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 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 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 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.

Mechanistic Studies on the Single-Turnover Yeast Thiamin Pyrimidine Synthase: Characterization of the Inactive Enzyme

“…Inspection of the THI5p-PLP structure and analysis of all available THI5p sequences does not reveal a canonical protein iron binding site close to the active site. Therefore, based on the extensive literature on the Fe­(II) binding to salicylaldimine Schiff base ligands, − we propose that the PLP imine and hydroxyl groups constitute two of the Fe­(II)-binding ligands of THI5p, and the required 10/12-electron oxidation can be achieved using three cycles of closely related Fe­(III) superoxide oxidation chemistry. , Analogous Fe­(III) superoxide chemistry has been proposed for tryptophan dioxygenase, extradiol dioxygenases, − 2-oxoacid dioxygenases, and Rieske dioxygenases . A striking feature of the THI5p structure is the existence of a highly conserved set of four cysteine residues (Cys195, 196, 197, and 199) providing a path for electron transfer from the buffer to the active site by disulfide interchange reactions analogous to those found in the Class I ribonucleotide reductases .…”

Binding of l-kynurenine to X. campestris tryptophan 2,3-dioxygenase

“…S3, and presumably will control release of product from the enzyme. These hydrogen bonding interactions between the substrate/product and the enzyme might well control the reactivity of the ternary Michaelis complex in the family of tryptophan dioxygenases [20]. Most of the reported inhibitor structures are for ferric enzymes (Table 2) which, as we show here, can be active towards the substrate (L-Trp).…”

A novel catalytic heme cofactor in SfmD with a single thioether bond and a bis-His ligand set revealed by a de novo crystal structural and spectroscopic study

“…Oxidative, mononuclear heme-based enzymes include cysteineligated superfamilies (cytochrome P450 and peroxygenase) and histidine-ligated superfamilies (peroxidase and dioxygenase). Our recent work on TDO and related heme-dependent enzymes, 10,23,[33][34][35][36][37][38] especially tyrosine hydroxylase (TyrH), prompted us to explore the structural and functional relationships of these enzymes, focusing on the question of whether or not a group of histidine-ligated heme-based enzymes can activate oxygen for monooxygenation and how this group may relate to other categories of mononuclear heme-based enzymes. In this work, integrated biochemical, spectroscopic, and structural studies have shown that (1) SfmD is a monooxygenase which can mediate an ascorbate/O 2 -dependent catalytic monooxygenation of its native substrate, (2) the de novo SfmD crystal structure and a partially reduced crystal structure reveal an unprecedented Hx n HxxxC (n $ 38) heme-binding motif of single thioether link and a bis-His ligand set where the rst histidine in the motif dissociates during catalysis, while the second histidine and cysteine residues (the HxxxC portion of the ligand set) remain bound to the prosthetic group, and (3) SfmD is shown to be a structurally validated prototypic monooxygenase in the superfamily currently known as the heme-dependent dioxygenase superfamily.…”