Induction of Extracellular Hydroxyl Radical Production by White-Rot Fungi through Quinone Redox Cycling

A simple strategy for the induction of extracellular hydroxyl radical (OH) production by white-rot fungi is presented. It involves the incubation of mycelium with quinones and Fe³⁺-EDTA. Succinctly, it is based on the establishment of a quinone redox cycle catalyzed by cell-bound dehydrogenase activities and the ligninolytic enzymes (laccase and peroxidases). The semiquinone intermediate produced by the ligninolytic enzymes drives OH production by a Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺). H₂O₂ production, Fe³⁺ reduction, and OH generation were initially demonstrated with two Pleurotus eryngii mycelia (one producing laccase and versatile peroxidase and the other producing just laccase) and four quinones, 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and 2-methyl-1,4-naphthoquinone (menadione MD]). In all cases, OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H₂O₂ levels and Fe³⁺ reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.White-rot fungi are unique in their ability to degrade a wide variety of organopollutants (36, 47), mainly due to the secretion of a low-specificity enzyme system whose natural function is the degradation of lignin (11). Components of this system include laccase and/or one or two types of peroxidase, such as lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP) (31). Besides acting directly, the ligninolytic enzymes can bring about lignin and pollutant degradation through the generation of low-molecular-weight extracellular oxidants, including (i) Mn³⁺, (ii) free radicals from some fungal metabolites and lignin depolymerization products (7, 22), and (iii) oxygen free radicals, mainly hydroxyl radicals (OH) and lipid peroxidation radicals (21). Although OH radicals are the strongest oxidants found in cultures of white-rot fungi (1), studies of their involvement in pollutant degradation are scarce. One of the reasons is that the mechanisms proposed for OH production still await in vivo validation.Several potential sources of extracellular OH based on the Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺) have been postulated for white-rot fungi. In one case, an extracellular fungal glycopeptide has been shown to reduce O₂ and Fe³⁺ to H₂O₂ and Fe²⁺ (45). Enzymatic sources include cellobiose dehydrogenase, LiP, and laccase. Among these, only cellobiose dehydrogenase is able to directly catalyze the formation of Fenton''s reagent (33). The ligninolytic enzymes, however, act as an indirect source of OH through the generation of Fe³⁺ and O₂ reductants, such as formate (CO₂⁻) and semiquinone (Q⁻) radicals. The first time evidence was provided that a ligninolytic enzyme was involved in OH production, oxalate was used to generate CO₂⁻ in a LiP reaction mediated by veratryl alcohol (4). The proposed mechanism consisted of the following cascade of reactions: production of veratryl alcohol cation radical (Valc⁺) by LiP, oxidation of oxalate to CO₂⁻ by Valc⁺, reduction of O₂ to O₂⁻ by CO₂⁻, and a superoxide-driven Fenton reaction (Haber-Weiss reaction) in which Fe³⁺ was reduced by O₂⁻. The OH production mechanism assisted by Q⁻ was inferred from the oxidation of 2-methoxy-1,4-benzohydroquinone (MBQH₂) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH₂) by Pleurotus eryngii laccase in the presence of Fe³⁺-EDTA. The ability of Q⁻ radicals to reduce both Fe³⁺ to Fe²⁺ and O₂ to O₂⁻, which dismutated to H₂O₂, was demonstrated (14). In this case, OH radicals were generated by a semiquinone-driven Fenton reaction, as Q⁻ radicals were the main agents accomplishing Fe³⁺ reduction. The first evidence of the likelihood of this OH production mechanism being operative in vivo had been obtained from incubations of P. eryngii with 2-methyl-1,4-naphthoquinone (menadione MD]) and Fe³⁺-EDTA (15). Extracellular OH radicals were produced on a constant basis through quinone redox cycling, consisting of the reduction of MD by a cell-bound quinone reductase (QR) system, followed by the extracellular oxidation of the resulting hydroquinone (MDH₂) to its semiquinone radical (MD⁻). The production of extracellular O₂⁻ and H₂O₂ by P. eryngii via redox cycling involving laccase was subsequently confirmed using 1,4-benzoquinone (BQ), 2-methyl-1,4-benzoquinone, and 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone), in addition to MD (16). However, the demonstration of OH production based on the redox cycling of quinones other than MD was still required.In the present paper, we describe the induction of extracellular OH production by P. eryngii upon its incubation with BQ, 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and MD in the presence of Fe³⁺-EDTA. The three benzoquinones were selected because they are oxidation products of p-hydroxyphenyl, guaiacyl, and syringyl units of lignin (MD was included as a positive control). Along with laccase, the involvement of P. eryngii VP in the production of O₂⁻ and H₂O₂ from hydroquinone oxidation has also been reported (13). Since hydroquinones are substrates of all known ligninolytic enzymes, quinone redox cycling catalysis could involve any of them. Here, we demonstrate OH production by P. eryngii under two different culture conditions, leading to the production of laccase or laccase and VP. We also show that quinone redox cycling is widespread among white-rot fungi by using a series of well-studied species that produce different combinations of ligninolytic enzymes.