Oxygen activation during oxidation of methoxyhydroquinones by laccase fromPleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-me thoxy-1,4-benzohydroquinone (MBQH(2)) and 2,6-dimethoxy-1,4-benzohydroquino ne (DBQH(2)) by lactase from Pleurotus eryngii was examined. Laccase oxidiz ed DBQH(2) more efficiently than it oxidized MBQH(2); both the affinity and maximal velocity of oxidation were higher for DBQH(2) than for MBQH(2). Au toxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q(.-) + O-2 <-> Q + O-2(.-)). As this reaction is reversible, its existence was first noted in studies o f the effect of systems consuming and producing O-2(.-) on quinone formatio n rates. Then, the production of H2O2 in laccase reactions, as a consequenc e of O-2(.-) dismutation, confirmed that semiquinones autoxidized, The high est H2O2 levels were obtained with DBQH(2), indicating that DBQ(.-) autoxid ized to a greater extent than did MBQ(.-). Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation an d laccase oxidation. Two ways of favoring semiquinone autooxidation over di smutation and laccase oxidation were increasing the rate of O-2(.-) consump tion,vith superoxide dismutase (SOD) and recycling of quinones with diaphor ase (a reductase catalyzing the divalent reduction of quinones), These two strategies made the laccase reaction conditions more natural, since O-2(.-) , besides undergoing dismutation, reacts with Mn2+, Fe3+, and aromatic radi cals. In addition, quinones are continuously reduced by the mycelium of whi te-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 mu M concentrations of MBQ(.-) and DBQ(.-) from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ(.-) autoxidation rose to 13.8% and that of DBQ(.-) increased to 39. 9%.