Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism

LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr¹⁸¹) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H₂O₂ and VA) lacked this lag, and H₂O₂-LiP (H₂O₂-treated LiP) was inactive. MS analyses revealed that VA-LiP includes one VA molecule covalently bound to the side chain of Tyr¹⁸¹, whereas H₂O₂-LiP contains a hydroxylated Tyr¹⁸¹. No adduct is formed in the Y171N variant. Molecular docking showed that VA binding is favoured by sandwich π stacking with Tyr¹⁸¹ and Phe⁸⁹. EPR spectroscopy after peroxide activation of the pre-treated LiPs showed protein radicals other than the tyrosine radical found in pristine LiP, which were assigned to a tyrosine–VA adduct radical in VA-LiP and a dihydroxyphenyalanine radical in H₂O₂-LiP. Both radicals are able to oxidize large low-redox-potential substrates, but H₂O₂-LiP is unable to oxidize high-redox-potential substrates. Transient-state kinetics showed that the tyrosine–VA adduct strongly promotes (>100-fold) substrate oxidation by compound II, the rate-limiting step in catalysis. The novel activation mechanism is involved in ligninolysis, as demonstrated using lignin model substrates. The present paper is the first report on autocatalytic modification, resulting in functional alteration, among class II peroxidases.