Kinetic and Spectroscopic Characterization of the Catalytic Ternary Complex of Tryptophan 2,3-Dioxygenase
Year: 2020
Type: article
Abstract: 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.
Source: Biochemistry
Language: English
Subfield: Inorganic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W3042300475