Veratryl alcohol-mediated oxidation of isoeugenyl acetate by lignin peroxidase

The mechanism of the veratryl alcohol (VA)-mediated oxidation of isoeugenyl acetate (IEA) by lignin peroxidase, and the subsequent spontaneous C(α)-C(β) cleavage of IEA to vanillyl acetate were studied, IEA oxidation only occurred in the presence of VA. It probably did not bind to lignin peroxidase as evidenced by an unaffected K(m) for VA in the presence of IEA, and by the fact that a 10-fold molar excess of the unreactive IEA counterpart, eugenyl acetate, did not affect the IEA oxidation rate. IEA was very efficient in recycling VA. Up to 34 mol of IEA were oxidized per mol VA. Formation of the predominant VA oxidation product, veratraldehyde, was postponed until IEA was almost completely oxidized. Together these findings suggest that IEA was oxidized by VA·⁺ rather than directly by lignin peroxidase. Thus, VA functioned as a redox mediator during IEA oxidation which is remarkable considering the high calculated ionization potential of 8.81 eV. Regardless of the presence of O₂, ≃ 2 mol of IEA were consumed per mol H₂O₂, which indicated that IEA was enzymatically oxidized by one electron to the putative radical cation (IEA·⁺). After formation of IEA·⁺, a series of O₂-dependent chemical reactions were responsible for C(α)-C(β) cleavage to the major oxidation product vanillyl acetate, as evidenced by the observation that an N₂ atmosphere did not inhibit IEA oxidation, but almost completely inhibited vanillyl acetate formation. GC-MS analyses revealed that under an air atmosphere 1-(4'-acetoxy-3'- methoxyphenyl)-2-propanone, 1-(4'-acetoxy-3'-methoxyphenyl)-1-hydroxy-2- propanone, and 1-(4'-acetoxy-3'-methoxyphenyl)-2-hydroxy-1-propanone were also formed. Formation of the latter two was diminished under an N₂ atmosphere.