Mechanism of oxidative C alpha-C beta cleavage of a lignin model dimer by Phanerochaete chrysosporium ligninase. Stoichiometry and involvement of free radicals

Abstract

The hemoprotein ligninase of Phanerochaete chrysosporium Burds. catalyzes the oxidative cleavage of lignin model dimers between C alpha and C beta of their propyl side chains. The model dimers hitherto used give multiple products and complex stoichiometries upon enzymatic oxidation. Here we present experiments with a new model dimer, 1-(3,4-dimethoxyphenyl)-2-phenylethanediol (dimethoxyhydrobenzoin, DMHB) which is quantitatively cleaved by ligninase in air to give benzaldehyde and veratraldehyde according to the stoichiometry: 2DMHB + O2----2PhCHO + 2Ph(OMe)2CHO. Catalytic amounts of H2O2 are required for this aerobic reaction. Under anaerobic conditions, ligninase uses H2O2 as the oxidant for cleavage: DMHB + H2O2----PhCHO + Ph(OMe)2CHO. Electron spin resonance experiments done in the presence of spin traps, 2-methyl-2-nitrosopropane or 5,5-dimethyl-1-pyrroline-N-oxide, show that C alpha-C beta cleavage yields alpha-hydroxybenzyl radicals as intermediate products. Under anaerobic conditions, these radicals react further to give the final aldehyde products. In air, O2 adds to the carbon-centered radicals, probably giving alpha-hydroxybenzylperoxyl radicals which fragment to yield superoxide, benzaldehyde, and veratraldehyde. These results lead us to propose a mechanism for C alpha-C beta cleavage in which attack by ligninase and H2O2 on the methoxylated ring of DMHB yields a cation radical, which then cleaves to give either benzaldehyde and an alpha-hydroxy(dimethoxybenzyl) radical or veratraldehyde and an alpha-hydroxybenzyl radical (cf. Kersten, P. J., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612; Snook, M. E., and Hamilton, G. A. (1974) J. Am. Chem. Soc. 96, 860-869). Similar mechanisms probably apply to the enzymatic C alpha-C beta cleavage of natural lignin.