Oxidation of Monomethoxylated Aromatic Compounds by Lignin Peroxidase: Role of Veratryl Alcohol in Lignin Biodegradation

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H₂O₂-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H₂O₂ concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H₂O₂ to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H₂O₂ and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H₂O₂. AA competitively inhibited the oxidation of VA by LiP; the K_i for AA inhibition was 32 μM. Addition of VA to LiPIII* resulted in its conversion to the native enzyme. In contrast, AA did not convert LiPIII* to the native enzyme; instead, LiPIII* was bleached in the presence of AA. Thus, AA does not protect LiP from inactivation by H₂O₂. These results support a mechanism whereby VA protects the enzyme from inactivation by H₂O₂, thus making more enzyme available for the oxidation of recalcitrant substrates, such as AA and probably polymeric lignin. The results do not support a mechanism whereby VA acts as a radical mediator in the LiP oxidation of AA and other monomethoxylated aromatics.