On the mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 by EDTA.

The mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 (LiPH2) by EDTA was investigated. It was found that EDTA was decarboxylated and that cytochrome c, nitro blue tetrazolium, ferric iron, and molecular oxygen were reduced in a reaction mixture containing LiPH2, H2O2, veratryl alcohol, and EDTA. The reductive activity observed with LiPH2 followed first order kinetics with respect to the concentration of EDTA. Stoichiometry studies showed that in the presence of sufficient EDTA, 1.7 mol of ferric iron were reduced per mole of H2O2 added to the reaction mixture. Superoxide- and EDTA-derived radicals were detected by ESR spin trapping upon incubation of LiPH2 with H2O2, veratryl alcohol, and EDTA. The Km values of veratryl alcohol and H2O2 remained the same for both the oxidative and reductive activities of LiPH2. Reductive activity was also observed with LiPH2 and EDTA using other free radical mediators in the place of veratryl alcohol, such as 1,4-dimethoxybenzene, 1,2,3- and 1,2,4-trimethoxybenzenes, and 1,2,4,5-tetramethoxybenzene. EDTA reduced the cation radical of 1,2,4,5-tetramethoxybenzene formed by LiPH2 in the presence of H2O2. Hence, it is proposed that the apparent inhibition of the veratryl alcohol oxidase activity of LiPH2 by EDTA is due to the reduction of the veratryl alcohol cation radical intermediate back to veratryl alcohol by EDTA. The reduction of cytochrome c, nitro blue tetrazolium, ferric ion, and molecular oxygen appears to be mediated by the EDTA radical formed by reduction of the veratryl alcohol cation radical.