Kinetic and spectroscopic characterization of the catalytic ternary complex of tryptophan 2,3-dioxygenase.

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 indole-amine 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 Mossbauer 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-Mossbauer 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 a-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.