Structure-function relationships of rat hepatic tryptophan 2,3-dioxygenase: Identification of the putative heme-ligating histidine residues

The liver cytosolic enzyme tryptophan 2,3-dioxygenase (TDO) catalyzes the o xidation of L-tryptophan to formylkynurenine and controls the physiological flux of tryptophan into both the serotonergic and kynureninic pathways. Th is hemoprotein enzyme is composed of four noncovalently bound subunits of e quivalent mass and contains two heme moieties per molecule. Electron parama gnetic resonance analyses have indicated that a histidyl nitrogen is involv ed in heme ligation [Henry et al., (1976) J. Biol. Chem. 251, 1578], but th e identity of the His residue(s) is unknown. In an attempt to characterize the active site of the enzyme we have substituted each of the 12 His residu es in the rat TDO subunit with Ala, to determine their relative importance in heme binding. Sequence alignment of the rat liver protein with that of k nown or putative TDO sequences from other organisms reveals that four of th e His residues are conserved in eukaryotes, two of which are also conserved in prokaryotes. Our findings indicate that replacement of the evolutionari ly conserved His 76 and 328 residues resulted in a dramatic reduction of TD O activity, whereas that of the eukaryotically conserved His70 resulted in a significant reduction relative to that of the wild-type enzyme. On the ot her hand, replacement of the other eukaryotically conserved His273 residue, while affecting the relative expression of the enzyme, had little effect o n its specific activity. Size-exclusion analyses revealed that the His76Ala and His328Ala mutants retained little or no heme, suggesting that these ma y be key residues in ligating the prosthetic heme moieties. Whether these H is residues are both provided by the same TDO subunit or a different TDO su bunit remains to be determined. (C) 2001 Academic Press.