Initial O₂Insertion Step of the Tryptophan Dioxygenase Reaction Proposed by a Heme-Modification Study

Abstract

L-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O₂ into the pyrrole ring of L-tryptophan. Although a ferrous-oxy form (Fe²⁺-O₂) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O₂ insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous-oxy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C-O stretch of the heme-bound CO and enhanced the dissociation of O₂ from the ferrous-oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O₂. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous-oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O₂ to the indole ring of L-Trp.

Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening

“…This could be accomplished by either an electrophilic addition or an iron-bound superoxide radical attack to the l -Trp, which requires further investigation despite extensive studies. 7, 10, 38 The second half involves a histidine-ligated ferryloxo (a compound II-like species) 8, 13 and an epoxyindole intermediate. The formation of a substrate-based epoxide intermediate has been found in other systems such as nonheme iron extradiol dioxygenase and Rieske dioxygenase.…”

Substrate Oxidation by Indoleamine 2,3-Dioxygenase

“…There is now general agreement that base-catalyzed proton abstraction does not occur ( 3 , 8 – 11 , 23 , 25 , 26 ), although the experimental evidence against it is still rather limited. Our data for S-Trp also provide convincing evidence against base-catalyzed abstraction because we observed Compound II formation ( Fig.…”

A short history of heme dioxygenases: rise, fall and rise again

“…Electrophilic addition from the ferrous oxy species, Fig. 6, is one possibility: recent evidence in TDO [98] (using modified hemes that were first used more than 30 years ago [99]) supports this. We have noted [74, 75] that oxygen may not be an especially good electrophile if it is bound to the heme as a ferric superoxide species, and there is spectroscopic evidence for a ferric superoxide species [97] from Raman’s work.…”