Evidence from spin-trapping for a transient radical on tryptophan residue 171 of lignin peroxidase

The heme enzyme lignin peroxidase contains a unique C beta-hydroxylated try ptophan residue (Trp171) on the surface of the enzyme. Mutagenetic substitu tion of Trp171 abolishes completely the veratryl alcohol oxidation activity of the enzyme. This led us to surmise that Trp171 may be involved in elect ron transfer from natural substrates to the heme cofactor. Here we present evidence for the formation of a transient radical on Trp171 using spin-trap ping in combination with peptide mapping. The spin-trap methyl nitroso prop ane forms a covalent adduct with Trp171 in the presence of hydrogen peroxid e which can be detected by its characteristic visible absorbance spectrum. A very similar chromophore can be obtained in a small molecular model syste m from N-acetyl tryptophanamide, the spin-trap, and a single-electron abstr acting system, The precise site the spin-trap is attached to could be ident ified in a crystal structure of spin-trap/ hydrogen peroxide-treated enzyme as the C6 atom of the indole ring of Trp171. These results indicate that T rp171 is redox-active and that it forms an indole radical by transfer of an electron to the heme of compound I and/or II. Apart from cytochrome c pero xidase and DNA photolyase, lignin peroxidase appears to be the third enzyme only which utilizes a tryptophan residue as an integral part of its redox catalysis. (C) 1999 Academic Press.