Tài liệu Báo cáo khoa học: Cleavage of nonphenolic b-1 diarylpropane lignin model dimers by manganese peroxidase from Phanerochaete chrysosporium Evidence for a hydrogen abstraction mechanism docx

Cleavage of nonphenolic b-1 diarylpropane lignin model dimers by manganese peroxidase from Phanerochaete chrysosporium Evidence for a hydrogen abstraction mechanism G. Vijay B. Reddy 1 , Malayam Sridhar 2 and Michael H. Gold Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering at OHSU, Beaverton, Oregon, USA Purified manganese peroxidase (MnP) from Phanerocha- ete chrysosporium oxidizes nonphenolic b-1 diarylpropane lignin model compounds in the presence of Tween 80, and in three- to fourfold lower yield in its absence. In the presence of Tween 80, 1-(3¢,4¢-diethoxyphenyl)-1-hydroxy-2-(4¢- methoxyphenyl)propane (I) was oxidized to 3,4-diethoxy- benzaldehyde (II), 4-methoxyacetophenone (III) and 1-(3¢,4¢-diethoxyphenyl)-1-oxo-2-(4¢-methoxyphenyl)pro- pane (IV), while only 3,4-diethoxybenzaldehyde (II) and 4-methoxyacetophenone (III) were detected when the reac- tion was conducted in the absence of Tween 80. In contrast to the oxidation of this substrate by lignin peroxidase (LiP), oxidation of substrates by MnP did not proceed under anaerobic conditions. When the dimer (I) was deuterated at the a position and subsequently oxidized by MnP in the presence of Tween 80, yields of 3,4-diethoxybenzaldehyde, 4-methoxyacetophenone remained constant, while the yield of the a-keto dimeric product (IV) decreased by approxi- mately sixfold, suggesting the involvement of a hydrogen abstraction mechanism. MnP also oxidized the a-keto di- meric product (IV) to yield 3,4-diethoxybenzoic acid (V) and 4-methoxyacetophenone (III), in the presence and, in lower yield, in the absence of Tween 80. When the reaction was performed in the presence of 18 O 2 , both products, 3,4-diethoxybenzoic acid and 4-methoxyacetophenone, contained one atom of 18 O. Finally, MnP oxidized the substrate 1-(3¢,5¢-dimethoxyphenyl)-1-hydroxy-2-(4¢-methoxyphenyl) propane (IX) to yield 3,5-dimethoxybenzaldehyde (XI), 4-methoxyacetophenone (III) and 1-(3¢,5¢-dimethoxyphe- nyl)-1-oxo-2-(4¢-methoxyphenyl)propane (X). In sharp contrast, LiP was not able to oxidize IX. Based on these results, we propose a mechanism for the MnP-catalyzed oxidation of these dimers, involving hydrogen abstraction at a benzylic carbon, rather than electron abstraction from an aromatic ring. Keywords: manganese peroxidase; hydrogen abstraction; diarylpropane dimers; Mn(III); radical mediator. Lignin is a complex, random, phenylpropanoid polymer that constitutes 15–30% of woody plant cell walls [1]. White-rot basidiomycetous fungi are primarily responsible for the initial decomposition of lignin in wood [2–5]. When cultured under ligninolytic conditions, the best-studied white-rot basidiomycete, Phanerochaete chrysosporium, produces two extracellular peroxidases, lignin peroxidase (LiP) and manganese peroxidase (MnP), which, along with an H 2 O 2 -generating system, appear to be the major components of its lignin degradation system [2,3,6–10]. LiP oxidizes a variety of lignin model compounds, including the most prevalent nonphenolic b-aryl ether (b-O-4 type) as well as diarylpropane (b-1 type) structures [10–14]. The enzyme abstracts one electron from the aromatic ring to form an aryl cation radical [3,11,15,16]. Chemical and ESR spectroscopic evidence have confirmed the formation of cation radical species in the LiP-catalyzed oxidation of alkoxybenzenes [17–21]. In contrast, MnP oxidizes Mn(II), its primary substrate, to Mn(III), which is chelated by organic acids such as oxalate or malonate [9,22,23]. The Mn(III)–organic acid complex, in turn, oxidizes monomeric phenols and phenolic lignin models via formation of a phenoxy radical [9,24–27]. MnP is also capable of oxidizing nonphenolic lignin model dimers and veratryl alcohol, in the presence of a radical mediator [28–30]. White-rot fungi, which produce MnP and laccase but not LiP, are still able to degrade lignin efficiently [31–33], suggesting that these fungi may produce mediators, enabling MnP and/or laccase to cleave nonphenolic lignin substructures. Both glutathione [28] and Tween 80 have been examined as possible mediators, and a peroxy radical has been implicated in the Tween 80 reaction [29,30]. In this study, we show that MnP oxidizes nonphenolic diarylpropane lignin models, in the presence and to a lesser extent in the absence of Tween 80. Previously, two mechanisms were considered for the oxidation and cleavage of nonphenolic lignin dimers by MnP, electron abstraction, and hydrogen abstraction [28–30]. In our current work, we have attempted to differentiate between these two Correspondence to M. H. Gold, Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering at OHSU, 20000 NW. Walker Road, Beaverton, OR 97006–8921, USA. Fax: + 1 503 7481464; Tel.: + 1 503 6460957; E-mail: mhgold@myexcel.com Abbreviations: LiP, lignin peroxidase; MnP, manganese peroxidase; THF, tetrahydrofuran. 1 Present address: Merck, PO Box 2000, RY80L-109, Rahway, NJ 07065, USA. 2 Present address: Department of Chemistry & Biochemistry, Texas Tech University, Lubbock, TX 79409, USA. (Received 1 August 2002, revised 31 October 2002, accepted 21 November 2002) Eur. J. Biochem. 270, 284–292 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03386.x mechanisms. The products formed from the oxidation and cleavage of several different diarylpropane substrates under both aerobic and anaerobic conditions were exam- ined. In addition, we have used 18 O 2 to follow the incorporation of molecular oxygen into the products. Based on the substrates used, the products identified, and the results of stable isotope studies, we propose a mechanism for C a –C b cleavage of the dimers involving hydrogen abstraction. Furthermore, our results do not support the alternative mechanism [30], involving electron abstraction from the aromatic ring. We also show that a-keto diaryl- propane lignin dimeric compounds are degraded by a hydrogen abstraction mechanism to produce benzoic acid derivatives. Materials and methods Synthesis of substrates and products The diarylethane model 1-(3¢,4¢-diethoxyphenyl)-1-oxo-2- (4¢-methoxyphenyl)ethane was prepared as described previ- ously [34,35]. This ketone was treated with methyl iodide in the presence of potassium tertiary butoxide and anhydrous dimethylsulfoxide to obtain the a-keto diarylpropane model 1-(3¢,4¢-diethoxyphenyl)-1-oxo-2-(4¢-methoxyphenyl)pro- pane (IV) [12]. The ketone dimer IV was reduced with either NaBH 4 or NaBD 4 to obtain 1-(3¢,4¢-diethoxyphenyl)-1- hydroxy-2-(4¢-methoxyphenyl)propane (I) or [C a - 2 H 1 ]I, I(D), respectively. Crude products were purified by silica gel preparative thin layer chromatography, using 5% ethyl acetate in hexanes. 1-(3¢,4¢-Diethoxyphenyl)-1,3-dihydroxy- 2-(4¢-methoxyphenyl)propane (VI), 1-(3¢,4¢-diethoxyphe- nyl)-1-oxo-2-(4¢-methoxyphenyl)-3-hydroxypropane (VIII) [9,26], 1,2-dihydroxy-1-(4¢-methoxyphenyl)ethane, and 1-(4¢-methoxyphenyl)-1-oxo-2-hydroxyethane (VII) were prepared as previously described [34,35]. 1-(3¢,5¢-Dimethoxyphenyl)-1-hydroxy-2- (4¢-methoxyphenyl)ethane This product was prepared by the Barbier reaction [36]. To a mixture of magnesium turnings (1.1 mg atom) and 3,5-dimethoxybenzaldehyde (1.1 mmol) in anhydrous tetra- hydrofuran (THF; 10 mL) was slowly added 4-meth- oxybenzyl chloride (1 mmol; 0.14 mL) using a syringe at room temperature under a nitrogen atmosphere and the mixture was refluxed for 24 h. The reaction was quenched with water (10 mL) and extracted with ether (3 · 5mL). The organic layer was washed with water (1 · 10 mL), dried over anhydrous sodium sulfate, rotary evaporated, and the crude preparation was purified by column chro- matography on neutral alumina, using 5% ethyl acetate in hexanes. 1-(3¢,5¢-Dimethoxyphenyl)-1-oxo-2- (4¢-methoxyphenyl)ethane The pyridinium chlorochromate-on-alumina reagent [37] (0.75 mmol; 0.8084 g) was added to a flask containing a solution of 1-(3,5-dimethoxyphenyl)-1-hydroxy-2-(4¢-meth- oxyphenyl)ethane (0.5 mmol) in hexanes (5 mL). After stirring for 4 h at room temperature, the solution was filtered, and washed with diethyl ether (3 · 5mL). The combined filtrates were evaporated to obtain the product. 1-(3¢,5¢-Dimethoxyphenyl)-1-oxo-2- (4¢-methoxyphenyl)propane (X) n-BuLi (0.48 mmol, 0.30 mL of 1.6 M solution in hexanes) was added to a solution of diisopropylamine (0.48 mmol, 0.05 g) in dry THF (2 mL) under a nitrogen atmosphere at 0 °C and the mixture was stirred for 0.5 h [38]. A solution of 1-(3¢,5¢-dimethoxyphenyl)-1-oxo-2-(4¢-methoxyphenyl)- ethane (0.4 mmol) in dry THF (3 mL) was added at 0 °C and stirred for 1 h. Iodomethane (1.6 mmol) was added at 0 °C, and the mixture was stirred for 8 h at room temperature. The reaction was quenched with water (5 mL), extractedwithether(3· 5 mL), dried over anhydrous sodium sulfate, and concentrated by evaporation. The crude preparation was purified by column chromatography on silica gel using 3% ethyl acetate in hexanes. 1-(3¢,5¢-Dimethoxyphenyl)-1-hydroxy-2- (4¢-methoxyphenyl)-1-propane (IX) To a solution of 1-(3,5-dimethoxyphenyl)-1-oxo-2-(4-meth- oxyphenyl)propane (X) (0.2 mmol, 0.06 g) in ethanol (5 mL) was added an excess of sodium borohydride (1.0 g) in three portions and the reaction mixture was stirred for 4 h at room temperature. The reaction mixture was neutralized with dilute hydrochloric acid, extracted with ether (3 · 5 mL), dried over anhydrous sodium sulfate and concentrated by rotary evaporation. The crude preparation of IX was purified by column chromatography on neutral alumina as described above. Chemicals 3¢,4¢-Diethoxybenzaldehyde (II), 4¢-methoxyacetophenone (III), 3¢,4¢-diethoxybenzoic acid (V) and 3¢,5¢-dime- thoxybenzaldehyde (XI) were obtained from Aldrich. 18 O 2 gas (99%) was obtained from Isotec Inc. (Miamisburg, OH, USA). Unless specified otherwise, other aromatic compounds were purchased from Aldrich. Enzymes Manganese peroxidase (MnP) isozyme 1 and lignin peroxi- dase (LiP) isozyme H8 were purified from the extracellular medium of acetate-buffered, agitated, aerobic cultures of P. chrysosporium OGC101 (ATCC 201542) as previously reported [39,40]. Purified MnP and LiP were electrophore- tically homogeneous and had an R z value of  5.0. Enzyme reactions Reactions with MnP were conducted at 28 °Cfor15hin 1mLof50m M sodium malonate, pH 4.5, containing the dimeric substrate (180 l M ), MnSO 4 (0.5 m M ), MnP (5 lg) and Tween 80 (polyoxyethylenesorbitan monooleate) (0.1%). LiP reactions were conducted at 28 °Cfor5min in 1 mL of 20 m M succinate, pH 3.0, containing the substrate (180 l M ) and enzyme (5 lg). The reactions were initiated by the addition of 100 l M H 2 O 2 and were Ó FEBS 2003 Mn peroxidase oxidation of diarylpropane dimers (Eur. J. Biochem. 270) 285 conducted under either aerobic or anaerobic conditions. For anaerobic experiments, reaction mixtures were evacu- ated and flushed with argon twice to ensure removal of oxygen. H 2 O 2 was evacuated and purged with argon separately before addition. Reaction products and unreact- ed substrates were extracted with ethyl acetate, dried over anhydrous sodium sulfate, evaporated under nitrogen, and analyzed directly by GC or as their (acetyl or trimethylsilyl) derivatives by GC or GC-MS, as described previously [18]. Reaction mixtures were also analyzed directly by HPLC. New substrates were also analyzed by NMR. Incorporation of 18 O from 18 O 2 Reactions were carried out in 2-mL vials fitted with rubber septa. All components of the reaction mixture, except H 2 O 2 , were added. The vials were evacuated, flushed with argon, reevacuated, and finally equilibrated with 18 O 2 . Reactions were initiated with the addition of a deoxygenated solution of H 2 O 2 , and incubated for 5 min (LiP reactions) or 15 h (MnP reactions) as described [16,27]. Reaction products were extracted and prepared for analysis as described above. Chromatography and mass spectrometry GC-MS was performed at 70 eV on a Finnigan 4500 mass spectrometer using a Galaxy data system and fitted with a Hewlett Packard (HP) 5790 A gas chromatograph and a 30-m fused silica column (DB-5; J & W Scientific, Folsom, CA, USA). The oven temperature was increased from 70 to 320 °Cat10°Cmin )1 . HPLC analysis of products was conducted with an HP Lichrospher 100 RP8 column, using a linear gradient of 0–100% acetonitrile in 0.05% phos- phoric acid over 10 min, with a flow rate of 1 mLÆmin )1 . Products were detected at 285 nm. Product yields on HPLC were quantitated using calibration curves obtained with standards. Results Oxidation of diarylpropane substrates by MnP Time courses for the oxidation of diarylpropanes I and VI by MnP, in the presence and absence of Tween 80, are shown in Fig. 1. After 12 h of incubation, over 90% of the added diarylpropane I was oxidized in the presence of Tween 80, while in the absence of Tween 80, 24% of the diarylpropane I was oxidized. In addition, while about 30% of the diarylpropane VI was oxidized in the presence of Tween 80 during the 12 h incubation, in the absence of Tween 80, only about 5% of the VI was oxidized. The products of the oxidation reactions are shown in Table 1 and Fig. 2. The products and percent yields of the MnP oxidation of the diarylpropane I in the presence of Tween 80 included the a-keto diarylpropane (IV, 58%), 3,4-dimethoxybenzaldehyde (II, 25%) and 4-methoxyaceto- phenone (III, 21%) (Fig. 2A, Table 1). A small amount ( 2%) of 3,4-diethoxybenzoic acid (V) was also detected (not shown). The oxidation products for diarylpropane I in the absence of Tween 80 included 3,4-diethoxybenzaldehyde (II, 20%) and 4-methoxyacetophenone (III, 14%) (Table 1). No detectable amount of a-keto diarylpropane (IV) was produced in the absence of Tween 80. For the oxidation of the diarylpropane VI, the products included the corres- ponding a-keto diarylpropane (VIII, 12%), 3,4-diethoxy- benzaldehyde (II, 13%) and 4-methoxyphenyl-ketol (VII, 8%) (Fig. 2C, Table 1). In the absence of Tween 80, only about 5% of the added diarylpropane VI was oxidized to yield 3,4-diethoxybenzaldehyde (II, 2.5%) and 4-methoxy- phenyl-ketol (VII, 1.5%) (Table 1). Again, no detectable amount of a-keto diarylpropane VIII was formed in the absence of Tween 80 (Table 1). To further examine the mechanism of diarylpropane oxidation by the MnP system, the diarylpropane IX was prepared. The products of the MnP oxidation of IX in the presence of Tween 80 included the a-keto diarylpropane (X, 32%), 3,5-diethoxybenzalde- hyde (XI, 9.7%) and 4-methoxyacetophenone (III, 11.5%) (Fig. 2E, Table 1). The products of the MnP oxidation of the diarylpropane IX in the absence of Tween 80 included 3,5-dimethoxybenzaldehyde (XI, 11.2%) and 4-methoxya- cetophenone (III, 14.4%). No detectable amount of the a-keto diarylpropane (X) was produced in the absence of Tween 80 (Table 1). To determine possible pathways for the oxidation of the diarylpropanes, the oxidation of the intermediate a-keto diarylpropanes, described above, also were examined. Oxidation of the a-keto diarylpropane IV by MnP, in the presence of Tween 80, yielded 4-methoxyac- etophenone (III, 22%) and 3,4-diethoxybenzoic acid (V, 13%) (Table 1). When the oxidation of the a-keto diarylpropane (IV) was carried out in the absence of Tween 80, yields of the products 4-methoxyacetophenone (III) and 3,4-diethoxybenzoic acid (V) were decreased by approxi- mately threefold (Table 1). In the presence of Tween 80, the a-keto diarylpropane (VIII) was oxidized to 3,4-diethoxybenzoic acid (V, 1.2%) and 4-methoxyphenyl-ketol (VII, 3%) (Table 1). Less than 1% yield of these products occurred when the reaction was Fig. 1. Oxidation of the diarylpropanes I (d,s)andVI(m,n)byMnP, in the presence (d,m) and absence (s,n)ofTween80.Enzyme reac- tions were carried out in 50 m M malonate for 12 h, extracted with ethyl acetate, and the amount of substrate remaining was quantitated by HPLC as described in the text and in the legend to Table 1. 286 G. V. B. Reddy et al. (Eur. J. Biochem. 270) Ó FEBS 2003 conducted in the absence of Tween 80. Finally, in the presence of Tween 80, oxidation of a-keto diarylpropane (X) resulted in the formation of 3,5-dimethoxybenzoic acid (XII, 5.0%) and the 4-methoxyacetophenone (III, 6.7%). When the reaction was conducted in the absence of Tween 80, the same products were formed but in lower yield (Table 1). Effect of deuterium on the oxidation of the diarylpropane (I) When the a-deuterated diarylpropane I(D) was oxidized by MnP in the presence of Tween 80, the yields of the products, 3,4-diethoxybenzaldehyde (II, 28%), and 4-methoxyaceto- phenone (III, 21%), were similar to those obtained with the undeuterated substrate I, whereas the a-keto diarylpropane product (IV, 9%) was formed in a sixfold lower amount (Table 1). Oxidation of diarylpropane substrates by LiP Oxidation of diarylpropane I by LiP yielded 3,4-diethoxy- benzaldehyde (II, 70%) and 4-methoxyacetophenone (III, 61%) in approximately 5 min (Fig. 2G). The a-keto diarylpropane IV product was not detected in this reaction. Identical results were obtained with a-deuterated diarylpro- pane I(D). When the diarylpropane (IX) was incubated with LiP, no products were formed and the amount of substrate remained unchanged (Table 1). Finally, when either of the a-keto diarylpropanes (IV or X) was incubated with LiP, no products were observed and the amount of the substrates remained unchanged (Table 1). Diarylpropane oxidation under either 18 O 2 or argon When the oxidation of the diarylpropane I, by MnP, was performed under 18 O 2 , 0.8 atoms of 18 O was incorporated into the 4-methoxyacetophenone (III), whereas 3,4-dieth- oxybenzaldehyde (II) did not contain detectable amounts of 18 O (Table 2). When the oxidation of a-keto diarylpropane (IV) by MnP was carried out under 18 O 2 , 18 Owas incorporated into both products. Approximately 0.8 atoms of 18 O were incorporated into 3,4-diethoxybenzoic acid (V) and 0.75 atoms into the 4-methoxyacetophenone (III) (Table 2). The percentage of 18 O incorporated was estima- ted by the ratio of the M + /(M + +2)peaksinthemass spectrum of the products. Neither the diarylpropane (I) nor the a-keto diarylpropane (IV) was oxidized by MnP when the reaction was carried out under argon, either in the presence or absence of detergent (data not shown). Discussion Under ligninolytic conditions, P. chrysosporium secretes two extracellular peroxidases, MnP and LiP, which are mainly responsible for the initial depolymerization of lignin in wood [2,3,22,41]. While both enzymes are iron heme- containing peroxidases, their detailed reaction mechanisms differ considerably. LiP abstracts an electron from the Table 1. Products obtained from the oxidation of nonphenolic diarylpropane substrates I, I(D),VI, IX and a-keto diarylpropane substrates IV, VIII, X by MnP a or LiP b . t ¼ trace. Tween 80 (+/–) Products formed (mol %) Dimeric substrate Enzyme II III IV V VII VIII X XI X Nonphenolic diarylpropane substrates I, I(D),VI, IX I MnP – 20 14 IMnP+2521582 I(D) MnP + 28 21 9 1 VI MnP – 2.5 1.5 VI MnP + 13 8 12 IX MnP – 14.4 11.2 IX MnP + 11.5 32 9.7 I LiP – 70 61 IX LiP – no reaction a-Keto diarylpropane substrates IV, VIII, XII by MnP a or LiP b IV MnP – 7 3.5 IV MnP + 22 13 VIII MnP – t t VIII MnP + 1.2 3 X MnP – 4.4 3.5 X MnP + 6.7 5.0 IV LiP – no reaction X LiP – no reaction a MnP reactions were conducted at 28 °C for 15 h in 50 m M malonate, pH 4.5, containing enzyme, substrate, MnSO 4 , and H 2 O 2 in the presence or absence of Tween 80, and the products were analyzed by HPLC and GC-MS as described in the text. Amount of products formed (mol percentage) are shown. Each reaction was run in triplicate; the results are the mean values. b Enzyme reactions were conducted at 28 °C for 5 min in 20 m M succinate, pH 3.0, containing enzyme, H 2 O 2 , and substrate, and the products were analyzed by HPLC and GC-MS as described in the text. Amount of products formed (mol percentage) are shown. Ó FEBS 2003 Mn peroxidase oxidation of diarylpropane dimers (Eur. J. Biochem. 270) 287 substrate aromatic ring, generating an aryl cation radical, which decomposes further by enzymatic and nonenzymatic processes [2,3,11–13,15,16,41,42]. In contrast, MnP oxidizes Mn(II) to Mn(III) and the latter oxidizes the aromatic substrate [2,22,24,39]. In the absence of radical mediators, MnP mainly oxidizes phenolic lignin substructures [9,25–27]. However, in the presence of mediators, MnP is able to oxidize nonphenolic lignin substructures [28,29]. In vitro experiments demon- strate that MnP cleaves nonphenolic b-arylether dimers in the presence of Tween 80, an unsaturated fatty acid containing detergent [29,30,43]. In contrast, Tween 20, which contains a saturated fatty acid, does not act as a mediator [29]. These studies suggest the involvement of lipid-derived peroxy radicals as mediators in the oxidation of nonphenolic lignin model compounds. However, the detergent-mediated mechanism of oxidation is not clearly understood. It has been proposed that fatty acid-based peroxy radicals can oxidize b-aryl ether lignin model compounds by abstracting either a hydrogen from the C1 position to produce a carbon-centered radical, or an electron from the aromatic ring to produce an aryl cation radical intermediate [29,30]. The C1-centered radical, resulting from a-hydrogen abstraction, has been proposed to undergo two subsequent reactions. The addition of oxygen at C1 followed by loss of HOO Æ yields an uncleaved a-keto dimer, which has been proposed to undergo homo- lytic C2–O fission to expel a phenoxy radical [30]. Alternatively, it has been proposed that a cation radical intermediate is formed, which subsequently undergoes C a –C b cleavage, similar to LiP-catalyzed oxidations [30]. However, the evidence in these studies for electron abstrac- tion by a peroxy radical is not convincing. Reduced glutathione also acts as a mediator in the oxidation of veratryl alcohol and nonphenolic b-ether structures by MnP. Enzymatically generated Mn(III) oxidizes the thiol to thiyl radical, which initiates substrate oxidation through hydrogen, but not electron, abstraction [28]. To determine the relative importance of a-hydrogen vs. electron abstraction, we studied the mechanism of oxidation of nonphenolic diarylpropane lignin model compounds by MnP, in the presence and absence of the unsaturated fatty acid-based detergent Tween 80. As the initial products of b-ether dimer cleavage undergo further reactions [30], elucidating the initial cleavage mechanism is difficult. Therefore, we selected b-1-type lignin model substructures as substrates, because they produce relatively stable primary products, which do not undergo extensive subsequent MnP- catalyzed oxidation. The diarylpropane I is oxidized by MnP in the presence and absence of the radical mediator Tween 80; however, the oxidation rates are approximately fourfold greater in the presence of Tween 80 than in its absence. In the presence of Tween 80, the diarylpropane I undergoes a–b cleavage to produce 3,4-diethoxybenzaldehyde (II) and 4-methoxy- acetophenone (III) as well as C a oxidation to produce the corresponding a-keto diarylpropane (IV), which is the dominant product. In contrast, in the absence of Tween 80, diarylpropane (I) is oxidized at a slower rate to produce only Table 2. Incorporation of 18 O during the oxidation of the nonphenolic diarylpropane I and the a-keto diarylpropane IV by MnP. Substrate Product m/z 18 O incorporated (%) a I II 196 (M + +2) – III 152 (M + +2) 70 I V 212 (M + +2) 80 III 152 (M + +2) 75 a18 O incorporation divided by the total oxygen incorporation · 100. Enzyme reactions were carried out in duplicate under 18 O 2 in 50 m M malonate, containing enzyme, substrate, Tween 80, MnSO 4 , and H 2 O 2 for 15 h at 28 °C. Products were extracted and quanti- tated by GC-MS as described in the text. Fig. 2. Products identified during the oxidation of nonphenolic diaryl- propanes (reactions A, C, and E) and a-keto diarylpropanes (reactions B, D, and F) by homogeneous MnP and LiP (G). MnP reactions were carried out in 50 m M malonate buffer in the presence or absence of Tween80for15hat28°C as described in the text. LiP reactions were carried out in 50 m M succinate at 28 °C for 5 min as described in the text. Products were extracted and analyzed by HPLC and GC-MS, as described in the text. Yields of products are presented in Table 1. 288 G. V. B. Reddy et al. (Eur. J. Biochem. 270) Ó FEBS 2003 the a–b cleavage products. Under these conditions, the a-keto diarylpropane product (IV) is not observed, suggest- ing that C1 oxidation does not occur. Although only about 20% of the diarylpropane (I) is oxidized by MnP in the absence of Tween 80, this finding is surprising, because it was previously thought that MnP is incapable of attacking nonphenolic lignin models in the absence of radical media- tors [28–30]. However, recent evidence suggests that metal– oxo complexes as well as Mn(III) complexes are able to abstract an H atom from certain aromatic compounds [44,45]. Corresponding products are formed during the oxidation of the diarylpropane VI, although at a lower rate. The lower reactivity of VI, when compared to I, may be due to the stearic hindrance offered by the hydroxyl group at C3, which may inhibit hydrogen abstraction at C2. When the MnP reaction was conducted under anaerobic conditions, in the presence or absence of the detergent, oxidation of these diarylpropanes does not occur, indicating that molecular oxygen is required. The LiP oxidation of diarylpropane substructures has been well studied and proceeds through an aryl cation radical intermediate [11,15,16]. Therefore, we compared the LiP and MnP oxidation of the diarylpropane I under a variety of conditions. LiP oxidizes over 70% of the diarylpropane I in 5 min to produce the C a –C b cleavage products 3,4-diethoxybenzaldehyde (II) and 4-methoxy- acetophenone (III). Furthermore, unlike the MnP reaction, the LiP-catalyzed oxidation of the diarylpropane I proceeds efficiently under both aerobic and anaerobic conditions [16]. Finally, the a-keto diarylpropane is not formed in the LiP- catalyzed oxidation of the diarylpropane I [12,16], whereas it is the major product in the MnP reaction. As the LiP oxidation of the diarylpropane I proceeds by the formation of an aryl cation radical [16], these results suggest that a cation radical is not an intermediate in the MnP-catalyzed reactions reported here. To pursue this question further, we examined the oxida- tion of 1-(3¢,5¢-dimethoxyphenyl)-1-hydroxy-2-(4¢-methoxy- phenyl)-1-propane (IX) by MnP and LiP. As expected, LiP was not able to oxidize the diarylpropane IX, owing to the lack of an electron-donating methoxy group at the para position of the A ring. This strongly suggests that a cation radical is difficult to produce with this substrate. In contrast, in the presence or absence of Tween 80, MnP oxidizes the diarylpropane (IX)-producing products corresponding to those produced during the oxidation of the diarylpropane (I). These results support our view that electron abstraction is an unlikely mechanism for the MnP-catalyzed oxidation of these diarylpropane substrates. The presence of deuterium at the C1 position of the diarylpropane (I) (Fig. 2A) had a strong influence on the yield of the a-keto diarylpropane (IV) formed. When deuterated diarylpropane I(D) was used as the substrate, the yield of the a-keto diarylpropane (IV) was decreased by about 6 fold, suggesting the involvement of H-abstraction. However, yields of the C a –C b cleavage products 3,4- diethoxybenzaldehyde (II) and 4-methoxyacetophenone (III) remained almost identical, for the deuterated and undeuterated substrates. This result is in contrast to that with the LiP-catalyzed oxidation of I(D), which is not affected by the presence of deuterium. Oxidation of the diarylpropane I to 3,4-diethoxybenzal- dehyde (II), 4-methoxyacetophenone (III), and a-keto diarylpropane (IV) can be explained on the basis of an initial hydrogen abstraction reaction at one of two posi- tions. A benzylic radical can be generated at C1, which is resonance stabilized by aromatic ring A, or at C2, which is stabilized by ring B, as well as by the adjacent methyl group through hyperconjugation. However, the presence of a hydroxyl group at C1 renders the C1–H bond more labile than the C2–H bond. A radical generated at C1 would add to O 2 to form a peroxyl radical, which would then eliminate HOO Æ to yield the a-keto diarylpropane IV, as shown in Fig. 3. This mechanism is similar to that reported earlier for the thiyl radical-mediated oxidation of nonphenolic lignin dimers by MnP [28]. Alternatively, a radical generated at C2 would add to O 2 and the resulting peroxy radical could abstract a hydrogen atom to form an unstable hydroper- oxide. The latter would undergo C a –C b cleavage with the elimination of H 2 O to form 3,4-diethoxybenzaldehyde (II) and 4-methoxyacetophenone (III) (Fig. 3). Addition of deuterium at the C1 position would slow the hydrogen abstraction at C1, but would have no effect on the formation of a radical at C2 as we observed. Fig. 3. Proposed hydrogen abstraction mechanism for the oxidative cleavage of the diarylpropane (I) by MnP in the presence of Tween 80. Ó FEBS 2003 Mn peroxidase oxidation of diarylpropane dimers (Eur. J. Biochem. 270) 289 When the oxidation of the diarylpropane I is performed in the presence of 18 O 2 , 18 O is incorporated only into the acetophenone (III). The benzaldehyde (II) did not contain any 18 O. This is similar to the LiP-catalyzed oxidation (electron abstraction), where 18 O is incorporated only into benzylic radical-derived products [16]. Therefore, hydrogen abstraction at C2 and electron abstraction from the aromatic ring result in C a –C b cleavage and 18 O incorpor- ation only into the acetophenone (III). The crucial differ- ence is that the hydrogen abstraction reaction catalyzed by MnP, unlike the LiP-catalyzed oxidation, does not proceed in the absence of O 2 [16]. The a-keto diarylpropane (IV) also is oxidized by MnP in the presence of Tween 80. The reaction products included 4-methoxyacetophenone (III) and 3,4-diethoxybenzoic acid (V). The benzaldehyde (II) is not detected as a product of the reaction. When the reaction is carried out in the absence of Tween 80, the oxidation rate is about 3-fold lower, but again, 4-methoxyacetophenone (III) and 3,4-diethoxyben- zoic acid (V) were detected. The a-keto dimer (IV) was not oxidized under anaerobic conditions, indicating that oxygen is required for this reaction as well. In contrast to the results with MnP, LiP is not able to oxidize the dimer (IX), nor any of the a-keto diarylpropane dimers. Furthermore, LiP oxidation of the diarylpropane (I) does not yield an a-keto diarylpropane product. Finally, the LiP oxidation of diarylpropanes occurs in the absence of molecular oxygen. Because LiP oxidizes these substrates by electron abstraction to form an aryl cation radical, these results strongly suggest electron abstraction is not occurring in the MnP reactions. The mechanism we propose for the MnP oxidation of the a-keto diarylpropane (IV) is shown in Fig. 4. The C2-centered radical generated after C2–H abstraction would add to oxygen to form a peroxy radical. The latter can react with the carbonyl group, producing an unstable dioxetane, which decomposes to 4-methoxyacetophenone (III) and 3,4-diethoxybenzoic acid (V) (Fig. 4). When the oxidation of a-keto diarylpropane (IV) by MnP is conducted in the presence of 18 O 2 , 18 O is incorpor- ated into 4-methoxyacetophenone (III) and 3,4-diethoxy- benzoic acid (V), supporting the mechanism shown in Fig. 4. MnP slowly oxidizes the a-keto diarylpropane (IV) even in the absence of Tween 80. Formation of aromatic acids from nonphenolic lignin model compounds has not been reported previously. In this study we also identified a small amount of 3,4-diethoxybenzoic acid during the oxidation of diarylpropane I by MnP in the presence of Tween 80. This is most likely due to the further oxidation of either 3,4-diethoxybenzaldehyde (II) or the a-ketodiaryl- propane (IV) produced during the oxidation of I. We have confirmed this secondary oxidation process in a separate reaction using 3,4-diethoxybenzaldehyde (II) as a substrate (data not shown). The oxidation of a benzaldehyde to benzoic acid by MnP has been discussed recently [29]. According to the mechanisms shown in Fig. 4, products formed from the C a –C b cleavage should be in equal molar ratio. However, we observe that yields of the acetophenone (III) are slightly lower than for the benzaldehyde (II). It is likely that some of the products are degraded further by the enzyme. We are investigating the nature of the oxidant in the reaction of the diarylpropane I by MnP in the absence of unsaturated detergent (Tween 80). However, both metal oxo complexes and Mn(III) complexes are capable of abstracting a hydrogen atom from organic compounds [44,45]. In addition, a malonic acid-generated free radical may be involved in the process [46]. In conclusion, the present study shows that MnP can catalyze C a oxidation and C a –C b cleavage of nonphenolic diarylpropane model compounds in the presence of Tween 80 and at a lower rate in its absence. Based on the products formed under various conditions, a mechanism based on electron abstraction can be ruled out; rather, these compounds apparently are oxidized solely via hydrogen abstraction mechanisms. This study also shows that the a-keto-1,2-diarylpropane is oxidized via hydrogen abstrac- tion to produce an aromatic acid and an acetophenone product. Acknowledgments This research was supported by Grants MCB-9808430 from the National Science Foundation and DE-FG03–96ER30325 from the Division ofEnergy Biosciences, U.S.Department of Energy (to M.H.G). Fig. 4. 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