CONICET | Buscador de Institutos y Recursos Humanos

Indoleamine 2,3-dioxygenase (IDO) and tryptophan diox-ygenase (TDO) are two heme proteins that catalyze the oxidation reactionof tryptophan (Trp) to N-formylkynurenine (NFK). Human IDO(hIDO) has recently been recognized as a potent anticancer drug target,a fact that triggered intense research on the reaction and inhibitionmechanisms of hIDO. Our recent studies revealed that the dioxygenasereaction catalyzed by hIDO and TDO is initiated by addition of the ferric iron-bound superoxide to the C2dC3 bond of Trp to form aferryl and TrpÀepoxide intermediate, via a 2-indolenylperoxo radical transition state. The data demonstrate that the two atoms ofdioxygen are inserted into the substrate in a stepwise fashion, challenging the paradigm of heme-based dioxygenase chemistry. In thecurrent study, we used QM/MM methods to decipher the mechanism by which the second ferryl oxygen is inserted into theTrpÀepoxide to form the NFK product in hIDO. Our results show that the most energetically favored pathway involves proton transferfrom TrpÀNH3+ to the epoxide oxygen, triggering epoxide ring opening and a concerted nucleophilic attack of the ferryl oxygen to the C2of Trp that leads to a metastable reaction intermediate. This intermediate subsequently converts to NFK, following C2ÀC3 bond cleavageand the associated back proton transfer from the oxygen to the amino group of Trp. A comparative study with Xantomonas campestrisTDO (xcTDO) indicates that the reaction follows a similar pathway, although subtle differences distinguishing the two enzyme reactionsare evident. The results underscore the importance of the NH3+ group of Trp in the two-step ferryl-based mechanism of hIDO andxcTDO, by acting as an acid catalyst to facilitate the epoxide ring-opening reaction and ferryl oxygen addition to the indole ring.