Open AccessKinetic and Spectroscopic Characterization of the Catalytic Ternary Complex of Tryptophan 2,3-Dioxygenase
Author(s) -
Jiafeng Geng,
Andrew C. Weitz,
Kednerlin Dornevil,
Michael P. Hendrich,
Aimin Liu
Publication year - 2020
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.0c00179
Subject(s) - chemistry , ternary complex , michaelis–menten kinetics , catalysis , reaction intermediate , substrate (aquarium) , dioxygenase , photochemistry , stereochemistry , enzyme , organic chemistry , enzyme assay , oceanography , geology
The first step of the kynurenine pathway for l-tryptophan (l-Trp) degradation is catalyzed by heme-dependent dioxygenases, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase. In this work, we employed stopped-flow optical absorption spectroscopy to study the kinetic behavior of the Michaelis complex of Cupriavidus metallidurans TDO (cmTDO) to improve our understanding of oxygen activation and initial oxidation of l-Trp. On the basis of the stopped-flow results, rapid freeze-quench (RFQ) experiments were performed to capture and characterize this intermediate by Mössbauer spectroscopy. By incorporating the chlorite dismutase-chlorite system to produce high concentrations of solubilized O 2 , we were able to capture the Michaelis complex of cmTDO in a nearly quantitative yield. The RFQ-Mössbauer results confirmed the identity of the Michaelis complex as an O 2 -bound ferrous species. They revealed remarkable similarities between the electronic properties of the Michaelis complex and those of the O 2 adduct of myoglobin. We also found that the decay of this reactive intermediate is the rate-limiting step of the catalytic reaction. An inverse α-secondary substrate kinetic isotope effect was observed with a k H / k D of 0.87 ± 0.03 when (indole- d 5 )-l-Trp was employed as the substrate. This work provides an important piece of spectroscopic evidence of the chemical identity of the Michaelis complex of bacterial TDO.