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Cationic cell wall-bound peroxidase (CWPO-C) has the capability to oxidize sinapyl alcohol, ferrocytochrome c, and synthetic lignin polymers, unlike most peroxidases that have been characterized in flowering plants, such as horseradish peroxidase and Arabidopsis thaliana peroxidase A2. It has been suggested that the oxidation site is located on the CWPO-C surface, and homology modeling and chemically modified CWPO-C studies suggest that Tyr74 and/or Tyr177 are possible participants in the catalytic site. The present study clarifies the importance of these Tyr residues for substrate oxidation, using recombinant CWPO-C and recombinant mutant CWPO-C with phenylalanine substitution(s) for tyrosine. Such recombinant proteins, produced in Escherichia coli as inclusion bodies, were successfully refolded to yield the active form, and purified recombinant protein solutions exhibited typical spectra of high-spin ferric protein and displayed H2O2-dependent oxidation of guaiacol, 2,6-dimethoxyphenol, and syringaldazine. Measurement of peroxidase activity with these guaiacyl and syringyl compounds as reducing substrates indicated that a single mutation, Y74F or Y177F, resulted in substantial loss of oxidation activity (˜ 40–60% and 82%, respectively). Also, over 95% of the oxidation activity was lost with a double mutation, Y74F/Y177F. These results indicated that Tyr74 and Tyr177, rather than the heme pocket, play a central role in the oxidation of these substrates. This is the first report of active residues on an enzyme surface being identified in a plant peroxidase. This study also suggests that sinapyl alcohol incorporation into lignin is performed by a peroxidase that generates Tyr radicals on its surface.Our present study provides the direct evidence that Tyr74 and Tyr177 located on the protein surface of cationic cell-wall-bound peroxidase (CWPO-C), instead of heme pocket, have a central role in oxidation of guiacyl and syringyl compounds. This study also suggests that peroxidases, which generate tyrosine radicals on the surface, are responsible for lignifications.