Quinone Redox Cycling in the Ligninolytic FungusPleurotus eryngiiLeading to Extracellular Production of Superoxide Anion Radical

Quinone redox cycling is generally known as an intracellular process that implies the reduction of quinones (Q) into semiquinones (Q−·) or hydroquinones (QH2), which autoxidize reducing oxygen to superoxide anion radical (O−2·). We demonstrate here for the first time the existence of quinone redox cycling in a ligninolytic fungus,Pleurotus eryngii,showing two particularities: extracellular production of O−2· and involvement of ligninolytic enzymes. Experiments were performed withP. eryngiicultures, showing laccase activity, and four quinones: 1,4-benzoquinone (BQ), 2-methyl-1,4-benzoquinone (MeBQ), 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ), and 2-methyl-1,4-naphthoquinone (menadione, MD). The overall process consisted of cell-bound divalent reduction of quinones, followed by extracellular laccase-mediated oxidation of hydroquinones into semiquinones, which autoxidized to a certain extent producing O−2· (at the pH values of natural degradation of lignin, some autoxidation of hydroquinones was observed only with DQH2and MDH2). The existence of a redox cyclic system involving quinones was evidenced by determining the chemical state of quinones along incubation under several conditions (either different O2concentrations and pH values or laccase amounts). Thus, QH2/Q ratios at system equilibrium decreased as either pH values and oxygen concentration (allowing hydroquinones autoxidation) or the amount of laccase increased. Once the cyclic nature of the system was demonstrated, special attention was paid to the production of O−2· during hydroquinone oxidation. Except in the case of BQH2, production of O−2· was found in samples containing hydroquinones and laccase. By the use of agents promoting the autoxidation of semiquinones (superoxide dismutase and Mn2+), production of O−2· during oxidation of BQH2could finally be demonstrated.