Reaction Mechanism of Heme-Based Dioxygenases

Abstract

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are two heme-containing enzymes that catalyze the oxidative cleavage of tryptophan (Trp) to N-formyl kynurenine (NFK), the initial and rate-limiting step of the kynurenine pathway. Until recently it was generally believed that the heme dioxygenase reaction follows a base-catalyzed mechanism. Based on this mechanism, the reaction is initiated by deprotonation of the indoleamine group of Trp by an active site base. It is followed by electrophilic addition of the heme-bound dioxygen to the C2=C3 bond of the indole moiety of Trp, leading to a heme-bound 3-indolenylperoxo intermediate, which subsequently converts to the product NFK, via a dioxetane intermediate or a Criegee type of rearrangement. In this work, we sought to use continuous-flow resonance Raman spectroscopy, combined with stopped-flow UV-Vis spectroscopy, to investigate the dioxygenase reaction carried out by IDO and TDO. Surprisingly, a ferryl intermediate was detected during the IDO reaction at 0.2 s. The presence of this intermediate supports a new mechanism, in which the two atoms of dioxygen are sequentially incorporated into the substrate via a two-step reaction. The ferryl intermediate is not observable during the TDO reaction, highlighting the structural differences between the two types of dioxygenases, as well as the importance of the stereoelectronic factors in modulating the reactions.