Laccase-Catalyzed Oxidation of Mn²⁺ in the Presence of Natural Mn³⁺ Chelators as a Novel Source of Extracellular H₂O₂ Production and Its Impact on Manganese Peroxidase

Abstract

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn²⁺ to Mn³⁺, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn³⁺ complexes were produced at 0.15, 0.05, and 0.10 μmol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H₂O₂ formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H₂O₂ production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn²⁺ oxidation and abiotic decomposition of these organic chelators by the resulting Mn³⁺, which leads to formation of superoxide and its subsequent reduction to H₂O₂. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn³⁺ complexes in assays containing 1 mM Mn²⁺ and 100 mM oxalate or malonate, but omitting an additional H₂O₂ source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn²⁺ oxidation in providing H₂O₂ for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.

Laccases (EC 1.10.3.2) are extracellular multicopper oxidases produced by different kinds of fungi (39), which oxidize lignin and many organic xenobiotics (7, 23, 27, 45). These enzymes couple four one-electron substrate oxidations to the four-electron reduction of dioxygen to water, without formation of free reduced oxygen species (7, 45).

Manganese peroxidases (MnP; EC 1.11.1.13) are part of the ligninolytic system of white rot and litter-decaying basidiomycetes. During the catalytic cycle, the active center is oxidized by H₂O₂. Reduction to the resting enzyme is achieved by two successive one-electron transfers, thereby oxidizing Mn²⁺ to Mn³⁺, respectively. This is facilitated by fungal organic acids such as oxalate or malonate upon chelation of the highly reactive Mn³⁺ state (4, 20-22, 40, 43). MnP catalyzes the oxidation of lignin, humic substances, and many organopollutants (16, 20, 29).

Extracellular H₂O₂ is required as a substrate for ligninolytic peroxidases. Extracellular enzymes like aryl alcohol oxidase (31) and glyoxal oxidase (19) produce H₂O₂. This compound is also formed upon oxidation of hydroquinones by ligninolytic enzymes and autoxidation of the resulting semiquinones concomitantly reducing O₂ to superoxide anion radical (13, 27). Mn²⁺ reduces superoxide to H₂O₂ and is thereby oxidized to Mn³⁺ (2, 27). Furthermore, superoxide may dismutate to H₂O₂ and O₂. Oxidation of oxalate, glyoxylate, and malonate by Mn³⁺ was also considered to be a source of H₂O₂ (16, 42). For oxalate, the following reactions are well established (41, 42):

(1)

(2)

(3)

(4)

For abiotic decomposition of malonate, the following reactions were proposed (16):

(5)

(6)

(7)

(8)

Superoxide and oxalate derived from reaction 7 subsequently can contribute to reactions 1 to 4. Autocatalytic generation of traces of Mn³⁺, which leads to H₂O₂ (16) and the release of small H₂O₂ concentrations from fungal mycelia (42), was considered to initiate MnP reactions, thereby enhancing the Mn³⁺ concentration and facilitating H₂O₂ production in the aforementioned follow-up reactions.

In a previous paper, we have demonstrated that a purified laccase from the white rot fungus Trametes versicolor oxidized Mn²⁺ to Mn³⁺ in the presence of the Mn³⁺ chelator pyrophosphate (15). Mn²⁺ oxidation involved concomitant reduction of laccase type 1 copper, thus providing evidence that Mn²⁺ oxidation occurs via one-electron transfer to type 1 copper as usual for substrate oxidation by blue laccases (7, 45). A Phellinus ribis laccase devoid of type 1 copper did not oxidize Mn²⁺ in the presence of pyrophosphate (25). The litter-decaying basidiomycete Stropharia rugosoannulata degrades chlorophenols (36), the fluoroquinolone antibacterial drug ciprofloxacin (44), 2,4,6-trinitrotoluene (33), and synthetic lignin (38) to CO₂ and H₂O. Here, we show that a catalytic system consisting of purified laccase from S. rugosoannulata, Mn²⁺, and organic Mn chelators such as oxalate and malonate generates H₂O₂. We further assessed key aspects of the catalytic mechanism underlying this new reaction and the impact on MnP produced along with laccase by S. rugosoannulata under certain culture conditions. This study was attempted to provide evidence for a physiological role of laccase-catalyzed Mn²⁺ oxidation and establishes a novel type of laccase-MnP cooperation with potential significance for lignocellulose breakdown and degradation of xenobiotics.

MATERIALS AND METHODS

Organism.

S. rugosoannulata DSM 11372 was an isolate of the Institute of Microbiology, University of Jena, Jena, Germany, and was maintained on malt agar plates (35).

Culture conditions and production of ligninolytic enzymes.

For laccase production, S. rugosoannulata was pregrown on liquid malt medium (44) and then transferred into defined medium (35) containing 56 mM glucose and 30 μM Mn²⁺ as MnSO₄, which was modified as follows. Diammonium tartrate was employed at 12 mM. 2,5-Xylidine and CuSO₄ were included at 200 and 50 μM, respectively. Culture flasks were incubated on a Multitron rotary shaker (INFORS, Bottmingen, Switzerland) at 24°C under agitation (60 rpm).

For MnP production, defined medium (35) containing 56 mM glucose and 1.2 mM diammonium tartrate was supplemented with additional MnSO₄ (final concentration, 100 μM) and directly inoculated with S. rugosoannulata pregrown on malt agar plates (35). Incubation was carried out at 24°C without agitation.

Enzyme isolation and purification.

Cell-free culture filtrates were concentrated as described in reference 15. Proteins in concentrates were separated on a Mono Q HR 5/5 anion-exchange column (Amersham Pharmacia Biotech, Freiburg, Germany) under the conditions described before (15). MnP elutions were monitored at 405 nm (heme). For further MnP purification, (NH₄)₂SO₄ at 35% saturation was added to enzyme pools derived from Mono Q separations. Precipitated proteins were removed by centrifugation (model 5415C centrifuge; Eppendorf, Hamburg, Germany) (14,000 rpm, 15 min), and MnP-containing supernatant was applied to a Phenyl Superose HR 5/5 hydrophobic interaction chromatography column (Amersham Pharmacia Biotech), preequilibrated with 10 mM Na-acetate buffer (pH 5.5) containing (NH₄)₂SO₄ at 60% saturation. Proteins were eluted at 1 ml/min with 10 mM Na-acetate buffer (pH 5.5) without (NH₄)₂SO₄. Enzyme-containing fractions (1 ml) were pooled, reconcentrated, and stored at −20°C (15). Protein concentrations were determined according to the method of Bradford (5).

Gel electrophoresis and staining.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE were performed as described in reference 15. Gels were stained with Coomassie brilliant blue R-250 (Serva Feinbiochemica, Heidelberg, Germany) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) for protein and laccase activities, respectively (15). Molecular weights of proteins were determined with commercial molecular weight markers (Bio-Rad, Munich, Germany).

Spectrophotometric determinations.

Routine determinations of ligninolytic enzyme activities are described in reference 15. Laccase was assessed with ABTS at pH 4.5. MnP, lignin peroxidase (LiP; EC 1.11.1.14), and manganese-independent peroxidase (MiP; EC 1.11.1.7) activities were monitored upon formation of Mn³⁺-malonate, oxidation of veratryl alcohol, and ABTS oxidation, respectively. Where indicated, 2,6-dimethoxyphenol (2,6-DMP) was additionally employed (15). Enzyme activities were expressed as units, where 1 U = 1 μmol of product formed per min.

Mn³⁺-oxalate (ɛ₂₇₀ = 5.5 mM⁻¹ cm⁻¹) (22) and Mn³⁺-malonate (ɛ₂₇₀ = 11.59 mM⁻¹ cm⁻¹) (43) concentrations were determined at 270 nm, and Mn³⁺-pyrophosphate (ɛ₂₅₈ = 6.2 mM⁻¹ cm⁻¹) (18) concentrations were monitored at 258 nm. Chelators were employed as sodium salts at concentrations and pH values (adjusted with phosphoric acid) indicated in the text. Mn²⁺ and Mn³⁺ were always applied as MnSO₄ and Mn³⁺-acetate, respectively.

H₂O₂ concentrations were determined with the horseradish peroxidase (HRP)-catalyzed oxidation of 4-hydroxyphenylacetic acid (PHPA) to the fluorescent product 2,2′-dihydroxyphenyl-5,5′-diacetic acid (17). Assays (final volume, 400 μl) contained up to 300 μl of enzyme-free sample, 15 U of HRP (type II; Sigma-Aldrich Chemie GmbH, Steinheim, Germany), and PHPA at 1.3 mM in 100 mM phosphate buffer (pH 7.4). Enzyme-free samples were obtained by ultrafiltration with 10-kDa-cutoff centrifuge filters (Sartorius, Göttingen, Germany). The samples from abiotic experiments were treated as the enzymatic ones. After incubation for 5 min and addition of NaOH (final concentration, 200 mM), assay mixtures were applied to a spectrofluorophotometer (34) operated at a 323-nm excitation wavelength. The fluorescence signal was integrated over an emission range from 335 to 550 nm. Calibration curves were established for each of the chelators employed with known concentrations of H₂O₂. Assays omitting HRP served as blanks.

The reduction of tetranitromethane (TNM) used to indicate superoxide anion radical was monitored at 350 nm (ɛ₃₅₀ = 14.6 mM⁻¹ cm⁻¹) (26). For this, 270 μl of ultrafiltrated sample was mixed with 30 μl of 10 mM TNM in methanol, and the initial linear increase in absorbance was used to calculate TNM-reducing activity.

All spectrophotometric determinations were carried out in air-saturated solutions at 35°C by using a double-beam spectrophotometer (15).

Absence of low-molecular-weight compounds in the purified enzyme fractions.

Since Mn³⁺ may also be formed during enzymatic oxidation of organic compounds (1, 27), the absence of hypothetical fungal redox mediators was ensured by directly applying purified enzyme fractions to high-performance liquid chromatography (HPLC) (15).

Chemicals.

All reagents were of analytical grade and were purchased from either Sigma-Aldrich Chemie GmbH, Merck, Darmstadt, Germany, or Fluka Chemie, Neu Ulm, Germany. Superoxide dismutase (SOD) (specific activity, 5,180 U/mg of protein) was obtained from Fluka.

RESULTS

Production and purification of laccase and MnP.

Culture filtrates of agitated S. rugosoannulata cultures expressed a laccase activity of 3,191 U/liter at the time point of harvest (culture day 7). Neither MnP, MiP, nor LiP was detectable. Culture filtrate (235 ml) was 23.5-fold concentrated and applied to a Mono Q HR 5/5 column. Laccase activity was recovered as a major peak in fractions eluted at 2 to 3 ml and two minor peaks at 8 to 11 and 11 to 15 ml of elution volume, respectively. Pooled major activity fractions were used for further experiments, corresponding to a specific activity of 158 U/mg of protein, a 3.7-fold purification, and a yield of 14%. This pool showed the typical absorbance maximum at nearly 610 nm indicative of type 1 copper (7) (an A₂₈₀/A₆₁₀ ratio of approximately 19) and did not contain any activity of MnP, MiP, or LiP. SDS-PAGE revealed one protein band with a molecular mass of approximately 66 kDa (Fig. 1A). Native PAGE and activity staining with ABTS also visualized a single band (not shown in Fig. 1). HPLC analysis of the preparation did not lead to any indication of a hypothetical natural redox mediator.

FIG. 1.

Coomassie brilliant blue R-250-stained gels (SDS-PAGE) containing Mono Q-separated laccase (L) (A) and phenyl Superose-separated (B) MnP. M, molecular mass markers.

Time courses of extracellular MnP and laccase activities in nonagitated S. rugosoannulata cultures are depicted in Fig. 2, where MnP was the predominant enzyme. No attempt was made to purify the laccase because of its low activity. After harvesting on culture day 19, culture filtrate (3,830 ml) was 106-fold concentrated and applied to a Mono Q HR 5/5 column. MnP activity was recovered as a single peak in the fractions between 15 and 20 ml of elution volume, together with a tailed 405-nm heme absorbance peak. Laccase and LiP were not detectable in any fraction. Pooled MnP fractions corresponding to an elution volume of 17 to 18 ml, which additionally contained 7% (as related to the MnP activity) MiP, were applied to a phenyl Superose HR 5/5 column. Again, MnP activity eluted as a single peak between 21 and 24 ml, together with a distinct main absorbance peak at 405 nm. Pooled fractions eluted at 20 to 23 ml were used for further experiments, achieving a specific activity of 187 U/mg of protein, a 5.5-fold purification, and a yield of 2.4%. This pool oxidized ABTS, 2,6-DMP, and veratryl alcohol neither with nor without H₂O₂, as proven over a pH range from 2.5 to 7.0. Traces of MiP (5% of the MnP pool activity) eluted at 18 to 19 ml, concomitant with a small A₄₀₅ peak. SDS-PAGE of the reconcentrated MnP pool revealed two protein bands with molecular masses of approximately 40 and 41 kDa (Fig. 1B). The absence of hypothetical fungal redox mediators was ensured as described above.

FIG. 2.

Time course of extracellular laccase (♦) and MnP (⋄) activities in nonagitated S. rugosoannulata cultures on defined medium containing 56 mM glucose, 1.2 mM diammonium tartrate, and 100 μM Mn²⁺. Symbols represent means ± standard deviations for triplicate cultures.

Laccase-catalyzed oxidation of Mn²⁺ to Mn³⁺ and formation of reduced oxygen species.

Representative kinetics of Mn³⁺-oxalate, -malonate, and -pyrophosphate formation catalyzed by S. rugosoannulata laccase are shown in Fig. 3. Corresponding UV-visible spectra revealed specific absorbance maxima at nearly 270 (22, 41) and 500 (43) nm for Mn³⁺-oxalate, 270 nm for Mn³⁺-malonate (43), and 258 (18) and 478 (1) nm for Mn³⁺-pyrophosphate. The spectra of synthetic Mn³⁺ complexes, prepared by dissolving 100 μM Mn³⁺-acetate in either 100 mM Na-oxalate, -malonate, or -pyrophosphate (pH 5.0) prior to use, were identical to those of enzymatically generated Mn³⁺ complexes. No Mn³⁺ complex formation was observed in assays containing heat-inactivated enzyme.

FIG. 3.

Typical time courses of Mn³⁺-oxalate (trace 1), -pyrophosphate (trace 2), and -malonate (trace 3) complex formation catalyzed by laccase. Assay mixtures (pH 5.0) contained 0.2 U of laccase/ml, 1 mM Mn²⁺, and 100 mM chelator, respectively.

Formation of Mn³⁺ complexes was dependent on both the kind and concentration of the respective chelator (Fig. 4). Saturation of Mn²⁺ oxidation was observed at 100 mM for both organic acids, since oxalate and malonate concentrations of 200 mM did not further enhance the Mn³⁺-oxalate and -malonate concentrations, respectively (not shown in Fig. 4). At 100 mM chelator and pH 5.0, formal enzyme activities of 0.15, 0.10, and 0.05 U/mg of protein were obtained for Mn³⁺-oxalate, -pyrophosphate, and -malonate production, respectively. Mn²⁺ oxidation was optimal at pH 5.0 (oxalate and pyrophosphate) and 4.5 (malonate) (Fig. 5A). The highest laccase activity was obtained with ABTS at pH 2.5, followed by 2,6-DMP at pH 3.5 (Fig. 5B).

FIG. 4.

Effect of oxalate, malonate, and pyrophosphate concentrations on laccase-catalyzed formation of Mn³⁺-oxalate (▪), -malonate (♦), and -pyrophosphate (□). Air-saturated reaction mixtures (pH 5.0) contained 0.2 U of laccase/ml, 1 mM Mn²⁺, and chelators as indicated. At chelator concentrations of 0, 25, and 50 mM, respectively, 100, 75, and 50 mM Na₂HPO₄ were additionally employed. Concentrations of Mn³⁺ complexes were determined after 2 h of incubation at 35°C in the dark. Symbols represent means ± standard deviations for triplicate experiments.

FIG. 5.

Effect of pH on laccase-catalyzed formation of Mn³⁺-oxalate (▪), -malonate (♦), and -pyrophosphate (□) at 100 mM chelator, respectively (A); oxidation of 1 mM ABTS (▿) and 1 mM 2,6-DMP (▵) in 100 mM pyrophosphate (B); and production of H₂O₂ at 1 mM Mn²⁺ and 100 mM oxalate (▴) (C). In panels A and C, 100% corresponds (except for malonate where 100% refers to a Mn³⁺-malonate concentration of 8.5 μM, according to the slightly diverging pH optimum of 4.5) to the corresponding values shown in Fig. 4 and Table 1, respectively, and all other conditions were as described there. In panel B, 100% corresponds to specific activities of 451.7 and 157.9 U/mg of protein for ABTS and 2,6-DMP oxidation, respectively. Symbols represent means ± standard deviations for triplicate experiments.

In enzyme assays containing oxalate or malonate, Mn²⁺ oxidation was unequivocally accompanied by formation of H₂O₂, whereas only insignificant levels were monitored in the absence of Mn²⁺ (Table 1). No remarkable H₂O₂ formation could be detected upon application of pyrophosphate. Employing oxalate, the H₂O₂ concentration was more than three times higher than in the presence of malonate. The pH dependency of H₂O₂ formation upon oxalate application was assessed and revealed an optimum at pH 5.0 (Fig. 5C), thus fitting the pH optimum determined for Mn³⁺-oxalate production (Fig. 5A). Both the Mn³⁺-oxalate and H₂O₂ concentration nearly linearly increased over a tested range of up to 0.5 U of laccase/ml (Fig. 6). TNM reduction used to indicate superoxide anion radical (26) was significantly higher in enzymatic assays containing the organic chelators and Mn²⁺ than in those omitting Mn (Table 1), which was not observed during application of pyrophosphate. Oxalate employment led to an approximately sevenfold-higher TNM-reducing activity than malonate application. These results are indicative of abiotic cleavage of oxalate and malonate by enzymatically formed Mn³⁺ according to reactions 1 to 7. In addition, H₂O₂ may have been decomposed to a certain extent upon reduction of Mn³⁺ in a reaction not generating free superoxide (2, 43). Certain amounts of reduced oxygen species further may have been produced during sample preparation and in Mn-omitting reaction mixtures upon autoxidation of contaminating Mn. Experiments omitting laccase confirmed that Mn³⁺ is the species responsible for H₂O₂ production and TNM reduction under conditions mimicking enzymatic reactions with respect to chelator and pH (Table 1). Addition of Mn³⁺ caused a significant higher H₂O₂ concentration and TNM-reducing activity in the presence of oxalate compared to those with malonate. This is qualitatively consistent with the results of the laccase experiments. Only very low levels of reduced oxygen species were detected upon addition of Mn²⁺ as well as in the absence of Mn. In confirmation, application of pyrophosphate led to similarly low values under any condition.

TABLE 1.

Effect of Mn and chelators on H₂O₂ production and TNM reduction in the presence and absence of laccase^a

Chelator and Mn addition	H2O2 production (μM)		TNM reduction (μmol/min/liter)
	+ Laccase	− Laccase	+ Laccase	− Laccase
Oxalate
Mn3+ added at 0.1 mM	ND	16.66 ± 2.9b,c	ND	3.46 ± 0.39b,c
Mn2+ added at 1 mM	14.90 ± 0.9b,c	2.09 ± 2.7	2.90 ± 0.09b,c	0.19 ± 0.04
Mn omitted	0.92 ± 0.5	2.26 ± 1.1	0.16 ± 0.05	0.18 ± 0.05
Malonate
Mn3+ added at 0.1 mM	ND	7.40 ± 0.4b	ND	1.08 ± 0.07b
Mn2+ added at 1 mM	4.57 ± 0.7b	1.29 ± 0.1	0.39 ± 0.05b	0.18 ± 0.03
Mn omitted	0.73 ± 0.4	0.59 ± 0.7	0.11 ± 0.06	0.12 ± 0.08
Pyrophosphate
Mn3+ added at 0.1 mM	ND	0.66 ± 0.3	ND	0.13 ± 0.04
Mn2+ added at 1 mM	0.52 ± 0.4	0.39 ± 0.1	0.11 ± 0.08	0.10 ± 0.02
Mn omitted	0.59 ± 0.3	0.43 ± 0.1	0.10 ± 0.09	0.30 ± 0.18

^aLaccase (+) was employed at 0.2 U/ml, and chelators were always applied at 100 mM. Air-saturated reaction mixtures (pH 5.0) were incubated at 35°C in the dark. H₂O₂ concentrations and TNM-reducing activities were determined after 2 (laccase assays) and 4 (assays without laccase) h of incubation. All values represent means ± standard deviations for triplicate experiments. ND, not done.

^bSignificantly different (P < 0.05) from corresponding values detected in the absence of Mn (laccase assays) or from corresponding values observed in the presence of Mn²⁺ as well as from those without Mn (assays omitting laccase), as obtained by Student's t test.

^cSignificantly different (P < 0.05) from the corresponding values obtained upon employment of malonate.

FIG. 6.

Dependence of Mn³⁺-oxalate (▪) and H₂O₂ (▴) formation on the amount of laccase. Air-saturated reaction mixtures (pH 5.0) contained 1 mM Mn²⁺, 100 mM oxalate, and laccase as indicated. Mn³⁺-oxalate and H₂O₂ concentrations were determined after 2 h of incubation at 35°C in the dark. Symbols represent means ± standard deviations for triplicate experiments.

Interaction of laccase and MnP.

In previous studies, essentially no MnP reactions were observed in enzymatic systems containing purified MnP, Mn²⁺, and 50 mM malonate (16) or 20 mM oxalate (41), but no additional source of H₂O₂. Lower oxalate concentrations ranging from 0.5 to 10 mM facilitated H₂O₂-independent MnP reactions (41). We therefore employed high chelator concentrations of 100 mM to demonstrate the effect of laccase on MnP reactions unequivocally. In experiments containing laccase, MnP, Mn²⁺, and organic chelators, but no additional H₂O₂, the Mn³⁺ complex formation was increasingly speeded up (traces 1 in Fig. 7A and B, respectively). After 90 min, approximately 22- and 2-fold-higher Mn³⁺-oxalate and -malonate concentrations were observed, respectively, compared to those in assays omitting MnP (Fig. 3). This indicates increasingly stimulated MnP reactions. No Mn³⁺ complex formation was found in assays containing MnP, Mn²⁺, and organic chelators, but omitting laccase (traces 3 in Fig. 7A and B, respectively). Thus, laccase was essential to initiate MnP reactions. In laccase-driven MnP reactions, Mn³⁺-oxalate formation occurred much faster than Mn³⁺-malonate production, which resulted in an approximately 30-fold-higher Mn³⁺-oxalate than Mn³⁺-malonate concentration after 90 min. No MnP reaction was observed at 100 mM pyrophosphate, 0.2 U of laccase/ml, 0.2 U of MnP/ml, and 1 mM Mn²⁺, where Mn³⁺-pyrophosphate formation was identical to that of assays containing laccase as the only enzyme (not shown in Fig. 7).

FIG. 7.

Kinetics of Mn³⁺-oxalate (A) and -malonate (B) formation in the presence of 0.2 U of laccase/ml, 0.2 U of MnP/ml, 1 mM Mn²⁺, and 100 mM chelator ( trace 1); 0.2 U of laccase/ml, 0.2 U of MnP/ml, 1,500 U of SOD/ml, 1 mM Mn²⁺, and 100 mM chelator (trace 2); and 0.2 U of MnP/ml, 1 mM Mn²⁺, and 100 mM chelator (trace 3) at pH 5.0.

According to the stoichiometry of reactions 3 and 4, SOD-catalyzed dismutation of superoxide produces 50% of the H₂O₂ that could be derived from superoxide reduction by Mn²⁺. Provided that in the absence of SOD H₂O₂ is generated from reduction of superoxide by Mn²⁺ at concentrations rate-limiting to MnP reactions, SOD would thus delay MnP-catalyzed Mn³⁺ production. This is evident from traces 2 in Fig. 7A and B, respectively. SOD inhibited Mn³⁺ production by approximately 50% upon application of oxalate as well as malonate, indicating that superoxide reduction by Mn²⁺ is the key process for H₂O₂ production in laccase-driven MnP reactions.

DISCUSSION

S. rugosoannulata produced large amounts of exclusively laccase when shaking-culture conditions together with 200 μM 2,5-xylidine, 30 μM Mn²⁺, 50 μM Cu²⁺, and 12 mM diammonium tartrate (providing 24 mM nitrogen) were applied. Copper, nitrogen, and 2,5-xylidine are known to enhance laccase gene transcription and extracellular enzyme titers in ligninolytic fungi (8). The presence of a chromatographic form with a molecular mass of 66 kDa and the absorbance peak at nearly 610 nm caused by laccase type 1 copper reveal that S. rugosoannulata produces a typical blue laccase (7, 15, 27, 40).

To date, no laccase has been described with a type 1 copper redox potential exceeding approximately +800 mV (versus a normal hydrogen electrode) (7, 45), whereas the potential of the aqueous Mn²⁺/Mn³⁺ couple at pH 7 is +1,510 mV (7). The redox potential difference between type 1 copper and substrate as the thermodynamically driving force for the electron transfer from substrate to type 1 copper is considered to be a major parameter in controlling the rate of laccase substrate oxidation under steady-state conditions (45). Thermodynamically, oxidation of substrates with higher redox potential than that of type 1 copper may become possible if a primary oxidation product would efficiently be removed from the reaction equilibrium (23). Compounds such as oxalate, malonate, and pyrophosphate rapidly chelate both the Mn²⁺ and Mn³⁺ states and form mononuclear complexes with various ligand ratios, depending on the respective equilibrium constant and chelator concentration (1, 9, 22). Increasing concentrations of oxalate, malonate, and pyrophosphate increasingly favor Mn complex formation (2, 9, 22). Thus, it remains to be elucidated whether free (hexa-aquo) or complexed Mn²⁺ acts as a laccase substrate. Free Mn²⁺ was reported to be the substrate for MnP compound II re-reduction (4). Mn³⁺ arising from oxidation of Mn²⁺ by laccase could be removed from the reaction equilibrium upon chelation, thus driving the reaction forward. Also, the redox potential of the Mn²⁺/Mn³⁺ couple commonly decreases on complexation (9), which could support Mn²⁺ oxidation by laccase. Our observation that Mn³⁺ formation was chelator-specifically enhanced with increasing chelator concentrations (Fig. 4) favors such effects.

The true Mn²⁺-oxidizing activities of laccase in the presence of oxalate and malonate may well be higher than the observed ones, which obviously reflect mixed kinetics involving simultaneous Mn³⁺ complex formation and decay. Potentially, the pH optima obtained for Mn³⁺-oxalate and -malonate formation (Fig. 5A) may have been faked by Mn³⁺ complex decomposition. Such decay processes lead to complex reaction equilibria, which are affected by several parameters, such as pH, Mn³⁺, Mn²⁺, and oxygen concentrations, as well as the kind and concentration of the respective chelator (9, 41). The net Mn³⁺-oxalate and -malonate decay rates are known to increase with decreasing pH, decreasing chelator, and increasing Mn³⁺ concentrations (9, 41). In contrast, Mn³⁺-pyrophosphate was reported to be stable for months at excess concentrations of pyrophosphate (1). The pH optimum of Mn³⁺-pyrophoshate production by S. rugosoannulata laccase is identical to that of T. versicolor laccase (15).

Nonagitated S. rugosoannulata cultures supplemented with 1.2 mM diammonium tartrate and 100 μM Mn²⁺ predominantly produced MnP concomitant with low levels of laccase, similar to previously published results (38). In Phanerochaete chrysosporium, the highest MnP transcript levels were observed in Mn²⁺-containing, nonagitated cultures upon nitrogen limitation (12), whereas MnP production shows a different response toward Mn²⁺ and nitrogen in other white rot fungi (24, 30). Since S. rugosoannulata MnP does not oxidize veratryl alcohol, ABTS, and 2,6-DMP in the absence of Mn²⁺, it resembles P. chrysosporium MnP in essentially strictly requiring Mn²⁺ as a substrate (29). Mn²⁺-independent oxidation of veratryl alcohol, ABTS, and 2,6-DMP was shown for Mn²⁺-oxidizing peroxidases from other white rot basidiomycetes (14, 24, 29).

Oxalate concentrations of up to 27.8 and 47.5 mM were found in white and brown rot fungi, respectively (37). H₂O₂-independent Cerporiopsis subvermispora MnP reactions clearly were speeded up by addition of 10 μM Mn³⁺ (41). Mn³⁺-oxalate production by S. rugosoannulata laccase thus meets physiologically relevant conditions (Fig. 4). For MnP, H₂O₂ K_m values still below the H₂O₂ concentrations derived from the laccase-Mn²⁺-oxalate system in this study (Table 1 and Fig. 6) were described (29). The pH profiles of laccase-catalyzed H₂O₂ production in the presence of oxalate (Fig. 5C) and Mn³⁺ formation upon oxalate and malonate (Fig. 5A) fit the pH activity profile of MnP (20, 24, 40). At 50 to 100 mM, malonate laccase produces Mn³⁺ at concentrations (Fig. 4) within the same order of magnitude of those previously shown to initiate H₂O₂-independent MnP reactions at 50 mM malonate (16). Only 20 to 30 μM malonate has been observed in ligninolytic cultures of P. chrysosporium (43). Laccase-driven MnP reactions were much faster in presence of 100 mM oxalate than malonate (Fig. 7), likely due to the higher H₂O₂ concentration resulting from Mn³⁺-oxalate decay (Table 1). Oxalate and malonate concentrations of 50 mM did not differentially affect MnP activities in a previous study (43), whereas MnP shows a different response at lower oxalate and malonate concentrations (20). Moreover, Mn²⁺ was found to enhance levels of laccase mRNA and extracellular laccase activities in litter-decomposing and white rot fungi (32), indicating a regulatory role in laccase expression. Hence, our results favor a physiological function of Mn²⁺ oxidation by laccase and are in further support of the role of oxalate as a natural chelator (21, 40). In ligninolytic fungi, laccase and MnP titers can considerably vary in terms of culture conditions and time (35), as also shown here. Consequently, we propose a novel cooperation between laccase and MnP according to the scheme shown in Fig. 8. In the presence of Mn²⁺ and oxalate, laccase produces Mn³⁺-oxalate. The latter initializes a set of follow-up reactions leading to H₂O₂ formation, which may initiate or support peroxidase reactions. This does not rule out other ways to generate H₂O₂ (13, 16, 19, 27, 31, 41, 42). Concerning biotechnological applications, laccase offers a simple and convenient alternative to supply peroxidases with H₂O₂, because laccases will become available at an economically feasible scale.

FIG. 8.

Proposed scheme for the extracellular generation of H₂O₂ as a result of laccase-catalyzed Mn²⁺ oxidation in the presence of oxalate and its impact on MnP. (The stoichiometry has not been balanced for oxalate, due to the possible involvement of complexes with various ligand ratios.)

H₂O₂ is also considered to be an oxidant in extracellular Fenton-type degradation mechanisms, implicated in white and brown rot basidiomycetes (3, 34, 44). Laccases were also described in brown rot fungi (10). We found a purified blue laccase from the brown rot basidiomycete Laetiporus sulfureus also active in Mn²⁺ oxidation (C. Höfer and D. Schlosser, unpublished data).

Moreover, microbial Mn²⁺ oxidation is an important biogeochemical process at present mainly attributed to bacterial activities (28), in which multicopper oxidases have been implicated (6, 11). Besides basidiomycetes, environmentally ubiquitous organisms such as ascomycetes, imperfect fungi, and yeasts are known to produce laccases (39). A contribution of laccase activities of such organisms to microbial Mn²⁺ oxidation in different environmental compartments has not been considered as yet, but seems reasonable.