Substrate specificity of Sphingobium chlorophenolicum 2,6- dichlorohydroquinone 1,2-dioxygenase

Article

pubs.acs.org/biochemistry

Substrate Specificity of Sphingobium chlorophenolicum 2,6-

Dichlorohydroquinone 1,2-Dioxygenase

Timothy E. Machonkin* and Amy E. Doerner

Department of Chemistry, Whitman College, 345 Boyer Avenue, Walla Walla, Washington 99362, United States

S

* Supporting Information

ABSTRACT: PcpA is an aromatic ring-cleaving dioxygenase that is

homologous to the well-characterized Fe(II)-dependent catechol

extradiol dioxygenases. This enzyme catalyzes the oxidative cleavage

of 2,6-dichlorohydroquinone in the catabolism of pentachlorophenol

by Sphingobium chlorophenolicum ATCC 39723. ¹H NMR and steady-

state kinetics were used to determine the regiospecificity of ring

cleavage and the substrate specificity of the enzyme. PcpA exhibits a high degree of substrate specificity for 2,6-disubstituted

app

hydroquinones, with halogens greatly preferred at those positions. Notably, the k_c^a_a^p_t^p/K of 2,6-dichlorohydroquinone is ∼40-

mA

fold higher than that of 2,6-dimethylhydroquinone. The asymmetric substrate 2-chloro-6-methylhydroquinone yields a mixture of

app

mO₂

1,2- and 1,6-cleavage products. These two modes of cleavage have different K

values (21 and 260 μM, respectively),

consistent with a mechanism in which the substrate binds in two catalytically productive orientations. In contrast,

monosubstituted hydroquinones show a limited amount of ring cleavage but rapidly inactivate the enzyme in an O₂-dependent

fashion, suggesting that oxidation of the Fe(II) may be the cause. Potent inhibitors of PcpA include ortho-disubstituted phenols

and 3-bromocatechol. 2,6-Dibromophenol is the strongest competitive inhibitor, consistent with PcpA’s substrate specificity.

Several factors that could yield this specificity for halogen substituents are discussed. Interestingly, 3-bromocatechol also

inactivates the enzyme, while 2,6-dihalophenols do not, indicating a requirement for two hydroxyl groups for ring cleavage and

for enzyme inactivation. These results provide mechanistic insights into the hydroquinone dioxygenases.

hydroxyl-substituted aromatic ring with a hydroxyl, amino, or

acteria use an astonishing range of molecules as carbon

B_{sources, a property that has been of interest for use in the}carboxylate at the ortho and/or para positions. Interestingly, all

of these enzymes appear to share essentially the same type of

metal-binding site employed by the EDOs: a single Fe(II)

center bound to the protein by a 2-histidine-1-carboxylate

facial-capping triad of ligands (or, in some cases, three

histidines). This would seem to imply that the factors

governing the substrate specificity among these ring-cleaving

dioxygenases lie in the active site pocket and the second

coordination sphere.

The dioxygenases that cleave hydroquinones lacking a

carboxylate functional group [hydroquinone dioxygenases

(HQDOs)] are unique among ring-cleaving dioxygenases in

that their substrates cannot bind to the Fe(II) center in a

bidentate fashion. However, they have been studied little, and

only five have been reported to date. Three of these are

homologues that are 40−50% identical in amino acid sequence:

2,6-dichlorohydroquinone 1,2-dioxygenase (PcpA) from the

pentachlorophenol degradation pathway of Sphingobium

chlorophenolicum ATCC 39723 (Scheme 1A),²⁸⁻³⁰chlorohy-

droquinone 1,2-dioxygenase (LinE) from the γ-hexachlorocy-

clohexane (lindane) degradation pathway of Sphingobium

japonicum (formerly Sphingomonas paucimobilis) UT26

(Scheme 1B),³¹and MnpC, an HQDO from a putative

bioremediation of xenobiotic compounds, including recalcitrant

pollutants such as chlorinated organic compounds.¹⁻⁵The

study of these pollutant-degrading catabolic pathways has

yielded a wealth of novel enzymes with interesting properties.

The group of bacterial enzymes involved in the catabolism of

aromatic hydrocarbons is one such example that has received

considerable attention. In most cases, aerobic catabolism of

aromatic hydrocarbons involves the oxidative ring cleavage of a

catechol. The substrate specificity of these catechol dioxyge-

nases can limit the range of aromatic hydrocarbons that can be

utilized by a particular catabolic pathway, in part because of the

buildup of toxic dead-end metabolites.³There are two types of

catechol dioxygenases: the Fe(III)-dependent intradiol dioxy-

genases (IDOs), which cleave the C−C bond between the

vicinal hydroxyl groups, and the Fe(II)-dependent extradiol

dioxygenases (EDOs), which cleave the C−C bond adjacent to

the vicinal hydroxyl groups.⁶⁻¹⁰Extensive studies of the EDOs

using a range of experimental approaches (including structures

of trapped intermediates) have yielded a generally accepted

mechanism^6,7,10−12supported by density functional theory

calculations.^13,14

In recent years, ring-cleaving dioxygenases that work on

noncatecholic substrates, including salicylates and genti-

sates,¹⁵⁻²¹homogentisate,^22,232-aminophenols,²⁴⁻²⁷and hy-

droquinones (that lack a carboxylate functional group), have

been described.²⁸⁻³⁴All of these substrates consist of a

Received: June 2, 2011

Revised: August 23, 2011

Published: August 26, 2011

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the regiospecificity of the enzyme and the tautomeric states of

the cleavage products. The results provide insights into the

mechanism of ring cleavage.

Scheme 1. Ring-Cleavage Reactions Catalyzed by the Known

HQDOs

EXPERIMENTAL PROCEDURES

■

Protein Production. The expression and purification of

PcpA were performed by the method of Xun and co-workers³⁰

with the modifications described previously.³⁵

Preparation of Potential Substrates and Inhibitors.

2,6-Dibromobenzoquinone was synthesized by the method of

Ungnade and Zilch.⁵¹The identity of this compound was

confirmed by ¹H NMR (CDCl₃): δ 7.33 (s). 2-Chloro-6-

methylhydroquinone was synthesized by the method of Burton

and Prail, with the following additional purification steps.⁵²The

product of the first step (1,4-diacetoxy-2-chloro-6-methylben-

zene) was purified by silica gel column chromatography. The

identity of this compound was confirmed by ¹H NMR

(CDCl₃): δ 6.99 (d, J = 2.2 Hz, 1H), 6.83 (d, J = 2.2 Hz,

1H), 2.30 (s, 3H), 2.22 (s, 3H), 2.13 (s, 3H). The final product

was recrystallized from hot dichloromethane. The identity of

the product was confirmed by ¹H NMR (CDCl₃): δ 6.71 (d, J =

2.8 Hz, 1H), 6.58 (d, J = 2.8 Hz, 1H), 2.26 (s, 3H). The purity

of both compounds was determined to be >98% by both thin

m-nitrophenol degradation pathway of Cupriavidus necator

JMP134.³⁴The two other HQDOs share no sequence identity

with PcpA but are >60% identical in sequence with each other

and cleave unsubstituted hydroquinone: HapCD from the 4-

hydroxyacetophenone degradation pathway of Pseudomonas

fluorescens ACB³²and PnpC1C2 from the p-nitrophenol

degradation pathway of Pseudomonas putida DLL-E4³³

(Scheme 1C). Thus, similar to the EDOs, which occur in

two different classes (type I and type II),^8,9there are two

distinct structural classes of HQDOs. PcpA and its homologues

(LinE and MnpC) and the type I EDOs are members of the

“vicinal oxygen chelate” (VOC) superfamily, as first recognized

by Xu et al.²⁹However, our experimentally verified structural

model of PcpA demonstrated that the four copies of the

βαββββ structural motif of the VOC superfamily are arranged

differently in this enzyme and that residues His11, His227, and

Glu276 are likely the ligands to the Fe(II) center.³⁵

A remarkable property of PcpA and LinE is their ability to

cleave chlorinated substrates. This contrasts with EDOs, which

are known to be subjected to mechanism-based inactivation by

chlorinated substrates.³⁶⁻³⁹However, whether PcpA and LinE

exhibit an actual preference for chloro substituents or merely

tolerate chloro substituents has not been previously established,

because very little about the substrate specificity of these

enzymes has been reported. Another HQDO, HapCD, has a

higher activity with methylhydroquinone than it does for its

native substrate, hydroquinone, and a lower activity toward

chlorohydroquinone and other halogenated hydroquinones.³²

Understanding how a naturally occurring ring-cleaving

dioxygenase can successfully utilize chlorinated substrates is

an important area for study. Considerable effort has been spent

in identifying EDOs capable of cleaving chlorinated sub-

strates³⁹⁻⁴⁶and engineering EDOs with improved abilities to

cleave chlorinated substrates and/or reduced sensitivity to

inactivation;⁴⁷⁻⁵⁰however, success on the latter front has been

limited.

1

layer chromatography and H NMR. Chlorobenzoquinone was

purchased from Alfa Aesar and repurified by silica gel column

chromatography. All other compounds were purchased

commercially, were reagent grade, and were used without

further purification.

Kinetic Studies. The rate of the enzymatic reaction was

determined by measuring product formation by a spectropho-

tometric assay developed by Xun and co-workers³⁰with the

modification (addition of imidazole) described previously.³⁵

The assay was performed on a Cary 50 Bio or Cary 5000 (for

the O₂dependence experiments) UV−visible spectrophotom-

eter (Varian). In some cases, the hydroquinones were formed

by in situ reduction of the substituted benzoquinone with

NaBH₄. This was done with 2,6-dichlorohydroquinone, 2,6-

bromohydroquinone, and 2,6-dimethylhydroquinone. To en-

sure that the absorbance change of the reaction mixture was

within a measurable range, either a 1.0 cm path length cuvette

with a 1.0 mL reaction volume or a 5.0 cm path length cuvette

with a 2.0 mL reaction volume was used. The 1.0 cm path

length cuvette was used for initial screening of possible

substrates, for all of the inhibitor studies, for the substrate

dependence steady-state kinetics of 2,6-dimethylhydroquinone,

and for the O₂dependence steady-state kinetics. In these

studies, 5−10 μL of 85−110 μM PcpA was used. The 5.0 cm

path length cuvette was used for the substrate dependence

steady-state kinetic studies of 2,6-dichlorohydroquinone, 2,6-

dibromohydroquinone, and 2-chloro-6-methylhydroquinone, in

which 2.5 μL of 110 μM PcpA was used. For the initial

screening of substrates, both high (200−400 μM) and low

(10−60 μM) concentrations of the compounds in question

were tested. Air-saturated 20 mM potassium phosphate buffer

(pH 7.0) was used throughout. All reactions were performed at

23 °C. Because of the use of NaBH₄for in situ reduction of the

substituted benzoquinones to the corresponding hydroqui-

nones, bubbles of H₂(g) formed during the course of the

reaction, thereby causing a small baseline shift in the

absorbance spectrum. To account for this absorbance shift,

the baseline absorbance, measured at 380 nm, was subtracted

from the product absorbance at each time point.

In this work, we investigated the range of molecules that can

act as substrates and inhibitors of PcpA, including structural

variants of the physiological substrate (2,6-dichlorohydroqui-

none) and substrates of other ring-cleaving dioxygenases.

Steady-state kinetic studies were used to establish the substrate

specificity of the enzyme as well as the mode and strength of

inhibition of various inhibitors. ¹H NMR was used to determine

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For the initial screening of possible substrates, the

spectrophotometric assay was conducted with a range of

compounds, and changes in the UV−visible absorbance

spectrum were monitored. For each compound tested, a

control experiment in the absence of PcpA was conducted to

ensure that enzymatic activity was the source of any spectral

changes. The appearance of a new transition in the PcpA-

containing reaction but not in the control was determined to be

representative of ring-cleavage activity. Molecules that were

determined to be cleaved by PcpA were further characterized

by steady-state kinetics. The initial velocities were measured,

divided by the enzyme concentration used, and plotted versus

substrate concentration, and the Michaelis−Menten equation

(1)

(2) PcpA was incubated both with and without the compound

in question in either air-saturated buffer or O₂-free buffer,

followed by addition of substrate (and more air-saturated

buffer, in the latter case). (3) The curvature in the product

formation of the native substrate when the compound in

question was used as an inhibitor was fit to the equation

(2)

was fit to the data with KaleidaGraph (Synergy) to yield the

to yield the rate of inactivation, j_s(P_t, P_i, and P_∞are the

amounts of product formed at time t, at the start of the assay,

and at time ∞, respectively). The apparent rate constant of

inactivation, j_i^a_n^p_a^p_ct, and the apparent binding constant of

inactivation, K_i^a_n^p_a^p_ct, were obtained by performing a global fit

of the values of j_sas a function of the inactivating compound

concentration, [I], at different substrate concentrations, [A],

with Solver:

app

K_m^ap_A^pand k values in air-saturated buffer (O₂concentration of

cat

267−275 μM). The dependence of the O₂concentration on

the rate in the presence of high concentrations (200 or 300

μM) of the substrates was determined by the same

spectrophotometric method, and the Michaelis−Menten

equation was fit to the data to yield the apparent K_mOand

2

k_catvalues. The O₂concentrations in the buffers were

determined with an O₂-sensitive electrode (Hansatech) in

separate control experiments without PcpA.

(3)

For the initial screening of inhibitors, the spectrophotometric

assay was conducted with the compound in question at 500 μM

and the native substrate at 50 μM, and the initial velocity

relative to that without inhibitor was calculated. Detailed

steady-state kinetic characterization was performed on three of

the strongest inhibitors. Likewise, the kinetics of the

monosubstituted hydroquinones were analyzed by treating

them as inhibitors of 2,6-dichlorohydroquinone. For each

inhibitor, the initial velocities were determined with five or six

different substrate concentrations at each of three or four

different inhibitor concentrations (and in the absence of

inhibitor). The inhibition constant, K_I, was determined by

performing a global fit to the data set using the Solver function

of Excel (Microsoft). Only the value of K_Iwas allowed to float.

The inhibitor studies were performed in the 1.0 cm path length

cuvette, which was more convenient to use and required less

material, whereas determination of the kinetic parameters of

2,6-dichlorohydroquinone required use of the 5.0 cm path

This procedure has been reported previously be Eltis and co-

workers.³⁸

Product Determination. PcpA reaction products were

identified in two different ways. First, the aqueous reaction

mixture (in an 86% H₂O/10% D₂O/4% CD₃OD mixture) was

1

directly characterized by H NMR spectroscopy, with sodium

3-(trimethylsilyl)-1-propanesulfonate (DSS) used as the

chemical shift standard. For these reactions, PcpA was buffer

exchanged into 20 mM potassium phosphate buffer (pH 7.0)

and reconstituted with iron(II) anaerobically (without the use

of imidazole) in a glovebox. The substrate concentration was

2.0 mM. Second, the aqueous reaction mixture was acidified to

pH <2 with 1.0 M HCl, extracted with CDCl₃, dried over

1

anhydrous Na₂SO₄, and characterized by H NMR spectrosco-

py. To ensure that degradation of the ring-cleavage products

1

did not occur, the H NMR spectra of the aqueous reaction

mixtures were recorded immediately after complete enzymatic

cleavage had occurred as determined by UV−visible spectro-

scopy. Likewise, acidification and extraction into CDCl₃were

length cuvette because of the low molar absorptivity of the

1

conducted the same day, and the H NMR spectra were again

app

mA

product and the low K for this compound. Use of only 1.0

1

recorded immediately. H NMR spectroscopy was performed

cm path length data consistently led to a large overestimate of

on a 400 MHz Bruker Avance III instrument.

app

mA

K_m^ap_A^p. Thus, in the fitting of the inhibitor data, the value of K

determined from the 5.0 cm path length data was kept fixed.

RESULTS

The uncertainty in K_Iwas estimated by a jackknife procedure⁵³

■

app

Identification of Substrates. Four classes of compounds

were tested as possible substrates of PcpA. The first class

consisted of compounds with substituents at the 2, 4, and 6

positions of the ring like the physiological substrate but varying

in the identity of those substituents: 2,6-dibromohydroquinone

(2,6-diBrHQ), 2,6-dimethylhydroquinone (2,6-diMeHQ), 2-

chloro-6-methylhydroquinone (2-Cl-6-MeHQ), 4-amino-2,6-

dichlorophenol, and 3,5-dichloro-4-hydroxybenzoate. The

second class consisted of monosubstituted hydroquinones

(HQs): chloro-, bromo-, methyl-, and hydroxyhydroquinone

(ClHQ, BrHQ, MeHQ, and HOHQ, respectively) and

gentisate. The third class consists of ortho-disubstituted

and propagated with the uncertainty in K

by standard

mA

methods.

Enzyme inactivation with different compounds was assessed

by several methods. (1) The curvature during product

formation was directly observed in the spectrophotometric

assay. Conditions under which the enzyme was completely

inactivated before the substrate was consumed were used (0.46

μM enzyme and 50 μM substrate for the monosubstituted

hydroquinones and 0.146 μM enzyme and 200−300 μM

substrate for the ortho-disubstituted hydroquinones), and this

was used to estimate the partition ratio:³⁸

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Table 1. λ_maxand Molar Absorptivity Values for the Reaction Products of Each of the Hydroquinone Substrates with PcpA in

a

Air-Saturated 20 mM Potassium Phosphate Buffer (pH 7.0)

substrate

reaction product

tautomer

λ_max(nm)

ε (M⁻¹cm⁻¹

)

b

2,6-dichlorohydroquinone (1)

2,6-dibromohydroquinone (5)

2,6-dimethylhydroquinone (9)

2-chloromaleylacetate (3)

2-bromomaleylacetate (7)

2-methylmaleylacetone (10)

keto

enol

keto

enol

keto

enol

253

259

254

315

250

314

248

240

310

9600

10600

7200

6300

8300

2500

c

2-chloro-6-methylhydroquinone (12)

mixture

d

chlorohydroquinone

bromohydroquinone

methylhydroquinone

maleylacetate

maleylacetone

∼5100

d

e

nd

a

1

For the ortho-disubstituted hydroquinones, the identities of the reaction products and their tautomeric states were determined by H NMR studies.

For the monosubstituted hydroquinones, the reaction products were tentatively assigned on the basis of the similarity of their UV−visible spectra

b

with those reported previously for unsubstituted maleylacetate and maleylacetone. In agreement with the value previously reported at pH 6.7 in

c

potassium phosphate buffer.³⁰The reaction products consist of a mixture of 2-methylmaleylacetate (14), which exists entirely in the keto form, and

2-chloromaleylacetone (16), which exists as a mixture of keto and enol forms. The molar absorptivity reported here is for the product mixture

d

e

obtained in air-saturated buffer. Reported previously: 4000 M⁻¹cm⁻¹at 243 nm.⁵⁴Reported previously at pH 6.43: 4400 M⁻¹cm⁻¹at 243 nm and

9300 M⁻¹cm⁻¹at 312 nm.⁵⁵

phenols and 3-substituted catechols: 2,6-dichlorophenol, 2,6-

dibromophenol, 2,6-dimethylphenol, 2-chloro-6-methylphenol,

2-bromo-6-methylphenol, 3-methylcatechol, 3-bromocatechol,

and pyrogallol. Lastly, catechol, 4-methylcatechol, 2,5-dichlor-

ohydroquinone, and unsubstituted hydroquinone were also

tested. Of these compounds, PcpA oxidatively cleaved 2,6-

diBrHQ, 2,6-diMeHQ, and 2-Cl-6-MeHQ (as well as 2,6-

diClHQ), and complete conversion to products (as shown by

absorbance at 248 nm in the control reaction without PcpA,

and estimating a molar absorptivity of the product when

complete conversion was obtained with larger amounts of PcpA

of ∼5100 M⁻¹cm⁻¹, we found that only ∼40% of the BrHQ in

the reaction shown in Figure 1 was converted to product. As

seen clearly in the plot of absorbance versus time (inset), most

of the product formation occurred during the mixing time of

the reaction, and the rate of reaction fell to zero well before the

substrate was depleted. Similar results were obtained with

ClHQ. With MeHQ, the difference spectrum showed

transitions at 240 and 310 nm in an approximately 3:4 ratio,

but complete conversion to products was never obtained. This

behavior indicates that PcpA was rapidly inactivated by these

substrates. Inactivation is a well-known phenomenon in EDOs

with certain substrates such as 3-chlorocatechol in which a

distinct curvature in the time course of product formation is

observed.³⁶⁻³⁹Inactivation has been thoroughly characterized

in Burkholderia xenovorans LB400 2,3-dihydroxybiphenyl 1,2-

dioxygenase (BphC), where it has been shown to be due to loss

of O₂from the active site as superoxide, leaving the iron center

in the inactive Fe(III) state.³⁸

1

complete loss of the substrate features by UV−visible and H

NMR spectroscopy) could be obtained at all substrate

concentrations. The λ_maxand molar absorptivity values for

these reaction products are summarized in Table 1.

PcpA also oxidatively cleaved ClHQ, BrHQ, and MeHQ.

However, complete conversion to products was not observed

even at low substrate concentrations (10−60 μM), except in

some cases with very large amounts of PcpA. An example with

BrHQ is shown in Figure 1. A difference spectrum shows that

λ_maxfor this cleavage product is 248 nm. Accounting for the

These results show that PcpA is a remarkably specific

enzyme: only ortho-disubstituted HQs are good substrates.

With monosubstituted HQs, ring cleavage is observed;

however, these substrates rapidly inactivate PcpA, leading to

only limited amounts of ring-cleavage product. PcpA did not

detectably cleave substrates bearing an amino or carboxylate

group at the 4 position. Other compounds that were not

substrates for PcpA include 2,5-dichlorohydroquinone, unsub-

stituted hydroquinone, catechols, and gentisate. Therefore,

PcpA requires hydroxyl groups at the 1 and 4 positions and

substituents at the 2 and 6 positions of the aromatic ring for

ring cleavage to occur without significant enzyme inactivation.

Characterization of the Ring-Cleavage Products. Xun

and co-workers demonstrated by mass spectrometry that the

ring-cleavage product of 2,6-diClHQ by PcpA was 2-

chloromaleylacetate, which is the result of a 1,2-oxidative

ring-cleavage reaction, followed by a rapid nonenzymatic

hydrolysis of the acid chloride (species 2 in Scheme 2). We

have characterized the ring-cleavage products of all four ortho-

disubstituted HQs by ¹H NMR. The substrate 2-Cl-6-MeHQ is

Figure 1. Absorbance spectrum of the ring cleavage of 50 μM BrHQ

by 0.46 μM PcpA. The dashed line shows data for a control reaction

without enzyme. The inset shows the time course for the absorbance

change at 248 nm.

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Scheme 2. Ring-Cleavage Products of PcpA Substrates, Showing the Various Isomers Possible under Different Conditions

Table 2. ¹H NMR Spectra (400 MHz) of the Ring-Cleavage Products of the Four Ortho-Disubstituted Hydroquinone Substrates

a

in an 86% H₂O/10% D₂O/4% CD₃OD Mixture at pH 7

substrate

product

2-Me

3-H

6.56 (s)

5-H

7-H

2,6-dichlorohydroquinone (1)

3b (80%)

3.48 (s)

3.75 (s)

3.46 (s)

3.73 (s)

5.69 (s)

3.82 (s)

3.39 (s)

3.67 (s)

5.70 (s)

3.89 (s)

b

3b (20%)

7b (79%)

7b (21%)

6.56 (s)

2,6-dibromohydroquinone (5)

2,6-dimethylhydroquinone (9)

2-chloro-6-methylhydroquinone (12)

6.74 (s)

b

6.73 (s)

10a (37%)

10b (63%)

14b (74%)

14b (13%)

2.02 (d, J = 1.6)

2.01 (d, J = 1.6)

2.03 (d, J = 1.6)

5.63 (q, J = 1.6)

6.00 (q, J = 1.6)

6.10 (q, J = 1.6)

6.06 (q, J = 1.6)

2.12 (s)

2.26 (s)

b

c

16a (6.5%)

16b (6.5%)

6.19 (s)

2.16 (s)

2.27 (s)

c

6.51 (s)

a

b

The chemical shifts are reported vs TSP. Scalar couplings are reported in hertz. Note that in all of the 2-substituted maleylacetates, a second form

is present at ∼20% that is also consistent with a keto tautomer. This may represent a second isomer of the same compound that is in slow exchange.

For example, partial double-bond character in the C3−C4 bond of these conjugated systems could lead to the observation of separate signals for the

c

s-cis and s-trans forms, although we cannot rule out other options. Although the integration values do not differ, we have offered tentative

assignments based on the similarities of the chemical shifts to those of the enol and keto forms of 2-methylmaleylacetone (10a and 10b,

respectively).

of particular interest, because cleavage between the hydroxyl

group and the ortho substituents could result in two different

products, and the ratio of these products could provide insight

into the factors that determine substrate specificity.

corresponded to those of the kinetic experiments and previous

literature reports: in an 86% H₂O/10% D₂O/4% CD₃OD

mixture at pH 7 (Table 2) and after acidification to pH <2 and

extraction into CDCl₃(Table S1 of the Supporting

Information).

These ring-cleavage products can exist as different isomers

under different conditions (Scheme 2) as discussed previously

in the literature for maleylacetates^56,57and maleylace-

tones.^55,58−60At approximately neutral pH in water, unsub-

stituted maleylacetate exists entirely in the keto form⁵⁷and

unsubstituted maleylacetone exists as a mixture of the keto and

enol forms.^55,58Under acidic conditions (and subsequent

extraction into organic solvents), these compounds cyclize to

form lactones. To determine both the regiospecificity of ring

cleavage and to identify the isomeric forms, we characterized

these ring-cleavage products by ¹H NMR under conditions that

1

The H NMR spectra of the ring-cleavage products in water

at pH 7 show that the oxidative ring cleavage of 2,6-diClHQ

and 2,6-diBrHQ yields 2-chloromaleylacetate and 2-bromoma-

leylacetate, respectively, and that these species exist exclusively

as the keto form [(E)-2-chloro-4-oxohex-2-enedioate (3b) and

(E)-2-bromo-4-oxohex-2-enedioate (7b), respectively]. The

ring-cleavage product of 2,6-diMeHQ exists as a mixture of

37% of the enol form and 63% of the keto form of 2-methyl-

maleylacetone [(2Z,4E)-4-hydroxy-2-methyl-6-oxohepta-2,4-di-

enoate (10a) and (Z)-2-methyl-4,6-dioxohept-2-enoate (10b),

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respectively]. In both forms, the C2 methyl group and the

3-H proton are cis to each other on the basis of the coupl-

ing constant. However, we cannot determine the orientation of

the C4C5 bond of the enol form. The H NMR spectrum

with at least predominant 1,2-cleavage of the monosubstituted

HQs.

Kinetic Parameters of Hydroquinone Substrates.

Results of steady-state kinetic studies of the four ortho-

disubstituted HQs in air-saturated buffer (267−275 μM O₂)

are shown in Figure 2. The steady-state kinetic parameters are

listed in Table 3.

1

of the ring-cleavage product of 2-Cl-6-MeHQ shows a mixture

of four species. Two species are the two different keto isomers

of 2-methylmaleylacetate (similar to what was observed with

the 2-halomaleylacetates), resulting from cleavage of the

substrate between the hydroxyl and chloro substituents (1,2-

cleavage). The other two species are the keto and enol forms of

2-chloromaleylacetone, resulting from cleavage of the substrate

between the hydroxyl and methyl group (1,6-cleavage). Overall,

the integrations of the signals yield 74 and 13% of the two keto

isomers of 2-methylmaleylacetate [(Z)-2-methyl-4-oxohex-2-

enedioate (14b)] and 6.5% each for the enol and keto forms of

2-chloromaleylacetone [(2E,4E)-2-chloro-4-hydroxy-6-oxohep-

ta-2,4-dienoate (16a) and (E)-2-chloro-4,6-dioxohept-2-enoate

(16b), respectively]. Thus, the reaction of 2-Cl-6-MeHQ with

PcpA yields 87% 1,2-cleavage and 13% 1,6-cleavage.

The UV−visible absorption spectra of the cleavage products

can be interpreted in the context of the NMR data in pH 7

water. The cleavage products of 2,6-diClHQ and 2,6-diBrHQ

show a single transition at 253 and 259 nm, respectively, and

the NMR spectra show that these species exist exclusively as

the keto tautomer. The 2,6-diMeHQ cleavage product shows

two transitions at 250 and 314 nm, and the NMR spectrum

shows that it exists as a mixture of the keto (63%) and enol

(37%) forms. Thus, the transition at 250−260 nm corresponds

to the keto form, and the transition at 314 nm corresponds to

the enol form, as expected from empirical rules for the UV−

visible absorption spectra of organic molecules.⁶¹The UV−

visible absorption spectrum of the cleavage products of 2-Cl-6-

MeHQ shows transitions at 250 and 314 nm, with the latter

much less intense than the former. The NMR spectrum

showed that it is a mixture of 87% keto form 2-

methylmaleylacetate (14b) and 13% 2-chloromaleylacetone

in a ∼1:1 keto:enol ratio (16a and 16b). However, the

conditions used for NMR differed from those used for the

kinetic experiments (higher substrate concentration and much

longer reaction time), and consequently, the 314 nm to 250

nm ratio was lower (for reasons explained in the subsequent

section on kinetics). When we account for this, the ratio of

products observed under the conditions used for kinetics in

air-saturated buffer can be estimated to be 68% 2-

methylmaleylacetate and 32% chloromaleylacetone. Pure 2-

methylmaleylacetate in water has been reported to have a

single transition at 250 nm with a molar absorptivity of 9600

M⁻¹cm⁻¹.⁵⁴From this, we can estimate the molar absorptivity

of chloromaleylacetone to be 7800 and 5500 M⁻¹cm⁻¹at 314

and 250 nm, respectively. This is in reasonable agreement with

our results for methylmaleylactone, considering the higher

enol:keto ratio.

Figure 2. Steady-state kinetics of PcpA with ortho-disubstituted

hydroquinones: (●) 2,6-diClHQ, (■) 2,6-diBrHQ, (◆) 2,6-

diMeHQ, and (▲) 2-Cl-6-MeHQ. The dependence of the hydro-

quinone substrate concentration on the initial velocity was determined

in air-saturated (267−275 μM O₂) buffer, and the curves are fits of the

Michaelis−Menten equation to the data.

PcpA exhibited the following trend in apparent specificity

app

constant (k_c^a_a^p_t^p/K ) among the ortho-disubstituted HQs: 2,6-

mA

diBrHQ ∼ 2,6-diClHQ > 2-Cl-6-MeHQ ≫ 2,6-diMeHQ.

While PcpA had a similar apparent specificity for 2,6-diClHQ

app

cat

and 2,6-diBrHQ, both the K and k

values for the latter

mA

were lower. 2,6-diMeHQ was turned over more slowly than the

dihalo-HQs and also had a significantly higher Michaelis

app

mA

constant. Thus, relative to that of 2,6-diClHQ, the k_c^a_a^p_t^p/K

was reduced ∼40-fold. As described above, 2-Cl-6-MeHQ

yields a mixture of cleavage products: the transition at 250 nm

comes from the keto forms of both 2-methylmaleylacetate (the

major product, 14b) and 2-chloromaleylacetone (16b), while

the transition at 314 nm is from the enol form of 2-

chloromaleylacetone (16a). Analysis of the rate data at the two

app

mA

wavelengths yielded K

values within the error bars.

Moreover, the ratio of products was invariant with substrate

app

cat

concentration. Thus, k was evaluated by treating the mixture

as a single product with the effective molar absorptivity

reported in Table 1. Relative to those of 2,6-diClHQ, the

apparent specificity constant of 2-Cl-6-MeHQ was approx-

Because only very limited amounts of the cleavage products

of the monosubstituted HQs could be produced, they were

characterized by only UV−visible absorption spectroscopy. The

cleavage products of ClHQ and BrHQ yielded identical

absorption spectra with a λ_maxat 248 nm and a molar

absorptivity of ∼5100 M⁻¹cm⁻¹, in reasonable agreement with

what has been reported for unsubstituted maleylactate.⁵⁴The

two transitions at 240 and 310 nm in the cleavage product of

MeHQ are consistent with what has been reported for

unsubstituted maleylacetone: 4400 M⁻¹cm⁻¹at 243 nm and

9300 M⁻¹cm⁻¹at 312 nm.⁵⁵Thus, these data are consistent

app

cat

app

mA

imately half, k

was somewhat lower, and K was only

slightly greater. Thus, the behavior of 2-Cl-6-MeHQ in air-

saturated buffer much more closely matches that of the dihalo-

HQs than it does that of 2,6-diMeHQ.

Partition ratios were estimated at high substrate concen-

trations (300 μM for 2,6-diMeHQ and 200 μM for the other

ortho-disubstituted HQs) and low enzyme concentrations, such

that the enzyme was completely inactivated before all of the

substrate was consumed (Table 3). Unlike with the

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Table 3. PcpA Steady-State Kinetic Parameters Measured with Hydroquinone Substrates in Air-Saturated Buffer

(s⁻¹

)

k

/K (s⁻¹μM⁻¹

)

partition ratio

j

(s⁻¹

)

app

inact

app

mA

app

cat

app

cat

app

substrate

K

(μM)

k

mA

a

2,6-dichlorohydroquinone (1)

2,6-dibromohydroquinone (5)

2,6-dimethylhydroquinone (9)

2-chloro-6-methylhydroquinone (12)

chlorohydroquinone

3.2 ± 0.3

1.3 ± 0.2

55 ± 6

3.05 ± 0.05

1.61 ± 0.03

1.25 ± 0.05

1.0 ± 0.1

1.2 ± 0.1

0.023 ± 0.003

0.54 ± 0.08

∼700

∼400

∼500

∼800

∼40

∼0.004

∼0.003

a

b

4.4 ± 0.7

2.41 ± 0.09

c

d

10 ± 1

∼2

∼1

∼0.2

∼0.1

0.046 ± 0.008

0.027 ± 0.003

c

d

e

bromohydroquinone

8.5 ± 1.0

∼40

∼8

c

e

methylhydroquinone

41 ± 5

nd

a

b

app

Estimated from the partition ratio and k (eq 1). Treating the product mixture as a single species with an effective molar absorptivity at 250 nm

cat

c

of 8300 M⁻¹cm⁻¹. K was determined by measuring the competitive inhibition of 2,6-diClHQ by these substrates (K_I). For ClHQ and BrHQ, it

app

mA

was also determined by fits to the curvature in the time course of product formation (K_i^a_n^p_a^p_ct). The value was the same within the error bars for ClHQ

d

e

app

inact

and somewhat lower (4.6 ± 1.1 μM) for BrHQ. Estimated from the partition ratio and j

(eq 1). Not determined.

Figure 3. Steady-state kinetics of PcpA with ortho-disubstituted hydroquinones. (A) Dependence of the O₂concentration on the initial velocity at

high substrate concentrations (300 μM for 2,6-diMeHQ and 200 μM for the others): (●) 2,6-diClHQ, (■) 2,6-diBrHQ, and (◆) 2,6-diMeHQ.

The curves are fits of the Michaelis−Menten equation to the data. (B) O₂dependence of the steady-state cleavage of 2-Cl-6-MeHQ (200 μM) by

PcpA as monitored at 250 (●) and 314 nm (■). The curves are simultaneous fits of both data sets accounting for different kinetic parameters for

the two cleavage modes and for the molar absorptivities of the two products.

Table 4. Steady-State Kinetic Parameters for the O₂Dependence of PcpA for the Ortho-Disubstituted Hydroquinones

a

substrate

K_mO(μM)

k_cat(s⁻¹

)

k_cat/K_mO(s⁻¹μM⁻¹

)

2

2,6-dichlorohydroquinone (1)

190 ± 40

120 ± 20

70 ± 10

21 ± 3

4.8 ± 0.5

2.2 ± 0.1

1.4 ± 0.2

1.58 ± 0.06

1.8 ± 0.2

0.026 ± 0.006

0.019 ± 0.003

0.020 ± 0.005

0.07 ± 0.01

2,6-dibromohydroquinone (5)

2,6-dimethylhydroquinone (9)

2-chloro-6-methylhydroquinone (12) 1,2-cleavage

2-chloro-6-methylhydroquinone (12), 1,6-cleavage

260 ± 60

0.007 ± 0.002

a

app

Because these data were obtained at a saturating concentration of the organic substrate (>40-fold above K ) for 2,6-diClHQ and 2,6-diBrHQ, the

mA

app

direct fit of the O₂dependence data with the Michaelis−Menten equation yields the true value of k_cat, and the K should be approximately equal to

the true K_mO(a 10-fold difference between K_dAand K_mAwould give a difference between K

obtained from the O₂dependence data was corrected for the nonsaturating substrate concentration, and K

those for the other substrates. For 2-Cl-6-MeHQ, these values are K

mO₂

app

mO₂

app

cat

and K_mOof <15%). For 2,6-diMeHQ, the k

2

app

and K_mOmay differ by more than

mO₂

2

app

mO₂

app

and k , rather than K_mOand k_cat.

cat

2

app

monosubstituted HQs, complete inactivation of PcpA by the

disubstituted HQs occurred slowly (∼10 min for the dihalo-

HQs and >1 h for 2-Cl-6-MeHQ and 2,6-diMeHQ). Given the

length of time required for inactivation, these values should be

viewed with some caution and may be lower limits on the actual

partition ratios. In particular, the dihalo-HQs air-oxidize to the

corresponding benzoquinones on this time scale, and the

presence of minute quantities of the benzoquinone was itself

found to reduce the rate of ring cleavage. The partition ratios

for the disubstituted HQs were all fairly similar. From the

partition ratios and the k

values, the rates of enzyme

cat

inactivation (j_i^a_n^p_a^p_ct) with these substrates were estimated. These

values were very similar for the four disubstituted HQs.

Because the monosubstituted HQs rapidly inactivate PcpA,

initial rate data could not be obtained easily. Therefore, 2,6-

diClHQ was used as a reporter substrate, treating the

monosubstituted HQs as competitive inhibitors. The resulting

K_Ifrom fits to these inhibition data yielded the K for these

substrates (Table 3). Also, the curvature in the time course of pro-

duct formation was fit, and the resulting exponential terms (j_s)

app

mA

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were plotted as a function of monosubstituted HQ

concentration at different 2,6-diClHQ concentrations and

fit with a competitive inhibition-type equation (eq 3), in

which 2,6-diClHQ inhibits the enzyme inactivation caused

by one of these compounds. The resulting apparent rate

constants of inactivation, j_i^a_n^p_a^p_ct, are listed in Table 3.

Qualitatively, MeHQ showed less curvature, thus precluding

mechanism in Scheme 3 (see the Supporting Information for

the derivations and for the definition of these coefficients). At a

Scheme 3. Mechanism by Which 2-Cl-6-MeHQ Binds to

PcpA in Two Different Orientations Yielding Two Different

Products

an accurate fit to the j_sdata. Partition constants were

app

estimated, and from the partition constants and j

values,

inact

app

k

cat

and in turn k /K

were estimated for ClHQ and

values were only somewhat lower than

cat

mA

app

BrHQ. The k

cat

those for the dihalo-HQs, and the specificity constants were

between those of the dihalo-HQs and 2,6-diMeQ. The most

notable difference between the dihalo-HQs and ClHQ and

fixed [O₂], because the denominator terms of V and V′ are

identical and the numerator is first-order in [A], then the ratio

of product formation as function of [A] is constant (in

agreement with experiment) and the initial rates should follow

Michaelis−Menten kinetics with the following equations for

BrHQ is that for the latter substrates the partition ratio was

app

inact

∼10-fold lower and, correspondingly, j

higher.

was ∼10-fold

Thus, overall, it appears that there is an interaction between

app

the ortho substituents of the substrate and the active site that

k

and K :

mA

cat

app

mA

app

cat

significantly affects k_c^a_a^p_t^p/K

and modestly affects k

,

indicating that this interaction leads to a much greater

specificity for HQs with halogen substituents versus methyl

substituents. Furthermore, the removal of one ortho substituent

greatly increases the rate of inactivation.

Kinetic Parameters of O₂. The O₂dependence on the

initial velocities for 2,6-diClHQ, 2,6-diBrHQ, and 2,6-diMeHQ

at high substrate concentrations is shown in Figure 3A, and the

(6)

(7)

(8)

resulting kinetic parameters are listed in Table 4. The k_cat/K_mO

2

values were remarkably similar for these three symmetric

substrates, and both the K_mOand k_catvalues for these substrates

shared the same trend: 2,²6-diClHQ > 2,6-diBrHQ > 2,6-

diMeHQ.

At a fixed [A], the initial rates do not follow true Michaelis−

Menten kinetics. However, if the terms in eqs 4 and 5 that are

second-order in [O₂] are small, the O₂dependence will still

show saturation behavior that can be fit to a Michaelis−Menten

In the presence of saturating amounts of 2-Cl-6-MeHQ, the

ratio of cleavage products was strongly dependent on O₂

concentration. Thus, the initial rates of the increase in

absorbance at 250 and 314 nm displayed different depend-

encies on O₂concentration (Figure 3B). Several mechanisms

were considered in which a single organic substrate could bind

to the enzyme to yield two different products. A mechanism

consistent with the observation that the product ratio is

invariant with respect to substrate concentration but varies with

O₂concentration is shown in Scheme 3. This yields the

following equations for the initial rates of formation for the two

products

app

type of equation; however, the resulting k and K

values

cat

mO₂

cannot be related back to the N_xand D_xterms. The ratio of the

initial rates of formation of the two products as a function of

[O₂] is

(9)

consistent with the experimentally observed nonlinear O₂

dependence on this ratio. Importantly, a mechanism in which

the asymmetric substrate is in rapid equilibrium between the

two binding modes without dissociating from the enzyme does

not yield the observed dependence of the O₂concentration on

the product ratio, nor does a mechanism in which the substrate

binds in a single orientation and then partitions between two

different ring-cleavage pathways. However, there may be other

mechanistic possibilities consistent with this observation, such

as one binding mode leading to partial enzyme inactivation.

This behavior in which a single substrate yields two different

(4)

app

mA

app

mO₂

products with the same K values but different K

values

has been observed before in a ring-cleaving dioxygenase. It was

first reported in Pseudomonas arvilla catechol 1,2-dioxygenase

(an IDO) with the substrate 3-methylcatechol, which led to a

mixture of 1,2- and 2,3-cleavage products, although a detailed

analysis of the kinetics was not performed.⁶²

The transition at 314 nm arises from just the 1,6-cleavage

product, while the transition at 250 nm arises from both

products. Therefore, the initial rates of increase at both

wavelengths were simultaneously fit with two Michaelis−Menten

(5)

where [A], [O₂], and [E_t] are the concentrations of the HQ

substrate, O₂, and enzyme, respectively, and the N_xand D_x

coefficients are functions of the rate constants from the

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equations (Figure 3, solid lines) with different k and K

Article

app

cat

app

mO₂

compounds bind at the active site in a manner similar to that of

the substrates and with a relatively high affinity, even though

they cannot be cleaved. The trend in inhibitor strength as a

function of ortho substituent mirrored the trend seen in the

values and with the reported molar absorptivity at 250 nm of

the 1,2-cleavage product (2-methylmaleylacetate)⁵⁴and the

estimated molar absorptivities of the 1,6-cleavage product

(chloromaleylacetone) at both 250 and 314 nm calculated

app

mA

app

K

and k /K values of the substrates. Bromo-substituted

cat mA

compounds were stronger inhibitors than the corresponding

chloro-substituted compounds, which in turn were much

stronger inhibitors than the methyl-substituted compounds.

Hydroquinone, which lacks ortho substituents, did not inhibit to

a detectable extent.

app

above. This yielded the k and K

values for 1,2- and 1,6-

cat

mO₂

app

cleavage listed in Table 3. Notably, the k values are similar,

cat

app

while the K

values differ ∼10-fold. Although there is

mO₂

considerable uncertainty in the molar absorptivity of chlor-

omaleylacetone at 250 nm, even a 50% error in this value

The capacity of the inhibitors 2,6-dichlorophenol, 2,6-

dibromophenol, and 3-bromocatechol to inactivate PcpA was

explored. PcpA was incubated for 15 s in air-saturated buffer

either with or without one of the compounds in question, and

then 2,6-diClHQ was added to a final concentration of 200 μM.

The concentration of the potential inactivator was kept

sufficiently low so that no diminution of the initial velocity

could be observed due to competitive inhibition. The initial

velocity was measured and compared to that obtained by the

usual method, in which PcpA is added last to air-saturated

buffer containing substrate and inhibitor. The monosubstituted

HQs (known inactivators of PcpA) were tested as well for

comparison. With 2,6-dibromophenol and 2,6-dichlorophenol,

no significant difference was observed in the relative initial

velocity (93−100%) compared to that for incubation with just

air-saturated buffer (93 ± 6%). With the monosubstituted HQs

and with 3-bromocatechol, a large amount of enzyme

inactivation was observed after just 15 s [relative initial velocities

of 26−47% (see Table S2 of the Supporting Information for

details)]. Control experiments in which PcpA was incubated

with these compounds under anaerobic conditions for several

minutes prior to being mixed with air-saturated buffer and

substrate showed no inactivation, indicating that the inactivation

is O₂-dependent. This suggests that inactivation of PcpA, either

by the monosubstituted HQs or by 3-bromocatechol, may arise

from oxidation of the Fe(II) center.

The potential of the three strongest inhibitors to inactivate

PcpA was also examined by measuring the curvature in the time

course of product formation (with 2,6-diClHQ as the

substrate) in the presence of each of the inhibitors, which

was reported above for the monosubstituted HQs. With 2,6-

dichloro- and 2,6-dibromophenol, the small increase in the

curvature with an increasing amount of inhibitor could be

entirely ascribed to substrate depletion, based on simulated data

from the integrated rate equation⁶³(see Figure S1 of the

Supporting Information for a comparison of ClHQ with 2,6-

dibromophenol). In contrast, 3-bromocatechol showed a much

larger degree of curvature, which was also seen with the

app

changes the K

values by <40% and preserves the ∼10-fold

mO₂

app

difference in K between the two binding modes. These K

mO₂

values cannot be directly compared to those obtained for the

symmetric substrates, because the value for the asymmetric

substrate has a complex dependence on the rate constants of

both pathways resulting from the two different binding modes.

Nonetheless, it appears that the substituent external to the site

of ring cleavage affects the O₂dependence of the kinetics. The

product resulting from cleavage between the chloro and

app

mO₂

hydroxyl groups has the lower K

(the methyl group is

external), and likewise, among the symmetric substrates, 2,6-

app

mO₂

diMeHQ has the lowest K

.

Identification and Kinetic Characterization of Inhib-

itors. The same molecules that were screened as possible

substrates for PcpA were also tested as inhibitors. For those

that were found to inhibit PcpA, the initial rate with 50 μM 2,6-

diClHQ and 500 μM inhibitor is reported as a percentage

relative to the rate without inhibitor in Table 5. The uncertainty

Table 5. Inhibitors of PcpA

a

inhibitor

relative initial rate (±4%)

K_I(μM)

13 ± 2

53 ± 7

2,6-dibromophenol

3-bromocatechol

30%

57%

b

3800 ± 800

48 ± 6

nd

2,6-dichlorophenol

66%

67%

c

4-amino-2,6-

dichlorophenol

c

pyrogallol

70%

83%

88%

nd

c

2-bromo-6-methylphenol

2,6-dimethylphenol

2-chloro-6-methylphenol

nd

c

nd

c

90%

nd

a

b

With 50 μM 2,6-diClHQ and 500 μM inhibitor. This compound is a

mixed-mode inhibitor. The first number gives the K_Ifor the

competitive inhibition component and the second that for the

c

uncompetitive inhibition component. Not determined.

monosubstituted HQs. From fits to the curvature data, the

was estimated to be approximately ±4%. Under these

conditions, gentisate, catechol, 3-methylcatechol, 4-methylca-

techol, and unsubstituted hydroquinone showed no evidence of

inhibiting PcpA. Even 5.0 mM unsubstituted hydroquinone did

not significantly inhibit 2,6-diClHQ cleavage (i.e., <3%). Thus,

although removing just one of the four substituents on the ring

results in a loss of PcpA’s ability to cleave it (without significant

inactivation), most of these compounds are still able to act as

inhibitors.

The three strongest inhibitors (2,6-dibromophenol, 2,6-

dichlorophenol, and 3-bromocatechol) were examined in more

detail. 2,6-Dibromo- and 2,6-dichlorophenol were competitive

inhibitors of PcpA versus 2,6-diClHQ, while 3-bromocatechol

was a mixed-mode inhibitor. This confirms that these three

app

K_i^a_n^p_a^p_ctand j

values of 3-bromocatechol were 40 ± 20 μM and

inact

0.04 ± 0.01 s⁻¹, respectively. This is consistent with a model in

which 3-bromocatechol competes with the substrate for the

active site, and some fraction of inhibitor binding events leads

to the irreversible inactivation of the enzyme.

DISCUSSION

■

Structural Requirements for Substrate and Inhibitor

Binding. The structural requirements for substituted arenes to

bind to the active site of PcpA are quite remarkable, and even

more striking are the stringent requirements for aromatic ring

cleavage (summarized in Figure 4). We will first address the

role of the para and ortho substituents and then discuss the

issue of catechol binding to PcpA.

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coordinated to a facial-capping tridentate ligand and o-chloro-

and bromophenolates designed as simple structural models of

PcpA, we observed metal−halogen distances that met this

criterion for secondary bonding.⁷³The energetics of metal−

halogen secondary bonding have not been explored. However,

it is thought that the strength of this interaction also increases

with the polarizability of the halogen.⁷⁰

In the EDOs, catechols are known to bind as monoanions

that coordinate the iron(II) as bidentate ligands. One might

expect that catechol derives a significant amount of its free

energy of binding from ligation of the o-hydroxyl group to the

iron(II) center, and that this interaction would be stronger than

the sort of noncanonical halogen interactions discussed above.

Thus, a bidentate ligand such as a catechol would have a higher

binding strength than monodentate ligands such as

o-dihalophenols. However, the opposite trend is observed in

PcpA: of the catechols tested, only 3-bromocatechol and

pyrogallol showed any evidence of inhibition of PcpA. 2,6-

Dibromophenol was a stronger inhibitor than 3-bromocatechol,

and even 2,6-dimethylphenol was a weak inhibitor; 3-

methylcatechol did not detectibly inhibit PcpA. In comparison,

while 2-halophenols are inhibitors of the well-studied EDO

P. putida mt-2 catechol 2,3-dioxygenase (XylE), the K_Ivalues of

these inhibitors are more than 10-fold higher than those of

inhibitors expected to be bidentate ligands, such as 2-

aminophenol and 2-hydroxypyridine N-oxide.⁷⁴⁻⁷⁶It could be

that ligation of the o-hydroxyl group to the iron(II) center

contributes less to the free energy of binding than specific

noncovalent interactions between the rest of the substrate or

inhibitor and the active site in a given enzyme (hydrogen

bonding to the o-hydroxyl group in EDOs vs halogen bonding

to the ortho substituents in the case of PcpA). Alternatively, the

shape of the substrate binding pocket in PcpA may force the

ortho substituents to be too distant from the iron(II) for an o-

hydroxyl group to coordinate, leaving noncovalent interactions

between the protein and the ortho substituents as the only

option.

Figure 4. Structures of the substrates and inhibitors of PcpA, ranked in

app

order of k_ca^a_t^pp/K (for substrates) and K_Ior inhibition strength at

mA

fixed concentrations (for inhibitors). Among the inhibitors, 2,6-

dibromo- and 2,6-dichlorophenol do not inactivate the enzyme but 3-

bromocatechol does, while the others have not been tested for

inactivation.

The p-hydroxyl group plays a key mechanistic role, because it

is an absolute requirement for ring cleavage. It also appears to

contribute to the binding strength, which in turn suggests

possible hydrogen bonding to this substituent from the protein.

Hydrogen bonding to the substrate p-hydroxyl group has been

implicated in gentisate²⁰and homogentisate dioxygenases.⁶⁴

However, a p-amino group does not have the same effect on

binding, as shown by the comparable amounts of inhibition

between 4-amino-2,6-dichlorophenol and 2,6-dichlorophenol,

which was also observed with the inhibition of gentisate

dioxygenase.¹⁶

Insights into the Mechanism of Ring Cleavage in

PcpA. A generally accepted mechanism exists for EDOs, based

upon experiments^6,7,10−12and DFT calculations.^13,14Substrate

binds to the Fe(II) as a monoanion, with the hydroxyl group at

the 2 position deprotonated. Next, O₂binds, which induces

deprotonation of the remaining substrate hydroxyl proton. The

ternary complex is best described as an Fe(II)−superoxide−

semiquinone ternary complex. The superoxide attacks at the 2

position, resulting in an alkylperoxide intermediate. A Criegee

rearrangement of this alkylperoxidespecifically, an alkenyl

migrationleads to a lactone, which is finally cleaved by

nucleophilic attack of the iron(II)-bound hydroxide. This

rearrangement may occur in multiple steps via epoxide and/or

gem-diol intermediates.^12,14It is believed that the structure of

the alkylperoxide intermediate and its rearrangement via an

alkenyl migration are the key features that lead to extradiol

catechol ring cleavage, whereas an acyl migration leads to

intradiol catechol ring cleavage in the Fe(III)-dependent

IDOs.⁶

The ortho substituents play a key role in the binding of both

substrates and inhibitors. Notably, unsubstituted hydroquinone

did not inhibit the enzyme to a measurable extent up to a

concentration of 5 mM (100-fold higher than the substrate

app

concentration). The k_c^a_a^p_t^p/K values of 2,6-diClHQ and 2,6-

mA

diBrHQ are ∼40-fold higher than that of 2,6-diMeHQ.

Likewise, the 2,6-dihalophenols are significantly stronger

inhibitors than 2,6-dimethylphenol. The importance of ortho

substituents for binding and the apparent trend in binding (Br >

Cl ≫ CH₃) among both substrates and inhibitors suggest a

specific interaction between ortho halo substituents and the

protein. Because methyl and chloro groups are similar in size, it

is not likely that this preference is solely due to steric

requirements. This trend could result from either halogen

bonding⁶⁵⁻⁶⁹or metal−halogen secondary bonding.⁷⁰In

halogen bonding, the halogen in a C−X bond interacts with

the partial negative charge on an oxygen or nitrogen

atom^65,67,69or with the π electrons from an aromatic

ring.^66,68Interestingly, the strength of the halogen bond

increases with halogen polarizability, which follows the same

trend observed in the binding of substrates and inhibitors in

PcpA. In o-chlorophenolate transition metal complexes, metal−

halogen secondary bonding has been observed at a metal−

chlorine distance of up to ∼1.0 Å beyond the normal metal−

chloride bond distance.^71,72In complexes of iron(II)

To date, essentially nothing is known about the mechanism

of ring cleavage in PcpA or any of the other HQDOs. Although

there must be important differences between the mechanism of

PcpA and that of the EDOs, given that both use an Fe(II)

ligated by a common facial-capping triad ligand set, it is likely

that there are also many similarities. Thus, the EDO mechanism

can be used to inform discussion of a mechanism for PcpA. As a

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a

Scheme 4. Plausible Mechanism of PcpA

a

Metal−ligand bonds are shown as dashed lines, and charges are shown explicitly. A dashed line is shown between one of the o-chloro substituents

and the Fe(II), to suggest that one of the substituents could be close enough to form a metal−halogen secondary bond; however, the mechanism in

no way requires this to be the case. Two possible alkenyl migrations are shown, distal and proximal. Only one of these is operative; however, the

available data cannot be used to determine which is operative.

result of this work, there are three key observations that must

be explained by any postulated mechanism for PcpA. First, 2,6-

dihalophenols are not substrates but are able to bind to the

active site as competitive inhibitors. Second, the heterosub-

stituted HQ, 2-Cl-6-MeHQ, binds in two different orientations

leading to distinct products. Third, monosubstituted HQs are

cleaved to an only limited extent by the enzyme but are potent

inactivators.

the hydroxyl group and either the ortho substituent proximal to

the Fe(II) center (akin to an intradiol-type cleavage) or the

substituent distal to the Fe(II) center (akin to an extradiol-type

cleavage). However, with only one ligating hydroxyl group, the

choice is not between an acyl or alkenyl migration, but instead

between two different types of alkenyl migrations (proximal

and distal). In the case of the symmetric substrates such as 2,6-

diClHQ, because both alkenyl migrations would yield identical

products, we cannot distinguish between these two options

with the available data.

With 2-Cl-6-MeHQ, there are two potential explanations for

how a mixture of products could arise. First, the substrate could

bind to the active site in two orientations (“chloro-in” and

“chloro-out”) with only one type of alkenyl migration of the

peroxide intermediate occurring; second, there could be only

one binding mode for the substrate, and the two products form

by partitioning between proximal and distal alkenyl migration.

The requirement for a p-hydroxyl group for ring cleavage can

be readily explained by postulating that the first steps of the

mechanism of PcpA (Scheme 4) are essentially the same as

those of EDO. In the first step, substrate binds to the Fe(II)

and is deprotonated to yield a monoanion. When O₂binds, the

p-hydroxyl group is deprotonated by an active site base,

yielding an Fe(II)−superoxide−semiquinone ternary complex.

The p-hydroxyl group is critical for stabilizing this ternary

complex, promoting binding of the O₂and its reduction to

superoxide and yielding an electronic structure that promotes

attack by O₂on the aromatic ring. The one-electron oxidation

of a phenolate would be far less favorable. Indeed, either an

o/p-hydroxyl or an o-amino substituent that can conjugate to

the ring is required for all ring-cleaving dioxygenases, except

salicylate 1,2-dioxygenase (and even in that case, the rate of ring

cleavage increases dramatically when a p-hydroxyl or amino

group is present).^17,18From this Fe(II)−superoxide−semi-

quinone ternary complex, attack by oxygen on the aromatic ring

could occur at either the C1 or C2 position. We favor the

former option. Formation of an alkylperoxide at C2 would

necessitate that the rearrangement to the lactone proceed via an

acyl migration, which is believed to be less favorable. Formation

of an alkylperoxide at C1 would allow rearrangement to occur

via an alkenyl migration, consistent with the EDO mechanism.

Bugg and co-workers have emphasized the importance of an

alkenyl migration from the alkylperoxide intermediate to yield

the observed extradiol cleavage in the EDOs, and that this is

greatly favored over the alternative (an acyl migration to yield

intradiol cleavage) because of both the orientation of the

alkylperoxide and the presence of a proton donor in the second

coordination sphere.⁶In PcpA, cleavage could occur between

The second option is ruled out by the kinetic data, which show

app

mO₂

that the two ring-cleavage modes have different K

values. As

discussed above, a mechanism in which there is a single binding

mode and a partitioning between products after that step does

not yield such behavior. Neither the favored binding orientation

nor the type of alkenyl migration is known; however, it is

known that cleavage between the chloro and hydroxyl groups is

preferred. Thus, either the substrate preferentially binds in the

chloro-in conformation and the enzyme promotes a proximal

alkenyl migration, or the substrate preferentially binds in the

chloro-out conformation and the enzyme promotes a distal

alkenyl migration. The minor product resulting from 1,6-

cleavage would then arise from the other binding mode but the

same type of alkenyl migration. Spectroscopic or structural

studies that define the preferred binding orientation for an

asymmetric substrate or inhibitor would thus define whether

the mechanism of PcpA proceeds via a proximal or distal

alkenyl migration.

Insights into the Mechanism of Inactivation in PcpA.

In PcpA, the monosubstituted HQs were substrates that

strongly inactivate the enzyme, while for the ortho-disubstituted

HQs, inactivation was at least 10-fold slower. Experiments in

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Scheme 5. Plausible Mechanism for How a Monosubstituted Hydroquinone (ClHQ) Leads to Rapid Enzyme Inactivation with

an Only Small Amount of Ring Cleavage

which PcpA was incubated with monosubstituted HQs in air-

saturated versus O₂-free buffer indicate that inactivation is O₂-

dependent, suggesting that oxidation of the Fe(II) center may

be the source of the inactivation, as is known to be the case in

the EDOs. Binding of O₂to the monosubstituted HQ-bound

Fe(II) center leads some ring cleavage and a significant amount

of inactivation, whereas binding of O₂to the disubsituted HQ-

bound Fe(II) center leads to ring cleavage most of the time and

inactivation only a small percentage of the time. Like 2-Cl-6-

MeHQ, a monosubstituted HQ (such as ClHQ) can also bind

in two different orientations: a chloro-in conformation in which

the o-chloro substituent is near the Fe(II) center (and may

weakly interact) and a chloro-out conformation in which the

substituent is pointed away from the Fe(II) and interacts with

some other part of the substrate binding pocket. One of the

binding modes could lead to productive ring cleavage, while the

other leads to efficient enzyme inactivation (Scheme 5). Which

binding mode leads to inactivation cannot be determined with

the available data, nor it is known what factors might cause

ClHQ bound in a certain conformation to yield rapid enzyme

inactivation.

The inhibitor 3-bromocatechol also inactivated the enzyme

in an O₂-dependent fashion but did not lead to any detect-

able ring cleavage. The fact that 3-bromocatechol inactivates

the enzyme but the o-dihalophenols do not highlights the

importance of an o/p-hydroxyl group, which appears to be

required for either ring cleavage or inactivation. Although

inactivation with substrates has been reported in many EDOs,

we are not aware of any studies of inactivation of EDOs with

inhibitors.

trend in K_Iamong the inhibitors and the trend in the substrate

specificity show a clear preference for halogen substituents at the

ortho position over methyl substituents, with Br binding more

tightly than Cl. A reason why catechols are not substrates for

PcpA, despite the apparent similarity of the active site to that of

the EDOs, can be partially established: most catechols bind (at

best) weakly to the Fe(II) center, and those that bind reasonably

well (3-bromocatechol) lead to rapid enzyme inactivation. These

results, combined with the precedent of what is already known

about the EDOs, allow several plausible features of the mechanism

for PcpA to be proposed: (a) binding of the substrate as a

monoanion and formation of a Fe(II)−superoxide−semiquinone

ternary complex, (b) attack by O₂at C1 to form an alkylperoxide

intermediate, and (c) akenyl migration to form a lactone followed

by its subsequent hydrolysis. The preferred mode of binding of

asymmetric substrates cannot be determined with the available

data. However, the preference for cleavage between the chloro and

hydroxyl groups in the substrate 2-Cl-6-MeHQ can be combined

with a future determination of substrate binding orientation to

define whether a proximal or distal alkenyl migration is occurring.

Future spectroscopic and structural studies will allow the

determination of the basis for the unique specificity of PcpA.

ASSOCIATED CONTENT

■

S

* Supporting Information

Derivation of the initial rate equations (eqs 4 and 5) for an

asymmetric substrate binding to a single enzyme active site in

two different orientations, EA and EA′, followed by a second

substrate (O₂) yielding two different products, P and Q; a table

1

of the H NMR data of the cyclized form of the ring-cleavage

products; a table of the relative initial rates of product

formation upon incubation in air-saturated buffer with various

compounds; and a figure showing the change in absorbance

from product formation over time with substrate (2,6-diClHQ)

and with varying concentrations of ClHQ or 2,6-dibromophe-

nol. This material is available free of charge via the Internet at

http://pubs.acs.org.

CONCLUSION

■

Steady-state kinetic studies have demonstrated that PcpA has a

high degree of substrate specificity: only 2,6-disubstituted HQs

are efficiently oxidatively cleaved, while monosubstituted HQs

show an only limited degree of ring cleavage but are potent

inactivators of the enzyme. Likewise, 3-bromocatechol can bind

and inactivate the enzyme but is not cleaved. This enzyme

inactivation is O₂-dependent, suggesting that it may arise from

irreversible oxidation of the Fe(II) center to an inactive Fe(III)

state, as in known for the EDOs. A variety of 2,6-disubstituted

phenols are good competitive inhibitors of PcpA. Both the

AUTHOR INFORMATION

■

Corresponding Author

*Phone: (509) 527-5799. Fax: (509) 527-5904. E-mail:

machonte@whitman.edu.

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(12) Kovaleva, E. G., and Lipscomb, J. D. (2008) Intermediate in the

O-O Bond Cleavage Reaction of an Extradiol Dioxygenase.

Biochemistry 47, 11168−11170.

(13) Deeth, R. J., and Bugg, T. D. H. (2003) A density functional

investigation of the extradiol cleavage mechanism in non-heme iron

catechol dioxygenase. J. Biol. Inorg. Chem. 8, 409−418.

(14) Siegbahn, P. E. M., and Haeffner, F. (2004) Mechanism for

Catechol Ring-Cleavage by Non-Heme Iron Extradiol Dioxygenase.

J. Am. Chem. Soc. 126, 8919−8932.

Funding

This research was supported by grants from the American

Chemical Society Petroleum Research Fund (ACS-PRF 44410-

G3), the M. J. Murdock Charitable Trust, and the National

Science Foundation (CHE-0951999), as well as funds from

Whitman College. The purchase of the 400 MHz Bruker

Avance III NMR spectrometer was supported by a grant from

the National Science Foundation (MRI-0922775).

(15) Harpel, M. R., and Lipscomb, J. D. (1990) Gentisate 1,2-

Dioxygenase from Pseudomonas. Purification, Characterization, and

Comparison of the Enzymes from Pseudomonas testosteroni and

Pseudomonas acidovorans. J. Biol. Chem. 265, 6301−6311.

(16) Harpel, M. R., and Lipscomb, J. D. (1990) Gentisate 1,2-

Dioxygenase from Pseudomonas. Substrate Coordination to Active-Site

Fe²⁺and Mechanism of Turnover. J. Biol. Chem. 265, 22187−22196.

(17) Hintner, J.-P., Lechner, C., Rigert, U., Kuhm, A. E., Storm, T.,

Reemtsma, T., and Stolz, A. (2001) Direct Ring Fission of Salicylate by

a Salicylate 1,2-Dioxygenase Activity from Pseudaminobacter salicyla-

toxidans. J. Bacteriol. 183, 6936−6942.

ACKNOWLEDGMENTS

■

We thank Prof. Marion Gotz (Whitman College) for help on

̈

the synthesis, purification, and characterization of some of the

substrates and reaction products and Prof. Jennifer Love

(University of British Columbia, Vancouver, BC) and her

students for use of their space and equipment for some of the

syntheses. We thank Anees Daud (Whitman College) for

synthesis of 2,6-dibromobenzoquinone. We thank Prof. Lindsay

Eltis (University of British Columbia) for use of the O₂-

sensitive electrode and other equipment, for advice on enzyme

inactivation and derivation of rate equations, for other insightful

discussions, and for a critical reading of the manuscript. We

thank John Zimmerman (Whitman College) for preparation of

some of the enzyme.

(18) Hintner, J.-P., Remtsma, T., and Stolz, A. (2004) Biochemical

and molecular characterization of a ring fission dioxygenase with the

ability to oxidize (substituted) salicylate(s) from Pseudaminobacter

salicylatoxidans. J. Biol. Chem. 279, 37250−37260.

(19) Adams, M. A., Singh, V. K., Keller, B. O., and Jia, Z. C. (2006)

Structural and biochemical characterization of gentisate 1,2-dioxyge-

nase from Escherichia coli O157: H7. Mol. Microbiol. 61, 1469−1484.

(20) Chen, J., Li, W., Wang, M. Z., Zhu, G. Y., Liu, D. Q., Sun, F.,

Hao, N., Li, X. M., Rao, Z. H., and Zhang, X. C. (2008) Crystal

structure and mutagenic analysis of GDOsp, a gentisate 1,2-

dioxygenase from Silicibacter pomeroyi. Protein Sci. 17, 1362−1373.

(21) Matera, I., Ferraroni, M., Burger, S., Scozzafava, A., Stolz, A.,

and Briganti, F. (2008) Salicylate 1,2-dioxygenase from Pseudamino-

bacter salicylatoxidans: Crystal structure of a peculiar ring-cleaving

dioxygenase. J. Mol. Biol. 380, 856−868.

ABBREVIATIONS

■

BrHQ, bromohydroquinone; ClHQ, chlorohydroquinone; 2-

Cl-6-MeHQ, 2-chloro-6-methylhydroquinone; DFT, density

functional theory; 2,6-diBrHQ, 2,6-dibromohydroquinone;

2,6-diClHQ, 2,6-dichlorohydroquinone; 2,6-diMeHQ, 2,6-

dimethylhydroquinone; DSS, sodium 3-(trimethylsilyl)-1-pro-

panesulfonate; EDO, extradiol dioxygenase; HOHQ, hydrox-

yhydroquinone; HQDO, hydroquinone dioxygenase; HQs,

hydroquinones; IDO, intradiol dioxygenase; MeHQ, methyl-

hydroquinone; VOC, vicinal oxygenase chelate.

́

(22) Titus, G. P., Mueller, H. A., Burgner, J., Rodriguez-de-Cor

̃

́

ba, S.,

Penalva, M. A., and Timm, D. E. (2000) Crystal structure of human

homogentisate dioxygenase. Nat. Struct. Biol. 7, 542−546.

(23) Veldhuizen, E. J. A., Vaillancourt, F. H., Whiting, C. J., Hsiao,

M. M. Y., Gingras, G., Xiao, Y. F., Tanguay, R. M., Boukouvalas, J., and

Eltis, L. D. (2005) Steady-state kinetics and inhibition of anaerobically

purified human homogentisate 1,2-dioxygenase. Biochem. J. 386, 305−

314.

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Substrate specificity of Sphingobium chlorophenolicum 2,6- dichlorohydroquinone 1,2-dioxygenase

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