Kinetic and Spectroscopic Characterization of the Catalytic Ternary Complex of Tryptophan 2,3-Dioxygenase

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 <i>Cupriavidus metallidurans</i> 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 O₂, 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 O₂-bound ferrous species. They revealed remarkable similarities between the electronic properties of the Michaelis complex and those of the O₂ 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 <i>k</i>_H/<i>k</i>_D of 0.87 ± 0.03 when (indole-<i>d</i>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.