Reappraising a Controversy: Formation and Role of the Azodication (ABTS2+) in the Laccase-ABTS Catalyzed Breakdown of Lignin

In fermentations of lignocelluloses, redox potentials (If not indicated otherwise, redox potentials in Volt are taken versus Normal Hydrogen Reference Electrodes (NHE).) E0 of laccases/plant peroxidases by 0.79/0.95 V enable oxidations of phenolic substrates and transformations of synthetic and substrate-derived compounds to radicals that mediate attacks on non-phenolic lignin (models) by 1.5 V. In consecutive one-electron abstractions, the redox mediator 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) is oxidized by electroor wet-chemistry to the green cation radical (ABTS•+, 0.68 V) and the red dication (ABTS2+, 1.09 V). The enzyme/ABTS couple generates the stable ABTS•+ whose low E0 cannot explain the couple’s contemporary attack on non-phenolic lignins. This paradoxon indicates the non-confirmed production of the ligninolytic ABTS2+ by the enzymes. During incubations of live sapwood chips in ABTS/H2O2 to prove their constitutive peroxidase, the enzyme catalyzed the formation of the expected green-colored ABTS•+ solution that gradually turned red. Its spectrophotometric absorbance peaks at λ = 515–573 nm resembled those of ABTS2+ at 518–520 nm. It is shown that portions of an ABTS•+ preparation with inactivated enzyme are reduced to ABTS during their abiotic oxidation of low-MW extractives from lignocelluloses to redox mediating radicals. The radicals, in turn, apparently transform the remaining ABTS•+ to red derivatives in the absence of functional oxidoreductases. Ultrafiltration and Liquid-Chromatography suggest the presence of a stable ABTS2+ compound absorbing at 515 nm, red protein/ABTS adducts, and further ABTS moieties. Therefore, ABTS mediated lignin degradations could result from chain reactions of ABTS•+-activated lignocellulose extractives and fissured rather than complete ABTS2+ molecules.

The manganese independent class III peroxidase represented by a set of isoforms [55] is constitutive to all terrestrial plants. Peroxidases are involved in lignification, cell elongation, stress response, and seed germination [56]. They have redox potentials of 0.89-0.95 V [19], oxidize phenolics up to 1.12 V [25], and are potential laccase substitutes in biotechnology [12,55]. Screenings for peroxidase activity were performed across the stemwood regions of European trees with wood chips incubated in ABTS/H2O2 reaction mixtures (unpublished). To the surprise, the normally stable green ABTS •+ solutions formed within 10-60 min were further oxidized and acquired red to brownish tones within 0. 5-8 (-24) h. The spectrophotometric absorbance peaks of ABTS •+ were replaced by single ones that resembled those reported for the red colored ABTS 2+ [30,45].
In this study, the production of the reddish ABTS derivatives was followed in ABTS solutions amended with wood chips, wheat straw, and green stem sections of grass. It was the goal to identify extractives of the respective plant tissues in their role as ubiquitous natural redox mediators and to characterize the ABTS derivatives by Liquid Chromatography, ultrafiltration, and UV-Vis and FT-IR spectrophotometry in partial comparison with an ABTS 2+ product generated with the oxidant K2O8S2. Consequences for ABTS applications in biotechnology are discussed.
The manganese independent class III peroxidase represented by a set of isoforms [55] is constitutive to all terrestrial plants. Peroxidases are involved in lignification, cell elongation, stress response, and seed germination [56]. They have redox potentials of 0.89-0.95 V [19], oxidize phenolics up to 1.12 V [25], and are potential laccase substitutes in biotechnology [12,55]. Screenings for peroxidase activity were performed across the stemwood regions of European trees with wood chips incubated in ABTS/H 2 O 2 reaction mixtures (unpublished). To the surprise, the normally stable green ABTS •+ solutions formed within 10-60 min were further oxidized and acquired red to brownish tones within 0. 5-8 (-24) h. The spectrophotometric absorbance peaks of ABTS •+ were replaced by single ones that resembled those reported for the red colored ABTS 2+ [30,45].
In this study, the production of the reddish ABTS derivatives was followed in ABTS solutions amended with wood chips, wheat straw, and green stem sections of grass. It was the goal to identify extractives of the respective plant tissues in their role as ubiquitous natural redox mediators and to characterize the ABTS derivatives by Liquid Chromatography, ultrafiltration, and UV-Vis and FT-IR spectrophotometry in partial comparison with an ABTS 2+ product generated with the oxidant K 2 O 8 S 2 . Consequences for ABTS applications in biotechnology are discussed.

Oxidation of ABTS in the Presence of Sapwood Chips
Triplicate plastic cuvettes (1 cm) were used to incubate 1-2 live sapwood chips in 0.6 mL each of ABTS (6.6 mg/10 mL; Fluka); 0.1 M KH 2 PO 4 buffer pH 4.5; and H 2 O 2 30% (15 mg/10 mL) prepared with bideionized water. Color reactions extending over 5 min to several h were followed at A 430 ; A 521 ; and A 550 as well as by scanning spectrophotometry (Helios Beta, Unicam UV-VIS, Cambridge, UK) after centrifugation of the supernatants at 14,000 g for 5 min. In the respective reference samples, H 2 O 2 solution was replaced by water. The test arrangement was also used to incubate live sapwood chips with 0.1 mL of the laccase solution from a late liquid idiophase culture of the basidiomycete Kuehneromyces mutabilis grown in 25 g malt extract (Merck) and 5 g casein peptone L −1 whereby H 2 O 2 solution was replaced by water. Laccase solution was applied, too, upon incubations of autoclaved sapwood chips with inactivated PO, of mature wheat straw, and of green stem sections of the grass Boa spp. Several reactions were re-examined with commercial Pyricularia oryzae laccase (Sigma).

Molecular Size Separation of the ABTS Derivative by Ultrafiltration
A reaction mixture as above was incubated with sapwood chips of European beech at 22 • C for 8 h, using the constitutive plant peroxidase as the catalyst. The red oxidation product was centrifuged at 14,000 g for 10 min and scanned in the wavelength range of 340-800 nm. A 25-mL aliquot of the solution was transferred to a Centrisart 13239 filter (Sartorius, Germany) with a molecular size cutoff of 10 kDa and centrifuged at 2000 g. Subsequently, scans were taken from both the bright-red filtrate and the remaining dark-red concentrate fractions.

Extraction of Peroxidase from the Outer Sapwood
Samples of 0.5 g in dry weight (DW) were rasped from vital and unbarked branches Ø 12-20 mm and submerged in 5 mL of 0.2 M KH 2 PO 4 buffer pH 4.5 at 6 • C for 4 h. Aliquots of the extracts were then transferred to 2-mL microtubes and stored at 6 • C. The peroxidase activity was determined by adding 0.1 mL of the centrifuged extract to 0.45 mL each of ABTS (6.6 mg/10 mL) and H 2 O 2 30% (15 mg/10 mL) solution prepared with 0.1 M KH 2 PO 4 buffer pH 4.5. The production of the green ABTS •+ cation radical to be expressed in µM min −1 was followed at A 420 for 12 min (¥ 420 = 36,000 M −1 cm −1 ) [57].

Extraction of Total Phenol from the Outer Sapwood
In quadruplicate test tubes, rasped outer sapwood samples 0.5 g in DW from vital branches of trees were suspended in 7 mL of bideionized water. Two samples were stored at 12 • C for 20 h before the aqueous extract was collected and centrifuged at 14,000 g for 5 min. The remaining two samples were boiled in a water bath at 98 • C for 1 h, compensated for weight losses with bideionized water, and stored up to 20 h at 12 • C.
A freshly prepared test solution of Fast Blue B Salt (O-dianisidine tetrazotized zinc chloride double salt; Acros) in 0.1 M KH 2 PO 4 buffer pH 4.5 (10 mg mL −1 ) was used as phenol reagent. Concentration values were spectrophotometrically determined. For a reaction mixture of 1 mL sapwood extract and 0.9 mL buffer, the initial A 500 value was recorded. After adding 0.1 mL of the phenol reagent, the spontaneous rise in the A 500 value was exactly read at 15 s. The test was repeated two times. From the increment in A 500 , the absorbance value of 0.1 mL reagent in 1.9 mL buffer, reduced by the value of the buffer in the glass cuvette, was subtracted. Total phenol concentrations expressed as pyrogallol equivalents were calculated via calibration curve.

Solvent Extraction of Potential Redox Mediator Substances from Sapwood of European Beech
The extraction of rasped beech sapwood samples (0.5 g DW) with 5-mL aliquots of bideionized water, 80% methanol, and 80% acetone, respectively, followed the protocol used for total phenol extraction. Aliquots of the extracts obtained at 12 • C were transferred to 2-mL test tubes after 20 h of incubation and stored at 2 • C. Alternative water, methanol, and acetone treated samples were boiled in a water bath at 98 • C for 60, 30, and 10 min, respectively, compensated for the losses in solvent, and left standing for up to 20 h at 12 • C. The extracts were tested for the presence of redox mediators in ABTS oxidation assays. Apart from the cold-water extract with its plant PO constituents, the reaction mixtures of the organic solvent and hot solvent extracts had to be amended with K.-mutabilis laccase.

Examination of ABTS Derivative by Liquid Chromatography
A non-purified sample of ABTS derivative generated with the low-MW root exudates of white mustard (Sinapis alba L.) was analyzed by Liquid Chromatography with MS detection (LC-MS) and pneumatically assisted Atmospheric Pressure Ionization (API). The Perkin Elmer Sciex API 16 S with Series 200 pump and autosampler was run with 5.2 KV ionization voltage, 5 µL sample injection, and a flow of 0.25 mL min −1 under isocratic conditions. The eluent was composed of 10% methanol, 85% AFFA A, and 5% AFFA B.

Oxidation of ABTS by Potassium Peroxodisulfate (K 2 O 8 S 2 )
A reaction mixture of ABTS (190 mg L −1 ; Fluka, > 99% HPLC grade; M 548.68) and K 2 O 8 S 2 (1.4 g L −1 ; Merck, p. a. quality; M 270.33; molar ratio 1:15) prepared with bideionized water was incubated in duplicate 1-cm glass cuvettes at 20 • C. The color changes from green (ABTS •+ ) and brownish black to the final deep lilac within 3 min to 96 h were followed by scanning spectrophotometry in the range of λ = 340-800 nm. The K 2 O 8 S 2 solution (1.4 g L −1 ) served as reference sample and did not show any peaks in the 400-800 nm range. Bulk samples of the deep lilac and insoluble ABTS oxidation product were prepared in 2-mL microtubes by dissolving 2-3 mg ABTS and 20 mg K 2 O 8 S 2 in 1.5-2 mL of bideionized water (pH 2.37). Following reaction times of 3-5 min, the product could be separated from the colorless supernatant by centrifugation at 14,000 g for 5 min. The resulting pellet was cleaned from K 2 O 8 S 2 traces down to 2 × 10 −6 and a final pH 3.5 by up to eight consecutive resuspensions and centrifugations in 1.8 mL batches of bideionized water. Stock solutions were prepared in 0.1 M KH 2 PO 4 buffer pH 4.5 for RBBR at 2.1 mg/10 mL; for ABTS at 6.6 mg/10 mL; and for H 2 O 2 30% at 5 mg/10 mL. The red ABTS derivative was obtained from incubations with sapwood chips of beech and corresponded with a maximum of 0.22 mg mL −1 of ABTS as the start substance. Combinations of the reaction mixtures are specified in Section 3. All incubations were performed at 22 • C.

Data Processing
In the case of numerical data, SPSS 8.0 software (Chicago, IL, USA) was used to calculate standard deviations (SD) of duplicate to quadruplicate results, linear correlations, and to perform one-way analyses of variance. To determine the positions of the comparatively flat peaks obtained by spectrophotometric scans of ABTS solutions in the A 340 to A 800 nm range, absorbance values of the curves were determined for 2-nm intervals. Peaks were accepted where the differences between neighboring intervals dropped to zero.

Formation of ABTS Derivatives
The oxidation of ABTS by plant PO of beech did not terminate with the formation of the green-colored cation radical (ABTS •+ ) if the live sapwood chip as the enzyme source was part of the reaction mixture. With the further oxidation to the ABTS derivative, the green color of the solution turned red within 1-8 h at room temperature. The product was stable and remained soluble by exposure to HCl and NaOH in the pH 0.5-14 range. Spontaneous reductions to the green ABTS •+ oxidation state upon applications of ABTS and the expected electron exchange by comproportionation [29] were not observed. The production of the ABTS derivative went along with the replacement of the peaks at A 420 , A 645 , and A 728 , which are ascribed to the electrochemically generated ABTS •+ (Figure 2) [41], by rather flat ones in the A 520 to A 550 range. Both the wavelength positions and the total absorbance values of the peaks changed with species and accession of the timber, with the kind of the oxidoreductase enzyme involved (Table 1; Figure 2) and with the activation, e.g., of the intrinsic PO by variable doses of H 2 O 2 ( Figure 3). No further peaks followed those displayed in the 400-600 nm scans.
applications of ABTS and the expected electron exchange by comproportionation [29] were not observed. The production of the ABTS derivative went along with the replacement of the peaks at A420, A645, and A728, which are ascribed to the electrochemically generated ABTS •+ (Figure 2) [41], by rather flat ones in the A520 to A550 range. Both the wavelength positions and the total absorbance values of the peaks changed with species and accession of the timber, with the kind of the oxidoreductase enzyme involved (Table 1; Figure 2) and with the activation, e.g., of the intrinsic PO by variable doses of H2O2 ( Figure 3). No further peaks followed those displayed in the 400-600 nm scans. (a) (b) (c) Figure 2. Oxidation of ABTS by sapwood internal peroxidase (PO)/external H2O2 systems and mushroom laccase (Lac), respectively, aided by redox mediators of beech (a), birch (b), and spruce (c). Scans A400 to A600 obtained for oak, sycamore, and pine the widest corresponded with those of beech, birch, and spruce, respectively. See Table 1 for positions of their absorbance peaks. Scans A 400 to A 600 obtained for oak, sycamore, and pine the widest corresponded with those of beech, birch, and spruce, respectively. See Table 1 for positions of their absorbance peaks.  Across the wood species, the reaction mixtures showed yellowish (hornbeam), red to red-brown (beech, oak), ruby (birch), and brown (spruce, pine) color tones. These may be influenced by concentration and composition of the easily extractable wood constituents, many of which may be phenolic in nature (Table 1) to yield colored compounds upon peroxidase and laccase catalyzed cooxidations or polymerizations. The inherent peroxidase activities of the sapwood samples collected from January to June 2014 generally peaked in April, were higher in sapwood from understory rather than from dominant tree sources, and failed to correlate with the A550 values (r = 0.033-0.364). Similarly, the drastic drop to 3-5% in the total sapwood phenol content between February and June did not interfere with the formation rate of the ABTS derivative (data not specified).
The red ABTS derivative was formed both with the live PO bearing sapwood and the heattreated one whose inactivated enzyme was compensated by commercial horseradish peroxidase or mushroom laccase supplements. Beech sapwood extracts drawn with water, methanol, and acetone at 12 or 98 °C were also able to replace the wood chip in the laccase supported catalysis of the ABTS derivative as they all contained the respective redox mediators. As shown in HPLC-MS assays, the compounds ranged 60 to 1400 in MW and reached from catechin and tryptophan to flavonoids (data not shown).
Incubation for 20-27 h at room temperature of 0.15 g mature wheat straw or of 0.2 g of the green stem tissue of Boa sp. in the ABTS/KH2PO4 reaction mixtures amended with fungal laccase solution resulted, too, in the formation of the red ABTS derivatives in the absence of external H2O2. The stable products showed single peaks at A550-A555 (straw) and A527-A531 (grass), respectively. Their handling in (possibly electrostatically loaded) vessels resulted sometimes in the transient appearance of ABTS •+ linked peaks at A430 and A728. This was combined with shifts of the derivative peaks by 10-30 nm to higher values. In the presence of beech timber pre-degraded by soft rot and amended with laccase, the formation of the ABTS derivative was delayed for 3-4 days. Moreover, the soft-rotten timber samples generated the red derivative even in the absence of laccase (and H2O2) amendments within 7-9 d from ABTS solution. To the utmost surprise, in the presence of the non-purified fungal laccase, a reaction mixture composed of ABTS/KH2PO4 without external H2O2, that normally persists in the green ABTS •+ state, was also found converted to the red ABTS derivative with a peak at A533-A535 after 9 d in all replicates.

Transformation of ABTS •+ to the ABTS Derivative in the Absence of Oxidoreductases
Amending the ABTS/KH2PO4 solution with fungal laccase yielded the stable ABTS •+ state at A420 > 4.0. The absorbance fell to 2.6-2.8 by boiling the solution in a water bath at 98 °C for 5 min to inactivate the laccase. Adding sapwood chips of beech whose enzyme had been inactivated by autoclaving resulted in the spontaneous abiotic-radicalic conversion of ABTS •+ to the red ABTS derivative within 6-8 s and the reduction of a portion of the cation radical to the initial ABTS. The Across the wood species, the reaction mixtures showed yellowish (hornbeam), red to red-brown (beech, oak), ruby (birch), and brown (spruce, pine) color tones. These may be influenced by concentration and composition of the easily extractable wood constituents, many of which may be phenolic in nature (Table 1) to yield colored compounds upon peroxidase and laccase catalyzed co-oxidations or polymerizations. The inherent peroxidase activities of the sapwood samples collected from January to June 2014 generally peaked in April, were higher in sapwood from understory rather than from dominant tree sources, and failed to correlate with the A 550 values (r = 0.033-0.364). Similarly, the drastic drop to 3-5% in the total sapwood phenol content between February and June did not interfere with the formation rate of the ABTS derivative (data not specified).
The red ABTS derivative was formed both with the live PO bearing sapwood and the heat-treated one whose inactivated enzyme was compensated by commercial horseradish peroxidase or mushroom laccase supplements. Beech sapwood extracts drawn with water, methanol, and acetone at 12 or 98 • C were also able to replace the wood chip in the laccase supported catalysis of the ABTS derivative as they all contained the respective redox mediators. As shown in HPLC-MS assays, the compounds ranged 60 to 1400 in MW and reached from catechin and tryptophan to flavonoids (data not shown).
Incubation for 20-27 h at room temperature of 0.15 g mature wheat straw or of 0.2 g of the green stem tissue of Boa sp. in the ABTS/KH 2 PO 4 reaction mixtures amended with fungal laccase solution resulted, too, in the formation of the red ABTS derivatives in the absence of external H 2 O 2 . The stable products showed single peaks at A 550 -A 555 (straw) and A 527 -A 531 (grass), respectively. Their handling in (possibly electrostatically loaded) vessels resulted sometimes in the transient appearance of ABTS •+ linked peaks at A 430 and A 728 . This was combined with shifts of the derivative peaks by 10-30 nm to higher values. In the presence of beech timber pre-degraded by soft rot and amended with laccase, the formation of the ABTS derivative was delayed for 3-4 days. Moreover, the soft-rotten timber samples generated the red derivative even in the absence of laccase (and H 2 O 2 ) amendments within 7-9 d from ABTS solution. To the utmost surprise, in the presence of the non-purified fungal laccase, a reaction mixture composed of ABTS/KH 2 PO 4 without external H 2 O 2 , that normally persists in the green ABTS •+ state, was also found converted to the red ABTS derivative with a peak at A 533 -A 535 after 9 d in all replicates.

Transformation of ABTS •+ to the ABTS Derivative in the Absence of Oxidoreductases
Amending the ABTS/KH 2 PO 4 solution with fungal laccase yielded the stable ABTS •+ state at A 420 > 4.0. The absorbance fell to 2.6-2.8 by boiling the solution in a water bath at 98 • C for 5 min to inactivate the laccase. Adding sapwood chips of beech whose enzyme had been inactivated by autoclaving resulted in the spontaneous abiotic-radicalic conversion of ABTS •+ to the red ABTS

Filtration of the Red ABTS Derivative Against a 10-kDa Cutoff
Filtration was terminated when the composite ABTS derivative (2.5 mL) produced with sapwood chips of beech had yielded a bright-red filtrate (molecular size ≤ 10 kDa) and a dark-red to brownish concentrate fraction (molecular size ≥ 10 kDa) in proportions of 73:27% (v/v; Figure 4). The derivative linked absorbance peak of the original composite fraction located at A 520 moved to A 515 in the filtrate and was lacking in the concentrate ( Figure 5). Whereas applications of ABTS to both fractions did not result in spontaneous comproportionation reactions, adding H 2 O 2 provoked the immediate greening in the concentrate, and a hesitative greening in the filtrate fraction due to contamination of the latter with PO traces in the close filter system. The respective solutions regained their red color within 2-4 days at room temperature. Applying the O 2 •− releasing KO 2 crystals known to reduce ABTS •+ [58] destroyed the red substances within 24 h.

Filtration of the Red ABTS Derivative Against a 10-kDa Cutoff
Filtration was terminated when the composite ABTS derivative (2.5 mL) produced with sapwood chips of beech had yielded a bright-red filtrate (molecular size ≤ 10 kDa) and a dark-red to brownish concentrate fraction (molecular size ≥ 10 kDa) in proportions of 73:27% (v/v; Figure 4). The derivative linked absorbance peak of the original composite fraction located at A520 moved to A515 in the filtrate and was lacking in the concentrate ( Figure 5). Whereas applications of ABTS to both fractions did not result in spontaneous comproportionation reactions, adding H2O2 provoked the immediate greening in the concentrate, and a hesitative greening in the filtrate fraction due to contamination of the latter with PO traces in the close filter system. The respective solutions regained their red color within 2-4 days at room temperature. Applying the O2 •− releasing KO2 crystals known to reduce ABTS •+ [58] destroyed the red substances within 24 h.

LC-MS Examination of a Non-Purified ABTS Derivative from White Mustard
Under the ionizing conditions of the weak acids in the eluent, the red ABTS derivative yielded major peaks at m/z 452.6 > 490.4 > 485.3. Among the numerous minor substances, m/z 514.1 to 514.2 represented the ABTS dianion molecule completed by two protons at the former NH4 binding sites.   red derivative displayed a pronounced absorbance peak of A535 = 0.174 at λ = 535-545 nm (scan not shown). Reoxidizing the portion of the reduced ABTS with subsequent laccase applications increased the final absorbance values to A535 = 0.42-0.45.

Filtration of the Red ABTS Derivative Against a 10-kDa Cutoff
Filtration was terminated when the composite ABTS derivative (2.5 mL) produced with sapwood chips of beech had yielded a bright-red filtrate (molecular size ≤ 10 kDa) and a dark-red to brownish concentrate fraction (molecular size ≥ 10 kDa) in proportions of 73:27% (v/v; Figure 4). The derivative linked absorbance peak of the original composite fraction located at A520 moved to A515 in the filtrate and was lacking in the concentrate ( Figure 5). Whereas applications of ABTS to both fractions did not result in spontaneous comproportionation reactions, adding H2O2 provoked the immediate greening in the concentrate, and a hesitative greening in the filtrate fraction due to contamination of the latter with PO traces in the close filter system. The respective solutions regained their red color within 2-4 days at room temperature. Applying the O2 •− releasing KO2 crystals known to reduce ABTS •+ [58] destroyed the red substances within 24 h.

LC-MS Examination of a Non-Purified ABTS Derivative from White Mustard
Under the ionizing conditions of the weak acids in the eluent, the red ABTS derivative yielded major peaks at m/z 452.6 > 490.4 > 485. 3. Among the numerous minor substances, m/z 514.1 to 514.2 represented the ABTS dianion molecule completed by two protons at the former NH4 binding sites.

LC-MS Examination of a Non-Purified ABTS Derivative from White Mustard
Under the ionizing conditions of the weak acids in the eluent, the red ABTS derivative yielded major peaks at m/z 452.6 > 490.4 > 485. 3. Among the numerous minor substances, m/z 514.1 to 514.2 represented the ABTS dianion molecule completed by two protons at the former NH 4 binding sites. A derivative appearing at m/z 486.3 points to additional demethylations that are replaced by two H + ( Figure 6). A derivative appearing at m/z 486.3 points to additional demethylations that are replaced by two H + ( Figure 6).

Chemical ABTS Oxidation
Molarities of 1:2 between ABTS and K2O8S2 in the reaction mixture completed the oxidation of the ABTS to the green cation radical with its slightly higher values shifted peaks at λ = 420/660/738 nm within 20-45 min. A black to blue-green color was observed at 4 h and did not show any further changes within 96 h. The green color spontaneously developed in dilute reaction mixtures with molarities of 1:15. Changes to brownish black and the disappearance of the A422 peak accompanied the formation of the final deep lilac reaction product within 2 h (Figure 7). Diluted aliquots were reddish violet and contained clouds of precipitates which formed deep lilac pellets in the colorless supernatants of non-depleted K2O8S2 solution of pH 2.15 upon centrifugation. Pellets obtained from bulk samples of 5 mg ABTS were separated from K2O8S2 residues by up to 10 consecutive resuspensions and centrifugations in 2-mL aliquots of bideionized water. The washing fluids increasing

Chemical ABTS Oxidation
Molarities of 1:2 between ABTS and K 2 O 8 S 2 in the reaction mixture completed the oxidation of the ABTS to the green cation radical with its slightly higher values shifted peaks at λ = 420/660/738 nm within 20-45 min. A black to blue-green color was observed at 4 h and did not show any further changes within 96 h. The green color spontaneously developed in dilute reaction mixtures with molarities of 1:15. Changes to brownish black and the disappearance of the A 422 peak accompanied the formation of the final deep lilac reaction product within 2 h (Figure 7). Diluted aliquots were reddish violet and contained clouds of precipitates which formed deep lilac pellets in the colorless supernatants of non-depleted K 2 O 8 S 2 solution of pH 2.15 upon centrifugation. Pellets obtained from bulk samples of 5 mg ABTS were separated from K 2 O 8 S 2 residues by up to 10 consecutive re-suspensions and centrifugations in 2-mL aliquots of bideionized water. The washing fluids increasing in pH from 2.7 to the stable 3.6 contained the green ABTS •+ at absorbance values of around A 420 = 0.8. This moderate release of ABTS •+ did not substantially increase in NaOH amended washing fluids of pH 8.0. In NaOH solutions of pH 11, the precipitates were dissolved by the consecutive reduction to the green ABTS cation radical and the final colorless ABTS within 1 h. Dispersing the separated and rinsed precipitates in aqueous ABTS solution resulted in the spontaneous appearance of green at absorbance values of A 420 > 2.5. Thus, the precipitates indicated their nature as active oxidants by comproportionation with ABTS. in pH from 2.7 to the stable 3.6 contained the green ABTS •+ at absorbance values of around A420 = 0.8. This moderate release of ABTS •+ did not substantially increase in NaOH amended washing fluids of pH 8.0. In NaOH solutions of pH 11, the precipitates were dissolved by the consecutive reduction to the green ABTS cation radical and the final colorless ABTS within 1 h. Dispersing the separated and rinsed precipitates in aqueous ABTS solution resulted in the spontaneous appearance of green at absorbance values of A420 > 2.5. Thus, the precipitates indicated their nature as active oxidants by comproportionation with ABTS.

Comparison of ABTS Derivatives by Fourier Transform Infrared Spectroscopy (FT-IR) Analyses
Differences in composition and stability of the red ABTS derivatives obtained by chemical or enzymatic oxidation were drastically illustrated by preliminary FT-IR analyses (Figure 8). The lilac precipitates of the completely by K2O8S2 oxidized monoazo compound (formed from 3.7 mg ABTS) showed major stretching vibrations at 1506.6, 1472.8, and 1441.4 cm −1 , i.e., in the range of (1555) 1525-1410 cm −1 associated with the presence of the -N=N-double bonds in the majority of azo dyes [59][60][61][62]. In this range, the band at 1472.8 cm −1 is lacking in the sample of the commercial ABTS. The compound generated by fresh beech wood chips (compare Section 2.2; 1.7 mg ABTS in 40 mg KH2PO4) showed then the azo group related band at 1473.77 cm −1 in an environment of a general structural decay, with the dominance of signals from P-O bonds around 859 cm −1 (Figure 8) [63].

Comparison of ABTS Derivatives by Fourier Transform Infrared Spectroscopy (FT-IR) Analyses
Differences in composition and stability of the red ABTS derivatives obtained by chemical or enzymatic oxidation were drastically illustrated by preliminary FT-IR analyses (Figure 8). The lilac precipitates of the completely by K 2 O 8 S 2 oxidized monoazo compound (formed from 3.7 mg ABTS) showed major stretching vibrations at 1506.6, 1472.8, and 1441.4 cm −1 , i.e., in the range of (1555) 1525-1410 cm −1 associated with the presence of the -N=N-double bonds in the majority of azo dyes [59][60][61][62]. In this range, the band at 1472.8 cm −1 is lacking in the sample of the commercial ABTS. The compound generated by fresh beech wood chips (compare Section 2.2; 1.7 mg ABTS in 40 mg KH 2 PO 4 ) showed then the azo group related band at 1473.77 cm −1 in an environment of a general structural decay, with the dominance of signals from P-O bonds around 859 cm −1 (Figure 8) [63]. in pH from 2.7 to the stable 3.6 contained the green ABTS •+ at absorbance values of around A420 = 0.8. This moderate release of ABTS •+ did not substantially increase in NaOH amended washing fluids of pH 8.0. In NaOH solutions of pH 11, the precipitates were dissolved by the consecutive reduction to the green ABTS cation radical and the final colorless ABTS within 1 h. Dispersing the separated and rinsed precipitates in aqueous ABTS solution resulted in the spontaneous appearance of green at absorbance values of A420 > 2.5. Thus, the precipitates indicated their nature as active oxidants by comproportionation with ABTS.

Comparison of ABTS Derivatives by Fourier Transform Infrared Spectroscopy (FT-IR) Analyses
Differences in composition and stability of the red ABTS derivatives obtained by chemical or enzymatic oxidation were drastically illustrated by preliminary FT-IR analyses (Figure 8). The lilac precipitates of the completely by K2O8S2 oxidized monoazo compound (formed from 3.7 mg ABTS) showed major stretching vibrations at 1506.6, 1472.8, and 1441.4 cm −1 , i.e., in the range of (1555) 1525-1410 cm −1 associated with the presence of the -N=N-double bonds in the majority of azo dyes [59][60][61][62]. In this range, the band at 1472.8 cm −1 is lacking in the sample of the commercial ABTS. The compound generated by fresh beech wood chips (compare Section 2.2; 1.7 mg ABTS in 40 mg KH2PO4) showed then the azo group related band at 1473.77 cm −1 in an environment of a general structural decay, with the dominance of signals from P-O bonds around 859 cm −1 (Figure 8) [63].  , and of the product generated by enzymatically active beech wood chips (c), red curve), paired with an ABTS-free reference sample (blue curve). The major peak at 858.86 cm −1 is not shown.

Discoloration of Remazol Brilliant Blue R
In two test arrangements, the promoting effect of the red ABTS derivative on the oxidative discoloration of the dye RBBR was significant (p ≤ 0.01). In the Table 2 test, applications of K.-mutabilis culture fluid solely exposed the RBBR solution to the fungal laccase activity. Adding H2O2 as an enzyme co-substrate activated the oxidative cycle of the fungal MnP in the narrow frame set by the presence of free Mn cations, whereas an equivalent of 0.044 mg ABTS was applied with the input of 0.2 mL of the red ABTS derivative generated with beech sapwood. The resulting absorbance values A595 for RBBR dropped in proportions of 1 (Lac):1.3 (Lac; MnP):3.3 (Lac; MnP; ABTS derivative). Additional applications of ABTS gave rise to the formation of the stable green ABTS •+ solution which masked any effects on the discoloration of RBBR. , and of the product generated by enzymatically active beech wood chips (c), red curve), paired with an ABTS-free reference sample (blue curve). The major peak at 858.86 cm −1 is not shown.

Discoloration of Remazol Brilliant Blue R
In two test arrangements, the promoting effect of the red ABTS derivative on the oxidative discoloration of the dye RBBR was significant (p ≤ 0.01). In the Table 2   The tests shown in Table 3

Discussion
The transformation of the green ABTS •+ solution to stable reddish products in the presence of lignocelluloses such as fresh and rotten timber, straw, and green herbage is possibly a hitherto non-reported phenomenon. The absorbance peaks of the transformation products appear at wavelength positions within λ = 513-573 nm and exceed the narrow span of 518-520 nm reported for the electrochemically [30,41] and wet-chemically [44,45] generated ABTS 2+ . Testing several inorganic peroxides yielded a further ABTS oxidation product that showed absorbance peaks at around A 420 and A 530 in scans [45] that resemble those given for the red ABTS derivatives in the Figures 3 and 5. The authors failed to purify the respective products and took them for sulfoxide and sulfone compounds obtained from ABTS •+ and the oxidants peroxomonosulfate and peroxodiphosphate. Majcherczyk et al. [46] obtained "a small amount of a reddish product" absorbing at λ = 560-570 nm upon the oxidation of veratryl alcohol with laccase/ABTS. A similar red product appearing at 562 nm was obtained from ABTS •+ in the presence of bovine serum albumin but also in Mes buffer solvent [64]. During repeated recoveries and re-oxidations with acetone and laccase, respectively, a purple by-product deduced to be azodication was gradually formed. Filtation against a 10 kDa cutoff separated a colorless filtrate from magenta colored substances the authors took for ABTS 2+ -laccase complexes [65].
The present tests with vital sapwood chips from seven European tree species show that color, wavelength position and total absorbance of the ABTS •+ transformation products vary with timber species and their accession, with the kind of the enzyme catalyst, and the amount of the applied H 2 O 2 as the exhaustible activator of the wood-intrinsic peroxidases ( Table 1, Figures 2 and 3). Co-oxidations and polymerizations of specific timber extractives as well as complex and adduct formations may then account for shifts of the absorbance peaks within the visible light spectrum.
Whereas the formation of the ABTS cation radical in the laccase-ABTS mediator system is enzyme catalyzed, its transformation to the reddish product is a spontaneous abiotic reaction with mediating extractives of lignocelluloses. Applying autoclaved beech sapwood chips with their heat-inactivated peroxidase to boiled ABTS •+ solution resulted in the formation of red products and the reduction of the remaining cation radical within 6-8 s in the absence of enzymes (Section 3.2). The reduction of ABTS •+ to ABTS comprises the one-electron oxidation of the lignocellulose extractives to radicals that overcome the potential of E 0 = 1.09 V during the formation of the azodication from ABTS •+ , with the promise to play a major part in the initial attacks on plant lignins, too. The respective extractives drawn with water or organic solvents from beech sapwood mimicked the mediating catalytic effect of the live and heat-killed timber and comprised molecules ranging 60 to 1400 in MW as shown by HPLC-MS analyses. Related extractives determined for wheat straw were N-heterocycles (16-29%), fatty acids (19-26%), and phenols (12-23%) beside carbohydrates, amino acids, n-alkyl benzenes, diols, sterols, steroids, and flavonoids in the m/z range 270-414 [66]. In the case of phenolics, redox mediating effects have long been known. Phenol, cresol and resorcinol enabled plant peroxidase to form the abiotic oxidant Mn 3+ [67]. Common phenolics with redox potentials E 0 between 0.54-1.23 V [14,19,32,34] but also the metabolite 3-hyroxyanthranilate of the wood-decay fungus Pycnoporus cinnabarinus [11] showed potential as laccase mediators.
Ultrafiltration against a 10 kDa cutoff separated a beech wood catalyzed red transformation product peaking at λ = 520 nm into a bright-red filtrate (peak at A 515 ) and a darker red concentrate without the typical ABTS derivative related peak (Figures 4 and 5). ABTS application to the filtrate (and the concentrate) did not result in a spontaneous comproportionation [41,46] of the red substance(s) although handling of comparable products from green herbage in presumably electrostatically loaded vessels gave rise to a transient re-appearance of ABTS •+ related peaks in spectrophotometric scans. The respective peaks disappeared again by the non-enzymatic transformation of ABTS •+ to the red derivative in the presence of (less efficient or lower concentrated?) lignocellulose extractives from grasses. In the concentrate fraction, application of ABTS plus H 2 O 2 led to the immediate formation of ABTS •+ by the enriched peroxidase constituents of 31.9-39.4 kDa in MW [55]. This was followed again by the formation of red products. The darker red tone of the concentrate compared to the filtrate fraction suggests a relative enrichment of the red derivatives (bound in complexes?) with high-MW compounds and especially an adduct constellation with (enzyme) proteins [30,65,68,69].
Liquid chromatograms of a non-purified red ABTS derivative generated by white mustard root exudates showed a peak at m/z 514.2 ( Figure 6). This corresponds with all oxidation states of ABTS M 548.68 deprived of both NH 4 moieties ( Figure 1) and re-complexed by two (H + ) protons. A peak at m/z 486.3 points to the replacement of both ethyl (C 2 H 5 ) by methyl (CH 3 ) groups of this compound. The main peaks appeared at m/z 490.4, 485.3, and 452.6. The substances should be ABTS derived but also linked with substrate components as they surpass by far the concentrations and the low-MW range of the carbohydrate and aliphatic and aromatic acid traces exuded by white mustard seedlings in axenic culture [70,71]. In comparable HPLC-MS assays, the radical content of vegetable extracts is determined by the quantitative discoloration (reduction) of a green ABTS •+ solution. Beside the m/z 514 peak valid for all protonated ABTS dianion moieties as above, the MS protocols show, e.g., adduct formations between ABTS( 2+ ) and the radical scavenging flavonols rutin, hyperoside, quercetin, and quercitrin compounds [72]. The FT-IR analysis of the enzymatically generated ABTS derivative indicated, beside the wide destruction of the molecule, conserved structures in the chromogenic environment of the -N=N-double bond. Its position at 1473.77 cm −1 corresponds with the vibration stretch at 1472.8 cm −1 of the K 2 O 8 S 2 oxidized product ( Figure 8). Nevertheless, in the case of the chemically identical structures of ABTS •+ at pH 1.5 and an ABTS 2+ preparation in concentrated HNO 3 , Raman spectroscopy yielded different spectra [43].
The red multi-component and water-soluble ABTS derivative pretended stability from pH 0.5 (HCl) to pH 14 (NaOH) and was destroyed by O 2 •− released from KO 2 . In contrast, the ABTS 2+ product presented, e.g., by Majcherczyk et al. [46] and generated with potassium peroxodisulfate formed double peaks at λ = 556 and 603 nm (Figure 7), precipitated from a supernatant pH 2.15, and was reduced to ABTS by comproportionation and higher alkaline conditions (pH 11, NaOH). It oxidized several non-phenolic alcohols [46]. Elemental analyses of the washed precipitate by ICP-MS suggested that the insolubility of the product was not due to the binding of K 2 O 8 S 2 derived K + to the so far complete ABTS 2− dianion ( Figure 1) in the pH range of up to 3.5 (data not shown). The catalytic potential of the red ABTS derivative needs further investigations. Its use in combination with the non-purified K. mutabilis laccase and with H 2 O 2 accelerated the bleaching of the dye RBBR significantly ( Table 2). Its bleaching was also stimulated when not only the live sapwood chip of beech with its natural mediators but also an amount of the red ABTS derivative was applied ( Table 3) that, it should not be concealed, contained active radicals derived from timber extracts.
The results invite to a reappraisal of an old controversy (e.g., [29,30]): is the cell-free and purified laccase/ABTS couple able to form ABTS 2+ in vitro? According to Christensen and Kepp [68], the turnover of ABTS is optimal by laccases with positively charged anchor points in their pockets near the T1 copper site. This positive charge should correspond with ABTS at pH values > 3 (pK a around 2) that appears as ABTS 2− dianion deprived of both NH 4 moieties (Figure 1). At pH 4.5, the enzyme (E 0 up to 0.79 V) would oxidize around 50% of the ABTS pool [73,74] to the green ABTS •− radical anion (E 0 = 0.68 V) [41] by one-electron abstraction. If the enzyme catalyzed accidental oxidations of the radical to dications in a numerical redox reaction it cannot afford (E 0 = 1.09 V) [41], the dications would comproportionate with ABTS back to the radical state. Indirect evidence for ABTS 2+ formations by laccase/ABTS is being derived from reactions of the couple with high redox potential substrates such as veratryl alcohol (3,4-dimethoxybenzyl alcohol) (E 0 = 1.4 V) and other benzyl alcohols that are frequently tested in the citrate and acetate buffers used by Cyclic Voltammetry [29,30,46,52,53]. The respective substrates could be contaminated by organic traces with mediator properties similar to those of wood extractives.
Amending an enzyme generated and subsequently boiled ABTS •+ solution with autoclaved beech wood caused its partial transformation to the red ABTS derivative within 6-8 s in an abiotic reaction with wood extractives and in the absence of functional enzymes (Section 3.2). The tests run over 1-2 h were repeated several times. This time span should not suffice that the red ABTS derivative had been formed with the aid of substrate contaminants as long as the enzyme had been active. Therefore, reactions of the laccase/ABTS •+ couple that suggest catalytic contributions of ABTS 2+ should be reconsidered. Organic trace contaminants of test substrate, redox mediator, buffer, and the enzyme itself could initiate the abiotic ABTS •+ to ABTS 2+ conversion by acting like wood extractives, with the dication generating further radicals from substrates and their contaminants in the stadium nascenti. Further dications may immediately be trapped and stabilized as "slowly generated red substances" [45,46,64]. The transformation of an ABTS •+ /KH 2 PO 4 solution to the red ABTS derivative by mushroom laccase added as part of a depleted and multi-contaminant malt/peptone nutrient solution as late as after 9 days may serve as a precedent. Bourbonnais et al. [29] recorded ABTS mediated lignin oxidation at the redox potential of laccase (585 mV) at a very slow rate. The oxidation of 2,4-dimethoxybenzyl alcohol by laccase/ABTS started first with the onset of a second and slower rate of oxygen consume the authors linked with the slow production of ABTS 2+ in the initially formed laccase/ABTS •+ solution [52]. Interactions of oxidoreductase enzyme/ABTS couples with the extractives of lignocelluloses drive these reactions to the utmost. In summary, it is uncontested that ABTS improves the efficacy of substrate conversions by oxidoreductases, irrespective of its presence as a transitory dication or a (red) multicomponent derivative.

Conclusions
There are no reasons to contest the importance of the ABTS dication in expanding the substrate ranges of laccases and plant peroxidases to higher-redox-potential substrates in vitro. Nevertheless, in the presence of lignocelluloses that contain a plethora of lower-MW extractives, the further conversion of the enzyme-catalyzed ABTS cation radical is a spontaneous and abiotic process that is complete within seconds. Its outcome may be the formation of radicals from lower-redox-potential extractives in a chain reaction initiated by ABTS •+ upon its reduction to functional ABTS, the apparent formation of a red ABTS derivative that possibly contains a stable form of ABTS 2+ beside high-MW adducts, and further ABTS moieties of MW > 400 Da, catalyzed by radicals derived from extractives or formed as artifacts under LC-MS conditions. Whether these ABTS moieties contribute more to oxidative degradation processes than the apparently to radicals transformed lignocellulose extractives is subject to further studies which may be rendered more difficult by the use of chemicals certified as "pure".