APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1984, p. 500-505 Vol. 47, No. 30099-2240/84/030500-06$02.00/0Copyright © 1984, American Society for Microbiology

Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonasputida mt-2 by 3-Halocatechols

IRIS BARTELS,lt HANS-JOACHIM KNACKMUSS,2* AND WALTER REINEKE1t

Gesellschaft fur Strahlen- und Umweltforschung mbH, Munich,1 and Lehrstuhlfiir Chemische Mikrobiologie derUniversitat-Gesamthochschule, D-5600 Wuppertal 1,2 Federal Republic of Germany

Received 23 May 1983/Accepted 1 December 1983

The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactiva-tion by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatecholswas only observed in the presence of oxygen and was largely irreversible. The rate constants forinactivation (K2) were 1.62 x 10-3 sec-1 for 3-chlorocatechol and 2.38 x 10-3 sec-1 for 3-fluorocatechol.The inhibitor constants (Ki) were 23 ,uM for 3-chlorocatechol and 17 ,uM for 3-fluorocatechol. The kineticdata for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivatedenzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chlorofor-myl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in thepresence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion,hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.

Metabolism of an aromatic compound in which a halogenhas been substituted for hydrogen may be fatal to a microbi-al cell. Thus, a halogenated aromatic compound, in itself nottoxic, may proceed along a normal metabolic pathway untilit is converted to a molecule which acts as a specific enzymeinhibitor. Such metabolic transformations have been termed"lethal synthesis" (42). Lethality could result from competi-tive inhibition of an essential enzyme, e.g., that of aconitaseby fluorocitrate (43), from removal of essential metal cofac-tors from catalytic sites of enzymes by a chelating agent, or

from an irreversible type of inhibition. Two types of mecha-nisms of specific irreversible enzyme inhibition can bedistinguished. The first type is the classical affinity labelingagents (52), which are substrate analogs containing chemi-cally reactive functional groups. In the second type, thesubstrate itself is chemically unreactive, but the product ofits enzymatic conversion is a highly reactive molecule.Inhibitors of the latter type have been termed K( a, inhibitorsbecause they require catalytic conversion by the enzyme

(45).Halocatechols are key metabolites formed during degrada-

tion of haloaromatics (5, 9, 11, 12, 15, 20, 27, 48, 56).Accumulation of these toxic intermediates or their coloredautoxidation products has frequently been observed (13, 16,17, 19, 24-26, 53, 58, 59), because classical ring cleavageenzymes are inefficient oxygenators when halocatechols are

substrates, for instance.The inefficiency of a classical ortho-pyrocatechase is due

to the steric and inductive effects of the halogen atom whichimpede ring cleavage by catechol 1,2-dioxygenase (10). In thepresent paper, we present evidence indicating that catechol2,3-dioxygenase (meta-pyrocatechase) from Pseudomonasputida mt-2 is irreversibly inactivated by 3-halocatechols.

* Corresponding author.t Present address: Institut fur Humangenetik der Universitat, D-

3400 Gottingen, Federal Republic of Germany.t Present address: Universitat-Gesamthochschule Wuppertal,

Fachbereich 9, D-5600 Wuppertal 1, Federal Republic of Germany.

500

We interpret this as the mechanism by which toluate-utilizing bacterial populations turn black during their expo-sure to 3-chlorobenzoate as a result of accumulating chloro-catechols.

MATERIALS AND METHODSOrganisms. The media and methods for cultivation of P.

putida mt-2 have been described by Dorn et al. (9).Chemicals. The biochemicals used were catalase (Boeh-

ringer Mannheim Biochemicals) and superoxide dismutase(Sigma Chemical Co.). 3-Chloro-, 4-chloro-, and 3-fluoroca-techol were prepared as described by Schreiber et al. (50)and were purified by sublimation before use. All otherreagents used were of analytical grade and obtained from E.Merck AG.Preparation of catechol 2,3-dioxygenase. Catechol 2,3-diox-

ygenase (EC 1.13.1.2) was purified from P. putida mt-2 by acombination of the procedure described by Nozaki et al. (38)and a heat treatment procedure at 55°C for 10 min (3) beforethe acetone fractionation. The enzyme preparations wereshown to be homogeneous by polyacrylamide gel electro-phoresis. Specific activities up to 141 pmol of product permin per mg of protein at 25°C were measured when assayedby the method of Nozaki (36). Since the specific activitiesdecreased considerably when stored at 4°C in phosphatebuffer containing 10% acetone, samples were reactivatedbefore each experiment by the following procedure. Reacti-vation was accomplished by incubation of the enzyme for 3 hunder anaerobic conditions in the presence of FeSO4 (1 mM)and dithioerythritol (1 mM). Excess of these reagents wasremoved by repeated concentrating and diluting the enzymesolution (four volume changes) in a diaflo chamber. Proteinwas estimated by the method of Bradford (6).

Inactivation kinetics. Inactivation experiments (three tofour experiments in each case) involved the following proto-col. At time zero, 50 RI of purified catechol 2,3-dioxygenase(the amounts of enzyme used in the experiments are given inthe figure legends and tables) was added to 450 [LI of asolution containing an appropriate concentration of 3-chloro-

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INACTIVATION OF CATECHOL 2,3-DIOXYGENASE 501

100

50

a

1-

cQ)cz

Q)S:c

. ,

_3

10

5 10 15 20 25 30

lime (mm)nFIG. 1. Kinetics of inactivation of catechol 2,3-dioxygenase by

Tiron and halocatechols. Catechol 2,3-dioxygenase (60 mU in 0.5 mlof potassium phosphate buffer [50 mM], pH 6.5) was incubated at15°C with 20 mM Tiron (A), 10 mM 4-chlorocatechol (O), 1 mM 3-chlorocatechol (0), or without additional compounds as the control(0). Inactivation by 0.1 mM 3-fluorocatechol (A) was measuredwith 72 mU of purified enzyme present per ml of assay mixture.

or 3-fluorocatechol in a final concentration of 50 mM potassi-um phosphate buffer, pH 6.5, at 15°C. At intervals, 10-p.lsamples were removed and assayed by addition to cuvettescontaining 2.99 ml of a solution (1 p.mol of catechol-150,umol of potassium phosphate [pH 7.5] in 3 ml), permittingcontinuous assay of the rate of formation of 2-hydroxy-6-oxohexa-2,4-dienoic acid by its absorption at 375 nm. Therate of inactivation was obtained from a semilogarithmic plotof percent activity remaining (abscissa) versus time (ordi-nate).

Reversibility of inactivation was examined by dialyzingthe samples overnight against a 3,000-fold volume of potassi-um phosphate buffer (50 mM), pH 7.5, containing 10%(vol/vol) acetone, at 4°C, followed by dialysis under anaero-bic conditions against the same acetone-phosphate buffercontaining ferrous iron (1 mM) and dithioerythritol (1 mM).

RESULTSOxidation of substituted catechols by catechol 2,3-dioxygen-

ase. The catechol 2,3-dioxygenase from P. putida mt-2showed high initial oxidation rates with 3-chloro- and 3-fluorocatechol when measured with an oxygen electrode.When the enzyme was exposed to these substrates, howev-er, its activity was almost completely destroyed within 1min.

Inactivation of catechol 2,3-dioxygenase. When purifiedcatechol 2,3-dioxygenase from P. putida mt-2 was treatedwith either 3-chloro- or 3-fluorocatechol, a time-dependentloss of activity ensued. Figure 1 shows a pseudo first orderinactivation of the enzyme by 3-chloro-, 4-chloro-, and 3-fluorocatechol and by Tiron. Notably in 3-position, the

fluorine substituent permitted considerably faster inactiva-tion than chlorine and at one-tenth the concentration. 4-Chlorocatechol or Tiron gave only slow inactivation whenpresent at much higher concentrations (10 and 20 mM,respectively).The inactivation mechanism of 3-chloro- and 3-fluoro-

catechol must be completely different from that of Tiron. Asa chelating agent, Tiron also acted as a slow inactivator inthe absence of oxygen. In contrast, inactivation of thedioxygenase by 3-chloro- and 3-fluorocatechol required oxy-gen (Fig. 2). Further, less than 10% of the original activitycould be restored when the dioxygenase was inactivated bythe 3-halocatechols, but 50% of the Tiron-inactivated en-zyme could be reactivated (Table 1).For the determination of the kinetic data of inactivation,

pseudo first order rate constants (Ki,iact) were plotted againstinhibitor concentrations by the method of Lineweaver andBurk (32). The vertical intercept in this reciprocal plot is the

100

50

a

cxE

.3:* _

cz

100

50

10

5

5 10 15 20 25 30

time (mmn)FIG. 2. Inactivation of catechol 2,3-dioxygenase in the presence

and absence of oxygen. Catechol 2,3-dioxygenase (in 0.5 ml ofpotassium phosphate buffer [50 mM], pH 6.5) was incubated at 25°Cwith 20 mM Tiron (0, 60 mU), 1 mM 3-chlorocatechol (0, 188 mU),0.5 mM 3-fluorocatechol (0, 36 mU), or without additional com-pounds as a control (A, 188 mU) in the absence of oxygen (0 to 15min). Anaerobic conditions were established by bubbling nitrogenthrough the assay mixture for 15 min before the test compoundswere added. Ordinary cuvettes were sealed by use of serum capsand equipped with syringes. The absence of oxygen was certified bytesting for ring cleavage activity by adding catechol as the assaysubstrate. For aerobic conditions, nitrogen was replaced by bub-bling air through the assay mixture.

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502 BARTELS, KNACKMUSS, AND REINEKE

TABLE 1. Reversibility of inactivation of catechol 2,3-dioxygenase after treatment with Tiron and 3-halocatecholsRelative activity (%)

Treatment After initial After After finalreactivationa inactivation' After dialysis' reactivationd

Control looe 81 90Tiron (23 mM) 100 6 17 523-Chlorocatechol (1.5 mM) 100 <1 <1 73-Fluorocatechol (0.8 mM) 100 <1 <1 6

a Activity (taken as 100%) present in the sample after incubation for 3 h in the presence of FeSO4 (1 mM) and dithioerythritol (1 mM) underanaerobic conditions (see text).

b Activity present in the samples after treatment with the inhibitor for 45 min.c Activity present after dialysis against 3,000-fold of Tris-hydrochloride buffer (50 mM, pH 6.8, 10% [vol/vol] acetone).d Activity present in the samples after anaerobic incubation for 3 h with FeSO4 (1 mM) and dithioerythritol (1 mM).Specific activity, 141 U mg of protein-l

limiting rate constant for inactivation, 1VK2, i.e., the ob-served inactivation rate if all the enzyme is in the E * Icomplex. The horizontal intercept is -1IKi, correspondingto the dissociation constant of the inactivator from the E * Icomplex. The value of K2 for 3-chlorocatechol was 1.62 x10-3 sec-1, whereas the inhibitor constant (Kj) determinedfrom the plot was 23 ,uM. For 3-fluorocatechol, the corre-

sponding kinetic parameters could only be determined whenthe enzyme was inactivated in the presence of 2-mercap-toethanol. The effect of 2-mercaptoethanol indicated an

additional inactivation mechanism, in which diffusable reac-tive species may be involved. From the plot, the K2 value for3-fluorocatechol was 2.38 x 10-3 sec -1, and the Ki deter-mined was 17 [tM.The presence of the natural substrate catechol retarded

the rate of dioxygenase inactivation by the halocatechols(Table 2). The trapping agent 2-mercaptoethanol, unlikecatechol, had no protecting effect on the 3-chlorocatechol-induced inactivation of catechol 2,3-dioxygenase, suggestingthat inactivation by this substrate must have occurred exclu-sively at the active site of the enzyme and that inhibitoryagents were not released from the active site into themedium. When inactivation was tested with crude extractfrom m-toluate-grown cells of P. putida mt-2, other enzy-

matic activities, such as 2-hydroxymuconic semialdehydehydrolase, were not affected during inactivation of catechol2,3-dioxygenase by the halocatechols. In contrast, 2-mer-captoethanol partly protected the catechol 2,3-dioxygenasefrom inactivation by the 3-fluorocatechol due to its scaveng-

ing of reactive intermediates diffusing into the medium(Table 2). This indicated a second and nonspecific mode ofinactivation.

Products from cleavage of 3-halocatechols by catechol 2,3-dioxygernase. With 3-methylcatechol as substrate, 2,3-cleav-age yielded 2-hydroxy-6-oxohepta-2,4-dienoic acid (39). Theanalogous conversion of 3-hatocatechols should generate 5-haloformyl-2-hydroxypenta-2,4-dienoic acids, which are

highly reactive acyl halides. When 3-chlorocatechol (0.1mM) was incubated with a large amount of pure catechol 2,3-dioxygenase (7 U ml of assay mixture-1), a product could bedetected which exhibited spectral properties identical tothose of 2-hydroxyhexa-2,4-dienedioic acid. Authentic 2-hydroxyhexa-2,4-dienedioic acid was generated by the meth-od of Saeki et al. (49) in a reaction mixture containingpyrogallol (0.3 mM) and 7 U of pure catechol 2,3-dioxygen-ase. Both the 3-chlorocatechol and the pyrogallol ring cleav-age products exhibited an absorption maximum at 290 nm

(pH 7.5). Under neutral conditions, these products were

chemically isomerized within 4 min, exhibiting an absorption

maximum at 239 nm. On acidification (pH 1.5), the absorp-tion maximum was shifted to 300 nm. Under alkaline condi-tions (pH >10), an absorption peak appeared at 352 nm.Structural interconversion among these forms was revers-ible, depending on the pH of the medium. In contrast, 2-hydroxyhexa-2,4-dienedioic acid as the product of hydroly-sis of 5-fluoroformyl-2-hydroxypenta-2,4-dienoic acid wasnot observed when 3-fluorocatechol was cleaved by catechol2,3-dioxygenase. Furthermore, a yellow reaction productwas produced by the addition of pure catechol 2,3-dioxygen-ase (7 U ml-') to a cuvette containing 3-fluorocatechol (0.5mM) in the assay mixture. Conversion of 3-fluorocatechol inthe reference was suppressed by bubbling nitrogen throughthe cuvette sealed by a serum cap. The spectral characteris-tics of the product were different from those of 2-hydroxy-hexa-2,4-dienedioic acid. At neutral pH, the. maximum ab-sorption peak was found at 384 nm. When the reactionmedium was acidified, the absorption maximum was shiftedto 306 nm. The peak returned to 384 nm on addition ofNaOH. This indicates a 2-hydroxy-6-oxohexa-2,4-dienoicacid probably carrying the fluoro substituent in the 3-position, which, because of the spatial similarity of C-F andC-H groups, would arise from 3-fluorocatechol being boundto the enzyme as a 6-substituted substrate yielding 2-hy-droxy-3-fluoro-6-oxohexa-2,4-dienoic acid. Approximatelyone-tenth of the available 3-fluorocatechol was convertedinto the semialdehyde before enzyme inactivation was com-plete. This calculation is based on a hypothetical extinction

TABLE 2. Reduction of inactivation rate of catechol 2,3-dioxygenase through 3-chloro- and 3-fluorocatechol by 2-

mercaptoethanol or catecholReduction of inactivation rate (%)a

Compound (concn in the assay caused by:mixture)

3-Chlorocatechol 3-FluorocatecholNone (control) ob 02-Mercaptoethanol (1 mM) <2 37Catechol (5 mM) 55 86Catechol (10 mM) 65 88

a Reduction of inactivation rate is given as percentage of thatmeasured without additional compounds (= 0% reduction). Theywere calculated from semilogarithmic plots of remaining enzymeactivities versus time. The incubation assay was contained in 0.5 mlof potassium phosphate buffer (50 mM, pH 6.5) and either 0.33 mM3-chlorocatechol-115 mU catechol 2,3-dioxygenase or 0.033 mM 3-fluorocatechol-169 mU catechol 2,3-dioxygenase, and additionalcompounds as indicated, incubated at 15°C.

b Half-life (t,12) value for 3-chlorocatechol inactivation, 4 min.C tp12 value for 3-fluorocatechol inactivation, I min.

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INACTIVATION OF CATECHOL 2,3-DIOXYGENASE 503

TABLE 3. Reduction of inactivation rate of catechol 2,3-dioxygenase by scavengers for active oxygen species

Reduction of inactivation rate (%)MCompound (concn in the assay caused by:

mixture)3-Chlorocatechol 3-Fluorocatechol

None (control) ob 0CBovine serum albumin <2 <2

(1 mg ml-1)Superoxide dismutase <2 32

(2,000 U ml-1)Catalase (2,600 U ml-') <2 22Superoxide dismutase <2 51

(2,000 U ml-') +catalase (2,600 U ml-')

Mannitol (1 mM) <2 8Formate (1 mM) <2 5Histidine (1 mM) <2 <2

a Rate determined as described in Table 2, footnote a.b t1/2 value for 3-chlorocatechol inactivation, 4 min.c t1/2 value for 3-fluorocatechol inactivation, 1 min.

coefficient of 4 x 105 at 384 nm for the 3-fluorocatecholcleavage product, which is in the range of that of 2-hydroxy-6-oxohexa-2,4-dienoic acid (38).

Influence of scavengers of active oxygen species on theinactivation. The results obtained with 3-fluorocatechol asthe putative suicide substrate indicated that, besides anacylfluoride as the likely actual inactivating species, anadditional one may exist not originating from the E Icomplex. The involvement of oxygen as the inorganic sub-strate of the catechol 2,3-dioxygenase and the observationthat no inactivation by the halocatechols took place anaero-bically justified the assumption that active oxygen speciescould participate in enzyme inactivation. Superoxide dismu-tase was found to inhibit inactivation of catechol 2,3-dioxy-genase by 3-fluorocatechol but not that initiated by 3-chlorocatechol (Table 3). Catalase also partly protected thering cleavage enzyme from suicide inactivation by 3-fluoro-catechol but not that caused by 3-chlorocatechol. Hydroxylradical scavengers such as mannitol and formate exhibitedslight protective effects on the inactivation by 3-fluorocate-chol but had no effect on the inactivation by the chlorinatedanalog. Apparently, singlet oxygen was not generated sincethe inactivation by either of the 3-halocatechols was unim-paired by a singlet oxygen scavenger, such as histidine.

DISCUSSIONCatechol 2,3-dioxygenase, first described by Dagley and

Stopher (7), catalyzes the conversion of catechol to 2-hydroxy-6-oxohexa-2,4-dienoic acid by the insertion of twoatoms of molecular oxygen. The enzyme is a typical extra-diol cleavage-type nonheme iron dioxygenase (37). Frombinding (40) and kinetic studies (23), a Bi Uni-orderedreaction mechanism has been proposed in which the enzymecombines first with the organic substrate, followed by theaddition of oxygen. Nozaki et al. (41) proposed that thebinding of substrate is stabilized through hydrophobic inter-actions rather than by iron chelation. However, the sub-strate combines with the enzyme at the site near the iron andinteracts with the iron. With 3-methylcatechol as the sub-strate, an extradiol proximal cleavage of the bond betweencarbon atoms 2 and 3 occurs to yield 2-hydroxy-6-oxohepta-2,4-dienoic acid (39). The analogous conversion of the 3-halocatechols would generate 5-haloformyl-2-hydroxypenta-2,4-dienoic acids, which are highly reactive acyl halides.

Several recent reports have discussed the concept ofsuicide enzyme inactivation and outlined various criteria bywhich this type of enzyme inhibition can be identified (1, 46,51, 60, 61). The central feature of such a process is thecatalytic conversion of a relatively nonreactive compoundproducing a reactive species, which then interacts with theenzyme in such a manner as to cause irreversible inactiva-tion. The present results demonstrated that 3-chlorocatecholwas a suicide substrate of catechol 2,3-dioxygenase probablydue to the formation of 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual deactivation species (Fig. 3, patha). Characteristically, this inactivation was irreversible, oxy-gen dependent, and followed pseudo first order kinetics.Inactivation was retarded in the presence of the naturalsubstrate catechol but unaffected by 2-mercaptoethanol as atrapping agent.An indication that 5-chloroformyl-2-hydroxypenta-2,4-

dienoic acid was the actual suicide product of 3-chlorocate-chol cleavage came from the identification of 2-hydroxy-hexa-2,4-dienedioic acid as a by-product of the reaction.Obviously, a minor part of the acylchloride product willacylate water instead of basic groups of the enzyme (Fig. 3,path b). That a minor part of the 3-chlorocatechol-inactivat-ed catechol 2,3-dioxygenase could be reactivated by ferrousiron in the presence of dithioerythritol (Table 1) indicatedthat inactivation might also partly result from chelating theiron from the active center of the enzyme. This assumptionhas been made by Hirata et al. (21) for the inactivation of thecatechol 2,3-dioxygenase by substrate analogs such as 3-methyl- or 4,5-dichlorocatechol. Similarly, Klecka and Gib-son (28) have recently reported the reversible inactivation by3-chlorocatechol of the catechol 2,3-dioxygenase from atoluene-grown P. putida, which differed, however, in severalrespects from the enzyme investigated here. Linear oxygenuptake for 30 sec was found with 3-chloro- and 3-fluorocate-chol (2 or 6%, respectively, of that activity with catechol)with the enzyme from P. putida mt-2. In contrast, no oxygenuptake was observed with the enzyme investigated byKlecka and Gibson with 3-chlorocatechol. In addition,

Pth

OH

> OH

XXC230 toctive \

path C

rOHI OX

LC2301(activeJ)I

HX

path a

path b

HO

HX

OH

eCO,H

- C230 (inactive)

6

OH

O07H

C230 (octive)

OH

HO

+C230 (active)

FIG. 3. Interactions between catechol 2,3-dioxygenase (C230)and halocatechols. 3-Chloro- and 3-fluorocatechol (X = Cl, F) arecleaved following path a. A minor product from 3-chlorocatechol is2-hydroxyhexa-2,4-dienedioic acid according to path b. In addition tothe 5-fluoroformylpenta-2,4-dienoic acid-induced inactivation (patha), 3-fluorocatechol yields 3-fluoro-2-hydroxy-6-oxohexa-2,4-dien-oic acid plus the reactive oxygen species superoxide anion, hydro-gen peroxide, and hydroxyl radical exhibiting additional suicideinactivation of catechol 2,3-dioxygenase.

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504 BARTELS, KNACKMUSS, AND REINEKE

whereas the P. putida mt-2 enzyme readily converted 4-substituted catechols, 4-chloro- and 4-fluorocatechol werecleaved by the other catechol 2,3-dioxygenase only at lowrates. Another mechanism of inactivation which cannot beequated with acyl halide formation must account for the lossof activity of catechol 2,3-dioxygenase by treatment with 4-isopropylcatechol (4) (which we have confirmed) or with 4-chlorocatechol treatment (54).

Kinetic measurements with 3-fluorocatechol differ signifi-cantly from those with the chloro analog, because 2-mercap-toethanol decreases the inactivation rate of catechol 2,3-dioxygenase by 3-fluorocatechol. Active oxygen species areknown to react with thiols in the medium as well as withsulfhydryl groups of enzymes (2, 22, 29-31, 35). Thus,presumably by removing likely active oxygen species in thepresence of 2-mercaptoethanol, the remaining inactivatingreaction must be the consequence of the formation of 5-fluoroformyl-2-hydroxypenta-2,4-dienoic acid (Fig. 3, patha). Under these conditions, inactivation rates and dissocia-tion constants were similar for both 3-chloro- and 3-fluoro-catechol as the suicide substrates. Because superoxide dis-mutase or catalase decreased inactivation rates, we concludethat, presumably, superoxide radical and hydrogen peroxidewere also reaction products formed during 3-fluorocatecholturnover. Mannitol and formate, scavengers of hydroxylradical (8, 34, 44), also exhibited some protecting influenceon catechol 2,3-dioxygenase, so formation of hydroxyl radi-cals may be involved in enzyme inactivation by 3-fluorocate-chol. Histidine, a scavenger of singlet oxygen (14, 33, 57,62), did not prevent inactivation by 3-fluorocatechol.The chloro analog binds to the enzyme as a 3-substituted

substrate to give 5-chloroformyl-2-hydroxypenta-2,4-dienoicacid. However, the spatial similarity of C-F and C-H groupsmay allow the 3-fluorocatechol to bind also as a 6-substitutedsubstrate indicated by the formation of a 2-hydroxyhexa-2,4-dienedioic acid, probably the 3-fluoro analog (Fig. 3, path c).The physicochemical similarity between C-F and C-OH isknown to permit fluorine to act as hydroxyl analog inbiochemical reactions (55). Unproductive binding of 3-fluor-ocatechol to catechol 2,3-dioxygenase may therefore occurso that activated oxygen cannot be transferred to the organicsubstrate and is released into the medium. On the otherhand, we observed that 2-fluorophenol is neither a substratenor an inhibitor of catechol 2,3-dioxygenase, suggesting thatboth OH groups are required for substrate binding.The present results demonstrate that extradiol cleavage of

3-halocatechols is a lethal catabolic step in P. putida mt-2 asa result of the likely formation of acyl halides. The biocidaleffect of those compounds on alkylarene-degrading popula-tions has been elegantly used for the selection of mutantsthat are blocked in the catabolic sequences before ringcleavage (63, 64).Because intradiol-cleaving dioxygenases in arene-utilizing

bacteria also fail to convert 3-halocatechols at sufficientrates (10), accumulation of halocatechols or their violet toblack autoxidation products results. As a consequence of thedestruction of meta-cleavage activity by the halocatecholsand their inefficient oxygenation by ortho-cleavage, thisaccumulation is a general phenomenon in bacterial popula-tions co-oxidizing haloaromatics and explains the instabilityand dark coloration of sewage suddenly loaded with haloaro-matics (18, 19). To what extent constructed hybrid strainsharboring the degradation sequence for chlorocatecholsfrom Pseudomonas sp. strain B13 (47, 48) might be success-ful in avoiding accumulation of halocatechols will be demon-strated in a later paper.

ACKNOWLEDGMENTSThis work was supported by the Deutsche Forschungsgemeins-

chaft.The technical assistance of Wiltrud Muller is gratefully acknowl-

edged. Experimental work for this study was carried out in theInstitut fur Mikrobiologie der Universitat Gottingen. We are gratefulto R. B. Cain, University of Kent at Canterbury, United Kingdom,for valuable discussion.

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