Lignin-degrading peroxidases, a group of biotechnologically interesting enzymes, oxidize high redox potential aromatics via an exposed protein radical. Low temperature EPR of Pleurotus eryngii versatile peroxidase (VP) revealed, for the first time in a fungalperoxidase,thepresenceofatryptophanylradicalinboth the two-electron (VPI) and the one-electron (VPII) activated formsoftheenzyme.Site-directedmutagenesiswasusedtosub-stitute this tryptophan (Trp-164) by tyrosine and histidine resi-dues.Nochangesinthecrystalstructurewereobserved,indicat- ing that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate for VPI reduction ( k 2app veratryl (VA), 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. first-order rate constant ( k 2 ) more affected than the dissociation constant ( K D 2 ). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant ( k 3 ) values were similar for the W164Y, W164H, and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed, revealing that Trp-164 is involved in this autocatalytic process.