Thymine hydroxylase is an a-ketoglutarate, non-heme iron-dependent dioxygenase that catalyzes the conversion of thymine to its corresponding alcohol, aldehyde, and carboxylic acid in three steps, each accompanied by the conversion of cl-ketoglutarate to succinate and CO2. Studies by Thornburg and Stubbe (Biochemistry 1993, 32, 14034) showed that incubation of thymine hydroxylase with 5-ethynyluracil resulted in the production of 5-(carboxymethyl)uracil and uracil-5-acetylglycine and inactivation of the protein by covalent modification. Tryptic digestion of the inactivated protein followed by isolation of the modified peptides and their analysis by mass spectrometry revealed sequences (N)SIAFXSNPSLR, in which X was proposed to be a modified tyrosine residue. Recent efforts to clone the gene for thymine hydroxylase fortuitously resulted in isolation of the unmodified peptide. Sequencing of this peptide established that the amino acid residue modified by 5-ethynyluracil is a phenylalanine and not the predicted tyrosine! Two sets of experiments have been carried out to reveal the structure of the 5-ethynyluracil-modified phenylalanine. Incubation of 5-ethynyluracil with thymine hydroxylase in the presence of O-18(2) revealed, subsequent to tryptic digestion and peptide isolation, that two atoms of oxygen derived from O-18(2) have been incorporated. A similar experiment using [2-C-14]-5-[1’, 2’-C-13(2)]ethynyluracil resulted in the isolation of a sufficient amount of modified peptide for analysis by 1D and 2D NMR spectroscopy. This analysis revealed a novel 7-carboxylated norcaradiene moiety. A mechanism involving partitioning of a carbene intermediate between insertion into the phenylalanine residue of the protein and rearrangement to generate a ketene is proposed to account for the structure of the peptide adduct and the previously characterized small molecule products (5-(carboxymethyl)uracil and uracil-5-acetylglycine). O-18(2)-Labeling experiments and the presence of a carboxylic acid in the adduct suggest that thymine hydroxylase carries out a second hydroxylation reaction while the oxidized inhibitor is covalently bound in the enzyme’s active site.