Reduction of the 2,2′-Azinobis(3-Ethylbenzthiazoline-6-Sulfonate) Cation Radical by Physiological Organic Acids in the Absence and Presence of Manganese

Laccase is a copper-containing phenoloxidase, involved in lignin degradation by white rot fungi. The laccase substrate range can be extended to include nonphenolic lignin subunits in the presence of a noncatalytic cooxidant such as 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), with ABTS being oxidized to the stable cation radical, ABTS^·+, which accumulates. In this report, we demonstrate that the ABTS^·+ can be efficiently reduced back to ABTS by physiologically occurring organic acids such as oxalate, glyoxylate, and malonate. The reduction of the radical by oxalate results in the formation of H₂O₂, indicating the formation of O₂^·− as an intermediate. O₂^·− itself was shown to act as an ABTS^·+ reductant. ABTS^·+ reduction and H₂O₂ formation are strongly stimulated by the presence of Mn²⁺, with accumulation of Mn³⁺ being observed. Additionally, 4-methyl-O-isoeugenol, an unsaturated lignin monomer model, is capable of directly reducing ABTS^·+. These data suggest several mechanisms for the reduction of ABTS^·+ which would permit the effective use of ABTS as a laccase cooxidant at catalytic concentrations.