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

Abstract

Abstract:In fermentations of lignocelluloses, redox potentials (If not indicated otherwise, redox potentials in Volt are taken versus Normal Hydrogen Reference Electrodes (NHE).) E 0 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 electro-or wet-chemistry to the green cation radical (ABTS •+ , 0.68 V) and the red dication (ABTS 2+ , 1.09 V). The enzyme/ABTS couple generates the stable ABTS •+ whose low E 0 cannot explain the couple's contemporary attack on non-phenolic lignins. This paradoxon indicates the non-confirmed production of the ligninolytic ABTS 2+ by the enzymes. During incubations of live sapwood chips in ABTS/H 2 O 2 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 ABTS 2+ 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 ABTS 2+ 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 ABTS 2+ molecules.

ABTS/PP Decolorization Assay of Antioxidant Capacity Reaction Pathways

“…The ABTS •+ radical cation can undergo further oxidation to form the dication ABTS 2+ with λ max at 513-520 nm, ε 36 000 L mol −1 cm −1 (see Figure 2a) [42]). The ABTS 2+ formation kinetics from ABTS •+ , when an excess of S 2 O 8 2− is applied, is approximately three times slower than the radical cation ABTS •+ formation from ABTS [47,48]. Apparently, the ABTS 2+ dication does not influence ABTS/PP assay measurements as it has rather low solubility in water and is relatively stable only in the excess of S 2 O 8 2− anions or in a very acidic medium, with any aqueous dilution resulting in a shift of the reaction balance back to ABTS •+ [41,[47][48][49][50][51].…”

Formation of the Azodication (ABTS2+) from ABTS [2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulphonate)] in Sterile Plant Cultures: Root–Exuded Oxidoreductases Contribute to Rhizosphere Priming

“…Comparable disintegrations of the red ABTS 2+ related product were also documented in preliminary Fourier-transform infrared spectrometry (FT-IR) analyses [62]. The lilac precipitates of a completely by K 2 O 8 S 2 oxidized monoazo compound of functional ABTS showed major stretching vibrations at 1506.6, 1472.8, and 1441.4 cm −1 .…”

“…Contemplating the treatments 1a and 4b with Cu(II) as the only efficient Fenton metal (Table 1), it is concluded that plants initiate oxidative activities in the rhizosphere by the release of peroxidases, carboxylates, and occasional traces of active oxygen species at least in alfalfa [38]. Azodication derivatives in the culture fluids confirm the presence of root-released phenolic molecules, too, that had been oxidized to aryloxy radicals with the respective redox mediator potential [62]. Their formation could mainly be ascribed to the PO/active-oxygen couple but also to the by-production of Mn 3+ with the aid of the radicals in turn.…”

“…The position of absorbance peaks in red culture fluids point to the presence of ABTS 2+ in the A 515 to A 520 range [56]. Modifications of the azo dication in solutions peak at A 542 to A 561 [62]. Heat-killed plant control cultures devoid of MnO 2 and PO activity did not oxidize ABTS.…”

“…The catalysis of the initial green oxidation stage of ABTS •+ was ascribed to the PO/H 2 O 2 system. The second oxidation step to ABTS 2+ only preceded with timber-released mediator molecules converted to radicals by ABTS •+ and possibly by the PO/H 2 O 2 couple, too [62].…”