Abstract: 4-Hydroxyphenylpyruvate (HPP) is converted to homogentisate by 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) as a part of tyrosine catabolism. The reaction involves oxidative decarboxylation of the side chain, hydroxylation of a phenyl ring, and substituent migration. We have sub-cloned and over-expressed HPPD from Steptomyces avermitilis in E. coli. This form of HPPD can be reliably purified to >98% purity in two chromatographic steps. The initial goal of our research has been to find evidence for the accumulation of intermediates using pre-steady state kinetic methods. The global analysis of spectrophotometric data indicates that three intermediates accumulate during a single turnover of the enzyme. 2,3,5,6-Tetra-deuterated HPP was synthesized from deuterated tyrosine, using cloned tyrosine amino transferase. We have measured the steady-state and pre-steady state isotope effects with both 2,3,5,6-tetra-deuterated HPP substrate and deuterated solvent, D₂O. Our data suggest that re-aromatization to form homogentisate and the release of this product are the last two processes observed in the pre-steady state. Hereditary type 1 tyrosinemia is a result of a deficiency of the enzyme that catalyzes the final step in the tyrosine catabolism pathway, fumarylacetoacetase. In the absence of treatment, patients who suffer from this disease generally die from either kidney failure or from primary liver cancer. A molecule, originally developed as an herbicide, NTBC inhibits HPPD, thereby shutting down four of the steps of tyrosine catabolism and alleviating the symptoms of T1Y. Treated with NTBC, tyrosinemia sufferers now live normal lives. Despite numerous studies on NTBC and similar inhibitors, the explicit binding mode was unknown. We have crystallized and solved the structure of HPPD in complex with NTBC. The accommodation of the inhibitor involves the pivoting of the C-terminal active site helix through 40 degrees. The inhibitor makes bidentate contact with the active site ferrous ion through its 5' and 7' oxygens and the aromatic ring is sandwiched in a staggered arrangement with two conserved active site phenylalanines, one of which is provided by the C-terminal alpha helix. Other than direct coordination to the metal ion, the inhibitor has no apparent hydrogen bonding or electrostatic interactions with the enzyme yet is bound essentially irreversibly.