Ferryl derivatives of human indoleamine 2,3-dioxygenase

Abstract

The critical role of the ferryl intermediate in catalyzing the oxygen chemistry of monooxygenases, oxidases, or peroxidases has been known for decades. In contrast, its involvement in heme-based dioxygenases, such as human indoleamine 2,3-dioxygenase (hIDO), was not recognized until recently. In this study, H(2)O(2) was used as a surrogate to generate the ferryl intermediate of hIDO. Spectroscopic data demonstrate that the ferryl species is capable of oxidizing azinobis(3-ethylbenzothiazoline-6-sulfonic acid) but not L-Trp. Kinetic studies reveal that the conversion of the ferric enzyme to the ferryl intermediate facilitates the L-Trp binding rate by >400-fold; conversely, L-Trp binding to the enzyme retards the peroxide reaction rate by ∼9-fold, because of the significant elevation of the entropic barrier. The unfavorable entropic factor for the peroxide reaction highlights the scenario that the structure of hIDO is not optimized for utilizing H(2)O(2) as a co-substrate for oxidizing L-Trp. Titration studies show that the ferryl intermediate possesses two substrate-binding sites with a K(d) of 0.3 and 440 μM and that the electronic properties of the ferryl moiety are sensitive to the occupancy of the two substrate-binding sites. The implications of the data are discussed in the context of the structural and functional relationships of the enzyme.

SCHEME 1.

Two-step dioxygenation mechanism of l-Trp catalyzed by hIDO (29, 30). The light and dark shading of the oxygen atoms denote the proximal and distal atoms of the heme-bound dioxygen.

FIGURE 1.

Time-resolved optical absorption spectra obtained following the mixing of substrate-free hIDO (2.2 μm) with H₂O₂ (2 mm) in the absence of l-Trp in a stopped-flow instrument at 20 °C (a) and the observed rate constant as a function of H₂O₂ concentration (b). The inset in a shows the exponential kinetic trace at 403 nm and the best fit of the data with a single exponential function.

FIGURE 2.

Time-resolved optical absorption spectra obtained following the mixing of l-Trp-bound hIDO (2. 6 μm) with H₂O₂ (2 mm) in a stopped-flow instrument at 20 °C (a) and the observed rate constant as a function of H₂O₂ concentration (b). The inset in a shows the exponential kinetic trace at 410 nm, and the best fit of the data with a single exponential function. The inset in b shows the expanded view of the linear region of the plot. To produce the l-Trp-bound hIDO, 25 mm l-Trp (final concentration) was used.

FIGURE 3.

Observed formation rate of the ferryl species derived from the reaction of substrate-free or l-Trp-bound hIDO with H₂O₂ as a function of temperature (a and b) and the associated Eyring plots (c). The numbers in a and b indicate the temperatures employed (in °C). The bimolecular rates shown in c were obtained from the slopes of the linear fits of the data shown in a and b. The numbers in the parentheses in c are (ΔH^‡ and −TΔS^‡) determined from the slope and intercept of the linear fits of the data (assuming T = 298 K). To produce the l-Trp-bound hIDO, 25 mm l-Trp (final concentration) was used.

FIGURE 4.

Absorption spectrum of the ABTS⨥ species derived from the peroxidase activity of hIDO (a) and its hIDO concentration dependence (b) and pH dependence (c). The structure of ABTS is shown in the inset of a. The absorption spectrum of ABTS⨥ shown in a was obtained by hand-mixing hIDO (0.52 μm) with H₂O₂ (0.39 mm) in the presence of ABTS (0.46 mm) in 100 mm, pH 7.4, phosphate buffer at room temperature; those from the control experiments were obtained under comparable conditions. The spectra were offset from each other for clarity. The initial rates of ABTS⨥ formation shown in b were obtained from steady-state kinetic measurements with 0.44 mm ABTS and 0.59 mm H₂O₂ in the presence of various amounts of hIDO. c, pH dependence of the relative initial formation rates of ABTS⨥ obtained from the steady-state kinetic measurements with 0.25 μm hIDO, 0.6 mm H₂O₂, and 0.44 mm ABTS (black circles) is overlaid with that of the relative ferryl formation rate obtained from stopped-flow mixing experiments with 1.8 μm hIDO and 81 μm H₂O₂ in the absence of ABTS (gray circles). All the reactions in c were performed in 200 mm phosphate buffer.

FIGURE 5.

Steady-state kinetics of ABTS⨥ formation as a function of ABTS and H₂O₂ concentration (a and b) and the inhibition effect of l-Trp (c). The Michaelis-Menten plots shown in a and b were obtained by hand-mixing hIDO (0.1 μm) with H₂O₂ (13 mm) and various amounts of ABTS (a) or hIDO (0.11 μm) with ABTS (1.2 mm) and various amounts of H₂O₂ (b). The ABTS⨥ formation rates shown in c were obtained from the reaction of hIDO (0.075 μm) with H₂O₂ (13 mm) and ABTS (1.0 mm) in the presence of various amounts of l-Trp. All the reactions were performed in 100 mm, pH 7.4, phosphate buffer at room temperature. The solid lines in a and b are the best fitted curves with the Michaelis-Menten model, whereas that in c is the best fitted curve for the data with a competitive inhibition model (see text).

FIGURE 6.

Optical absorption spectra of the substrate-free (SF), single l-Trp bound (1×Trp) and double l-Trp-bound (2×Trp) ferryl derivative of hIDO (a), the kinetic traces associated with the reaction of ferric hIDO with H₂O₂ (b), and the binding affinity of the ferryl species toward l-Trp (c). The optical absorption spectra shown in a were obtained 1.0, 1.9, or 8.0 s following the mixing of hIDO (2.2 μm) with a mixture of H₂O₂ (2 mm) and l-Trp (0 and 36 μm or 15 mm l-Trp, respectively) in a stopped-flow instrument at 20 °C. The visible regions of the spectra were amplified by a factor of 4 and offset for clarity. The inset in b shows the difference spectrum between those of the substrate-free and 1×Trp-bound hIDO. The kinetic traces in b were obtained at 412 nm following the initiation of the mixing of ferric hIDO with H₂O₂ (2 mm) in the absence or presence of 36 μm l-Trp. The data shown in c were fitted with a two-substrate binding site model with K_d¹ and K_d² of 0.3 and 440 μm, respectively, as indicated by the solid line.

FIGURE 7.

High frequency resonance Raman spectra of the ferric (a) and ferryl (b) derivatives of hIDO. The spectra were obtained with 19 μm hIDO in the presence of ∼0.2 mm H₂O₂ and various amounts of l-Trp in 100 mm, pH 7.4, phosphate buffer. The bands indicated by asterisks are associated with the excess of l-Trp in the solutions.

FIGURE 8.

Low frequency resonance Raman spectra of the ferric (a) and ferryl (b) derivatives of hIDO. The spectra were obtained with 19 μm hIDO in the presence of ∼0.2 mm H₂O₂ and various amounts of l-Trp in 100 mm, pH 7.4, phosphate buffer. The bands indicated by asterisks are associated with the excess of l-Trp in the solutions.

FIGURE 9.

Low frequency resonance Raman spectra (a) and H₂¹⁶O₂-H₂¹⁸O₂ isotope difference spectra (b) of the ferryl derivatives of hIDO. The spectra in a were obtained with 19 μm hIDO in the presence of ∼0.2 mm H₂¹⁶O₂ or H₂¹⁸O₂ and various amounts of l-Trp in 100 mm, pH 7.4, phosphate buffer. The H₂¹⁶O₂-H₂¹⁸O₂ isotope difference spectra in b were obtained from the data shown in a. The w value indicated in b is the full-width at the half-maximum of each spectral band.