Peroxidase versus Peroxygenase Activity: Substrate Substituent Effects as Modulators of Enzyme Function in the Multifunctional Catalytic Globin Dehaloperoxidase

Article abstract of DOI:10.1021/acs.biochem.8b00540

The dehaloperoxidase-hemoglobin (DHP) from the terebellid polychaete Amphitrite ornata is a multifunctional hemoprotein that catalyzes the oxidation of a wide variety of substrates, including halo/nitrophenols, haloindoles, and pyrroles, via peroxidase and/or peroxygenase mechanisms. To probe whether substrate substituent effects can modulate enzyme activity in DHP, we investigated its reactiviy against a panel of o-guaiacol substrates given their presence (from native/halogenated and non-native/anthropogenic sources) in the benthic environment that A. ornata inhabits. Using biochemical assays supported by spectroscopic, spectrometric, and structural studies, DHP was found to catalyze the H₂O₂-dependent oxidative dehalogenation of 4-haloguaiacols (F, Cl, and Br) to 2-methoxybenzoquinone (2-MeOBQ). ¹⁸O labeling studies confirmed that O atom incorporation was derived exclusively from water, consistent with substrate oxidation via a peroxidase-based mechanism. The 2-MeOBQ product further reduced DHP to its oxyferrous state, providing a link between the substrate oxidation and O₂ carrier functions of DHP. Nonnative substrates resulted in polymerization of the initial substrate with varying degrees of oxidation, with 2-MeOBQ identified as a minor product. When viewed alongside the reactivity of previously studied phenolic substrates, the results presented here show that simple substituent effects can serve as functional switches between peroxidase and peroxygenase activities in this multifunctional catalytic globin. More broadly, when recent findings on DHP activity with nitrophenols and azoles are included, the results presented here further demonstrate the breadth of heterocyclic compounds of anthropogenic origin that can potentially disrupt marine hemoglobins or function as environmental stressors, findings that may be important when assessing the environmental impact of these pollutants (and their metabolites) on aquatic systems.

Full text of DOI:10.1021/acs.biochem.8b00540

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Article
Peroxidase vs. Peroxygenase Activity: Substrate
Substituent Effects as Modulators of Enzyme Function
in the Multifunctional Catalytic Globin Dehaloperoxidase
Ashlyn Henson McGuire, Leiah M. Carey, Vesna S de Serrano, Safaa Dali, and Reza A. Ghiladi
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00540 • Publication Date (Web): 27 Jun 2018
Downloaded from http://pubs.acs.org on July 2, 2018
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Peroxidase vs. Peroxygenase Activity:
Substrate Substituent Effects as Modulators of
Enzyme Function in the Multifunctional Catalytic
Globin Dehaloperoxidase
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Ashlyn McGuire, Leiah M. Carey, Vesna de Serrano, Safaa Dali 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
(
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Abbreviations
,4-DNP, 2,4-dinitrophenol; 2-methoxy-1,4-benzoquinone (2-MeOBQ); 4-Br-G, 4-
2
bromoguaiacol; 4-BP, 4-bromophenol; 4-Cl-G, 4-chloroguaiacol; 4-F-G, 4-fluoroguaiacol; 4-
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MeO-G, 4-methoxyguaiacol; 4-Me-G, 4-methylguaiacol; 4-NO -G, 4-nitroguaiacol; 4-NP, 4-
2
nitrophenol; 5-Br-G, 5-bromoguaiacol; 5,5-dimethyl-1-pyrroline N-oxide (DMPO); 6-Br-G, 6-
bromoguaiacol; Compound I, two-electron oxidized heme cofactor compared to the ferric form,
IV
commonly as an Fe =O porphyrin π-cation radical; Compound II, one-electron oxidized heme
IV
IV
cofactor when compared to the ferric form, commonly as an Fe =O or Fe –OH; Compound ES,
IV
two-electron-oxidized 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; Hb, hemoglobin; HSM,
horse skeletal muscle; Mb, myoglobin; TBP, 2,4,6-tribromophenol; TCP, 2,4,6-trichlorophenol
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ABSTRACT: The dehaloperoxidase-hemoglobin (DHP) from the terebellid polychaete
Amphitrite ornata is a multifunctional hemoprotein that catalyzes the oxidation of a wide variety
of substrates, including halo-/nitro-phenols, haloindoles, and pyrroles, via peroxidase and/or
peroxygenase mechanisms. To probe whether substrate substituent effects can modulate enzyme
activity in DHP, we investigated its reactivity against a panel of o-guaiacol substrates given their
presence (from native/halogenated and non-native/anthropogenic sources) in the benthic
environment that A. ornata inhabits. Using biochemical assays supported by spectroscopic,
spectrometric, and structural studies, DHP was found to catalyze the H O -dependent oxidative
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dehalogenation of 4-haloguaiacols (F, Cl, Br) to 2-methoxybenzoquinone (2-MeOBQ). O-
labeling studies confirmed that O-atom incorporation was derived exclusively from water,
consistent with substrate oxidation via a peroxidase-based mechanism. The 2-MeOBQ product
further reduced DHP to its oxyferrous state, providing a link between the substrate oxidation and
O -carrier functions of DHP. Nonnative substrates resulted in polymerization of the initial
2
substrate with varying degrees of oxidation, with 2-MeOBQ identified as a minor product. When
viewed alongside the reactivity of previously studied phenolic substrates, the results here show
that simple substituents effects can serve as functional switches between peroxidase and
peroxygenase activities in this multifunctional catalytic globin. More broadly, when including
recent findings on DHP activity with nitrophenols and azoles, the results here further
demonstrate the breadth of heterocyclic compounds of anthropogenic origin that can potentially
disrupt marine hemoglobins or function as environmental stressors, findings that may be
important when assessing the environmental impact of these pollutants (and their metabolites) on
aquatic systems.
Keywords: globin; guaiacol; heme; peroxidase; peroxygenase; structure-function
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INTRODUCTION
Guaiacol (methoxyphenol), a major component of wood lignin, is released into the
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environment from both anthropogenic and natural sources, some examples of which include: i)
biomass burnings, where during the combustion of wood lignin the semi-volatile guaiacol-
derived pollutants can be aerosolized, enter the troposphere, and can further react with
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atmospheric nitrogen-containing species to yield nitroguaiacols, which are generally considered
to be more toxic than the parent compound, ii) the paper-pulp industry, where during the
bleaching of wood pulp the bleaching agents (molecular chlorine, hypochlorite, etc.) react with
lignin, resulting in chlorinated phenolic derivatives (i.e. chloroguaiacols, chlorocatechols), and
iii) as derivatives of polybrominated diphenyl ethers that originate both artificially (from
chemical transformations of polybrominated flame-retardant chemicals) and naturally (as
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biosynthetic compounds produced by marine bacteria). These compounds are then released into
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the environment through wastewater effluent or via precipitation, leading to contamination
of both water and soil. With recent reports demonstrating that guaiacol pollutants are acutely
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toxic to a level that meets the European toxicity classification of ‘harmful’, there is an
increasing need to assess the impact of these compounds, including their metabolites, on the
environment.
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Although enzyme-catalyzed guaiacol oxidation has been extensively examined,
most
studies have focused on enzymes from microbial or plant origins. As such, the mechanisms by
which infaunal organisms cope with guaiacol pollutants as environmental stressors, produced
either natively as halometabolites by other organisms or from anthropogenic sources, remains
relatively understudied by comparison. Our platform for exploring the interactions of such
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environmental pollutants with infaunal organisms is Amphitrite ornata, a sediment-dwelling
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marine polychaete. Inhabiting coastal mudflats that are rich in biogenically produced
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halometabolites excreted from other organisms, A. ornata faces stressors that include
brominated indoles, pyrroles, phenols, and – of particular relevance to the present study –
guaiacols. To overcome the challenges posed from this diverse array of toxic haloaromatic
compounds, Amphitrite ornata employs its hemoglobin to function both as a detoxification
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enzyme and as an O -transport protein.
Named dehaloperoxidase (DHP), this coelomic
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hemoglobin possesses an unusually broad substrate profile encompassing halophenols,
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haloindoles, and (halo)pyrroles, and as such is the first known multifunctional catalytic globin
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to possess biologically-relevant peroxidase
and peroxygenase
activities, as well as an
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oxidase activity that leads to the formation of indigo derivatives. DHP also exhibits activity
against compounds of anthropogenic origin, including the H O -dependent oxidation of mono-
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and di-nitrophenols, as is also capable of binding azole species, including imidazole,
benzotriazole, benzimidazole, and indazole.
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DHP has two isoenzymes encoded by separate genes, A and B, which differ by only five
amino acid substitutions (note – isoenzyme A is listed first): I/L9, R/K32, Y/N34, N/S81, and
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S/G91. Although these two isoenzymes are structurally identical, DHP B consistently exhibits a
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greater reactivity than isoenzyme A, and as such recent studies have focused on this variant.
Mechanistic studies have shown that both isoenzymes appear to function via a Poulos−Kraut
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type mechanism : ferric DHP reacts with H O to form DHP Compound I, an iron(IV)-oxo
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porphyrin π-cation radical species. In WT DHP, Compound I rapidly converts in DHP to an
32, 33, 40
iron(IV)-oxo heme center with a tyrosyl radical that has been termed Compound ES
by
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analogy with cytochrome c peroxidase, whereas in mutants such as DHP B (Y28F/Y38F), the
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Compound I species can be investigated directly. These peroxide-activated forms of DHP
initially return to the ferric resting state upon substrate oxidation, although further reduction to
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oxyferrous DHP has been noted under some conditions.
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Given the diverse functions (O -carrier, peroxidase, peroxygenase, oxidase) exhibited by
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DHP across this broad panel of substrates, we have previously suggested that the substrate itself
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can act to modulate enzyme activity in DHP,
with binding/orientation (relative to the ferryl
iron), pKa, and redox potential all being key determinants as to which mechanism leads to
substrate oxidation. More interestingly, however, it also appears that substituent effects may play
a pseudo-allosteric role (Figure 1): the peroxidase substrate phenol is converted to a
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peroxygenase substrate upon addition of a 4-nitro substituent, whereas 4-Br-phenol is an
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inhibitor of peroxidase and peroxygenase activities in DHP. These substituent effects suggest
that it may be possible to modulate DHP activity by switching the substrate oxidation
mechanism, not through traditional mutagenesis or heme cofactor modification, but through
selection of the substrate itself.
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Peroxidase
Inhibitor
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o-Guaiacol Substrates
Peroxygenase
Figure 1. DHP peroxidase (blue), peroxygenase (red), and oxidase (green) substrates and o-
guaiacol structures (black); X = F, Cl, Br, I.
To investigate how guaiacol pollutants may undergo biotransformations by infaunal
organisms, and to probe the role of substituent effects as modulators/regulators of the multiple
activities in DHP, we evaluated a panel of 4-X-guaiacol (X = -F, -Cl, -Br, -NO , -OMe, -Me)
2
substrates using a combination of spectroscopic methods, biochemical assays, and isotope
labelling studies. The basis for selecting guaiacols (Figure 1) within the context of
substituent/electronic effects is: i) the addition of the methoxy substituent to phenol (blue box),
4
-nitrophenol (red box), and 4-Br-phenol (green box) will enable us to probe its effect on the
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peroxidase, peroxygenase,
guaiacol substrates, ii) haloguaiacols are native to the benthic environment of A. ornata,
likely represent physiologically-relevant substrates, while anthropogenic analogs, such as 4-
and inhibition pathways of DHP relevant to the putative
18, 43
and
4
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nitroguaiacol and 4-chloroguaiacol , are relevant as environmental stressors, and iii) guaiacol
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oxidation is well understood with respect to monofunctional heme proteins,
but is
unknown with respect to multifunctional catalytic globins. As will be shown, the results establish
that guaiacols are indeed substrates for DHP, and demonstrate that simple substituents effects
can be used as a functional switch between peroxidase and peroxygenase activities in modulating
the activity of this multifunctional catalytic globin. More broadly, when viewed along with our
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recent findings employing nitrophenols and azoles, the results further demonstrate the breadth
of heterocyclic compounds of anthropogenic origin that can potentially disrupt marine
hemoglobins or function as environmental stressors, findings that may be important when
assessing the environmental impact of these pollutants (and their metabolites) on aquatic
systems.
EXPERIMENTAL
Materials. Ferric samples of WT DHP B were expressed and purified as previously
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reported.
Oxyferrous DHP B was prepared by the aerobic addition of excess ascorbic acid to
41
ferric DHP B, followed by removal of the reducing agent via a PD-10 desalting column.
-1
-1 40
Enzyme concentration was determined spectrophotometrically using εsoret=116,400 M cm .
Horse skeletal muscle (HSM) myoglobin and horseradish peroxidase (HRP) were purchased
−1
−1
from Sigma-Aldrich and enzyme concentrations were determined using εsoret = 188,000 M cm
4
5
-1
-1 46
and 102,000 M cm , respectively. Solutions of H O were prepared fresh daily in 100 mM
2
2
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KP (pH 7) and kept on ice until needed. Isotopically labeled H O (90% O-enriched) and
i
2
2
18
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H O (98% O-enriched) were purchased from Icon Isotopes (Summit, NJ). Stock solutions of
2
guaiacols (10 mM) were prepared in MeOH and stored in the dark at -80 °C until needed.
Acetonitrile (MeCN) was HPLC grade, and all other reagent-grade chemicals were purchased
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from VWR, Sigma-Aldrich or Fisher Scientific and used without further purification.
Guaiacol Binding Studies. The guaiacol substrate dissociation constants (K ) were
d
determined in triplicate for DHP in 100 mM KP (pH 7) containing 6.25% MeOH at 25 °C using
i
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a Cary 50 UV-vis spectrophotometer per previously published protocols.
The UV-visible
spectrometer was referenced with 50 µM WT DHP B in 100 Mm KP (pH 7) in 6.25% MeOH.
i
Spectra were acquired in the presence of guaiacol substrate concentrations (3.75-50 equiv) while
maintaining constant DHP B (50 µM) and methanol concentrations. Analyses of the Q-band
region (450-600 nm) was performed using the ligand binding function in Grafit (Erithacus
Software Ltd.).
HPLC Activity Studies. Reactions were performed in triplicate at pH 7 (unless otherwise
indicated) in 100 mM KP containing 5% MeOH at 25 °C. Assay components (250 µL final
i
volume) included 10 µM enzyme, 500 µM substrate (in MeOH), and the reaction was initiated
by the addition of 500 µM H O . Experiments were also performed in the presence of 500 µM 4-
2
2
bromophenol, 500 µM D-mannitol, 100 mM DMPO or 10% v/v DMSO, which were added prior
to the addition of H O . The reactions were quenched after 5 min with excess catalase. A 100 µL
2
2
aliquot was diluted 10-fold with 900 µL 100 mM KP (pH 7). The samples were analyzed using a
i
Waters 2796 Module coupled with a Waters 2996 photodiode array detector and equipped with a
Thermo Fisher Scientific ODS Hypersil (150 mm × 4.6 mm) 5 µM particle size C column.
1
8
Separation was performed using a linear gradient of binary solvents (solvent A, H O + 0.1%
2
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.
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6
Product Determination by LC-MS. Analysis was carried out on a Thermo Fisher Scientific
Exactive Plus Orbitrap mass spectrometer using a heated electrospray ionization (HESI) probe.
The samples were introduced via LC injection into the mass spectrometer at a flow rate of 250
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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µL/min (solvent A, H O + 0.1% formic acid; solvent B, acetonitrile + 0.1% formic acid) after
2
elution from a Thermo Hypersil Gold (50 x 2.1 mm, particle size 1.9 µm) C column. The mass
18
spectrometer was operated in positive and negative modes. Spectra were collected while
scanning from 100-1500 m/z. Data analysis was performed using Thermo Xcalibur software. The
assay included 10 µM DHP B, 500 µM substrate and 500 µM H O in buffer (5 mM KP , pH 7)
2
2
i
with a final volume of 250 µL, and was allowed to react for 5 min before quenching with
1
8
catalase. A 10 µL aliquot of the undiluted reaction was injected for LC-MS analysis. For the O-
18
labeling studies, unlabeled H O was replaced with H O , and/or the KP buffer was replaced
2
2
2
2
i
18
18
with H O to ensure >90% labeled H O.
2
2
Stopped-Flow UV-Visible Spectroscopy 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. Protein and hydrogen peroxide solutions were prepared in 100
mM KP (pH 7), and the guaiacol substrates were dissolved in buffer containing 4% methanol.
i
Data were collected (900 scans total) over a three-time domain (1.5, 15, 150 ms) 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. Kinetic data were baseline
corrected using the Specfit autozero function when appropriate.
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Double-mixing experiments were performed using an aging line prior to the second mixing
step to observe Compound I/ES/II reactivity with the guaiacol substrates (5-50 equiv): i)
Compound I was pre-formed by the reaction of ferric DHP B (Y28F/Y38F) with 10 equiv of
31
1
1
1
1
1
1
1
1
1
1
2
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2
2
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2
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3
4
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4
4
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5
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5
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5
6
0
1
2
3
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5
6
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1
2
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5
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9
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1
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0
H O in an aging line for 85 ms prior to mixing with the substrate; ii) Compound ES was pre-
2
2
40
formed by reaction of ferric WT DHP B with 10 equiv of H O in an aging line for 500 ms ; and
2
2
iii) Compound II was formed from oxyferrous DHP B pre-incubated with 1 equiv of TCP and
4
1
reacted with 10 equiv of H O in an aging line for 3 s.
2
2
Protein Crystallization and X-ray Diffraction Studies. Non-His tagged DHP B was
2
9, 30, 37
overexpressed and purified per literature.
hanging-drop vapor diffusion method. The crystals were grown from mother liquor solutions of
2% PEG 3350 and 0.2 M ammonium sulfate at pH 6.4, and were equilibrated against identical
DHP B crystals were obtained through the
3
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 soaked for 12 hours in
substrate enhanced reservoir solutions identical to the mother liquor. Substrate final
concentrations were all 100 mM (5% DMSO v/v). The crystals were cryo-protected by briefly
dipping them in reservoir solution containing 20% glycerol and then cryocooled in liquid N2.
Data were collected at 100 K on the SER-CAT 22-BM beamline at the APS synchrotron facility,
48
utilizing a wavelength of 1.00 Å. All data were scaled and integrated using HKL2000,
4
9
50
51
molecular replacement was performed with Phaser-MR from the PHENIX and CCP4 suite
3
7
of programs using 3IXF as the search model. Model building and manual placement of waters
52
53
54
utilized COOT and refinement was carried out using refmac and phenix.refine.
RESULTS
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6
DHP-Catalyzed Guaiacol Reactivity with H O . The hydrogen peroxide-dependent reaction
2
2
of ferric DHP B with guaiacol substrates at pH 7 was monitored via HPLC, and the
corresponding substrate conversion percentages are reported in Table 1. Reactions were initiated
upon addition of 500 ꢀM H O to a solution containing 10 ꢀM enzyme and 500 ꢀM guaiacol,
0
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7
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7
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2
incubated at 25 °C for 5 min, and then quenched with catalase. With the exception of o-guaiacol
32.4%), all substrates exhibited very high conversion, with the 4-X-guaiacol series (X = F, Cl,
(
Br) essentially being fully oxidized (>99.7%), and 5- and 6-Br-guaiacol only slightly diminished.
Non-native guaiacol substrates (4-NO -, 4-Me-, and 4-MeO-guaiacol) were also found to be very
2
reactive (92.8-98.6%). No turnover of substrate was observed in the absence of H O (non-
2
2
oxidant control) or enzyme (non-enzymatic control).
Using 4-Br-guaiacol as a representative substrate owing to its similarity to previously studied
5
5
halophenols, the following observations were noted: i) a minimal pH effect was observed (pH 5
< pH 7 ≈ pH 8); ii) substrate conversions were also found to be identical when the assays were
initiated from the oxyferrous (‘hemoglobin-like’) or ferric (‘peroxidase-like’) oxidation states, in
41
line with previous observations for DHP for the oxidation of TCP via a peroxidase mechanism,
2
4
34
25
and the oxidation of haloindoles, nitrophenols, and pyrroles via a peroxygenase cycle; iii)
both DHP B and the canonical peroxidase HRP yielded complete conversion of the substrate to
product(s), although the product distributions were found to be different (vide infra); iv) horse
skeletal muscle myoglobin exhibited a 3.4-fold attenuation in reactivity. These results support
that DHP reactivity with guaiacols is on a par with classical peroxidase enzymes, yet is greater
than other members of the globin family.
Mechanistic studies were also employed to elucidate key details of the reaction of 4-Br-
guaiacol with DHP. Under anaerobic conditions, substrate conversion (99.7%) was identical to
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the aerobic reaction, showing no dependence on molecular oxygen. The addition of the hydroxyl
radical scavengers D-mannitol (99.7%) and dimethyl sulfoxide (DMSO; 99.6%) also exhibited
no effect on the oxidation of 4-Br-guaiacol, suggesting that freely diffusible hydroxyl radicals
are not involved in the reaction. Similarly, the radical scavenger 5,5-dimethyl-1-pyrroline N-
oxide (DMPO) did not show reduced 4-Br-guaiacol reactivity (99.4%). However, DMPO did
significantly inhibit substrate conversion of 5-Br-guaiacol (27.6 ± 1.1% vs. 90.0 ± 1.0%) and 4-
1
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NO -guaiacol (28.5 ± 1.3% vs. 92.8 ± 0.2%), indicating a difference between these substrates
2
pertaining to the diffusibility of substrate radicals (vide infra). When the peroxidase and
4
2, 56
peroxygenase inhibitor 4-bromophenol (K = 1.15 mM
) was added, substrate conversion was
d
minimally decreased by 1.2-fold, suggesting that 4-bromophenol has only a slight inhibitory
effect on 4-Br-guaiacol oxidation.
a
Table 1. DHP-catalyzed oxidation of substituted guaiacols.
Substrate
Conversion (%)
Substrate Variation
Ferric WT DHP B, pH 7
o-guaiacol
+ 4-F-guaiacol
+
32.4 (±0.2)
>99.9
+
+
+
4-Cl-guaiacol
4-Br-guaiacol
5-Br-guaiacol
99.7 (±0.1)
99.7 (±0.2)
90.0 (±1.0)
88.8 (±0.2)
92.8 (±0.2)
94.6 (±1.2)
98.6 (±0.3)
+ 6-Br-guaiacol
+ 4-NO -guaiacol
+
+
2
4-MeO-guaiacol
4-Me-guaiacol
pH Studies (4-Br-guaiacol)
pH 5
pH 8
90.5 (±0.2)
99.6 (±0.1)
Enzyme Variation
Oxyferrous WT DHP B
4-Br-guaiacol
+ 5-Br-guaiacol
HRP
+
99.7 (±0.1)
84.8 (±2.0)
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+
4-Br-guaiacol, pH 6
Myoglobin (HSM)
4-Br-guaiacol
+ 5-Br-guaiacol
>99.9
+
29.3 (±2.8)
32.9 (±2.0)
Mechanistic Probes (Ferric WT DHP B)
4-Br-guaiacol
500 µM mannitol
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+
99.7 (±0.2)
99.6 (±0.1)
99.4 (±0.2)
84.8 (±0.1)
99.7 (±0.2)
n.d.
+ 10% DMSO
+ 100 mM DMPO
+
500 µM 4-BP
anaerobic
H O
-
2
2
- enzyme
-Br-guaiacol
n.d.
5
+
-NO -guaiacol
100 mM DMPO
27.6 (±1.1)
28.5 (±1.3)
4
2
+
100 mM DMPO
a
Reaction conditions: [enzyme] = 10 µM, [guaiacol] = [H O ] = 500 µM 5% MeOH/100 mM
2
2
,
KP (v/v), pH 7 unless otherwise noted; n.d. = no reactivity detected; DMPO: 5,5-dimethyl-1-
i
pyrroline N-oxide; 4-BP: 4-bromophenol.
Identification of Reaction Products by HPLC and LC-MS. The HPLC chromatograms for the
reaction of ferric WT DHP B with guaiacol substrates are shown in Figure 2 and Figure S1, and
a list of the products by their retention times and masses is provided in Tables S1 and S2.
Although identification of the exact chemical structures was not pursued, the products generally
consisted of dimers, trimers, and tetramers with varying degrees of oxidation, in line with
1
0, 57-59
previous reports for heme enzyme catalyzed oxidation of guaiacol.
X = F, Cl, Br), dehalogenation products were also identified, an expected result given the DHP-
catalyzed oxidative dehalogenation of 2,4,6-trihalophenols to their corresponding 2,6-
For the 4-X-guaiacols
(
1
9, 55
dihaloquinones has been well established.
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2
-MeOBQ
-Me-guaiacol
60 nm
4
2
*
4-MeO-guaiacol
2
80 nm
*
4
2
-NO -guaiacol
2
60 nm
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4
5
6
7
8
9
0
*
6-Br-guaiacol
300 nm
*
*
5
2
-Br-guaiacol
80 nm
4
3
-Br-guaiacol
60 nm
*
*
o-guaiacol
60 nm
2
3
4
5
6
7
8
9
10
Time, min
Figure 2. HPLC chromatograms at the indicated detection wavelength depicting the reaction of
DHP B with guaiacol substrates. Reaction conditions: [DHP B] = 10 µM, [guaiacol] = [H O ] =
00 µM; 5% MeOH/100 mM KP (v/v) at pH 7. The reaction was quenched after 5 minutes with
the addition of excess catalase (t = 9 min). The 2-MeOBQ product is highlighted at t = 4.5 min.
2
2
5
i
R
R
*
= retention time of initial substrate.
Interestingly, a common product with a characteristic retention time of t = 4.5 min was
R
observed in the oxidation of six substrates: o-guaiacol, 4-X-guaiacol (X = F, Cl, Br), 4-MeO-
guaiacol and 4-NO -guaiacol. This product exhibited an identical retention time, UV−visible
2
+
spectrum (Figure S2), and mass (m/z 139.04, [M + H] ) as an authentic commercial sample of 2-
methoxy-1,4-benzoquinone (2-MeOBQ; see Figure S3 for the corresponding calibration curve).
The amount of 2-MeOBQ produced varied 10-fold across these six substrates: 4-F-guaiacol (398
± 14 µM), 4-Cl-guaiacol (397 ± 23 µM), 4-Br-guaiacol (374 ± 3 µM), 4-MeO-guaiacol (231 ± 1
µM), 4-NO -guaiacol (42 ± 3 µM), and o-guaiacol (35 ± 1 µM), representing 80%, 79%, 75%,
2
4
6%, 8.3% and 7.0% of the starting material being converted to 2-MeOBQ, respectively.
‒
‒
‒
Formation of 2-MeOBQ implies dehalogenation (-HX, X = F , Cl , Br ), loss of methanol (-
MeOH) or loss of nitrite (HNO ) from the aforementioned substrates, although no attempts to
2
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identify the leaving groups were made. No such 2-MeOBQ product was seen in the reactions
with 4-Me-guaiacol, 5-Br-guaiacol, or 6-Br-guaiacol. In the presence of the radical scavenger
DMPO, the 2-MeOBQ product was unaffected for the 4-Br-guaiacol reaction, however no 2-
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MeOBQ was observed when DMPO was included in the reaction with 4-NO -guaiacol, and
2
fewer product peaks were noted in the reaction with 5-Br-guaiacol (data not shown).
Isotopically-labeled Oxygen Studies. To determine the origin of the O-atom incorporated into
18
18
18
the 2-MeOBQ product, studies employing labeled H O and H O (98% and 90% O-atom
2
2
2
enriched, respectively) were performed with 4-Br-guaiacol as the substrate, and subsequently
analyzed by LC-MS. The corresponding background-subtracted total ion chromatograms (TICs)
1
8
are shown in Figure 3. In the presence of unlabeled H O and H O , no significant increase in
2
2
2
mass was observed (m/z: 139.04; Figure 3A), when compared to the unlabeled H O/H O
2
2
2
conditions (m/z: 139.04; data not shown), suggesting that the source of O-atom incorporation
1
8
was not H O . The reaction employing H O and unlabeled H O resulted in an increase of 2
2
2
2
2
2
and 4 Da in 2-MeOBQ (m/z: 139.04, 4.9%; 141.04, 38.1%, 143.04, 57.0%; Figure 3B),
suggesting scrambling from solvent was occurring for both O atoms in 2-MeOBQ. Importantly,
18
18
the H O/H O double-label reaction produced similar results (m/z: 139.04, 6.1%; 141.04,
2
2
2
1
8
16
4
0.3%, 143.04, 53.6%; Figure 3C) as the H O/H O reaction, providing unequivocal evidence
2 2 2
that the O-atom was derived from exclusively from H O with no O-atom incorporation from
2
H O , consistent with a peroxidase mechanism for guaiacol oxidation by DHP.
2
2
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0
Figure 3. ESI-MS total ion chromatogram for the DHP B and 4-Br-guaiacol reaction product. A)
16
18
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16
18
18
H O/H O , B) H O/H O , and C) H O/H O . Reaction conditions: [DHP] = 10 µM, [4-
2
2
2
2
2
2
2
2
2
Br-guaiacol] = [H O ] = 500 µM, 5% MeOH/100 mM KPi (v/v) at pH 7 and 25 °C.
2
2
Guaiacol Binding Studies. The electronic absorption spectra of ferric DHP B in the presence
of guaiacol substrates were recorded in 100 mM KP (pH 7) containing 6.25% MeOH (v/v). No
i
significant systematic changes in the Soret band were observed upon ligand binding (data not
shown), thus precluding its use for substrate binding studies. The Q-band region, however,
showed well-behaved optical difference spectra (Figures S4 and S5) upon titration of the
guaiacol substrates (3.75-100 eq), enabling the determination of the corresponding apparent
dissociation constants (K values). In general, the substituted guaiacols exhibited weaker binding
d
2
4
34
affinities to ferric DHP B than other substrates/ligands, including haloindoles, nitrophenols,
3
5
and azoles (Table 2). For the 4-X-guaiacol panel of substrates, a trend in K was observed of
d
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increasing binding affinity as the size of the halogen atom increased: H < F < Cl < Br. An
identical trend has been previously noted for the 5-X-indole series (F < Cl < Br < I) as observed
2
4
in both substrate binding and resonance Raman studies, and correlates well with the increased
affinity of the halogen for occupying the Xe1 binding site (a hydrophobic cavity surrounded by
amino acids L100, F21, F24, F35, and V59) as determined from crystallographic studies of 4-
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42
halophenols binding to DHP. When compared to the binding of the known DHP B inhibitor
5
6
4
-Br-phenol (1.15 mM ), the 4-Br-guaiacol binding affinity is significantly stronger, which
rationalizes the minimal impact this inhibitor had on 4-Br-guaiacol conversion (vide supra).
Table 2. K Values for Substrate Binding to Ferric WT DHP B at pH 7.
d
Substrate
Guaiacol
o-guaiacol
K (µM) at pH 7 Reference
d
a
14712 ± 714
2438 ± 207
493 ± 53
374 ± 42
n.d.
a
4
-F-G
a
4
-Cl-G
a
4-Br-G
a
4
4
4
5
6
-MeO-G
-Me-G
a
1433 ± 97
1341 ± 26
1745 ± 340
a
-NO -G
2
a
-Br-G
-Br-G
a
1332 ± 342
Phenol
4-BP
5
3
3
6
4
4
1150
262 ± 23
105 ± 21
4
2
-NP
,4-dNP
Indole
2
2
2
4
4
4
5
5
-Cl-indole
-Br-indole
317 ± 23
150 ± 10
62 ± 10
5-I-indole
Azole
3
5
imidazole
benzotriazole
52 ± 2
82 ± 2
110 ± 8
35
35
benzimidazole
a
this work; n.d., no detectable change in absorbance after addition of 100 equiv 4-MeO-G.
X-ray Crystallographic Studies of WT DHP B Complexed with Guaiacols. Crystal structures
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were determined to atomic resolution for DHP in complex with five different guaiacol substrates.
X-ray data collection and refinement statistics are provided in Table S3, and selected distances
are provided in Table 3. For each structure, WT DHP B crystallized with two molecules (A and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B) per asymmetric unit, in agreement with all previously reported dehaloperoxidase crystal
19
structures. Each guaiacol substrate (4-Br-G, 5-Br-G, 6-Br-G, 4-NO -G and 6-MeO-G) was
2
found to bind in the distal pocket (Figure 4) with partial occupancy, sharing the distal pocket
with a water molecule ligated to the heme Fe. As such, these DHP–guaiacol complexes are the
first examples observed of ligand binding in DHP without the displacement of the distal water
molecule. With the exception of 5-Br-G, all guaiacols exhibited a single binding site that was
virtually identical for 4-Br-G (Figure 4A), 4-NO -G (Figure 4E) and 4-MeO-G (Figure 4D): the
2
substrates were oriented for favorable π-stacking interactions with F21, their methoxy
substituents were found in the Xe1 binding site (a hydrophobic cavity surrounded by amino acids
6
0
L100, F21, F24, F35, and V59 that was shown to bind Xe), the 4-X substituents (Br, NO or
2
MeO groups) were directed away from the heme in a hydrophobic region deeper in the distal
cavity (reorienting F60 to accommodate the bulkier NO and MeO substituents), and the phenolic
2
OH for each was directed toward the heme Fe and within H-bonding distance to both H55 (Nε)
and the heme-bound distal water. Two conformations were observed for 5-Br-G, assigned as 5-
Br-G and 5-Br-G : the binding orientation of 5-Br-G (Figure 4B) is similar to the 4-X-G (X =
A
B
A
Br, NO or MeO) binding sites, however the Br atom resides closer to the cavity entrance. In
2
addition, the 4-X-G molecules are better positioned for stronger π-stacking interactions with F21
than 5-Br-G , which is reflected in i) the 4.6-fold and 1.3 fold stronger binding affinities of 4-Br-
A
G and 4-NO -G, respectively, when compared to 5-Br-G, and ii) higher occupancies of the 4-X-
2
G substrates compared to 5-Br-G (4-Br-G, 90% > 4-MeO-G, 75% > 4-NO -G, 70% > 5-Br-G,
2
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1
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2
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4
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5
5
5
5
5
5
6
3
5% /30% ). In contrast, a rotation of the guaiacol molecule in the distal pocket was observed
A
B
for 5-Br-G and 6-Br-G (Figures 4B and 4C), with the Br atoms located in the Xe1 binding site,
B
the OH and methoxy groups oriented toward the top of the distal pocket, reduced π-stacking
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
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9
0
1
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3
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5
6
7
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4
5
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7
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0
interactions with F21, and the unsubstituted region of the ring directed toward the heme cofactor.
H55 is out of optimal H-bonding distance with regard to 5-Br-G (Table 3), yet is well-
B
positioned
to
interact
with
the
MeO
oxygen
of
6-Br-G.
Figure 4. X-ray crystal structures of DHP B complexed with A) 4-Br-guaiacol (gray; 6CKE), B)
5
6
-Br-guaiacol (green; 6CRE), C) 6-Br-guaiacol (yellow; 6CO5), D) 4-methoxy-guaiacol (cyan;
CH6), and E) 4-nitro-guaiacol (purple; 6CH5). The Br atom for each substrate in panels A-C is
colored magenta for clarity. F) The bis-histidine hexacoordinated form of DHP.
As a side note, each guaiacol structure exhibited one of the two protomers in a bis-histidine
3
5
ligated (hexacoordinated) heme orientation (Figure 4F). We have previously observed such a
hemichrome species in DHP B when complexed with benzotriazole using crystals grown from
PEG 4000 (as opposed to PEG 3350 here). Other DHP complexes (unpublished results) have
exhibited the bis-histidine ligation in a single protomer when PEG 3350 was used, and as such
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we surmise that this coordination is attributable to the use this precipitant during crystallization,
and is not exclusive to the guaiacol complexes. The functional consequences (if any) of the bis-
histidine ligation were not further explored here.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 3. Selected distances (Å) for DHP B-guaiacol complexes (protomer A).
b
b
4
-Br-G
6CKE
90%
5-Br-G
6CRE
6-Br-G
6CO5
40%
4-NO -G
6CH5
4-MeO-G
6CH6
75%
2
PDB accession code
Substrate occupancy
c
A: 35% / B: 30%
58%
70%
100%
Distal H O occupancy
100%
34%
90%
2
ε
int
int
int
2
ext
ext
ext
int
ext
int
ext
H55 N ꢁꢁꢁOH (guaiacol)
2.64
4.62
2.47
4.42
A
/6.52
/5.51
B
4.78
6.38
2.61 /8.35
4.55 /7.34
2.66 /9.54
δ
int
ext
int
ext
int
ext
H55 N ꢁꢁꢁOH (guaiacol)
A
B
4.65 /7.55
ζ
1
3
4
F21 C ꢁꢁꢁC (guaiacol)
3.76
3.97
4.85
4.17
3.67
A
(C )/4.36
B
(C ) 4.05 (C )
3.71
3.78
4.75
3.73
3.93
4.74
γ
4
5
6
1
F21 C ꢁꢁꢁC (guaiacol)
FeꢁꢁꢁOH (guaiacol)
A
(C )/3.76
B
B
B
(C ) 3.71 (C )
6.75
4.78 /7.28
A
C2
FeꢁꢁꢁO (guaiacol)
4.62
4.71 /6.08
6.96
4.71
4.68
A
C4
C5
C6
C4
C4
FeꢁꢁꢁX (guaiacol)
Br : 8.43
2.85
Br : 8.21 /5.43
Br : 5.61
----
N : 8.04
2.76
O : 7.85
2.72
A
B
guaiacol
OHꢁꢁꢁdistal H O
2.37A
2
Feꢁꢁꢁdistal water
2.18
6.68
6.01
2.15
2.52
2.29
6.54
5.68
2.27
2.13
2.20
δ
int
ext
int
ext
int
ext
FeꢁꢁꢁH55 N
FeꢁꢁꢁH55 N
6.38 /8.98
6.44 /8.80
6.64 /9.73
ε
int
ext
int
ext
int
ext
5.84 /10.17
2.23
5.88 /10.18
5.91 /11.59
ε
FeꢁꢁꢁH89 N
2.14
2.12
Fe to pyrrole-N plane
0.062
0.063
0.062
0.059
0.059
a
the two 5-Br-G conformations are denoted as subscripted A or B as shown in Figure 4B, and
the two H55 conformations are designated as interior ( ) or exterior ( ). for 4-Br-G and 6-Br-
G, H55 was found to be exclusively in the interior and exterior conformations, respectively. 4-
int
ext
b
c
NO –G also exhibited an additional slightly shifted conformation, but of minor occupancy, and
2
therefore not described.
Stopped-flow UV-visible Spectroscopic Studies with 4-Br-guaiacol. Single and double-
mixing stopped-flow UV-visible spectroscopic methods were used to investigate the reaction of
4
-Br-guaiacol (as a representative 4-X-guaiacol substrate) with DHP B. Studies were performed
3
1
with H O -activated DHP B (i.e., pre-formed as Compound I [DHP B(Y28F/Y38F)],
2
2
32, 40
41
Compound ES,
or Compound II ), or by pre-incubating DHP B with 4-Br-guaiacol followed
by addition of H O . For the majority of the studies described below, the data did not properly fit
2
2
one-step/two species or two-step/three species irreversible mechanisms, and as such
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2
2
2
2
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3
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4
4
4
4
4
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5
5
5
5
5
5
5
6
experimentally obtained spectra at selected time points detailed in the figure legends are shown.
Compound I Reactivity – Generated in an initial mixing step of ferric DHP B (Y28F/Y38F)
3
1
with 10 equiv H O in an aging line for 85 ms, Compound I was reacted with 10 equiv 4-Br-
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
guaiacol at pH 7. The activated enzyme, Compound I [406 (Soret), 528, and 645 nm], was not
observed (Figure 5A). Rather, ferric-like DHP B (Y28F/Y38F) [406 (Soret), 513, 640 (sh) nm,
black] was the first spectrum recorded, and suggested that Compound I was reduced by the
substrate within the mixing time (1.5 ms) of the stopped-flow apparatus. The rapid reaction of
24
Compound I with 4-Br-guaiacol is virtually identical to its reactivity seen with 5-Br-indole, 4-
3
4
25
nitrophenol and pyrrole in that these substrates were also able to reduce Compound I to the
ferric enzyme within the stopped-flow mixing time. The ferric enzyme then converted to a new
DHP B species [427 (Soret), 556, and 578 (sh) nm, blue; t = 246 s] that appeared to be a mixture
4
1
of both ferrous and oxyferrous DHP.
Compound ES Reactivity – Ferric WT DHP B was rapidly mixed with 10 equiv of H O at
2
2
pH 7, incubated for 500 ms to allow for the maximum accumulation of Compound ES (ferryl
40
heme + tyrosyl radical), and subsequently mixed with 10-50 equiv 4-Br-guaiacol. As a
representative reaction, the preformed Compound ES [418 (Soret), 546, 582 nm, black; t = 0 s]
was converted in the presence of 10 equiv 4-Br-guaiacol to ferric DHP B [407 (Soret), 505, 640
(
sh) nm, red; t = 8.8 s] that underwent further reduction to the resting oxyferrous state after 500 s
Figure 5B, Table 4). The progression of initial Compound ES reduction to the ferric enzyme,
(
followed by a slower reduction to oxyferrous DHP, mirrors previous observations of Compound
24
40
ES reactivity with haloindoles and halophenols, suggesting the similar product-driven
reductions that occurred in those studies may also be present here with the guaiacol substrates.
Compound II Reactivity – Formed after 3 s in an ageing line from an initial mixing step of 10
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equiv H O with oxyferrous WT DHP B that was pre-incubated with 1 equiv 2,4,6-
2
2
4
1
trichlorophenol, Compound II was reacted with 10 equiv 4-Br-guaiacol. The preformed
Compound II [420 (Soret), 546, 584 nm, black; t = 0 s] initially converted to ferric DHP B [407
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Soret), 505, 640 (sh) nm, red; t = 17.4 s], was further reduced to oxyferrous DHP B [416
Soret), 542, 578 (sh) nm, blue; t = 246 s], and remained stable as this species for the duration of
(
the 500 s observation time (Figure 5C). The reduction of Compound II to ferric DHP by 4-Br-
41
24
guaiacol here parallels the same observations made when employing TCP, haloindole, and
25
pyrrole substrates, as does the further reduction (presumably product-driven) to oxyferrous
DHP, albeit virtually complete formation (in lieu of a mixture of ferric/oxyferrous DHP) was
observed here (vide infra).
Oxyferrous Reactivity – It has been previously shown that oxyferrous DHP can be activated
4
1, 61, 62
24
25
by H O in the presence of halophenol,
haloindole, and pyrrole substrates, whereas in
2
2
their absence, only a slight bleaching of the Soret band and/or long time scale conversion to
Compound RH is observed. To investigate if guaiacols are able to facilitate this substrate-
dependent activation of DHP, stopped-flow methods were employed to rapidly mix H O (10
2
2
equiv) with a solution of oxyferrous DHP B that was preincubated with 10 equiv of 4-Br-
guaiacol. The following spectral changes were noted: oxyferrous DHP B [416 (Soret), 542, 578
nm, black; t = 0 s] was oxidized to ferric DHP B [407 (Soret), 501, 640 (sh) nm, red; t = 7.3 s],
followed by a gradual reduction back to the oxyferrous state [413 (Soret), 541, 578 nm, blue; t =
500 s] (Figure S6). Interestingly, no transiently-observed ferryl species were formed, which
41
24
25
differs from the reaction performed in the presence of halophenol, haloindole and pyrrole
substrates where the Compound II intermediate was observed.
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Kinetic data for the reaction of H O -activated DHP B with 4-Br-guaiacol. A) DHP B
2
2
(Y28F/Y38F): top panel, stopped-flow UV−visible spectra of the double-mixing reaction of
preformed Compound I (10 ꢀM) with a 10-fold excess of 4-Br-guaiacol at pH 7.0 (900 scans
over 500 s); inset: the single wavelength (408 nm) dependence on time obtained from the raw
data; bottom panel, experimentally obtained spectra for Compound I reacted with 4-Br-guaiacol
(black, t = 0 s), and its reduction to a ferrous-like species of DHP B was observed in the presence
of 4-Br-guaiacol (blue, t = 246 s). B) Ferric DHP B/Compound ES: top panel, stopped-flow
UV−visible spectra of the double-mixing reaction between ferric DHP B (10 µM) premixed with
1
0 equiv of H O (500 ms) and then reacted with 10 equiv 4-Br-guaiacol at pH 7.0 (900 scans
2 2
over 500 s); inset: the single wavelength (408 nm) dependence on time obtained from the raw
data; bottom panel, experimentally obtained spectra of Compound ES (black, t = 0 s) reacted
with 4-Br-guaiacol, resulting in ferric DHP B (red, t = 8.8 s) and further reduction to oxyferrous
DHP B (blue, t = 500 s). C) Ferric DHP B/Compound II: top panel, stopped-flow UV−visible
spectra of the single-mixing reaction between Compound II and 4-Br-guaiacol. Compound II
was formed from an initial reaction between oxyferrous DHP B (10 µM) preincubated with 1
equiv of trichlorophenol and 10 equiv of H O and reacted for 3 s prior to the second mix with 4-
2
2
Br-guaiacol at pH 7.0 (900 scans over 500 s); inset: the single wavelength (408 nm) dependence
on time obtained from the raw data; bottom panel, experimentally obtained spectra of Compound
II (black, t = 0 s) reacted with 4-Br-guaiacol, resulting in ferric DHP B (red, t = 17.4 s) and
further reduction to oxyferrous DHP B (blue, t = 246 s).
Double-mixing stopped-flow UV-visible spectroscopic methods were also used to investigate
the reactivity of the remaining guaiacol substrates (10-50 equiv) with pre-formed Compound ES
in a manner identical to that described above for 4-Br-guaiacol. Figures S7 and S8 display
representative reactions of preformed Compound ES [418 (Soret), 546, 582 (sh) nm, black; t = 0
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s] with 10 equiv of each of the guaiacol substrates, and Table 4 summarizes the optical data.
Although the time required to do so varied over 40-fold (4.37 – 186 s), all substrates were found
to reduce Compound ES to ferric DHP B. However, while four (o-guaiacol, 4-Br-G, 4-MeO-G,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
-NO -G) of the seven guaiacol reactions were found to further reduce the resultant ferric
2
enzyme to the oxyferrous state, two were unable to do so: 4-Me-guaiacol (Figure S8A) and 5-Br-
guaiacol (Figure S8B). Rather, the enzyme converted to a mixture of ferric DHP and the
31, 32, 40
peroxidase-attenuated state Compound RH.
Importantly, the formation of oxyferrous DHP
in these stopped-flow studies strongly correlated with the observation of 2-MeOBQ as identified
in the HPLC studies above, suggesting that 2-MeOBQ could serve as a reducing agent of ferric
DHP (vide infra). In the case of 6-Br-guaiacol (Figure S8C), the formation of a product that
exhibited a strong absorption band ~450 nm that overlapped with the Soret and Q bands
prevented any conclusive spectral assignments.
The main observations from these stopped-flow studies were i) H O -activated enzyme was
2
2
reduced to ferric DHP by 4-Br-guaiacol, with the rate of reduction following the order:
Compound I > Compound ES > Compound II, ii) the oxyferrous enzyme reacted with H O in
2
2
the presence of 4-Br-guaiacol, however without the formation of an observable Compound II
species, iii) all of the reactions with 4-Br-guaiacol yielded oxyferrous DHP as a final observable
species, and iv) 2-MeOBQ was identified as the putative reductant.
Table 4. Summary of Stopped-Flow UV-vis Spectroscopic Data for the Reaction of Compound
ES with Guaiacol Substrates at pH 7.
Compound ES →
Ferric, time (s)
26
Observed ferric
Ferric →
Final observed species
Substrate
species λmax (nm) Oxyferrous, time (s)
409, 504, 640
λ
max (nm)
o-guaiacol
> 500
411, 540, 578
(
oxy/ferric mixture)
4
5
-Br-G
-Br-G
9
5
407, 505, 640
411, 505, 640
> 500
n/a
418, 542, 578 (oxy)
411, 505, 640
(
Compound RH-like)
420, 455 (product)
6-Br-G
n/o
n/o
n/o
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-NO -G
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186
410, 539 (sh)
> 500
412, 539, 576
oxy/ferric mixture)
418, 543, 578 (oxy)
409, 503, 640
Compound RH-like)
(
4
-MeO-G
-Me-G
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30
409, 503, 640
409, 503, 640
250
n/a
4
(
n/a: not applicable (i.e., oxyferrous DHP was not formed)
n/o: not observable owing to product formation at 455 nm
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0
Reduction of Ferric DHP B by 2-MeOBQ. Stopped-flow UV/visible spectroscopic methods
were employed to explore if the 2-MeOBQ product was capable of the aerobic reduction of ferric
DHP to yield the oxyferrous enzyme observed in the above studies. A solution of 8 ꢀM ferric
DHP B was rapidly mixed with authentic 2-MeOBQ (62.5 ꢀM, Figure 6; 0.625, 6.25, and 625
ꢀ
M, Figure S9) at pH 7, and monitored for 50 s. For 2-MeOBQ concentrations of 6.25-625 ꢀM,
the data were best fit to a two-step, three species irreversible mechanism: the first step (k1)
yielded an initial oxyferrous-like species whose spectral features were similar, but not identical,
to oxyferrous DHP B [UV-visible spectrum: 419-20 (Soret), 542-4, 578 nm] that was
subsequently fully formed in the second step (k ). Interestingly, the reaction exhibited saturable
2
kinetics with respect to k and 2-MeOBQ, whereas k was [2-MeOBQ]-independent (Figure
1
2
S10). Even at a sub-stoichiometric concentration of 0.625 µM 2-MeOBQ (Figure S9A), a partial
formation of oxyferrous DHP was observed. Overall, the reduction observed here with 2-
MeOBQ was appreciably faster than that observed previously with 2,6-dichloro-1,4-
-1 40
dibenzoquinone (DCQ: kobs = 0.085 s ), which is the product of the DHP-catalyzed oxidative
dehalogenation of 2,4,6-trichlorophenol. These results support a “product-driven” reduction
wherein the 2-MeOBQ product of guaiacol-oxidation via a DHP peroxidase activity reduces the
peroxidase-active ferric DHP B to its hemoglobin-active oxyferrous state.
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4
20
A 1.0
0.85
.75
0.65
Data
Fit
0
.8 408
0
0.6
0
10 20 30 40 50
0.4
0.2
0.0
Time (s)
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578
4
90
3
00
400
500
Wavelength (nm)
600
700
800
420
Ferric DHP
Oxyferrous-like
Oxyferrous
B 1.0
420
0.8 408
0
0
0
.2
542
578
578
4
90
0.6
0.4
0.2
.1
.0
5
44
4
50 500 550 600 650 700
0.0
300
400
500
600
Wavelength (nm)
700
800
Figure 6. Kinetic data for the reaction of 2-MeOBQ with ferric DHP B. A) Stopped-flow
UV−visible spectra of the single-mixing reaction between ferric DHP B (10 µM) reacted with
2.5 µM 2-MeOBQ at pH 7.0 (900 scans over 50 s); inset: the single wavelength (418 nm)
dependence on time obtained from the raw data (black) and the corresponding fit (red). B)
Calculated spectra of the three reaction components derived from the SVD analysis: ferric DHP
B (black), an apparent mixture of ferric and oxyferrous DHP B (red), and oxyferrous DHP B
6
(blue).
DISCUSSION
The activity studies reported here demonstrate that DHP B was able to catalyze the
conversion of all guaiacols studied under physiological conditions. With the exception of o-
guaiacol that only had a 30% conversion to products, both monosubstituted native-halogenated
guaiacols (-F, -Cl, -Br) and nonnative guaiacols (-Me, -OMe, -NO ) were converted in high
2
yields (>90%) to monomeric and oligomeric product(s). Isotopic labeling studies unequivocally
demonstrated that the oxygen atom incorporated into the products for the 4-Br-guaiacol reaction
originated exclusively from H O, while radical scavengers were shown to inhibit substrate
2
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conversion. Together, these results provide support that guaiacol oxidation follows a radical-
based peroxidase mechanism. Moreover, the oxidation of the guaiacol substrates could be
initiated from either the traditional peroxidase ferric oxidation state, or from the oxyferrous form
0
1
2
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9
0
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0
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9
0
of the enzyme that is relevant to the O -transport function of DHP as the coelomic hemoglobin of
2
A. ornata.
These results are consistent with the well-established peroxidase activity of DHP B with
1
1
trihalophenol substrates, as well as with previous studies of guaiacol oxidation by other
peroxidase enzymes, specifically myoglobin and other heme peroxidases where it has been used
as a classic substrate in kinetic assays owing to the formation of the highly colored (λmax = 470
5
8, 63
nm) 3,3’-dimethoxy-4,4’-biphenoquinone reaction product.
Some notable examples of
guaiacol oxidation in these systems include i) monitoring the peroxidase activity of myoglobin
6
4-66
57,
mutants via guaiacol oxidation,
and ii) the oxidation of guaiacol by horseradish peroxidase,
5
9
10
67
lignin peroxidase, and manganese peroxidase. In those systems, the products were
identified as guaiacol dimers (i.e., 3,3’-dimethoxy-4,4’-biphenoquinone and its reduced analog
,3’-dimethoxy-4,4’-diphenol), trimers, and tetramers, with the guaiacol moieties linked through
3
5
7
the carbon ortho to the phenol group. The monomer, 2-MeOBQ, however was not observed in
those studies, but it has been previously identified in the case of laccase-catalyzed phenol
6
8
dehydrogenation, and in studies of guaiacol oxidation employing small molecule catalysts (i.e.,
69, 70
silver oxide, ferric chloride and chromium trioxide).
demonstrate that DHP B is capable of oxidizing 4-R-guaiacols (with the exception of R = Me) to
-MeOBQ, either as the major product (R = F, Cl, Br), or as a minor product (R = H, MeO,
The results presented herein
2
NO2).
The formation of 2-MeOBQ further establishes the ‘product-driven’ reduction observed in
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III
II
DHP that appears to be a hallmark of this catalytic hemoglobin: at +206 ± 6 mV, the Fe /Fe
71
redox couple for DHP B is higher than most other hemoglobins (e.g., human Hb, + 158 mV;
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74
horse heart Hb, + 152 mV; horse heart Mb, + 46 mV; sperm whale Mb, −43 mV ), and
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significantly more so than other peroxidases (e.g., myeloperoxidase, −5 mV; cytochrome c
7
6
77
peroxidase, −182 mV; HRP, −266 mV ). Demonstrated first with 2,6-dichloroquinone
4
0
(
DCQ), the oxidation product of its peroxidase activity with 2,4,6-trichlorophenol, and later
2
4
with 5-Br-3-oxindole, the oxidation product of its peroxygenase activity with 5-Br-indole, DHP
has been shown (almost counterintuitively) to be susceptible to reduction by the oxidation
products of its catalytic activities. Here, we provide strong evidence that the ‘product-driven’
reduction is attributable to 2-MeOBQ, which may serve as a ‘rescue’ of the enzyme to return to
its oxyferrous state, thereby enabling this multifunctional hemoglobin to maintain its O2-
transport function after cycling peroxidase activity.
Importantly, activation of oxyferrous DHP in the presence of guaiacol substrate and H O
2
2
was also demonstrated, suggesting that the entire catalytic cycle can be initiated from, and
ultimately return to, the hemoglobin-active O -carrier state. How such a substrate-dependent
2
activation of oxyferrous DHP occurs has been the subject of several studies, and two possible
mechanisms have been suggested: i) trace amounts of the ferric enzyme react with H O and
2
2
substrate, thereby generating diffusible substrate radicals that oxidize oxyferrous DHP to the
6
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catalytically-active ferric form,
or ii) substrate binding to DHP destabilizes the oxyferrous
state in the presence of hydrogen peroxide, leading to a reaction between ferrous DHP and H O2
2
that activates the enzyme via a Compound II intermediate that is subsequently quickly reduced to
41
the ferric enzyme. Here, we employed the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)
to scavenge diffusible radicals, and noted that the 4-Br-guaiacol reaction was unaffected by
2
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