Chemical Rescue of the Distal Histidine Mutants of Tryptophan 2,3-Dioxygenase

Abstract

Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent enzyme that catalyzes the oxidative degradation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK). A highly conserved histidine residue in the distal heme pocket has attracted great attention in the mechanistic studies of TDO. However, a consensus has not been reached regarding whether and how this distal histidine plays a catalytic role after substrate binding. In this study, three mutant proteins, H72S, H72N, and Q73F were generated to investigate the function of the distal histidine residue in Cupriavidus metallidurans TDO (cmTDO). Spectroscopic characterizations, enzymatic kinetic analysis, and chemical rescue assays were employed to study the biochemical properties of the wild-type enzyme and the mutant proteins. Rapid kinetic methods were utilized to explore the molecular basis for the observed stimulation of catalytic activity by 2-methylimidazole in the His72 variants. The results indicate that the distal histidine plays multiple roles in cmTDO. First, His72 contributes to but is not essential for substrate binding. In addition, it shields the heme center from nonproductive binding of exogenous small ligand molecules (i.e., imidazole and its analogs) via steric hindrance. Most importantly, His72 participates in the subsequent chemical catalytic steps after substrate binding possibly by providing H-bonding interactions to the heme-bound oxygen.

Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening

“…16 After incubating with TDO, each probe generated a new peak centered near 321 nm similar to that observed from the reaction with l -Trp, suggesting that both probes are alternate substrates. The catalytic rate using these probes as the substrate in place of l -Trp was compared to that with l -Trp (Figure 2).…”

“…Recently, compelling evidence from multiple groups supports the stepwise oxygen insertion mechanism. 8–9, 11, 13–14, 15,16–17 …”

Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions

“…Fu et al (81) proposed that TDO reactivation by H 2 O 2 involves a two-electron reduction of a Fe IV ϭO intermediate by L-Trp to form a Fe II -Trp adduct indicated by the formation of a distinct peak at 432 nm by optical absorption spectroscopy (81). Also, for TDO it was not possible to detect a Fe IV ϭO species in the presence of H 2 O 2 and L-Trp (81), which has been recently reported to exhibit a ␥-Soret maxima at 414 nm and peaks in the visible region with reduced intensity at 504 and 630 nm, and increased peak intensities at 526 and 548 nm (82). In marked contrast, our optical absorption and RR spectroscopy data on IDO show that H 2 O 2 in the presence of L-Trp forms an Fe IV ϭO species as the major detectable IDO heme species, which exhibits a ␥-Soret peak at 412 nm and visible region peaks at ϳ541 and ϳ576 nm (Figs.…”

Heterolytic OO bond cleavage: Functional role of Glu113 during bis-Fe(IV) formation in MauG

“…5A, the black trace) [29, 32, 39]. The identity of this freezing-induced artifact is assigned as a hydroxide-bound hexacoordinate low-spin Heme 5C species, based on the similarity of its g -values to those of the hydroxide-bound hexacoordinate low-spin heme species reported in other hemoproteins [40, 41]. The hydroxide ligand is believed to be derived from a Heme 5C -bound water molecule shown in the crystal structure of MauG [22].…”