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-alpha -C-beta cleavage of IEA to vanillyl acetate were studied. IEA oxidation onl y occurred in the presence of VA. It probably did not bind to lignin peroxi dase as evidenced by an unaffected K-m for VA in the presence of IEA, and b y the fact that a 10-fold molar excess of the unreactive IEA counterpart, e ugenyl acetate, did not affect the IEA oxidation rate. IEA was very efficie nt in recycling VA. Up to 34 mol of IEA were oxidized per mol VA. Formation of the predominant VA oxidation product, veratraldehyde, was postponed unt il 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 remarkabl e considering the high calculated ionization potential of 8.81 eV. Regardless of the presence of O-2, approximate to 2 mol of IEA were consume d 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 O-2-dependent chemical reactions were responsible for C-alph a-C-beta cleavage to the major oxidation product vanillyl acetate, as evide nced by the observation that an N-2 atmosphere did not inhibit IEA oxidatio n, but almost completely inhibited vanillyl acetate formation. GC-MS analys es 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. Formatio n of the latter two was diminished under an N-2 atmosphere.