 2
7
9
81
Da in the catechol mass [m/z: 200.96, 7.2%; 202.96 ( Br), 48.6%; 204.96 ( Br), 44.2% (Figure
C)], which strongly suggests that the origin of the O-atom incorporated into the catechol
product was derived from hydrogen peroxide. Further, the results obtained for the reaction
4
18
18
79
employing both labeled H O and H O [m/z: 200.96, 1.8%; 202.96 ( Br), 48.3%; 204.96
2
2
2
8
1
18
(
2 2 2
Br), 49.9% (Figure 4D)] were virtually identical to those of the H O/H O reaction, and
provides unequivocal evidence that the oxygen atom was derived exclusively from hydrogen
peroxide, with no incorporation of oxygen from water, and consistent with a peroxygenase
mechanism for cresol oxidation by DHP for catechol formation.
2
3

Figure 4. ESI-MS total ion chromatograms obtained in negative ion mode for the 5-Br-3-Me-
,2-benzenediol (catechol) product observed in the reaction of 4-BrC with DHP B: (A)
1
18
18
2
18
18
H O/H O , (B) H O/H O , (C) H O/H O , and (D) H O/H O . Reactions conditions: 10
2
2
2
2
2
2
2
2
2
2
2
µM DHP B, 500
µ
M 4-Br-
o
-cresol, 500
µ
M H O , 5% MeOH / 5 mM KP (v/v), pH 7, 25 °C, 10
2
2
i
minutes.
Substrate Binding Studies. While the titration of substrates (0.5-150 equiv; Figures S7-S9)
to ferric WT DHP B in 100 mM KP / 5% MeOH (v/v) showed no significant changes to the
i
Soret band (data not shown), the Q-band region (450-700 nm) showed well-behaved optical
difference spectra, thereby allowing for the determination of the apparent substrate binding
constant (Kd). On a whole, the EPA pollutants exhibited a stronger binding affinity to DHP B
when compared to the corresponding substituted guaiacols [23], halophenols [70] and pyrroles
[
36], but relatively similar
substrates (Table 3). Across the halogenated 4-X-
affinity with increasing halogen size was noted, H < F < Cl < Br. This same trend has been
previously reported for the 4-X- -guaiacol [23] and 5-X-indole [35] series, and is attributed to
K
d
values as the nitrophenol [25], haloindole [35] and azole [24]
o
-cresol series, a trend of increasing binding
o
the Xe1 binding pocket in DHP B (hydrophobic cavity surrounded by amino acids L100, F21,
F24, F35 and V59) having a higher affinity for larger halogen atoms, as deduced from
crystallographic studies of 4-X-phenol binding to DHP [81]. The binding affinity of 4-
d d
bromophenol (K = 1.15 mM [70]), is 13-fold weaker than the affinity of 4-Br-o-cresol (K =
2
4

0
.086 mM), which likely explains why the reactivity of this substrate was only partially affected
decreased 1.7-fold) in the presence of the inhibitor (Table 2).
Table 3. Kd Values for Substrate Binding to Ferric WT DHP B at pH 7.
(
Substrate
Kd
(
µM)
Ref
EPA Pollutants
a
a
a
a
a
a
a
a
a
a
o
p
-Cresol
-Cresol
4594 (± 393)
2682 (± 381)
6721 (± 582)
309 (± 26)
2970 (± 391)
130 (± 4)
86 (± 14)
155 (± 5)
629 (± 36)
79 (± 9)
m
-Cresol
-Me- -cresol
-F- -cresol
-Cl- -cresol
-Br- -cresol
-NO2- -cresol
-Cl- -cresol
4
4
4
4
4
4
o
o
o
o
o
m
Pentachlorophenol
Substituted Guaiacols
o
4
4
4
4
4
-guaiacol
-Me- -guaiacol
-F- -guaiacol
-Cl- -guaiacol
-guaiacol
-guaiacol
14712 (± 714)
1433(± 97)
2438 (± 207)
493 (± 53)
374 (± 42)
1341 (± 26)
[23]
[23]
[23]
[23]
[23]
[23]
o
o
o
o
-Br-
-NO
2
-o
Phenol
4
4
-bromophenol
-nitrophenol
1150
262 (± 23)
[70]
[25]
Indole
5
5
5
-Cl-indole
-Br-indole
-I-indole
317 (± 23)
150 (± 10)
62 (± 10)
[35]
[35]
[35]
Azole
Imidazole
52 (± 2)
82 (± 2)
110 (± 8)
[24]
[24]
[24]
Benzotriazole
Benzimidazole
This work.
a
X-Ray Crystallographic Studies. The structures of DHP B in complex with
pentachlorophenol, 4-R- -cresol (R = H, F, Cl, Br, Me, NO ) and -cresol were determined by
o
p
2
X-ray crystallography to near atomic resolution (1.35-2.1 Å). Selected distances for substrate
interactions and occupancy in the distal pocket can be found in Table 4, and the crystallographic
data collection, processing, and structure refinement results are given in Table S6. The protein
2
5

1 1 1
crystallized in the space group P2 2 2 with two molecules in the asymmetric unit (protomer A
and protomer B), as was reported in our previous studies [23, 25, 48, 49, 71]. The substrates
were found to bind in the distal heme cavity, positioned ~3.2–5 Å above the heme moiety, with
the phenol ring substituents directing substrate orientation in the hydrophobic binding cavity
above the heme (Figure 5).
2
6

Table 4. Selected Distances (angstroms) for DHP B-EPA Pollutants Complex (Protomer A).
a
b
c
c
c
4
-F-o-cresol 4-Cl-o-cresol
4-Br-o-cresol
4-NO -o-cresol
o-cresol
6OO1
p-cresol
6OO6
4-Me-o-cresol
Pentachlorophenol
6OO8
2
PDB Entry
Substrate
6ONG
6ONK
6ONX
6ONZ
6ONR
A: 50% B:
20%
8
0%
100%
70%
70%
100%
5.7
3.7
4.4
4.8
7.1
4.4
-
A: 20%, B: 60%
A: 40%, B: 15%
occupancy
ε
H55 N ...-OH
ext
int
int
ext
ext
A
B
B
B
A
B
A
B
B
B
B
B
7
.3
.6
2.7
4.7
3.8
4.0
4.9
5.1
8.6
2.4/ 9.1
5.9
5.7 /7.1
7.3 /7.0
5.1 /5.2
(
substrate)
δ
H55 N ...OH
substrate)
ext
ext
ext
A
A
B
A
5
4.4/ 8.9
4.1
3.8 /5.0
5.4 /4.9
4.9 /5.3
(
ζ
1
F21 C …C
substrate)
A
A
3
4
6
4
4
.5
3.8
4.0
4.8
5.1
8.7
3.8
3.8
7.3
4.6
4.5 /3.7
4.0
3.1
4.4 /4.5
(
γ
4
F21 C …C
A
B
B
B
A
.6
.9
.7
.8
3.8 /2.9
4.7 /5.9
(
substrate)
Fe…OH
A
A
B
B
B
A
7.3 /4.5
7.0 /4.9
7.5 /7.6
(
substrate)
o-CH3
Fe…C
A
A
4.8 /7.3
4.9 /7.3
-
(
substrate)
Fe…X
d
N
O1
5.4 , 5.1 ,
A
A
B
O2
-
4.8 /7.3
3.7 /2.2
(
substrate)
6.2
ε
ext
int
ext
ext
int
ext
Fe…H55 N
10.3
5.9
6.4
2.1
5.6/ 9.8
10.0
8.1
10.2
8.3
2.2
10.3
8.5
2.2
10.4
8.5
2.1
4.7/ 9.4
δ
ext
int
ext
ext
int
ext
Fe…H55 Nε
Fe…H89 N
Fe to pyrrole N
plane
8.5
6.0/ 10.0
2.1
6.2/ 9.7
2.1
2.2
2.2
0.274
0.254
0.188
0.175
0.193
0.158
0.262
0.0942
a
b
For 4-Cl-C, numbers shown for protomer B. For 4-Br-C, H55 was found to be both in the interior and exterior conformations,
designated as interior (int) or exterior (ext). The alternate substrate conformations are denoted with superscripts A and B X refers to
c
d
the substituent on para position.
2
7

F60
2.7
F60
Y38
A
E
T56
.8
B
F
F21
C F21
D
F21
Y38
.7
3
.1
F21
2.6
H55
2
3
.0
4-Cl-C
H55
H55
3.0
H55
2
2
.5
4
.9
4-Br-C4.8
4-NO -C
2
4
-F-C
2
.1
2
.1
H89
2
.1
2.2
H89
H89
H89
Y38
3.1
G
H
T56
.5
T56
Y38
Y38
F21
F21
2.7
H55
F21
2.5
H55
2.5
F21
Y38
H55
2
3.1
A
H55
2
.5
2.5
B
2
.8
2.2
o-cresol 2
2.6
4-Me-C
.9
p-cresol
3.4
2.5
PCP
4
.9
3
.3
2
.2
H89
2
.2
H89
2
.1
H89
2
.2
H89
Figure 5. X-ray crystal structures obtained for DHP B in complex with (A) 4-F-o-cresol [cyan,
PDB 6ONG], (B) 4-Cl-o-cresol [pink, PDB 6ONK], (C) 4-Br-o-cresol [green, PDB 6ONX], (D)
4
6
6
-NO -o-cresol [blue, PDB 6ONZ], (E) o-cresol [orange, PDB 6OO1], (F) p-cresol [purple, PDB
OO6], (G) 4-Me-o-cresol [yellow, PDB 6ONR], and (H) pentachlorophenol [burgundy, PDB
OO8]. Panels provide atomic distances and interactions between the substrate and amino acids
2
and/or heme macrocycle.
For the halogenated cresols 4-X-o-cresols (X = F, Cl, Br; Figure 5A-C), bulky atoms such as
Br were found to bind deeper in the hydrophobic region, closer to residues L100 and F60 in the
hydrophobic cavity above the
accordance with previously published structures [81]. When compared with 4-Br-o-guaiacol
PDB 6CKE [23]), the halogen atoms of 4-Cl/Br-o-cresol superimpose exactly with the p-Br
α heme edge (Xe1 hydrophobic binding pocket), which is in
(
substituent of the guaiacol (Figure S10A) in the Xe1 hydrophobic cavity, while the o-OMe group
coincides with the o-Me group. Additionally for the 4-Cl/Br-o-cresol structures, H55 was found
positioned inside the heme cavity, which enables hydrogen bonding interactions with the
hydroxyl group of the substrate (Figure 5B and 5C). The overall strong affinity of these two
substrates with the enzyme (4-Cl-o-cresol, K = 130
µ
M; 4-Br-o-cresol, K = 86
µ
M; Table 3) is
-stacking of the
substrate aromatic ring with F21. By contrast, 4-F-o-cresol was found to bind closer to the heme
d
d
a reflection of these interactions, with an additional stabilization arising from
π
2
8

γ
edge and the heme propionate D due to hydrogen bonding interactions of the hydroxyl group
with both T56 and Y38, mediated by a water molecule (Figure 5A), which could reflect in its
weaker binding affinity (K = 2970 M) compared to 4-Cl-o-cresol and 4-Br-o-cresol. Two
d
µ
distinct conformations were observed for 4-F-o-cresol and 4-Cl-o-cresol on protomers B and A,
respectively (Figure S11A-B), which affected whether H55 was in the open or closed
conformation, as well as substrate occupancy (4-F-o-cresol: A 55%, B, 20%; 4-Cl-o-cresol: A
5
8% and 20%).
The cresol substrates with less bulky substituents in the para position were found to bind in a
similar position as 4-F-o-cresol. Two conformations were observed for o-cresol (protomer A)
and 4-Me-o-cresol, where the hydroxyl group is either facing up towards Y38 or facing down
towards the heme. When facing down, o-cresol (Figure 5E) has a hydrogen bonding interaction
to a water molecule positioned 3.3 Å away from the iron center, and when facing up the
hydroxyl group of o-cresol interacts with propionate D and Y38. Additionally, o-cresol showed
the presence of only one substrate conformation in protomer B (Figure S11C), oriented such to
be able to hydrogen bond with Y38 and heme propionate D. The hydroxyl group of 4-Me-o-
cresol (Figure 5G) has two different hydrogen bonding interactions when facing up: i) an
interaction with T56 and, ii) an interaction with propionate D and Y38 mediated by a water
molecule, which together can account for relatively strong affinity of this substrate with the
protein (K = 309 µM). The p-cresol (Figure 5F) isomer, for which only one conformation was
d
observed in both chains and with the methyl group facing towards the Xe1 hydrophobic cavity,
shows H-bonding interactions of the hydroxyl group with the heme propionate D and Y38,
which together correlate to a stronger binding affinity compared to o-cresol (o-cresol, K
d
= 4594
µ
M vs. p-cresol, K = 2682 M). When comparing the structures of o-cresol, p-cresol, and 4-Me-
µ
d
2
9