4oxid de pbenzodioxina lip

7/28/2019 4oxid de Pbenzodioxina LiP

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Biochemistry 1994, 33, 10969-10976 10969

Oxidation of Dibenzo-p-dioxin by Lignin Peroxidase from the BasidiomycetePhanerochaete chrysosporium?Dinesh K. Joshi and Michael H. Gold '

Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology,Portland, Oregon 97291 -1000Received M ay 4 , 1994; Revised M anuscript Received June 17, 1994"

ABSTRACT: Dibenzo-p-dioxin (I)was rapidly degraded in ligninolytic cultures of the basidiomycetePhanerochaete chrysospor ium. Lignin peroxidase (Lip) oxidized I to generate the following products:catechol (V),ibenzo-p-dioxin-2,3-quinoneVIII),-hydroxy-5-( 2-hydroxyphenoxy)- 1 Cben zoquinon e(IX),4,5-dihydroxy- 1,2-benzoquin one (X), -(2-hydroxyphenoxy)-1,4-benzoquinone XI), -hydroxy-l ,2-ben-zoquinone (XII), nd 1,2-benzoquinone (XIII). Ident ical products were formed when the reaction wasconducted under argon. N o incorporat ion of l 8 0 nto products was observed when the reaction was conduc tedunder 1802.xidation of I n H2180 resulted in incorporation of two atoms of I8O into the quinone VIII.None nzyma tic hydrolysis of the quinone (VIII) ielded catechol (V), X nd X. Hydro lysis of VI11 n H2180resulted in incorporation of l 8 0 atoms into IX and X, hereas no incorporation of l 8 0 atoms into V wasobserved. The se results ar e explained by mecha nisms involving the one-electron oxidation of I by Lip toproduce the corresponding cat ion radical . Nucleophi l ic at tac k of water on the cat ion radical generates a2-hydroxydibenzo-p-dioxinadical, which is oxidized to a delocalized cation. Th e attack of water a t positionC-4a of the 2-hydroxydibenzo-p-dioxin ation, followed by oxidation and C-0-C bond cleavage, lead toformation of the quinone (XI), hich undergoes 1,Ca ddition of water and cleava ge of the second C-0-Cbond to generate V nd XII. Similarly, the at t ack of water on C-3 of the delocal ized c at ion and subsequentoxidation generates the quinone VIII,which undergoes nonenzymatic 1,4-addition of water, followed byC-0-C bond cleavage to generate IX. IX also undergoes a w ater-addition reaction followed by C-0-Cbond cleavage to generate V and X. Alternatively, the quinones IX and XI could undergo enzymaticoxidation of phenolic functions to gene rate their correspo nding phe noxy radicals, which would be in resonancewith carbon-centered radicals. Oxidation of these radicals to cations, with subsequent water attac k andC-0-C bond cleavage, would yield 1,2-benzoquinone (XIII) nd 2,5-dihydroxy- 1,4-benzoquinone (XIV)from IX, nd XI11 and 2-hydroxy- 1,4-benzoquinone (XV) rom XI, espectively.

White-rot basidiomycetous fungi are primarily responsiblefor initiating the depolymerization of lignin, which is a keystep in the earth's carbon cycle (Buswell &Odier, 1987; Goldet al., 1989; Higuchi, 1990; Kirk & Farrell, 1987). The best-studied lignin-degrading fungus, Phanerochaete chrysospo-r ium, secretes two extracellular heme peroxidases-ligninperoxidase (Li p) and manganese peroxidase (MnP)-which,along with an H zOz-generating system, are apparently m ajorcomponents of th e organism's extracellular lignin degradativesystem (Gold et al., 1989; Ham mel et al., 1993; Kirk &Farrell,1987; Wariishi et al., 1991b).Nucleotide sequences of several LiP cD NA s and gene s (Gold

& Alic, 1993), as well as the recently published Lip X -raycrystal structures (Ed wards et al., 1993; Piontek et al., 1993;Poulos et al., 1993), demonstrate that important catalyticresidues, including the proximal and the distal His, the distalThis work was supportedby Grants MCB-9207997 from th e NationalScience Foundation, R-82-1269 from th e U S . Environmental ProtectionAgency, and DE-FG06-92ER20093 rom th e U S . Departmentof Energy,Division of Energy Biosciences.

* Towhom correspondence should be addressed at th e Department ofChemistry, Biochemistry, an d Molecular Biology, Oregon GraduateInstituteof Science&Technology, P.O. Box 91000,Portland, OR 9729 1-1000.Telephone: 503-690-1076. Fax: 503-690-1464.Abstract published in Advance ACS Abstracts, August 15, 1994.Abbreviations: PCDDs, polychlorinated dibenzo-p-dioxins; Lip,lignin peroxidase; MnP, manganese peroxidase;HPLC, high-performanceliquid chromatography; GC, gas chromatography; GCMS, gas chro-matography-mass spectrometry.

Arg, and an H-bo nded Asp, are all conserved within the hemepocket. Li p also shares mechanistic features with other plantand fungal peroxidases. Characterization of the formationand reac tions of the oxidized intermediate L ip compounds I,11, and I11 indica tes tha t the ca taly tic cycle of L ip is similarto that of horseradish peroxidase (Dunford &Stillman, 1976;Gold et al., 1989; Ma rque zet al., 1988; Renganathan &Gold,1986). Yet L ip has several unique features including a verylow pH optim um of -3.0 (Renganatha n et al., 1987; Tien etal., 1986) and an unusually high reactivity of compound I1with Hz02 (Wariishi et al., 1991a). In particular, Lip is ableto oxidize nonphenolic compounds with redox potentialsbeyond the reach of other peroxidases (Gold et al., 1989;Higuchi, 1990; Kirk & Farrell, 1987; Valli et al., 1992b;Schoem aker, 1990; Ham mel et al., 1986).Under ligninolytic conditions, P . chrysosporium also iscapable of mineralizing a wide range of I4C -labeledaromaticpollutants (Bumpus & Aust, 1987; Ham mel, 1989; Joshi &Gold, 1993; Spa daro et al., 1992; Valli & Gold, 1991; Valliet al., 1992a,b). Recently, the pathways for the fungaldegradation of 2,4-dichlorophenol (Valli &Gold, 1991), 2,4-dinitrotoluene (Valli et al., 1992a), 2,7-dichlorodibenzo-p-dioxin (Valli et al., 1992b), 2,4,5-trichlorophenol (Joshi &Gold, 1993), and anthracene (H am me let al., 1991) have beenelucidated, demonstrating that Lip and MnP as well asintracellular enzymes a re involved in the degradative process.Owing to their acute toxicity in anim al tests, polychlorinateddibenzodioxins (PCDDs) have been recognized as environ-

0006-2960/94/0433- 10969$04.50/0 0 994 American Chemical Society


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10970 Biochemistry, Vol.33, No. 36 , I994mental hazards for several decades (Hansen, 1991; Rappe,1980; Schwe tz et al., 1973). Th e solubility of PCD Dsdecreases with the increasing number of chlorine atoms(Rappe, 19 80). Th e insolubility of PCD Ds and the formationof reactive intermediates durin g in vivo peroxidase-catalyzedreactions has complicated efforts to study the mechanisticaspects of their degradation. Since dibenzo-p-dioxin (I ) isrelatively more w ater soluble than its chlorinated derivatives,we examined its oxidation by Lip . In this report, we describethe reactions involved in the Lip oxidation of I.

Joshi and Gold

MATERIALS AN D METHODSChemicals. Dibenzo-p-dioxin (I) was obtained from Ch emService (West Chester, PA) . Catechol (V), 2,5-dihydroxy-1 Cbenzoquinone (XIV), 1-bromo-2,5-dimethoxybenzene,guaiacol, potassium iodate, and sodium periodate wereobtained from Aldrich (Milwaukee, WI). 1,2,4-Trihydroxy-benzene (VI) was obtained from Lancaster Synthesis(Windham, NH ). Aromatic compounds obtained com-mercially were further purified by recrystallization.Preparation of Substrates. Dibenzo-p-dioxin-2,3-quinone(VIII): Catechol was oxidized with potassium iodate asreported (Forsyth et al., 1960). Th e product (approxim ately

10% yield) was purified by silica gel column ch romatog raphyusing ch1oroform:ethyl acetate (9 55 ) as the eluant.2,3-Dihydroxydibenzo-p-dioxin11): Sodium dithionite (4 5mg) in water (1 0 mL) was added to a solution of dibenzo-p-dioxin-2,3-quinone (MII) (25 mg) in methanol (20 mL),and the mixture was stirred a t room temperature for 10 min.The mixture was extracted with ethyl acetate, dried oversodium sulfate, and evapo rated under reduced pressure, andthe product was purified by silica gel column chromatographyusing ethyl acetate:hexane (2:3) as the eluant.2,2,4,5-Tetrahydroxydiphenyl ther (111): A mixture ofdibenzo-p-dioxin-2,3-quinoneMII) (25 mg) and 6 N hy-drochloric acid (4 mL) in methanol (30 mL) was stirred atroom temperature for 10 min. Th e reaction mixture was

brought to pH 7.0 with 3 N NaOH, treated with sodiumdithionite (50 mg), stirred for an additional 5 min, andextracted with ethyl acetate. Th e organic phase was driedover sodium sulfate and evaporated un der reduced pressure.The crude product mixture was derivatized with aceticanhydride and pyridine (2:l) and analyzed by GC MS . 2,3-Diacetoxydibenzepdioxin, 2,2,4,54etraacetoxydiphenyl ether,1,2-diacetoxybenzene,an d 1,2,4,5-tetraacetoxybenzene ereproduced in 58,19 ,7, and l%yields, respectively. The productswere separated by silica gel column chromatography usingethyl acetate and hexane (2:3) a s the eluant.2,2,5-Trihydroxydiphenyl ether (IV) was prepared bycoupling guaiacol and l-brom0-2,5-dimethoxybenzene,ol-lowed by demethylation with AlBr3 as described (Ungnade

&Otey, 195 1). Th e product was purified by silica gel columnchromatography using ethyl acetate:hexane (2:3) as the eluent.1,2,4,5-Tetrahydroxybenzene (VII) was prepared by thequantitative reduction of 2,5-dihydroxy-1,4-benzoquinone(XIV) using sodium dithion ite as previously described (Josh i& Gold, 1993). 4,5-Dihydroxy-l ,Zbenzoq uinone (X ) an d1,2-benzquinone (XIII) were prepared from 1,2,4,5-tetrahy-droxybenzene (VII) and catechol (V), respectively, using 1molar equiv of sodium period ate in water (Alder &Magnusson,1959). Th e o-quinone products, compounds X an d XIII, wereconverted to their phenazine d erivatives as described (Valliet al., 1992b).Fungal Degradation of Dibenzo-p-dioxins. P. chryso-sporium strain O GC lOl (Alic et al. , 1 987) was grown from

10-

0 5 10 1 5 2 0

Time (days)5

F I G U R E: Comparison of P. hrysosporium degradation of dibenzo-p-dioxin (0) nd 2,7-dichlorodibenzo-p-dioxin0 ) nder ligninolyticconditions. Stationar y cultures containing 1.2mM ammonium tartratewere inoculated with conidia and incubated for 6 days at 38 OC, afterwhich the substrate was added. Flasks were purged with 100%,Ozperiodically. The cultures were harvested, and the substrate remainingwas determined as described in the text.a conidial inoculum at 38 OC in stationary cu lture (25 m L)as described (Gold et al., 1982) using a medium describedpreviously (Goldetal ., 198 2;K irke tal., 1978) with 2%glucoseand 1.2 mM ammonium ta rtra te as the carbon and nitrogensources, respectively, and with 20 mM sodium 2,2-dimeth-ylsuccinate, pH 4.5, as the buffer. Cultures were incubatedunder air for 3 days, after which they were purged with 0 2every 3 days. After 6 days of incubation , the subs trate inN,N-dimethylformamide (20 pL) was added to cultures to afinal concentration of 25 pM . At the indicated intervals, anentire culture was homogenized in a blender (1 min), sa turatedwith NaC1, and extracted with ethyl acetate (3 X 50 mL).The organic fraction was dried over sodium sulfate andevaporated under reduced pressure, and the disappearance ofsubstrate was determined by G C analysis. The substrate alsowas added to uninoculated control cultures.Enzymes. Li p isozyme I1 was purified from the extra-cellular medium of an acetate-buffered, agitated culture ofP. chrysosp orium a s described previously (Gold et al., 1984;Wariishi & Gold, 1990). The Li p concentration wasdetermined a t 408 nm using an extinction coefficient of 133mM-l cm-l (Gold et al., 1984).Enzym e Reactions. Reaction mixtures (2 mL) consistedof Lip (10 pg/mL), substrate (0.2 mM), H202 (0.1 mM),and Tween 80 (0.1%) in 20 mM sodium succinate, pH 3.0.Veratryl alcohol (0.2 mM ) was added to stimu late the reactionand to prevent enzyme inactivation (W ariishi &Gold, 1990).The enzyme and H202 were added twice, at 0 an d 5 min, andthe reaction was carried out a t 28 C for 10 min. Reactionproducts were reduced with sodium dithion ite, extracted withethyl acetate a t pH 2, dried over sodium sulfate, evaporatedunder nitrogen, acetylated, and analyzed by G C or GC MS .Control reactions were conducted in the absence of enzymeor H202 or with boiled enzyme. 1,2-Benzoquinones wereconverted to their phenazine derivatives using 1 Zphen ylene-diamine in acetic acid and methanol prior to reduction asdescribed (Valli et al., 1992b) and analyzed by GCMS.

80- ncorporation Stu dies. For experiments conducted inH21S0, reaction m ixtures (0.5 mL) as described above wereenriched with H2180 83%). H2180 (97%) was obtained fromIso-Yeda Co., Ltd., Israel.


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Lignin Peroxidase Oxidation of Dibenzo-p-dioxin Biochemistry, Vol. 33, No. 36, 1994 10971

H H

I V (0.1)OH

O HV I (0.1)

I1 1 (0.2)

H @OHO H

V I 1 (0.1)FIGURE: Products identified from th e oxidation of dibenzo-p-dioxin (I) by Lip in the presence of H2O2,ollowed by reduction with sodiumdithionite. Reaction conditions and identification of products were as described in the text. Mole percent yields of oxidation products andremaining substrate ar e indicated in parentheses.

For experiments conducted under 1802, he reaction vesselwas evacuated, flushed with argon three times, and equilibratedwith 1 8 0 2 (99.1%; Isotec Inc., Miam isburg, O H ) as described(Kuw ahara et al., 1984; Renganath an et al. , 1986). Thereaction was initiated by the additio n of 02 -free H20 2 via asyringe. In each case, the reaction mixture was reduced,derivatized, and analyzed as described above. Th e l 8 0incorporation was determined by com paring intensities of theion peaks.Nonenzymatic Decay of Dibenzo-p-dioxin-2,3-quinone(VrII). The quinone (VIII) (0.2 mM) was incubated in 20mM sodium succinate buffer, pH 3, for 1 0 min. The reactionmixture was reduced with sodium dithion ite, extracted, driedover sodium sulfate, evaporated, and analyzed as describedabove. The quinone (X) was derivatized with 1,2-phenylene-diamin e prior to reduction as described above. Th e phenazinederivative of th e quinone (X ) was acetylated and analyzed byG C M S .The above reaction also was performed by incubatingdibenzo-p-dioxin-2,3-quinoneVIII) (0.2 mM ) in 1% sulfuricacid for 3 min under air or under argon. The same hydrolysisreaction under air was conducted in 1% sulfuric acid enrichedwith 93 atom %H2180. Th e products of all the reactions werereduced with sodium dithionite, acetylated, and analyzed asdescribed above.l S 0 - sotope- Exchan ge Reaction between l S 0 -EnrichedWater and Quinone Oxygens. Freshly prepared 1,2-benzo-quinone (XIII) was incubated in H 2l8O-enriched (90 atom %)20 mM sodium succinate (pH 3) for 20 min. Th e productmixture was reduced, extracted, dried, and derivatized asdescribed above. Similarly, freshly prepared 4,s-dihyd roxy-1,2-benzoquinone (X ) was incubated in H2l80-enriche d (90atom %) 1% sulfuric acid for 3 min.Gas Chromatography and Mass Spectrometry. GC MSwas performed at 70 eV on a VG Analytical 7070E massspectrometer fitted with an HP 5790A gas chromatographand a 30-m fused silica column (J & W Science, DB-5) . G Canalysis was performed on a similar gas chroma tograph and

column. The oven temperature was programmed from 70 to320 "Ca t 1 0 OC/min. Products wereidentified by comparisonof their retention times on GC and their mass fragm entationpatterns with chemically prepared standards . Yields werequantitated on G C by using calibration curves obtained withstandards using a flame ionization detector.RESULTS

Fung al Degradation. Time courses for the degradation ofdibenzo-p-dioxin (I) an d 2,7-dichlorodibenzo-p-dioxin ncultures of P. chrysosporium are shown in Figure 1. Thehalf-life of dibenzo-p-dioxin (I) in nitrogen-limited cultureswas approximately 1 day, whereas only about 43% of 2,7-dichlorodibenzo-p-dioxinwas degraded during the 24-dayperiod of the ex periment, confirming our earlier results (Valliet al., 1992b). Thesubstratewasstablein uninoculatedcontrolcultures during the length of the experiment.Enzym atic Oxidation ofDibenzo-p-dioxin (I). Oxidationof dibenzo-p-dioxin I) by Li p, followed by ch emical reduction,yielded 2,3-dihydroxydibenzo-p-dioxin11), 2,2',4,5-tetrahy-droxydiphenyl ether (111), 2,2',5-trihydroxydiphenyl ether(IV),catechol (V), 1,2,4-trihydroxybenzene(VI), and 1,2,4,5-tetrahydroxybenzene (VII). When the products of the reactionwere analyzed without subsequent redu ction, 4,5-dihydroxy-1,2-benzoquinone (X ) and 1,2-benzoquinone (XIII) also wereidentified. Th e yields of the products formed are shown inFigure 2. These yields are underestimates, owing to thepolymerization of quinones. All products were identified bycomparing their GC retention times and mass spectra withthose of chemically prepared s tandards (T able 1). The sameproduct pattern was obtained when the reaction was conductedunder either aerobic or anaerobic conditions. No productswere obtained when the reaction was performed in the absenceof either enzyme or H202 or with boiled enzyme.leg- ncorporation Studies. When substrate I was incu-bated with Lip under 1 8 0 2 n H2160,no incorpora tion of labeledoxygen into any of the produ cts was observed. Whe n thereaction was conducted in H2180, one (1 5.5 atom %,m/z 112)


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10972 Biochemistry, Vol. 33, No . 36, 1994 Joshi and GoldTable 1: Mass Spectra of Substrate (Dibenzo-p-dioxin) and Derivatives of Enzyme Oxidation Productsa

GC retentionsubstrate or metabolite time (min) mass spectrum m / z (re1 int)dibenzo-p-dioxin (I) 12.492,3-diacetoxydibenzo-p-dioxin 20.532,2,4,5-tetraacetoxydiphenyl ther 22.12

184 (loo), 155 (7.29), 28 (34.78), 02 (13.69), 2 (19.90),300 (6.2), 258 (8.98), 16 (loo), 187 (9.99, 71 (1.89), 42 (2.24),402 (3.8), 60 (17.15), 18 (37.25), 76 (84.71), 234 (loo),

76 (6.62), 3 (15.76)131 (2.79), 02 (1.67), 92 (1.71). 7 (9.81)215 (4.41), 87 (2.79), 61 (2.73), 41 (4.39), 26 (11.85),110 (20.39), 93 (9.11)

344 (2.64), 02 (41.29), 60 (71.38), 218 (loo), 199 (2.02),171 (3.26), 147 (2.22), 25 (8.65), 10 (31.86), 4 (36.88)194 (3.41), 52 (26.82), 10 (loo), 92 (1.39), 1 (5.81), 63 (3.30)252 (2.15), 10 (13.41), 68 (30.61). 126 (100). 7 (3.52).2,2,5-triacetoxydiphenylether1,2-diacetoxybenzene1,2,4-triacetoxybenzene

17.249.7914.5617.58

. . . .69 (2.36), 5 (2.35)113 (ll.l), 69 (20.2)310 (10.5), 68 (20.1), 26 (69.3), 184 (84.1), 142 (loo),1,2-benzoquinone (phen azine

4,5-diacetoxy-l,2-benzcquinone11.9821.90

180 (loo), 153 (9.75), 27 (4.59, 10 (3.70), 102 (9.15),296 (14.2), 54 (333,212 loo), 183 (13.8), 166 (8.1), 102 (8.9)derivative)(phenazine derivative)

90 (7.63), 6 (18.37), 3 (8.69)

@ Products identified from the oxidation of dibenzo-p-dioxin by L ip. Reactio n conditions and analysis were as described in the text. In all cases,the retention time and mass spectra of standard compounds were essentially identical to those of the oxidation products.Table 2: Relative Intensities (%) of the Molecular Ion Region ofMass Spectra of Products of Enzymatic Oxidations andNonenzymatic Reactions@

A. Enzy matic Oxidatio n of Dibenzo-p-dioxin (I)product m / z 1 8 0 2 b H2l8OC H21602,3-dihydroxydibenzo-p-dioxin11) 216 100 7.3 100218 0.8 6.7 0.9220 0 100 0catechol (V) 110 100 100 100112 0.7 23.1 0.7114 0 55.0 0

B. Nonenzymatic Decay of Dibenzo-p-dioxin-2,3-quinonedproduct m / z H2180d H2160218 1.0 0.9220 0 02,2,4,5-tetrahydroxydiphenyl ther (111) 234 6.18 100236 67.33 2.3

2,3-dihydroxydibenzo-p-dioxin11) 216 100 100

2382401121141,2,4,5-tetrahydroxybenzeneVII) 142144146148150

catechol (V) 11010058.831000.802.7613.6768.6232.09100

001000.701000.3000

C. Isotope-Exchange Reaction between H2I8O and Quinone O xygensproduct m / z H2I80 HJ60catechol (V)e 110 100 100112 21.1 0.83114 3.1 01,2,4,5-tetrahydroxybenzeneVIIY 142 100 100144 90.3 0.31146 30.7 0@ Reaction conditions and analysis were as described in the text.Reaction under 99.1% l*O-enriched 0 2 . Reaction in 83% H2180-containing buffer. Reaction in 93% H2180-containing 1% sulfuric acid.

e Reaction under 90% H2180-containingbuffer. f Reaction under 90%H2180-containing 1% sulfuric acid.ortwo(36 .7atom% ,m/z 114) atomsof l8Owereincorporatedin catechol (Ta ble 2). Similarly, compound I1 incorporatedone (6.0 atom %, m/z 218) or two (89.4 atom %, m/z 220)atoms of l*O.Nonenzymatic Decay of Dibenzo-p-dioxin-2,3-quinone(VIII). When dibenzo-p-dioxin-2,3-quinoneVIII) was in-

cuba ted in 20 mM succinate buffer (p H 3) for 20 min followedby reduction, 2,2,4,5-tetrahydroxydiphenyl ther (111) ( l a ) ,catechol (V) (3%), and 1,2,4,5-tetrahydroxybenzene M I)(trac e) were obtained. The hydrolysis of VI11 occurred morerapidly in 1% sulfuric acid, wherein 2,2/,4,5-tetrahydroxy-diphenyl ether (111) (6%), catechol (V ) (lo%), and 1,2,4,5-tetrahydroxybenzene ( M I ) (0.5%) were formed af ter 3 min.Derivatization of the product mixture with 1 Zphe nylene -diamine, prior to reduction with sodium dithionite, yieldedthe phenazine derivative of 4,5-dihydroxy- 1 Zbenzoq uinone(XIWhen dibenzo-p-dioxin-2,3-quinoneW I ) was hydrolyzedin H2180-enriched 1% sulfuric acid followed by reduction,1,2,4,5-tetrahydroxybenzeneM I) incorporatedone (6.3 atom%,m/z 144),two(31.65atom%,m/z 146),three(46.14atom%, m/z 148), or four (14.84 atom %, m/z 150) atoms of l 8 0(Table 2). Similarly, 2,2/,4,5-tetrahydroxydiphenyl ther (III)incorporated one (29.5 atom %, m/z 236), two (43.3 atom %,m/z 238), or three (24.8 atom %, m/z 240) atom s of l 8 0 .However, no incorporation of l80nto 2,3-dihydroxydibenzo-p-dioxin (11) or catechol (V) was observed.

Isotope- Exch ange Reactions between leg- nriched Waterand Quinone Oxygens. Whe n 1 Zbenzoquinone was incubatedin H;*O, incorporation of one (20 atom %, m/z 112) or two(2.7 atom %, m/z 114) atom s of l 8 0 Table 2) into the quinonewas observed. Whe n 4S-dihydroxy- 1 2-benzoquinone(X) asincubated in H 2I80 , incorporation of one (44.4 atom %, m/z144)ortwo(15.2atom%,m/z 146)atomsof 180wasobserved.DISCUSSION

Un der ligninolytic conditions, P. hrysosporium is capableof mineralizing many persistent arom atic pollutants (Bumpus&Aust, 1987; Ham mel, 1989; Joshi &Gold, 1993; Spa daroet al., 1992; Valli & Gold, 1991; Valli et al., 1992a,b).Recently, the P. hrysosporium degradative pathways for thepollutants 2,4-dichlorophenol (Valli & Gold, 1991), 2,4-dinitrotoluene (Valli et al., 1992a), 2,7-dichlorodibenzo-p-dioxin (Valli et al., 19 92b), 2,4,5-trichlorophenol (Joshi &Gold, 1993), and anthrac ene (Ha mm elet al., 1991) have beenexamined. These studies demonstrate that both Lip and Mn Pplay importa nt roles in the P. hrysosporium degradation ofthese pollutants. To our knowledge P. hrysosporium andpresumably o ther white-rot fungi ar e the only microorganisms


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Lignin Peroxidase Oxidation of Dibenzo-p-dioxin Biochemistry, Vol. 33, No. 36 , 1994 10973

I A B1 -e-D1+H?O

L I "VI11IIX

l+H2O

J +H20[ @ : P O HI "CXo:XJoXI

J + H 2 0

V X I 1

V XFIGURE: Proposed mechanism for lignin peroxidase-catalyzed oxidation of dibenzo-p-dioxin(I), showing the involvement of water additionto quinone intermediates.known to d egrade polychorinated dibenzo-p-dioxin pollutants(Valli et al., 1992b ). Thu s a study of the mechanisms involvedin these degradations may have practical significance.Th e results in Figure 1 show that , under nutrient nitrogen-limiting conditions, P. hrysosporium degrades dibenzo-p-dioxin (I ) considerably faster than 2,7-dichlorodibenzo-p-dioxin (Valli et al., 199 2b), suggesting that chlorine substitutionon the aromatic ring retards the degradation rate. Thisdecrease in degradation rate is probably due to two factors.First, th e solubility of chlorinated dibenzo-p-dioxinsdecreaseswith an increasing numbe r of ring chlorine substituents (Rappe ,1980). In addition, chlorine substitution on the arom atic ringraises the redox potential, making the substrate m ore resistantto oxidation. W e have selected dibenzo-p-dioxin (I ) to studythe mechanistic aspects of dioxin oxidation because I issufficiently soluble in water and is a relatively good Lipsubstrate.Lignin peroxidase catalyzes the oxidation of a variety ofnonphenolicaromatic compounds o their correspondingcationradicals (Gold et al., 1989; Higuchi, 1990; Kirk & Farrell,

1987; Kersten et al., 1985; Schoem aker, 1990; Ham mel e t al.,1986). Indeed, the Li p oxidation of dibenzo-p-dioxin (I ) toits cation radical has been demonstrated by electron spinresonance spectroscopy (Ham mel et al., 1 986). However, nodegradation products were identified in that study. The cationradical of dibenzo-p-dioxin also has been generated chemicallyand electrochemicallyand identified by electron spin resonancespectroscopy (Tomita &Ueda, 1964; Shine & Shade, 1974;Cauquis & Maurey-Mey, 1972).

Th e Lip-catalyzed oxidation of dibenzo-p-dioxin (I), fol-lowed by chemical reduction of the quinone products, yieldsthe following products: 2,3-dihydroxydibenzo-p-dioxin 11),2,2',4,5-tetrahydroxydiphenyl ether (111), 2,2',5-trihydroxy-diphenyl ether (IV), catechol (V), 1,2,4-trihydroxybenzene(VI), an d 1,2,4,5-tetrahydroxybenzeneVII) (Figure 2). Thenature of the products generated by th e oxidation of dibenzo-p-dioxin (I ) suggests the initial formation of an aryl cationradical, which undergoes further reactions with water to formthe variety of products observed in Figure 3.


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10974 Biochemistry, Vol. 33 , No. 36, 1994aooXXoOHIX1Lip

Joshi and Gold

GI

J/ iHZO

K

01

UO0D0XII1 ip

a0:XJoLI

0

XI11 x IV XI11 x vFIGURE: Proposed mechanism for lignin peroxidase-catalyzed cleavage of C-0-C bonds in the quinones IX and XI, howing the possiblefurther oxidation of phenolic intermediates.As shown in Figure 3, the initial nucleophilic attack of HzOat position C-2 of the dibenzo-p-dioxin cation radical (A )would generate a delocalized 2-hydroxydibenzo-p-dioxinradical (B), which loses another electron, either by adisproportionation reaction or by L ip oxidation, to form the

delocalized cation C r D. Th e attack of water on cation C,followed by cleavage of the C-0-C bond, would gene rate2-(2-hydroxyphenoxy)- 1,4-benzoquinone (XI), which wasidentified a s 2,2',5-trihydroxydiphenyl ether (IV). Quinonesare known to undergo 1,4-addition reactions with water(Finley, 1974). Our results suggest that the quinone XIundergoes a spontaneous addition of water w ith subsequentcleavage of the remaining C-0-C bond to form catechol (V )and 4-hydroxy- 1,2-benzoquinone XII). The quinone XI1 wasidentified as 1,2,4-trihydroxybenzene (VI) after reductiveacetylation. Catech ol was identified as its acetylated deriva-tive.The attack of HzO at position C-3 of thecation intermediateD (Figure 3) and further oxidation would lead to the formation

of dibenzo-p-dioxin-2,3-quinoneVIII), presumably via thedihydrodiol nterm ediate(E). he quinone VIII was identifiedas 2,3-dihydroxydibenzo-p-dioxin11) after reductive acety-lation. Attempts by other researchers to identify this quinoneas a product of th e reaction of th e dibenzo-p-dioxin cationradical (A ) with water were unsuccessful (Shine & Shade,1974;Cauquis &Maurey-Mey, 1972). The dibenzo-p-dioxin-2,3-quinone (VIII) would undergo addition of water (atposition C-4 a), followed by cleavage of the first C-0-C bond,to generate 2-hydroxy-5-(2-hydroxyphenoxy)- 4-benzoquino-ne (IX). This product also was identified after reductiveacetylation as 2,2',4,5-tetrahydroxydiphenyl ether (111). Thequinone IX also undergoes an addition rea ction with a secondmolecule of HzO, followed by cleavage of the second C-0-Cbond, to form catechol (V ) and 4,5-dihydroxy- 1,2-benzo-quinone (X). The quinone X was identified both as itsphenazine derivativeand as 1,2,4,5-tetrahydroxybnzene(WI)after reductive acetylation. Catechol was identified as itsacetylated product. Th e direct observation of the quinones


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Lignin Peroxidase Oxidation of Dibenzo-p-dioxinVI11 and XI indicates that the attack of water on the2-hydroxydibenzo-p-dioxin ation is occurring at both the C- 3and C-4 a positions. Th e relative yields of these quinones (VnIan d XI ) depends on the contribution of structures C an d Dto the resonance hybrid.

We have confirmed the mechanism discussed above byconducting the reaction in lsO-enriched water. Eighty-ninepercent incorporation of two atoms of l 8 0 ( m / z 220) fromlabeled water into 2,3-dihydroxydibenzo-p-dioxinII) uggeststh at both quinone oxygens in compound VI11 ar e derived fromwater. Wh en the reaction was performed under argon,identical products were obtained. Furthermore, when thereaction was condu cted und er 1802, no incorporation of I8Oin the products was observed. These results suggest that thecleavage of C-0-C bonds occurs via the nucleophilic att ackof water on the cation rather than via the scavenging of aradical by molecular oxygen.

Formatio n of considerable amoun ts of unlabeled catechol(V) (47.8%) during the L ip oxidation of dibenzo-p-dioxin (I)in I80-labeled water supports a mechanism whereby thequinones VI11 and XI undergo nonenzymatic reaction withH2 0. To further confirm the mechanism of the nonenzymaticreaction of the quinone VI11 with water, we chemicallysynthesized dibenzo-p-dioxin-2,3-quinoneVIII). When thequinone VI11 was hydrolyzed in succinate buffer (pH 3),catechol (V), 2-hydroxy-5-(2-hydroxyphenoxy)-l4-benzo-quinone (IX), an d 4,5-dihydroxy-l,2-benzoquinoneX) wereformed. The quinone X was identified as its phenazinederivative and also as 1,2,4,5-tetrahydroxybenzene VII) afterreductive acetylation, whereas the quinone IX was identifiedas 2,2,4,5-tetrahydroxydiphenyl ther (111) after reductiveacetylation. Detection of 2-hydroxy-5-(2-hydroxyphenoxy)-1,4-benzoquinone (IX) suggests that dibenzo-p-dioxin-2,3-quinone (VIII) undergoes stepwise cleavage of C-0-C bondsto form catechol (V) an d 4 ,Sdihydrox y- 1,2-benzoquinone (X).Cauquis and co-workers reported the formation of 2,5-dihydroxy- 1,4-benzoquinone (XIV) from the reaction of thedibenzo-p-dioxin cation radical with water (Cauquis &Maurey -Mey, 1972 ). Under the conditions used in tha t study,the initially formed 4,5-dihydroxy- 1,2-benzoquinone (X ) mayhave undergone rapid isomerization to form the thermody-namically more stable 2J-dihydroxy- 1 4-benzoquinone (XIV).We also have observed tha t th e hydrolysis reaction is catalyzedby acid. Wh en the reaction was performed in 1% sulfuricacid, the yields of the short-lived quinone X were improved,suggesting that t he polymerization of quinone X occurs overtime. Whe n the hydrolysis of th e quinone VI11 was conductedin H21 80, incorporation of l8Oatoms was observed in bothquinones IX an d X, suggesting that the quinone oxygens arederived from wat er. Excess incorpo ration of labeled oxygensin quinones IX an d X s presumably du e to the rapid exchangereaction between 180-enrich edwater a nd quinone oxygens asshown by the isotope-exchange reaction between t he quinoneX oxygen and water. However, no exchan geof labeled oxygenswas observed with the quinone VIII.

An alternative mechanism for the oxidation of dibenzo-p-dioxin is shown in Figure 4. In this mechanism, the quinonesIX an d XI undergo enzymatic oxidation of the phenolicfunction to generate their corresponding phenoxy radicals, Gor L (Figure 4). Oxidation of the intermediate IX generatesthe phenoxy rad ical, G (Figu re 4), which is in resonance withthe carbon-centered radical, H . Further oxidation of thisradical, followed by attack of H2O on the cation, results inthe cleavage of the C-0-C bond and generation of 1,2-benzoquinone (XIII) an d 2,5-dihydroxy-l,4-benzoquinone

Biochemistry, Vol. 33, No. 36, 1994 10975(XIV). Previously, we proposed this mechanism for the L ipoxidation of 2,7-dichlorodibenzo-p-dioxinValli et al., 1992b).Similarly, 2-(2-hydroxyphenoxy)- 1,4-benzoquinone (XI)can undergo enzymatic oxidation to generate the phenoxyradical L,which is in resonance with the carbon-centeredradical M (Figu re 4). Furth er oxidation of this radical withsubsequent attack of H20 on t he cation results in th e cleavageof th e C-0-C bond to gene rate 1,Zbenzo quinon e (XIII) an d2-hydroxy- 1,4-benzoquinone (XV).Th e phenoxy radical mechanism predicts the incorporationof one 1 8 0 atom from labeled water into 1,2-benzoquinone.However, catecho l (V ) derived from the nonenzymatic reactionof the quinones IX an d XI with water would be expected toundergo subsequent oxidation by Lip, resulting in theformation of 1 Zb enzcquin one, with incorporation of I8O ro mlabeled water. Incorporation of l8Odu e to quinone isotopeexchang e reactions also is expected. Thus, it is difficult todifferentiate th e phenoxy radical mechanism fro m these otherpossibilities.On the basis of the products detected during L ip oxidationof dibenzo-p-dioxin (I), we conclude that the reaction isinitiated by the one-electron oxidation of dibenzo-p-dioxin(I) to its corresponding cation radical. Th e cation radicalundergoes reactions with water to yield various quinoneintermediates (VIII, IX, or XI). The phenoxyquinone inter-mediates may undergo nonenzymatic as well as enzymaticreactions. M nP efficiently oxidizes phenols (Wariishi et al.,1989; Tuo r et al., 19 92). Thu s in culture, M n P is likely tocatalyze the oxidation of these phenoxyquinone intermediates,accelerating the degradative process.As we have shown (Valli et al., 1992b), similar mechanismsare involved in the oxidation of 2,7-dichlorodibenzo-p-dioxinby Lip, including an initial enzyme-catalyzed one-electronoxidation to generate a cation radical. However, the natu reof the final products is determined by th e chlorine substitutionpattern on the substrate. In the case of asymmetricallysubstituted ch lorinated dibenzo-p-dioxin isomers, the initialattack of w ater on thecorresponding cation radical could occuron either of the aromatic rings, leading to the formation ofa large variety of products. W e are continuing to study themechanisms involved in th e fungal deg radation of polychlo-rinated dibenzo-p-dioxins.REFERENCESAlder, E., &Magnusson, R. (1959) Acta Chem. Scand. 12,505-Alic, M., Letzring, C., & Gold, M. H . (1987) Appl. Environ.Bumpus, J. A., & Aust, S . D. (1987) Bioessays 6, 166-170.Buswell, J . A., &Odier, E. (1987) Crit. Re v. Biotechnol. 6 , 1 4 0 .Cauquis, G., & Maurey-Mey, M. (1972) Bull. SOC . him. Fr.,Dunford, H . B., & Stillman, J . S. 1976) Coord. Chem. Rev. 19,Edwards, S. ., Raag, R ., Wariishi, H ., Gold, M . H., & Poulos,T. L. (1993) Proc. Natl. Acad. Sci. U.S.A .90, 750-754.Finley, K. T. (1974) in The Chemistry of the QuinonoidCompounds, Part 2 (Patai, S. , Ed.) pp 877-1 144, John W iley& Sons, London.Forsyth, W. G. C., Quesnel, V. C., & Roberts, J . B. (1960)Biochim. Biophys. Acta 37, 322-326.Gold, M. H. , & Alic, M. (1993) Microbiol. Rev. 57, 605-622.Gold, M . H., Mayfield, M. B., Cheng, T. M., Krisnangkura, K.,Enoki, A., Shimada, M., & Glenn, J. K. (1982) Arch.Microbiol. 132, 115-122.Gold, M. H ., Kuw ahara, M., Chiu, A. A., &Glenn, J. K. (1984)Arch. Biochem. Biophys. 234, 353-362.

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4oxid de pbenzodioxina lip

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