2oxid de clorof ppor lip (2)

7/28/2019 2oxid de Clorof Ppor LiP (2)

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Biochemistry 1988, 27 , 6563-6568 6563

Th e O xidative 4-Dechlorination of Polychlorinated Phenols Is Catalyzed byExtracellular Fungal Lignin Peroxidases+

Kenne th E. Ha m m e l* a nd P a u l J. Ta r doneDepartment of Chemistry, College of Environmental Science and Forestry, State University of New York,Syracuse, New York 13210

Received February 18, 1988; Revised Man uscript Received April 20, 1988

ABSTRACT: Th e extracellular l ignin peroxidases ( l igninases) of Phanerochaete chrysosporium catalyzedHzOz-dependent spectral changes in several environmentally significant polychlorinated phenols: 2,4-dichloro-,2,4,5-trichloro-, 2,4,6-trichloro-, and pentachlorophenol. G as chro mat ogr aph y/m ass spectrometry of reducedand acetylated reaction products showed that, in ea ch case, l ignin peroxidase catalyzed a 4-dechlorinationof the star t ing phenol to yield a p-benzoquinone. Th e oxidation of 2,4-dichlorophenol a lso yielded adechlorinated coupling dim er, tentatively identif ied as 2-chloro-6-(2,4-dichlorophenoxy)-p-benzoquinone.Experim ents on the stoichiometry of 2,4,6-tr ichlorophenol oxidation showed tha t this sub strate was qu an-ti ta tively dechlorinated to give the quinone and inorganic chloride. Hz '*O-labeling experiments on 2,4,6-trichlorophenol oxidation demonstrated that water was the source of the new 4-OXO substituent in 2,6-di-chloro-p-benzoquinone. O ur results indicate a mechanis m whereby lignin peroxidase oxidizes a 4-chlorinatedphenol to a n electrophilic intermediate , perhaps the 4-chlorocyclohexadienone ation. Nucleophilic a ttackby water and elimination of HCl then ensue at the 4-position, which produces the quinone. Lignin peroxidaseshave previously been implicated in the degradation by Phanerochaete of several nonphenolic aromaticpollutants [cf . Haemmerli , S. D., Leisola , M. S. A ., Sangla rd , D., & Fiechter , A . (1986) J . Bi o l . Chem.2 6 1 , 6900-6903. Ham mel , K. E., Kalyanaraman, B., & Kirk , T. K. (1986) J . B i o l. Chem. 261 ,16948-169521. I t app ears l ikely from our results that these peroxidases could also catalyze the init ia ldechlorination of certain polychlorinated phenols in vivo.

L i t t l e is known about biochemical mechanisms for thedegradation of polychlorinated phenols, a major class of en-vironmental pollutants. Som e of these phenols are producedfor use as broad-spectrum biocides or as precursors for theman ufacture of chlorophenoxyacetate herbicides (Alexander,1974; Reineke, 1984). Others, notably 2,4,6-trichlorophenol,are contaminan ts of kraft paper mill effluents (Hu ynh et al.,1985). The microbial catabolism of chlorophenoxyacetatesfurther contributes to the environmental accumulation ofcertain isomers, especially 2,4-dichlorophenol and 2,4,5-tri-chlorophenol (Alexander, 1974). In general, the resistanceof chloroarom atics to biodeg radation increases with the de greeof ring halogenation (Reineke, 1984).Previous work has shown that Phanerochaete chrysospo-r ium, a ligninolytic basidiomycete, can deg rade a variety ofaromatic pollutants, including certain polycyclic aromatichydrocarbons (Bumpu s et al., 1985; Ham mel et al. ; 1986b,Sanglard et al., 1986) and chlorinated phenols (Huy nh et al.,1985). P. chrysosporium is notable for its production of ex-tracellular lignin peroxidases (ligninases), normally active inthe oxidative depolymerization of lignin, that catalyze se-quential one-electron oxidations of numerous aromatic sub-strates. Th e reactions catalyzed include the Cl-C2 cleavageof lignin model dim ers (Gold et al., 1 984; Tien & Kirk, 1984;Ham mel e t al., 1986a; Kirk et al., 1986b; Miki et al., 1986),the oxidation of alkoxylated benzyl alcohols to aldehydes (Goldet al., 1984; Tien & Kirk, 1984), the oxidation of certainalkoxybenzenes to quinones (K ersten et al., 1985b; PaszczyiiskiThis work was supported in part by US. nvironmental ProtectionAgency Cooperative Research Agreement 813530. It has not beensubjected to the Environme ntal Protection Agency's peer and adminis-trative review and therefore may not necessarily reflect the views of theAgency, and no official endorsement should be inferred.*Author to whom correspondence should be addressed.

et al., 1986), and haloperoxidase-type oxidations of bromideand iodide (Rengana than et al., 1987). Recently, both ligninperoxidases and whole cultures of P. chrysosporium wereshown to oxidize certain polycyclic aromatic hydrocarbons topolycyclic quinones, which indicates a biodegradative role forthese enzymes in at least some of the pollutant oxidationsaccomplished by Phanerochaete (Haemmerli et al. , 1986;Ham mel et al. , 1986b). W e are interested in the possibilitythat these peroxidases might be involved in the bio degradatio nof polychlorinated phenols and now report that they catalyzethe oxidative 4-dechlorination of these pollutants.EXPERIMENTALROCEDURES

Enzyme. Lignin peroxidases were obtained from trans-aconitate-buffered agitated cultures of P. chrysosporium(AT CC 24725) (Jag er et al. , 1985; Kersten & Kirk, 1987).The enzymes were purified by anion-exchange high-perform-ance liquid chromatography (HPLC)' on a 0.46 X 25 cmSynchropak 4300 (quaternary aminoethyl) column (Syn-chrom, Lafayette, IN ), using a 60-mL linear gradient of KCl(0-0.5 M, 1.5 mL min-') in 10 mM potassium phosphate (pH6.5). When chloride analyses of 2,4,6-trichlorophenol reactionswere to be done, the KCl gradient was replaced with a 60-mLgradient of sodium acetat e (0.01-1.0 M, 1.5 mL min-', pH6.0). Thes e techniques afforded resolution at least as goodas that obtained previously on Pharmacia Mono Q (Kirk etal. , 1986a).Chemicals . Polychlorinated phenols were obtained fromUltra Scientific (H ope, RI) and were pure as judged by gaschromatography (GC ). Chlorohydroquinone and 2,3,5,6-tetrachloro-p-benzoquinone ere purchased from Aldrich (St.

Abbreviations: HPLC, high-performance liquid chromatography;GC , gas chromatography; GC/MS, gas chromatography/mass spec-trometry.0006-2960/88/0427-6563$01S O / O 0 1988 American Chemical Society


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6564 B I oc H E M I S T R Y H A M M E L A N D T A R D O N ETable I: Mass S pectr a of p-Diacetoxybenzenes P roduced by Derivatization of the Quinones Formed during Lignin Peroxidase CatalyzedPolychlorophenol O xidations"

subs trate derivatized product mass spectrum m l z (re1 intensitvl. - I2,4-dichlorophenol 2-chloro-p-diacetoxybenzene2,4,5-trichloropheno1 2,5-dichloro-p-diacetoxybenzene 230 (0.6), 228 (2.3), 188 (5.2), 186 (16.9), 146 (35.9), 144 (100.0)264 (0.6), 262 (1.8), 224 (0.8), 222 (7.1) , 220 (10.5), 182 (9.2), 18 0 (60.3), 178I1 00.0)\ - - - . - ,2,4,6-trichlorophenoI 2,6-dichloro-p-diacetoxybenzenepentachlorophenol

264 (0.9) , 262 (1.3), 224 (1.7), 222 ( l l . O ) , 220 (17.1), 182 (10.7), 180 (64.3), 1782,3,5,6-tetrachloro-p-diacetoxybenzene 36 (0.2), 334 (2.1), 332 (5.2), 330 (3.9), 294 ( l . l ) , 292 (4.6), 290 (10.8), 288(1 00.0)(7.9), 254 (0.7), 252 (8.5), 250 (49.7), 248 (100.0), 246 (80.0)

Ions due to " C are not shown.Louis, MO). 2,5-Dichloro-p-benzoquinone as purchasedfrom Kodak (Rochester, N Y ) and 2,6-dichloro-p-benzoquinonefrom Pfaltz & Bauer (Waterbury, CT). All other chemicalswere of reagent grade.Reaction Co nditions. Typical enzym atic reaction mixtures(1 .O-5.0mL, room t emp erature) contained a polychlorinatedphenol (75-200 pM), P. hrysosporium lignin peroxidase H 1or H2 (K irk et al., 1986a) (0.3-2.0 pM ), sodium tartra te (20mM , pH 3.0), and Tween 80 (0.1% w/v). Th e reactions wereinitiated with H2 0 2 400 pM final concentration). Substrateoxidation was observed by repeated scans on a VarianD M S 100 absorption spectrophotometer. When the relativeturnover numbers of different lignin peroxidases for 2,4,6-trichlorophenol oxidation were to be determined, the reactionmixtures contained 100 pM 2,4,6-trichlorophenol and 400 p M(saturating) H202. The reactions were monitored spectro-photometrically a t 272 nm (see below). Relative turnovernumbers for veratryl alcohol oxidation were obtained from thechange in absorbance at 308 nm in reaction mixtures withoutTween 80 that contained 1.0 mM veratryl alcohol and 400 pMH202 Tien & Kirk, 1984). Lignin peroxidase concentrationswere calculated by using an extinction coefficient of 168 mM-'cm-' at 409 nm (Tien et al., 1986).Product IdentiJications. Th e completed enzymatic reactionswere purged with argon for several minutes, treated withsodium dithionite, and then extracted three times with 1volume of CH2C 12. Th e pooled organ ic phases were dried oversodium su lfate, concentrated to ca. 10 pL under a stream ofdry argon, and acetylated in 20 pL of 1:1 acetic anhydride/pyridine. Th e reduced and acetylated reaction products wereanalyzed by gas chromatography/electron impact massspectrometry (G CI M S) a t 70 eV in a Finnegan 4500 instru-ment fitted w ith an 8-m nonpolar fused silica capillary column(SPB- 1, Supelco). Th e gas chromatograph was programmedto rise from 50 to 150 OC at 5 "C min-' and then from 150to 300 OC at 10 OC min-'. Au then tic stand ards of chlorin atedp-diacetoxybenzenes were prepared from chlorinated quinonesor hydroquinones in a scaled -up version of the procedureoutlined above. Th e hydroquinones were recrystallized fromglacial acetic acid before acetylation.

Stoichiometry of 2,4,6-Trichlorophenol Oxidation. Theproduction of 2,6-dichloro-p-benzoquinoneas monitored a t272 nm. Th e extinction coefficient of auth enti c 2,6-di-chloro-p-benzoquinone in 20 mM sodium tartrate/O. 1% (w/v)Tween 80 was determ ined to be 14.0 mM-' cm-'. Th e pro-duction of C1- was determined by a modified mercuric thio-cyanate method (Florence & Farrar, 1971) in 0.4-mL samplestha t were withdrawn periodically from a 3.2-mL reaction. Th ereaction was stopped in each samp le by pipetting it into 0.3mL of 5.25 M perchloric acid l0.3 75 M ferric nitrate. Satu -rated mercuric thiocyanate in ethanol (0.3 mL) was thenadded, and th e absorbance of the sample was measured after5 min at 460 nm. Th e chloride concentration in each samplewas then determined from a linear calibration plot of chloride

standards that had been made up in tartrate/Tween 80 re -action mixture.Source of th e 4 0 x 0 Sub stitu ent in 2,6-Dichloro-p-benzo-quinone. H2180-labelingexperiments were done in enzymaticreaction mixtures that were enriched to 50 atom % excessH2 18 0 by dilution of 97.4% H2180 (MS D Isotopes). Thereactions were terminated with sodium dithionite, and theresulting 2,6-dichlorohydroquinonewas extracted (2X) withCH2C 12. Th e pooled organic ex tracts w ere dried over sodiumsulfate, concentrated by ro tary evaporation, an d analyzed byG C /M S as outlined above. The l80 ptake a t the 4-positionof 2,6-dichloro-p-benzoquinone as calculated from the in-tensitie s of the @-chlorocyclopentadienone ons detected at m/z114, 116, and 118.RESULTS N D DISCUSSION

Prod uct Identifications. Th e lignin peroxidases of P.chrysosporium catalyzed H20 2-depen dent spectral changesin all of the 4-chlorinated phenols we examined (Figure 1).With each substrate, these spectral changes were the same forall of the lignin peroxidases tested . For the di- and tri-chlorophenols, the spectra showed several near isosbestic points,which indicates that these substrates were oxidized to endproducts with negligible accumulations of intermediate species.The UV absorption spectra of the products from 2,4,5- and2,4,6-trichlorophenoloxidations (A- =272-273 nm) matchedthose we obtained for standards of 2,s- and 2,6-dichloro-p-benzoquinone dissolved in reaction mixture. Similarly, the UVabsorption maximum for the products of pentachlorophenoloxidation (290 nm) was the sam e as the max imum we observedfor a standard of tetrachloro-p-benzoquinone p-chloranil).G C /M S of the reduced and acetylated products showed tha tall of the polychlorophenols we examined had been enzy-matically dechlorinated. Th e only derivatized products de-tected from oxidations of the trichlorophenols and penta-chlorophenol were di- and tetrachlorinated p-diacetoxy-benzenes, whose GC retention times and m ass spectra matchedthose obtained for authentic standards of the derivatizedquinones as shown in Table I. In the case of pentachloro-phenol oxidation, the increase in absorbance observed at250-260 nm (Figure 1C) suggests the formation of additionalproducts, since p-chloranil does not exhibit a maxim um in thisregion, but these products were not observed in the GC/MSexperiments. Th e reduced and acetylated products of 2,4-dichlorophenol ox idation included 2-chloro-p-diacetoxybenzene(Table I ) . Control experiments, in which the pa rent phenolswere worked up in the same manner as the reaction mixtures,showed that dechlorination was not an artif act of the deriva-tization procedure. In some experiments, diazomethane ratherthan acetic anhydridelpyridine was used for derivatizationafter dithionite treatment, and this method gave p-dimeth-oxybenzenes that corresponded to the p-diacetoxy derivativesshown in Table I (data not shown). We conclude that P.chrysosporium lignin peroxidases catalyze the oxidative 4-


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D E C H L O R I N A T I O N O F P H E N O L S B Y L I G N I N P E R O X I D A S E S V O L . 27, N O . 1 7 , 1 9 8 8 6565

C

Wavelength, nmI , A '

00 0 1220 240 260 280 300 320Wavelength, nmFIGURE : Spectral changes in polychlorinated phenols upon incubationwith P. hrysosporium lignin peroxidase and HzOz.he reactionswere initiated with H202nd followed in repetitive scans between320 and 220 nm, at 100 nm m i d . (A) 2,4-Dichlorophenol(160pM):The spectrum of the sta rting material was recorded, and scans werethen taken at 0, 1.1, 2.3, 4.4, 7.4, and 14.5 min after initiation. Theenzyme concentration was 1.6pM. (B) 2,4,6-Trichlorophenol (100pM): he spectrum of the starting material was recorded, and scanswere then taken a t 0, 1.1, 2.3, 5.4, 8.4, nd 15.5 min after initiation.The enzyme concentration was 0.3 pM. (C) Pentachlorophenol (160pM): The spectrum of the starting material was recorded, and scanswere then taken at 0, 1.1, 3.3,6 .4, and 11.6 min after initiation. Theenzyme concentration was 0.8 p M . See Experimental Procedures.The spectral changes in 2,4,5-trichlorophenol (not shown) resembledthose obtained for 2,4,6-trichlorophenol.dechlorination of these polychlorophenols to give p-benzo-quinones.

0 1 2 3 4 5T ime (min)FIGURE 2: Time course of 2 ,4,6-trichlorophenol oxidation by ligninperoxidase (1 O pM) nd HzOz.he open circles indicate productionof 2,6-dichloro-p-benzoquinone. he open squares indicate productionof inorganic chloride. The black arrow indicates the starting con-centration of 2,4,6-trichlorophenol (96 p M) . See ExperimentalProcedures.

The lignin peroxidase catalyzed oxidation of 2,4-dichloro-phenol also yielded a dim er that h ad lost one chlorine. It wastentatively identified after derivatization as reduced andacetylated 2-chloro-6-(2,4-dichlorophenoxy)-p-benzoquinone:mass spectrum m / z (relative intensity) 388 (M', 0.7), 35 0(4.8), 348 (19.3), 346 (22.0), 310 (2.2), 308 (30.0), 306 (88.8),304 (10 0) (ions due to 13C not shown). Th e Occurrence of thisproduct is consistent with the expected one-electron per-oxidative mechanism for phenol oxidation by lignin peroxidase(Tien & Kirk, 1984): Th e 0- C coupling of two 2,4-di-chlorophenoxy radicals would give 2,4-dichloro-6-(2,4-di-chlorophenoxy)phenol, which itself would be a sub strate foroxidative dechlorination by the enzyme to yield th e quinone.Although 2,4,5-trichlorophenol could yield an analogous dimerby the same mechanism, since it has a nonchlorinated orthoposition, we did not observe this product. Th e 2,4,5-tri-chlorophenoxy radical is evidently hindered enoug h tha t de-chlorination is favored over intermolecu lar coupling under ourreaction conditions (aqu eous media, low concentratio ns of theradicals).Characteristics of 2,4,6-Trichlorophenol Oxidation. Of thepolychlorinated phenols we investigated, 2,4,6-trichlorophenolwas oxidized most rapidly by lignin peroxidase. Th e oxidativedechlorination of this sub strate was essentially quantitative:In the presence of excess H202,0.93 2,6-dichloro-p-benzo-quinone and 1.06 C1- were produced per 2,4,6-trichlorophenolsupplied (Figure 2). Th e enzyme showed no evidence ofsaturation by its phenolic substrate at 2,4,6-trichlorophenolconcentrations u p to 0.5 mM , the highest we could obtain inour reaction mixture. The optimal pH range for 2,4,6-tri-chlorophenol oxidation by lignin peroxidase was 2.5-3 -0,asit is for nonphenolic substrates (Tien et al., 1986).We consistently observed t hat the lignin peroxidase isozymesthat eluted earlier during anion-exchange H PL C were moreactive in 2,4,6-trichlorophenol oxidation than the later elutin gisozymes were. Purified fractions corresponding to the lessacidic isozymes H1 and H2 of Kirk et al. (1986a), whichoxidized the standard lignin peroxidase substrate veratrylalcohol (1.0 mM) with turnover numbers of 16-20 s-l underour conditions, oxidized 2,4,6-trichlorophenol (0.1 m M ) a trates of 3-5 s-'. By contrast, purified acidic isozymes thatcorresponded to lignin peroxidases H6-H 10 oxidized veratrylalcohol with comparable turnover numbers (14-26 s-l), yetoxidized the trichlorophenol more slowly (ca. 1 s-l). T hecapacity to oxidize 2,4,6-trichlorophenol was no t limited onlyto those peroxidases with lignin peroxidase activity. Ot herextracellular fun gal peroxidases, Le., the P . chrysosporium


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6566 B I O C H E M I S T R Y H A M M E L A N D T A R D O N EH3-H5 fractions (Kirk et al. , 1986a) and an enzyme fromGeotrichum candidum (A TC C 26195; Bordeleau & Bartha,1972 ), as well as a plant (horserad ish) peroxidase, all oxidizedthis substrate to 2,6-dichloro-p-benzoquinone.

Source of the 4 0 x 0 Subs ti tuent in 2,6-Dichloro-p-benzo-quinone. Th e reduced but underivatized product of 2,4,6-trichlorophenol oxidation, 2,6-dichlorohydroquinone, hro-matographed satisfactorily by GC, and we exploited thisfinding in H2180-labeling studies of the dechlorination re action .Figure 3A shows the results obtained when a lignin peroxidasecatalyzed trichlorophenol oxidation was done in naturalabundan ce reaction mixture. The products were treated withdithionite and extracted, and th e resulting hydroqu inone wasanalyzed by G C/ M S. The ions observed at m / z 142 and 144indicate the loss of H Cl from the molecular ions, whereas theions at m / z 114 and 116 show the further loss of C O to yield,presumably, monochlorinated cyclopentadienone radical cat-ions.Figure 3B shows the results obtained in a control experi-ment, in which an authentic standard of 2,6-dichloro-p-benzoquinone was incubated for 4.0 min in reaction m ixturethat had been made up in 50 atom % excess H2180. Thehydroquinone was then obtained and subjected to GC /M S as

outlined above. The relative intensities of th e ions at m / z 114,116, and 118 demo nstrate th at on e of the two dichloroquinoneoxygens, Le., the oxygen th at was not subsequently lost as C Ofrom the hydroquinone, did not exchange rapidly with solventwater. Th e calculated amou nt of exchange under these con-ditions was 20%. By cont rast, the other carbon yl oxygen, Le.,the one to be lost from th e molecular ion of the hy droquinoneas CO , exchanged completely with water durin g the same time,as can be seen from the relative intensities of the molecularions at m / z 178, 180, 182, and 184. Prom inent ions indicatingthe loss of HCl an d C O in the mass spectrometer are char-acteristic of a phenol with two ortho chlorines, but not of on ewith two meta chlorines (Heller & Milne, 1978), which leadsus to the conclusion that the oxygen at C1 of 2,6-dichloro-hydroquinone is the first to be lost upon ionization. Moreover,exchange of the oxo substituent at C1 of the quinone is ex-pected to be more rapid than at C4, because the inductiveeffect of two ort ho chlorines would favor nucleophilic att ackby water. These points accord with our observation that thefirst oxygen to be lost from the hydroquinone molecular ionin the mass spectrometer is the sam e oxygen t hat exchangesrapidly with water in the quinone. Therefore, we concludethat the peaks at m / z 114, 116, and 118 represent 0-chloro-cyclopentadienone ions tha t still contain the 4-oxo substituentof the original quinone.

This 4-oxo substituent is the one introduced du ring oxidative4-dechlorination of 2,4,6-trichlorophenol by lignin peroxidase.W e therefore asked whether the enzymatic oxidation of thisphenol in 50 atom 5% excess H2180would lead to significantlabeling of the cyclopentadienone ions seen at m/z 114, 116,and 118. This experiment, done simultaneously with theabove-mentioned control (to tal reaction time 4.0 min), showedthat 100% of the newly introduced quinone oxygens come fromwater (Figure 3C).

Our H2180-labeling experiments indicate tha t this reactio n involves attackby water on an electrophilic intermediate, perhaps the 4-chlorocyclohexadienone cation. Th e resulting 4-chloro-4-hydroxycyclohexadienone would eliminate HCl, in agreementwith our observed stoichiometry, to give the final p-benzo-quinone (Figure 4). A chlorinated phenoxy radical is shownas an intermediate in Figure 4 because phenoxy radicals are

Mechanism of Oxidative Dechlorination.

I' 4488?I

182184

r I 7 7 2 4 8

FIGURE : Mass spectrum of 2,6-dichlorohydroquinone btained afterreduction of the quinone. (A ) The quinone was obtained by ligninperoxidase catalyzed oxidation of 2 ,4,6-trichlorophenol in natu ralabundance reaction mixture and then reduced and extract ed. Theratio of th e peaks at m/z 114 and 116, both theoretical and found,is 1.00:0.33. The asterisks on the chem ical structures depicted showthe oxygen atom that is introduced during the lignin peroxidasecatalyzed oxidation of 2,4,6-trichlorophenol to 2,6-dichloro-p-benzoquinone. (B ) An authentic sta ndard of the quinone was incu-bated in 50 atom % excess H2'80-contain ing eaction mixture for 4.0min and then reduced an d extracted. Given 20% exchange of thequinone's 4-oxo substituent with 50 atom '3% Hzl*O, he theoreticalratio of the peaks at m / z 114, 116, and 118 is 1.00:0.44:0.03. Theratio found was 1.00:0.44:0.02. (C) The quinone was obtained bylignin peroxidase catalyzed oxidation of 2,4,6-trichlorophenol n 50atom % excess H2'*0-conta ining eaction mixture (total reaction time4.0 min) and then reduced and extracted. Given quantitative in -corporation of 50 atom % H2I 80 nto the new 4-oxo substituent, thetheoretical ratio of the peaks at m / z 114, 116, an d 11 8 is0.76: 1.00:0.24. The ratio found was 0.70:1.00:0.25. See ExperimentalProcedures.


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C I8 C lHl i gn inase+ h H + , e -H202

H2 0 2

o+ 0p-20

C I CI0

H+, -

ClJJJCl0

FIGURE 4: Hypothet ical scheme for the mechanism of 2,4,6-tri-chlorophenol oxidation by lignin peroxidase an d H2O2.known to be intermediates in other peroxidase-catalyzed phenoloxidations, and the occurrence of a dimeric product from thelignin peroxidase catalyzed oxidation of 2,4-dichlorophenoldoes indirectly in dicate the production of phenoxy radicals.However, our data do not establish that the dechlorinationpathway that yields p-benzoquinones must occur in one-electron rather than two-electron steps, and we have not sofar been able to detect any phenoxy radicals by ESR tech-niques (B. Kalyanaraman and K. Hammel, unpublished ob-servations). It was recently suggested that phenoxy radicalintermediates from peroxidase-catalyzed phenol oxidationsmight react at their ortho or para positions with molecularoxygen to yield peroxy radicals, which would then react furtherto give catechols, hydroquinones, or quinones (Ortiz deMontellano & Grab, 1987). Although phenoxy radicals arelikely intermediates in the reactions we have studied, ourH28 0-lab eling experiments do not support a role for dioxygenin the lignin peroxidase catalyzed oxidation of 4-chlorinatedphenols.CONCLUSIONS

The lignin peroxidase catalyzed 4-dechlorination of poly-chlorophenols bears some resemblance to th e reaction of thesecompounds with certain inorganic oxidants, e.g., lead dioxide(Hunte r & Morse, 1926). The possibility that peroxidasesmight catalyze such dechlorinations was noted by Saundersand Stark (1967), who were, however, unable to detect 2,6-dichloro-p-benzoquinone as a product when 2,4,6-trichloro-phenol was incubated for three days with turnip peroxidasean d H202.hloroquinones have been reported t o be unstablein water (Han cock et al., 1962; Saunders & Stark , 1967), andwe also have observed that 0.1 mM aqueous solutions of

2,6-dichloro-p-benzoquinone and 2,3,5,6-tetrachloro-p-benzoquinone develop a purple color with concomitant dis-appearance of the quinones (as determined by HP LC , datanot shown) within ca. 1 h. Ou r success in identifying thechloroquinones as products is probably attributable to shortreaction times (


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6568 Biochemistry 1988, 27 , 6568-6573chloro-6-(2,4-dichlorophenoxy)-p-benzoquinone,76540-52-8; -chloro-p-benzoquinone,695-99-8.REFERENCESAlexander, M. (1974) Adv. Appl. Microbiol. 18, 1-73.Bollag, J.-M., Sjoblad, R. D., & Minard, R. D . (1977) E x -Bordeleau, L. M ., & Bartha, R. (1 972) Can. J. Microbiol. 18,Bumpus, J. A., Tien, M., Wright, D., & Aust, S. D. (1985)Science (Washington, D.C.) 228, 1434-1436.Evans, W. C., Smith, B. S. W., Fernley, H. N ., & Davies, J.I. (1971) Biochem. J. 122, 543-551.Florence, T. M ., & Farrar, Y. J. (1971) Anal . Chim. Acta54, 373-377.Gold, M. H., Kuwahara, M., Chiu, A. A., & Glenn, J. K.(1984) Arch. Biochem. Biophys. 234, 353-362.Haemmerli, S . D., Leisola, M. S.A., Sanglard, D., & Fiechter,A. (1986) J. Biol . Chem. 261, 6900-6903.Hammel, K. E., Kalyanaraman, B., & Kirk, T. K. (1986a)Proc. Nat l . Acad. Sci . U.S.A . 83, 3708-3712.Hammel, K. E., Kalyanaraman, B., & Kirk, T. K. (1986b)J . Biol. Chem. 261, 16948-16952.Hancock, J. W ., Morrell, C. E., & Rhum , D. (1962) Tetra-hedron Lett. 22, 987-989.Heller, S. R., & Milne, G. W. A. (1978) E P A INZH M a ssSpectral Data Base, Vol. 1, p 628, U S . GovernmentPrinting Office, Washington, DC.Hunter, W. H., & Morse, M. (1926) J . Am. Chem. SOC. 8,Husain, M., Entsch, B., Ballou, D. P., Massey, V., & Chap-Huynh, V.-B., Chang, H.-M., Joyce, T. W ., & Kirk, T. K.Jager, A., Croan, S . , & Kirk, T. K. (1985) Ap pl. Enuiron.Kaufman, S . (1961) Biochim. Biophys. Acta 51, 619-621.

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Inhibition of Ca thep sin B by Peptidyl Aldehydes and Ketones: Slow-BindingBehavior of a Trifluoromethyl Ketone?Roger A. S mith ,* Lesl ie J. Copp, Sheila L. Donnelly, Robin W . Spencer, and Al len Kran tz

Syn tex Research (Ca nada), 2100 Sy ntex Court, Mississauga, Ontario, Canada LS N 3 x 4Received January 8, 1988; Revised M anuscript Received March 29, 1988

ABSTRACT: Inhibit ion of the cysteine proteinase cathepsin B by a series of N-benzyloxycarbonyl-L-phenylalanyl-L-alanine ketones an d the analog ous aldehyde has been investigated. Surprisingly, whereasthe aldeh yde was found to be alm ost as potent a competitive reversible inhibitor as the na tural peptidylaldehyde, leupeptin, the corresponding trifluoromethyl ketone showed comparatively weak (and slow-binding)reversible inhibit ion. Evaluatio n of competit ive hydration a nd hem ithioketal formation in a model systemled to a structure-activity correlation sp anni ng several orders of mag nitu de in both cathepsin B inhibitionconstants (Ki) and mode l sys tem equil ibrium d a ta (K R~H , ap p aren t ) .

I t as been known for some time that peptidyl aldehydes areeffective, reversible inhibitors of serine (Thompson, 1973;Thompson & Bauer, 1979; Dutta et al. , 1984; Stein &Strim pler, 1987) and cysteine (Aoyagi et al., 1969; Westerik

& Wolfenden, 1 972; Matti s et al., 1977; Knight, 1980; Baici& Gyger-M arazzi, 1982; Ogura et al. , 1985; Mackenzie etal., 1986) proteinases by forming hemiacetals and hemithio-acetals at the active site (Scheme I) . In recent years interesthas focused on the potential of trifluoromethyl ketones toinhibit proteinases in a similar fashion, by the formation ofContribution No. 27 5 from th e Insti tu te of Bio-Organic Chemistry.

0006-2960/88/0427-6568$01.50/0 0 988 American Chemical Society

2oxid de clorof ppor lip (2)

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