Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii

Transcription

1 Eur. J. Biochem. 268, (2001) q FEBS 2001 Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii H 2 O 2 generation and the influence of Mn 21 Víctor Gómez-Toribio, Angel T. Martínez, María J. Martínez and Francisco Guillén Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Formation of H 2 O 2 during the oxidation of three ligninderived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn 31 formed from Mn 21 oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all ph 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn 21 -mediated activity of VP in the absence of exogenous H 2 O 2 (unlike glyoxylate and oxalate buffers). In the absence of Mn 21, efficient hydroquinone oxidation by VP was dependent on exogenous H 2 O 2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O 2 ) that spontaneously dismutated to H 2 O 2 and O 2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H 2 O 2 (a quinone/h 2 O 2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn 21, exogenous H 2 O 2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H 2 O 2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O 2 production through semiquinone autooxidation and O 2 reduction by Mn 21. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn 21 oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn 21 greatly increased hydroquinone oxidation efficiency. Keywords: versatile ligninolytic peroxidase; hydroquinone oxidation; hydrogen peroxide production; Pleurotus eryngii. The ligninolytic system of white-rot fungi is composed of a variety of oxidative enzymes, i.e. lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase [1,2]. Moreover, the existence of a versatile peroxidase (VP) sharing LiP and MnP catalytic properties has been recently reported [3 5]. The H 2 O 2 required by ligninolytic peroxidases is generated by several direct and indirect enzymatic mechanisms. Direct reduction of O 2 to H 2 O 2 is catalyzed by the extracellular enzymes glyoxal and aryl-alcohol oxidases [6,7]. The other mechanisms that provide H 2 O 2 involve the production and reduction of superoxide anion radicals (O 2 ) that are generated by autoxidation of some reaction products of ligninolytic enzymes when acting on several fungal metabolites and lignin degradation intermediates [8 10]. Among Correspondence to F. Guillén, Centro de Investigaciones Biológicas, CSIC, Velázquez 144, E Madrid, Spain. Fax: , Tel.: , guillen@cib.csic.es Abbreviations: BQ, 1,4-benzoquinone; BQH 2, 1,4-benzohydroquinone; DBQ, 2,6-dimethoxy-1,4-benzoquinone; DBQH 2, 2,6-dimethoxy- 1,4-benzohydroquinone; HRP, horseradish peroxidase; LiP, lignin peroxidase; MBQ, 2-methoxy-1,4-benzoquinone; MBQH 2, 2-methoxy- 1,4-benzohydroquinone; MnP, manganese peroxidase; SOD, superoxide dismutase; VP, versatile peroxidase Enzymes: catalase (EC ); horseradish peroxidase (EC ); manganese peroxidase (EC ); superoxide dismutase (EC ). (Received 21 February 2001, revised 1 May 2001, accepted 13 July 2001) ligninolytic enzymes, most studies on H 2 O 2 production via O 2 have been carried out using MnP. Like other peroxidases [11,12], MnP is able to oxidize several substrates without the need of exogenous H 2 O 2 as this compound is produced during the reaction. Such substrates include NADPH, thiols, and several organic acids produced by white-rot fungi, e.g. oxalic, glyoxylic, and malonic acids [9,13 17]. The peroxidation cycle of MnP is similar to that of other peroxidases but it is unique in its dependence on Mn 21 [18]. The reaction of the resting enzyme with H 2 O 2 produces the two-electron oxidized species compound I. Stepwise reduction of compound I by two substrate-derived electrons produces compound II, and subsequently the resting enzyme. Whereas reducing substrates of compound I are, apart from Mn 21, phenols and arylamines, the conversion of compound II to the resting enzyme is strictly dependent on Mn 21. The Mn 31 produced in these reactions oxidizes in turn the typical substrates of peroxidases, including those mentioned above supporting H 2 O 2 generation. The existence of different H 2 O 2 -generating mechanisms in white-rot fungi is of interest because lignin degradation depends on the presence of H 2 O 2, not only for peroxidase activities but also for the generation of hydroxyl radicals, and not all the fungal species produce extracellular oxidases [19]. Furthermore, the composition of the ligninolytic system differs qualitatively and quantitatively depending on the fungal species and the conditions of growth [20 22]. In this respect, we have recently reported that laccase, which is widely distributed among white-rot fungi, can participate in

2 4788 V. Gómez-Toribio et al. (Eur. J. Biochem. 268) q FEBS 2001 H 2 O 2 production through the oxidation of hydroquinones to semiquinones, reducing O 2 to O 2 that dismutates to H 2 O 2 and O 2 [10]. A similar H 2 O 2 -producing mechanism could involve ligninolytic peroxidases as hydroquinones are substrates of these enzymes [23,24]. Hydroquinones are produced by white-rot fungi by reducing quinones that derive from both lignin degradation and the oxidation by ligninolytic enzymes of the large amount of aromatic metabolites produced by these fungi [25,26]. Oxidation of hydroquinones by plant and animal peroxidases in the absence of added H 2 O 2 has been reported previously [27,28]. Based on these facts, we decided to investigate the production of H 2 O 2 during the oxidation of lignin-derived hydroquinones by the VP secreted by Pleurotus eryngii [3]. This third type of ligninolytic peroxidase, like LiP and MnP, has the capacity to oxidize nonphenolic aromatic compounds and Mn 21, respectively. Unlike MnP, VP does not require Mn 21 to close its catalytic cycle, being able to oxidize phenolic compounds both directly and through Mn 31 generation. As Mn 21 considerably increases the efficiency of VP to oxidize phenols [3], and promotes both hydroquinone oxidation and H 2 O 2 production by laccase [29,30] and HRP [27], we investigate here the effect of this ion on the production of H 2 O 2 during the oxidation of hydroquinones by VP. MATERIALS AND METHODS Chemicals and enzymes H 2 O 2 (Perhydrol 30%) was obtained from Merck. 1,4-Benzohydroquinone (BQH 2 ), 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzohydroquinone (MBQH 2 ) and 2,6- dimethoxy-1,4-benzoquinone (DBQ) were purchased from Aldrich. 2,6-Dimethoxy-1,4-benzohydroquinone (DBQH 2 ) was prepared from DBQ by reduction with sodium borohydride [31] and 2-methoxy-1,4-benzoquinone (MBQ) was produced from MBQH 2 by oxidation with silver oxide [32]. Stock solutions of hydroquinones were prepared in nitrogen-saturated deionized water acidified with 2 mm HCl, and kept frozen until use. All other chemicals used were of analytical grade. VP allelic variant PL1 from P. eryngii liquid culture [5] and MnP isoenzyme 1 from Phanerochaete chrysosporium were produced and purified as previously reported by Martínez et al. [3] and Palma et al. [33], respectively. HRP (type II), bovine liver superoxide dismutase (SOD) and catalase were obtained from Sigma. Enzymatic assays Unless otherwise stated, peroxidase activities were assayed in 20 mm sodium tartrate buffer, ph 5, using 500 mm MBQH 2 or 100 mm MnSO 4 as substrates. Reactions were startedbyadditionof100mmh 2 O 2. The production of MBQ and Mn 31 tartrate complex was followed spectrophotometrically (1 360 ¼ 1252 M:cm 21 and ¼ 6500 M:cm 21, respectively). International units (mmol:min 21 ) of peroxidase enzymatic activity were used. Production of quinone during the peroxidative oxidation of BQH 2 and DBQH 2 was followed spectrophotometrically (1 247 ¼ M:cm 21 and ¼ 562 M:cm 21, respectively). These assays were performed at room temperature ( C). Anaerobic enzymatic experiments were carried out by purging with nitrogen during the whole incubation period. Analytical procedures Protein concentration was determined using the Bradford reagent (Biorad) and bovine serum albumin as standard. Total H 2 O 2 production during nonenzymatic oxidation of MBQH 2 was estimated by using HRP and phenol red [34]. The reaction mixture contained an appropriate amount of sample, 2.5 U HRP (as specified by the manufacturer), 2.8 mm phenol red and 100 mm phosphate buffer, ph 6. After oxidation of phenol red by HRP (5 min) NaOH was added (200 mm final concentration) and the absorbance was read at 610 nm. Samples preincubated with 100 U:mL 21 catalase were used as blanks. A standard curve of H 2 O 2 was prepared with dilutions of Perhydrol 30% processed in the same way. The H 2 O 2 concentration in the commercial solution was calculated spectrophotometrically (1 230 ¼ 81 M:cm 21 ). Time course of H 2 O 2 production during the Mn 21 -dependent oxidation of organic acids by VP was also determined through the oxidation of phenol red present in the reaction mixtures. Phenol red oxidation was stopped by adding NaOH to samples, that were processed spectrophotometrically as described above. RESULTS In order to select a buffer for studies on H 2 O 2 production from oxidation of hydroquinones by VP in the presence of Mn 21, the possibility that H 2 O 2 might be produced from the Mn 21 -dependent oxidation of glyoxylate, oxalate, succinate and tartrate was investigated. Glyoxylate and oxalate have been reported to support the activity of MnP from the whiterot fungi P. chrysosporium [9,15] and Ceriporiopsis subvermispora [16] in the absence of exogenous H 2 O 2, using succinate as buffer. Tartrate was evaluated because it has been used in most studies on P. eryngii VP [3,24,35]. H 2 O 2 production was monitored for 210 min in reactions containing the above organic acids (as buffers, ph 5), VP, Mn 21 and phenol red. Both glyoxylate and oxalate supported phenol red oxidation by VP in the absence of exogenous H 2 O 2, demonstrating the production of this compound during the reaction (Fig. 1). In agreement with the results reported for MnP: (a) glyoxylate was more Fig. 1. H 2 O 2 production during the Mn 21 -dependent oxidation of organic acids by VP. Reactions contained 80 nm VP (25 mu on MBQH 2 ), 100 mm Mn 21, 2.8 mm phenol red, and 20 mm either glyoxylate (G), oxalate (O), tartrate (T), or succinate (S) buffer, ph 5. When indicated, reactions contained 4 mm H 2 O 2. Means of three replicates are shown (95% confidence limits were less than 5% of the mean).

3 q FEBS 2001 H 2 O 2 generation by Pleurotus veringii peroxidase (Eur. J. Biochem. 268) 4789 Table 1. VP activity on lignin-derived hydroquinones. Reactions were performed in 20 mm tartrate buffer, ph 5, and contained 32 nm VP, 500 mm hydroquinone, and 100 mm H 2 O 2. Means of three replicates and 95% confidence limits are shown. Hydroquinone VP (mu) Wavelength (nm) 1 (M:cm 21 ) BQH ^ ,028 MBQH ^ ,252 DBQH ^ reactive than oxalate, (b) H 2 O 2 production from oxalate was preceded by a lag period that was shortened by the presence of exogenous H 2 O 2, and (c) the reactions were strictly dependent on Mn 21. It is likely that the lag period was not observed in the case of glyoxylate because it was shorter than 15 min (the time at which the first sample was taken). Neither succinate nor tartrate supported H 2 O 2 generation. Even in the presence of 4 mm H 2 O 2, demonstrated to produce 8 mm Mn 31 in parallel experiments lacking phenol red, no more H 2 O 2 was detected than added. Based on these results, tartrate buffer was selected for experiments involving hydroquinone oxidation. In the absence of Mn 21, VP catalyzed the oxidation of the three hydroquinones derived from p-hydroxyphenyl, guaiacyl and syringyl units of lignin, i.e. BQH 2, MBQH 2 and DBQH 2, respectively. Oxidation of these hydroquinones (50 mm) by VP in the presence of stoichiometric amounts of H 2 O 2 was evaluated by changes in the absorption spectrum of the reaction mixtures, which gave rise to spectra identical to those of authentic BQ, MBQ and DBQ (data not shown). Table 1 shows the wavelengths of new peaks that were used to calculate the molar extinction coefficient of quinones under the reaction conditions used, and to determine initial quinone production rate. It was observed that this rate slightly increased with the number of methoxyl substituents of hydroquinones. Kinetic constants of MBQH 2 oxidation by VP have been previously reported [24,35] The production of H 2 O 2 during the oxidation of MBQH 2 by VP in the presence of substoichiometric amounts of exogenous H 2 O 2 was investigated by studying the effect of O 2 on the MBQ/H 2 O 2 molar ratio. It was expected that under anaerobic conditions semiquinone radicals generated by VP (reaction 1) were converted into quinone mainly via disproportionation (reaction 2) [36]. Thus, 1 mol of quinone should be produced for every mol of H 2 O 2 consumed. In the presence of O 2, a second pathway of quinone formation would involve semiquinone autoxidation (reaction 3) with the concomitant production of O 2 and, by its dismutation, H 2 O 2 (reaction 4). Due to H 2 O 2 formation, the MBQ/H 2 O 2 molar ratio in the peroxidase reaction consuming a limited amount of H 2 O 2 should be higher than 1. 2MBQH 2 1 H 2 O 2!2MBQ 2 1 2H 2 O 1 2H 1 2MBQ 2 1 2H 1 $ MBQ 1 MBQH 2 ð2þ MBQ 2 1 O 2 $ MBQ 1 O 2 2 ð3þ 2O H 1! O 2 1 H 2 O 2 ð4þ Figure 2A shows the amount of quinone produced in reactions containing 100 mm MBQH 2 and 10 mm H 2 O 2, ð1þ from which MBQ/H 2 O 2 ratio was calculated. Under anaerobic conditions 10 mm H 2 O 2 gave rise to 10 mm MBQ, whereas in the presence of O 2 the MBQ/H 2 O 2 ratio increased from 1.0 to 2.2. Omitting any component from the reaction mixture resulted in a complete prevention of MBQH 2 oxidation. It was clear therefore that H 2 O 2 was generated during the reaction as MBQ was produced in an amount greater than equimolar to the H 2 O 2 added. In order to confirm H 2 O 2 production, the effect of factors promoting semiquinone autoxidation was evaluated. The removal of O 2 from the reaction mixture due to either its dismutation catalyzed by SOD or its reduction by Mn 21 (reaction 5) would shift the equilibrium of semiquinone autoxidation (reaction 3) towards the right. This way, production of quinone by the latter reaction would be favoured against semiquinone disproportionation and consequently H 2 O 2 and quinone levels should increase. O Mn H 1 $ H 2 O 2 1 Mn 31 ð5þ As shown in Fig. 2A, no significant effect on MBQ levels was caused by SOD and Mn 21 in reactions lacking O 2. However, under aerobic conditions SOD and Mn 21 increased the MBQ/H 2 O 2 ratio from the anaerobic control experiment 6.1- and 10-fold, respectively (it should be noted that MBQH 2 was completely oxidized in reactions with Mn 21 ). Figure 2B shows the time course of MBQ production under aerobic conditions, including control reactions lacking H 2 O 2. The presence of SOD and Mn 21 raised the initial quinone production rate 1.4- and 17.5-fold, respectively. The greater increase produced by Mn 21 can be Fig. 2. Influence of O 2, SOD, and Mn 21 in the oxidation of MBQH 2 by VP. Reactions contained 20 mm tartrate buffer, ph 5, 160 nm VP (50 mu on MBQH 2 ), 100 mm MBQH 2, 10 mm H 2 O 2, and when indicated 100 U:ml 21 SOD, and 100 mm Mn 21. (A) Total amount of quinone produced under anaerobic and aerobic conditions (error bars represent ^ 95% confidence limits). (B) Time course of quinone production under aerobic conditions (the results shown are from one experiment typical of three).

4 4790 V. Gómez-Toribio et al. (Eur. J. Biochem. 268) q FEBS 2001 explained by taking into consideration that it is possible to produce twice as much H 2 O 2 from O 2 with Mn 21 as with SOD (reactions 5 and 4, respectively), and additionally that some portion of the hydroquinone could also be oxidized by the Mn 31 (reaction 6) generated through the oxidation of Mn 21 by O 2 (reaction 5). MBQH 2 1 Mn 31! MBQ 2 1 Mn H 1 ð6þ Contrary to that observed under anaerobic conditions, the absence of H 2 O 2 in reactions containing O 2 did not prevent MBQH 2 oxidation. Over the 4-min timespan of the assays, no quinone was produced in controls lacking both H 2 O 2 and VP (buffered solutions of MBQH 2 ). However, in reactions containing MBQH 2 and VP, MBQ was slowly produced at a rate of 0.4 mm:min 21 (Fig. 2B). Promotion of semiquinone autoxidation, caused by the presence of SOD in the reaction, increased MBQ production rate 2.5-fold. Except for the existence of a distinct lag period of a few seconds, MBQ production in H 2 O 2 -lacking blanks containing Mn 21 was similar to that observed in complete reactions carried out in the presence of 10 mm H 2 O 2, with MBQH 2 also being fully oxidized. These results demonstrated MBQH 2 oxidation by VP in the absence of exogenous H 2 O 2, which was dramatically increased by Mn 21. One interesting finding was observed in long-term aerobic controls lacking both H 2 O 2 and VP. Near complete oxidation of MBQH 2 (89%) was achieved after 100 min in reactions containing 100 mm Mn 21 (Fig. 3A). The production of MBQ began after a lag period of 15 min, describing a sigmoidal curve. By raising the concentration of Mn 21, MBQH 2 was fully oxidized, the lag period shortened, and the maximal MBQ production rate increased. In order to get reproducible results in these experiments, it was essential to start the reaction just after MBQH 2 was thawed, because the lag period shortened as the time MBQH 2 was kept in solution at room temperature increased. In this respect, Fig. 3A also shows chemical oxidation of MBQH 2 in reactions lacking Mn 21, with 3 mm MBQ produced over the 100-min reaction time. From these results it was apparent that after some MBQH 2 was oxidized to semiquinone radicals, probably by O 2 in a reaction catalyzed by free metal ions present in trace amounts (sum of reactions 7 and 8), the Mn 31 produced by the sequence of reactions 3 and 5 propagated MBQH 2 oxidation (reaction 6). MBQH 2 1 Me n! MBQ 2 1 Me n H 1 Me n 21 1 O 2! Me n 1 O 2 2 ð8þ As these reaction mixtures did not contain VP, the H 2 O 2 generated through reactions 4 and 5 was accumulated. Figure 3B shows the H 2 O 2 levels in reactions containing Mn 21, estimated after complete oxidation of MBQH 2 to avoid underestimation caused by the likely reaction of HRP with MBQH 2 instead of phenol red. H 2 O 2 levels ranged from 90.8 to 79.9 as the concentration of Mn 21 increased from 100 to 1000 mm. This negative correlation could be explained considering that reaction 5 is reversible [37], and/ or that the conversion of semiquinone radicals into MBQ could be caused by Mn 31. As observed by comparing the quinone production curve from the nonenzymatic reaction shown in Fig. 3A, containing 100 mm Mn 21 with that obtained in the equivalent enzymatic reaction shown in Fig. 2B (VP 1 Mn 21 H 2 O 2 ), the presence of VP dramatically decreased the lag period and increased the maximal quinone production rate. These results indicated that shortly after a minor amount of H 2 O 2 was produced (probably by the sequence of reactions 7, 8, 3 and 5), VP started to oxidize MBQH 2 quite efficiently. As mentioned above, VP has the unique ability to oxidize phenolic compounds both directly (reaction 1) and through ð7þ Fig. 3. Influence of Mn 21 in chemical oxidation of MBQH 2. The composition of the reaction mixture was as follows: 20 mm tartrate buffer, ph 5, 100 mm MBQH 2, and mm Mn 21. (A) Time course of quinone production (the results shown are from one experiment typical of three). (B), H 2 O 2 levels after full oxidation of MBQH 2 (error bars represent ^ 95% confidence limits). Fig. 4. Comparison of VP, MnP, and HRP activities on MBQH 2 in the absence of exogenous H 2 O 2 and presence of Mn 21. Reactions were carried out in 20 mm tartrate buffer, ph 5, and contained 100 mm MBQH 2,20mMMn 21, and 64 nm VP showing an activity of 10 mu on MBQH 2 (assayed with 100 mm hydroquinone and H 2 O 2 ) and 508 mu on Mn 21. The amount of HRP and MnP used in these reactions was that showing the same activity than VP on MBQH 2 and Mn 21, respectively, assayed under the same reaction conditions. Means of three replicates are shown (95% confidence limits were less than 5% of the mean).

5 q FEBS 2001 H 2 O 2 generation by Pleurotus veringii peroxidase (Eur. J. Biochem. 268) 4791 Mn 21 oxidation (sum of reactions 9 and 6). 2Mn 21 1 H 2 O 2 1 2H 1!2Mn H 2 O ð9þ In order to determine whether the oxidation of hydroquinone by VP was carried out through Mn 21 oxidation and its effect on hydroquinone oxidation, VP activity on 100 mm MBQH 2 was compared to that of HRP and MnP. To obtain comparative results, the amounts of HRP and MnP used in these reactions was that showing the same activity in the presence of 100 mm H 2 O 2 than 64 nm VP when acting on 100 mm either MBQH 2 or Mn 21, respectively. As shown in Fig. 4, production of MBQ in the reaction catalyzed by HRP, which can use the H 2 O 2 produced from semiquinone radicals autoxidation to oxidize MBQH 2 but not Mn 21, was preceded by the largest lag period and proceeded at the lowest rate. The oxidation of MBQH 2 catalyzed by VP greatly shortened the lag period and increased MBQ production rate. Obviously, these effects could only be caused by the ability of VP to catalyze Mn 21 oxidation. Finally, an identical MBQ production curve to that observed with VP was obtained during the Mn 21 -dependent oxidation of MBQH 2 catalyzed by MnP, showing that VP activity on MBQH 2 was mainly carried out through Mn 21 oxidation. In addition to MBQH 2, VP also catalyzed the oxidation of BQH 2 and DBQH 2 in the absence of exogenous H 2 O 2 and the presence of Mn 21. The time course of BQ, MBQ and DBQ production during the 4-min reactions is compared in Fig. 5. Oxidation of BQH 2 was preceded by the longest lag period and took place at the lowest rate. By increasing the number of methoxyl substituents in the hydroquinone aromatic ring, which decreases the redox potential of hydroquinone, the lag period was shortened and maximal quinone production rate increased. Although no quinone was produced from BQH 2 and MBQH 2 in control reactions blanks lacking VP, 2 mm quinone was found in blanks containing DBQH 2 after 4 min (data not shown). These results are in agreement with those from Table 1, showing a faster oxidation by VP of the hydroquinone containing the higher number of methoxyl substituents, and those reported previously showing that DBQH 2 semiquinone radicals autoxidized better than MBQH 2 semiquinone radicals [10]. DISCUSSION Peroxidases are known to have a very low specificity for Fig. 5. Oxidation of BQH 2, MBQH 2 and DBQH 2 in the absence of exogenous H 2 O 2 and presence of Mn 21. Reactions contained 20 mm tartrate buffer, ph 5, 50 mm hydroquinone, 20 mm Mn 21, and 160 nm VP (50 mu on MBQH 2 ). The results shown are from one experiment typical of three. their reducing substrates, catalyzing the oxidation by H 2 O 2 of a wide number of organic and inorganic compounds. Some of these compounds, including organic acids, hydroquinones, NADH, and thiols, have been shown to support peroxidase activity in the absence of exogenous H 2 O 2 [12,27,38,39]. Autoxidation of the substrate, which in the case of thiols and BQH 2 has been shown to be catalyzed by free metal ions [40,41], leads to the production of a minor amount of H 2 O 2 initiating the peroxidation cycle of the enzyme. Then, the substrates are oxidized to free radicals that autoxidize, reducing O 2 to O 2, the latter regenerating the H 2 O 2 needed to propagate the peroxidase reaction. If Mn 21 is present, oxidation of the substrate is activated because spontaneous dismutation of O 2 is replaced by its reduction by Mn 21, rendering higher levels of H 2 O 2 and the oxidant Mn 31 [42]. This oxidase-like reaction of peroxidases, is shown in the present paper for Pleurotus VP with glyoxylic and oxalic acids and hydroquinones as substrates. Whereas the oxidation of organic acids leading to H 2 O 2 production by ligninolytic peroxidases has been previously reported [8,9,16,17], the oxidation by these enzymes of ligninderived hydroquinones in the absence of exogenous H 2 O 2 and presence of Mn 21 is for the first time described here. Oxidation of glyoxylic and oxalic acids by VP was dependent on Mn 21, even in the presence of H 2 O 2 (Fig. 1). Therefore, the mechanism of the overall reaction should not be different to that proposed for MnP [9,16]. To summarize the results obtained with hydroquinones, Fig. 6 shows a diagram of the most likely reactions occurring during the oxidation of these substrates by VP in the absence and presence of Mn 21. Exogenous H 2 O 2 was required to oxidize MBQH 2 by VP in reactions lacking Mn 21 (Fig. 2). Under these conditions, VP oxidized hydroquinone to semiquinone radicals (reaction 1). In the absence of O 2 these radicals were transformed into quinone via disproportionation (reaction 2), whereas with O 2 autoxidation also took place generating O 2 (reaction 3) and then H 2 O 2 (reaction 4). The use of this peroxide by VP to oxidize hydroquinone explained the different quinone/h 2 O 2 ratios obtained under aerobic and anaerobic conditions (Fig. 2A). In the presence of Mn 21, both H 2 O 2 and Mn 31 were produced (reaction 5) to such an extent that MBQH 2 was fully oxidized (reaction 6) even in the absence of exogenous H 2 O 2 (Fig. 2B). As inferred above (results shown in Fig. 4), most H 2 O 2 produced in reaction 5 was used by VP to oxidize Mn 21 (reaction 9) instead of MBQH 2 (reaction 1). Based on the results shown in Fig. 4, it can be concluded that the ability of VP and MnP to oxidize Mn 21 is the reason why these ligninolytic enzymes are more efficient than HRP for the oxidation of hydroquinones in the absence of exogenous H 2 O 2. Besides the reactions depicted in Fig. 6, further research would be needed to determine whether O 2 and/or semiquinone radicals could react with native VP leading to enzyme intermediates that are outside the peroxidation cycle, as described previously for HRP [43,44], myeloperoxidase [28,45], and LiP [46]. With regards to the origin of the H 2 O 2 required to initiate the peroxidation cycle of VP, the results shown in Fig. 3 demonstrated H 2 O 2 production in buffered solutions containing MBQH 2 and Mn 21. Although in the present in vitro experiments hydroquinone autoxidation could be mediated by traces of free metal ions, conversion of hydroquinone into

6 4792 V. Gómez-Toribio et al. (Eur. J. Biochem. 268) q FEBS 2001 Fig. 6. Scheme of the reactions involved in H 2 O 2 production during the oxidation of hydroquinones by VP in the absence and presence of Mn 21 (solid and dashed arrows, respectively). The schemes resulting from the elimination of reactions 1 or 9 are valid to illustrate the mechanism of hydroquinone oxidation by MnP and HRP, respectively. semiquinone radicals under more natural conditions can be catalyzed by laccase [10] and/or the initial H 2 O 2 required for VP activity can be provided by aromatic aldehyde redox cycling involving aryl alcohol oxidase [47]. Despite these facts, chemical oxidation of hydroquinones promoted by Mn 21 could be relevant during the initial stages of wood biodegradation because ligninolytic enzymes are too large to penetrate into nonmodified wood cell walls [48]. Mn 31 and hydroxyl radical derived from H 2 O 2 are considered to be two of the oxidizing agents involved in the initial attack on lignocellulose by white-rot fungi [49,50]. ACKNOWLEDGEMENTS This research was funded by the projects Fungal metalloenzymes oxidizing aromatic compounds of industrial interest (QLK ) of the European Union, Novel peroxidases and oxidases from Pleurotus: structural studies and heterologous expression related to biodegradation of aromatic compounds with industrial or environmental interest (Bio ) of the Spanish Biotechnology Programme, and Biodegradation of soil aromatic pollutants by Pleurotus species (07M/0051/1998) of the Environmental Programme from the Comunidad Autónoma de Madrid. The stay of V. Gómez-Toribio at the Centro de Investigaciones Biológicas was supported by a fellowship from the Comunidad Autónoma de Madrid. REFERENCES 1. Buswell, J.A. & Odier, E. (1987) Lignin biodegradation. Crit. Rev. Biotechnol. 6, Shimada, M. & Higuchi, T. (1991) Microbial, enzymatic and biomimetic degradation of lignin. In Wood and Cellulosic Chemistry (Hon, D.N.S. & Shiraishi, N., eds), pp Marcel Dekker, NY, USA. 3. Martínez, M.J., Ruiz-Dueñas, F.J., Guillén, F. & Martínez, A.T. (1996) Purification and catalytic properties of two manganeseperoxidase isoenzymes from Pleurotus eryngii. Eur. J. Biochem. 237, Mester, T. & Field, J.A. (1998) Characterization of a novel manganese peroxidase-lignin peroxidase hybrid isozyme produced by Bjerkandera species strain BOS55 in the absence of manganese. J. Biol. Chem. 273, Ruiz-Dueñas, F.J., Martínez, M.J. & Martínez, A.T. (1999) Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii. Mol. Microbiol. 31, Kersten, P.J. & Kirk, T.K. (1987) Involvement of a new enzyme, glyoxal oxidase, in extracellular H 2 O 2 production by Phanerochaete chrysosporium. J. Bacteriol. 169, Guillén, F., Martínez, A.T. & Martínez, M.J. (1992) Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii. Eur. J. Biochem. 209, Popp, J.L., Kalyanaraman, B. & Kirk, T.K. (1990) Lignin peroxidase oxidation of Mn 21 in the presence of veratryl alcohol, malonic or oxalic acid, and oxygen. Biochemistry 29, Kuan, I.C. & Tien, M. (1993) Glyoxylate-supported reactions catalyzed by Mn peroxidase of Phanerochaete chrysosporium: Activity in the absence of added hydrogen peroxide. Arch. Biochem. Biophys. 302, Guillén, F., Muñoz, C., Gómez-Toribio, V., Martínez, A.T. & Martínez, M.J. (2000) Oxygen activation during the oxidation of methoxyhydroquinones by laccase from Pleurotus eryngii. Appl. Environ. Microbiol. 66, Svensson, B.E. (1988) Thiols as myeloperoxidase-oxidase substrates. Biochem. J. 253, Scheeline, A., Olson, D.L., Williksen, E.P., Horrass, G.A., Klein, M.L. & Larter, R. (1997) The peroxidase oxidase oscillator and its constituent chemistries. Chem. Rev. 97, Glenn, J.K., Akileswaran, L. & Gold, M.H. (1986) Mn (II)

7 q FEBS 2001 H 2 O 2 generation by Pleurotus veringii peroxidase (Eur. J. Biochem. 268) 4793 oxidation is the principal function of the extracellular Mnperoxidase from Phanerochaete chrysosporium. Arch. Biochem. Biophys. 251, Paszczynski, A., Huynh, V.-B. & Crawford, R. (1986) Comparison of ligninase-i and peroxidase-m2 from the white-rot fungus Phanerochaete chrysosporium. Arch. Biochem. Biophys. 244, Kuan, I.C. & Tien, M. (1993) Stimulation of Mn-peroxidase activity: a possible role for oxalate in lignin biodegradation. Proc. Natl Acad. Sci. USA 90, Urzúa, U., Kersten, P.J. & Vicuña, R. (1998) Manganese peroxidase dependent oxidation of glyoxylic and oxalic acids synthesized by Ceriporiopsis subvermispora produces extracellular hydrogen peroxide. Appl. Environ. Microbiol. 64, Hofrichter, M., Ziegenhagen, D., Vares, T., Friedrich, M., Jager, M.G., Fritsche, W. & Hatakka, A. (1998) Oxidative decomposition of malonic acid as basis for the action of manganese peroxidase in the absence of hydrogen peroxide. FEBS Lett. 434, Gold, M.H., Youngs, H.L. & Gelpke, M.D. (2000) Manganese peroxidase. Met. Ions Biol. Syst. 37, Peláez, F., Martínez, M.J. & Martínez, A.T. (1995) Screening of 68 species of basidiomycetes for enzymes involved in lignin degradation. Mycol. Res. 99, Keyser, P., Kirk, T.K. & Zeikus, J.G. (1978) Ligninolytic enzyme system of Phanerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation. J. Bacteriol. 135, Hatakka, A. (1994) Lignin-modifying enzymes from selected white-rot fungi production and role in lignin degradation. FEMS Microbiol. Rev. 13, Martínez, M.J., Böckle, B., Camarero, S., Guillén, F. & Martínez, A.T. (1996) MnP isoenzymes produced by two Pleurotus species in liquid culture and during wheat straw solid-state fermentation. In Enzymes for Pulp and Paper Processing (Jeffries, T.W. & Viikari, L., eds), pp ACS, Washington, USA. 23. Chung, N.H., Shah, M.M., Grover, T.A. & Aust., S.D. (1993) Reductive activity of a manganese-dependent peroxidase from Phanerochaete chrysosporium. Arch. Biochem. Biophys. 306, Heinfling, A., Ruiz-Dueñas, F.J., Martínez, M.J., Bergbauer, M., Szewzyk, U. & Martínez, A.T. (1998) A study on reducing substrates of manganese-oxidizing peroxidases from Pleurotus eryngii and Bjerkandera adusta. FEBS Lett. 428, Schoemaker, H.E., Meijer, E.M., Leisola, M.S.A., Haemmerli, S.D., Waldner, R., Sanglard, D. & Schmidt, H.W.H. (1989) Oxidation and reduction in lignin biodegradation. In Plant Cell Wall Polymers (Lewis, N.G. & Paice, M.G., eds), pp American Chemical Society, Washington, USA. 26. Shimada, M., Ohta, A., Kurosaka, H., Hattori, T., Higuchi, T. & Takahashi, M. (1989) Roles of secondary metabolism of wood rotting fungi in biodegradation of lignocellulosic materials. In Plant Cell Wall Polymers (Lewis, N.G. & Paice, M.G., eds), pp American Chemical Society, Washington DC, USA. 27. Klapper, M.H. & Hackett, D.P. (1963) The oxidatic activity of horseradish peroxidase. I. Oxidation of hydro- and naphthohydroquinones. J. Biol. Chem. 238, Kettle. A.J. & Winterbourn, C.C. (1992) Oxidation of hydroquinone by myeloperoxidase. Mechanism of stimulation by benzoquinone. J. Biol. Chem. 267, Guillén, F., Martínez, M.J., Muñoz, C. & Martínez, A.T. (1997) Quinone redox cycling in the ligninolytic fungus Pleurotus eryngii leading to extracellular production of superoxide anion radical. Arch. Biochem. Biophys. 339, Muñoz, C., Guillén, F., Martínez, A.T. & Martínez, M.J. (1997) Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties and participation in activation of molecular oxygen and Mn 21 oxidation. Appl. Environ. Microbiol. 63, Baker, W. (1941) Derivatives of pentahydroxybenzene, and a synthesis of pedicellin. J. Chem. Soc Haemmerli, S.D., Schoemaker, H.E., Schmidt, H.W.H. & Leisola, M.S.A. (1987) Oxidation of veratryl alcohol by the lignin peroxidase of Phanerochaete chrysosporium. FEBS Lett. 220, Palma, C., Martínez, A.T., Lema, J. & Martínez, M.J. (2000) Different fungal manganese-oxidizing peroxidases: a comparison between Bjerkandera sp and Phanerochaete chrysosporium. J. Biotechnol. 77, Pick, E. & Keisari, Y. (1980) A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J. Immunol. Methods 38, Camarero, S., Sarkar, S., Ruiz-Dueñas, F.J., Martínez, M.J. & Martínez, A.T. (1999) Description of a versatile peroxidase involved in natural degradation of lignin that has both Mnperoxidase and lignin-peroxidase substrate binding sites. J. Biol. Chem. 274, Sawada, Y., Iyanagi, T. & Yamazaki, I. (1975) Relation between redox potentials and rate constants in reaction coupled with the system oxygen-superoxide. Biochemistry 14, Archibald, F.S. & Fridovich, I. (1982) The scavenging of superoxide radical by manganous complexes: in vitro. Arch. Biochem. Biophys. 214, Kenten, R.H. & Mann, P.J.G. (1953) The oxidation of certain dicarboxylic acids by peroxidase systems in presence of manganese. Biochem. J. 53, Burner, U. & Obinger, C. (1997) Transient-state and steady-state kinetics of the oxidation of aliphatic and aromatic thiols by horseradish peroxidase. FEBS Lett. 411, Misra, H.P. (1974) Generation of superoxide free radical during the autoxidation of thiols. J. Biol. Chem. 249, Li, Y. & Trush, M.A. (1993) Oxidation of hydroquinone by copper: chemical mechanism and biological effects. Arch. Biochem. Biophys. 300, Yamazaki, I. & Piette, L.H. (1963) The mechanism of aerobic oxidase reaction catalyzed by peroxidase. Biochim. Biophys. Acta 77, Yamazaki, I. & Yokota, K. (1973) Oxidation states of peroxidase. Mol. Cell. Biochem. 2, Dordick, J.S., Klibanov, A.M. & Marletta, M.A. (1986) Horseradish peroxidase catalyzed hydroxylations: mechanistic studies. Biochemistry 25, Burner, U., Krapfenbauer, G., Furtmüller, P.G., Regelsberg, G. & Obinger, C. (2000) Oxidation of hydroquinone, 2,3-dimethylhydroquinone and 2,3,5-trimethylhydroquinone by human myeloperoxidase. Redox Reports 5, Schmall, M.W., Gorman, L.S. & Dordick, J.S. (1989) Ligninasecatalyzed hydroxylation of phenols. Biochim. Biophys. Acta 999, Guillén, F., Martínez, A.T., Martínez, M.J. & Evans, C.S. (1994) Hydrogen peroxide-producing system of Pleurotus eryngii involving the extracellular enzyme aryl-alcohol oxidase. Appl. Microbiol. Biotechnol. 41, Flournoy, D.S., Paul, J.A., Kirk, T.K. & Highley, T.L. (1993) Changes in the size and volume of pores in sweetgum wood during simultaneous rot by Phanerochaete chrysosporium Burds. Holzforschung 47, Evans, C.S., Dutton, M.V., Guillén, F. & Veness, R.G. (1994) Enzymes and small molecular mass agents involved with lignocellulose degradation. FEMS Microbiol. Rev. 13, Joseleau, J.P., Gharibian, S., Comtat, J., Lefebvre, A. & Ruel, K. (1994) Indirect involvement of ligninolytic enzyme systems in cell wall degradation. FEMS Microbiol. Rev. 13,