The crystal structure of lignin peroxidase at 1.70 angstrom resolution reveals a hydroxy group on the C-beta of tryptophan 171: A novel radical site formed during the redox cycle

The crystal structure of lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium was refined to an X-factor of 16.2% utilizing s ynchrotron data in the resolution range from 10 to 1.7 Angstrom. The final model comprises all 343 amino acid residues, 370 water molecules, the heme, four carbohydrates, and two calcium ions. Lignin peroxidase shows the typi cal peroxidase fold and the heme has a close environment as found in other peroxidases. During refinement of the Lip model an unprecedented modificati on of an amino acid was recognized. The surface residue tryptophan 171 in L iP is stereospecifically hydroxylated at the CP atom due to an autocatalyti c process. We propose that during the catalytic cycle of Lip a transient ra dical at Trp171 occurs that is different from those previously assumed for this type of peroxidase. Recently, the existence of a second substrate-bind ing site centered at Trp171 has been reported, by us which is different fro m the "classical heme edge" site found in other peroxidases. Here, we repor t evidence for a radical formation at Trp171 using spin trapping, which sup ports the concept of Trp171 being a redox active amino acid and being invol ved in the oxidation of veratryl alcohol. On the basis of our current model , an electron pathway from Trp171 to the heme is envisaged, relevant for th e oxidation of veratryl alcohol and possibly lignin. Beside the opening lea ding to the heme edge, which can accommodate small aromatic substrate molec ules, a smaller channel giving access to the distal heme pocket was identif ied that is large enough for molecules such as hydrogen peroxide. Furthermo re, it was found that in Lip the bond between the heme iron and the N-epsil on 2 atom of the proximal histidine residue is significantly longer than in cytochrome c peroxidase (CcP). The weaker Fe-N bond in LiP renders the hem e more electron deficient and destabilizes high oxidation states, which cou ld explain the higher redox potential of Lip as compared to CcP. (C) 1999 A cademic Press.