Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

Rong Fu,Rupal Gupta,Jiafeng Geng,Kednerlin Dornevil,Siming Wang,Yong Zhang,Michael P Hendrich,Aimin Liu

doi:10.1074/jbc.m111.253237

Abstract

An intriguing mystery about tryptophan 2,3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2,3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔE(Q) = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. In the presence of L-Trp, the reactivation was demonstrated by enzyme assays and by various spectroscopic techniques. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism. These results may shed light on how a metalloenzyme maintains its catalytic activity in an oxidizing environment.

Highlights

Reaction of Oxidized TDO and H2O2 in the Absence of L-Trp— The reaction of ferric TDO with H2O2 was examined with an oxygen electrode in a stirred cell at 25 °C
When ferric TDO was added to the buffer containing H2O2, similar O2 production was observed (Fig. 1B), indicating no dependence on the order of additions
Addition of either the protein or peroxide alone, as shown in Fig. 1, did not produce O2. These results demonstrate that O2 is produced from H2O2 and lead to the conclusion that Fe(III)-TDO possesses a catalase-like activity with H2O2 in the absence of L-Trp

Summary

Present address

Indoleamine 2,3-dioxygenase (IDO), was isolated 31 years later from tissues other than the liver [10] Both enzymes catalyze the same reaction, TDO is highly substrate-specific with L-Trp, whereas IDO presents a more relaxed specificity. Hydrogen peroxide (H2O2) was implicated as an activator by the finding that the resting ferric TDO becomes active toward L-Trp after treatment with H2O2 [22] This was later confirmed by independent optical spectroscopic studies from various laboratories [14, 17, 23] and by the observations that the overall enzyme reactivation was inhibited by catalase and that the addition of peroxide relieved the reactivation inhibition [14]. We provide unequivocal evidence in support of the formation of the ferrous form of the enzyme by reaction of ferric TDO with peroxide and L-Trp. A previously unknown twophase enzyme reactivation mechanism is proposed based on the chemical identification of most of the intermediates and products. We further hypothesize that the physiological significance of the peroxide reactivity is to allow for the reactivation of the enzyme in an oxidizing environment

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION