LIGNIN PEROXIDASES CAN ALSO OXIDIZE MANGANESE

The peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium include lignin peroxidases and manganese-dependent peroxidases. The major isozymes, called lignin peroxidases, are thought to oxidize chemicals directly. The manganese-dependent peroxidases (H3, H4, H5, and H9) are relatively minor, making up only a fraction of the total peroxidase protein. However, we have found that lignin peroxidases will also catalyze the H2O2-dependent oxidation of Mn2+ to Mn3+. We have used lignin peroxidase isozyme H2 (LiPH2) to characterize the manganese peroxidase activity of lignin peroxidases. Transient state kinetic studies were used to obtain a second-order rate constant of 4.2 x 10(4) M(-1) s(-1) for the reaction of LiPH2-compound I with free or chelated Mn2+ at pH 6.0. This reaction was too fast to monitor at pH 4.5. Only chelated Mn2+ could reduce LiPH2-compound II to ferric enzyme. The Mn2+-chelate (oxalate) first bound LiPH2-compound Il with a Kd Of (1.5 +/- 0.3) X 10(-5) M and then reduced LiPH2-compound IT to ferric enzyme with a first order rate constant of 215 +/- 6 s(-1). Steady-state kinetic studies on LiPH2 were performed by directly monitoring the formation of Mn3+-oxalate. These results show that oxidation of Mn2+ by a lignin peroxidase does not occur through free radical mediation as proposed previously [Popp et al. (1990) Biochemistry 29, 10475-10480). Electron spin resonance and oxygen evolution studies also indicate that Mn2+ is directly oxidized by LiPH2. In addition, veratryl alcohol oxidation by LiPH2 was inhibited in the presence of Mn2+, suggesting that both Mn2+ and veratryl alcohol are substrates for LiPH2.