AUTOCATALYTIC FORMATION OF A HYDROXY GROUP AT C-BETA OF TRP171 IN LIGNIN PEROXIDASE

In the high-resolution crystal structures of two lignin peroxidase iso zymes from the white rot fungus Phanerochaete chrysosporium a signific ant electron density at single bond distance from the C beta of Trp171 was observed and interpreted as a hydroxy group. To further clarify t he nature of this feature, we carried out tryptic digestion of the enz yme and isolated the Trp171 containing peptide. Under ambient conditio ns, this peptide shows an absorbance spectrum typical of tryptophan, A t elevated temperature, however, the formation of an unusual absorbanc e spectrum with lambda(max) = 333 nm can be followed that is identical to that of N-acetyl-alpha,beta-didehydrotryptophanamide, resulting up on water elimination from beta-hydroxy tryptophan. The Trp171 containi ng tryptic peptide isolated from the recombinant and refolded lignin p eroxidase produced from Escherichia coli does not contain the characte ristic 333 nm absorbance band at any temperature. However, treatment w ith 3 equiv of H2O2 leads to complete hydroxylation of Trp171, Reducin g substrates compete with this process, e.g., in the presence of 0.5 m M veratryl alcohol, about 7 equiv of H2O2 is necessary for complete mo dification. We conclude that the hydroxylation at the C beta of Trp 17 1 is an autocatalytic reaction which occurs readily under conditions o f natural turnover, e.g., in the ligninolytic cultures of P, chrysospo rium, which are known to contain an oxidase-based H2O2-generating syst em. No dependence on dioxygen was found for this oxidative process, Ch emical modification of fungal lignin peroxidase with the tryptophan-sp ecific agent N-bromo succinimide leads to a drastically reduced acitiv ity with respect to the substrate veratryl alcohol. This suggests that Trp171 is involved in catalysis and that electron transfer from this surface residue to the oxidized heme cofactor is possible under steady -state conditions.