EPR and Mössbauer spectroscopy show inequivalent hemes in tryptophan dioxygenase

Abstract

Tryptophan 2,3-dioxygenase (TDO) is an essential enzyme in the pathway of NAD biosynthesis and important for all living organisms. TDO catalyzes oxidative cleavage of the indole ring of L-tryptophan (L-Trp), converting it to N-formylkynurenine (NFK). The crystal structure of TDO shows a dimer of dimer quaternary structure of the homotetrameric protein. The four catalytic sites of the protein, one per subunit, contain a heme that catalyzes the activation and insertion of dioxygen into L-Trp. Because of the alpha(4) structure and because only one type of heme center has been identified in previous spectroscopic studies, the four hemes sites have been presumed to be equivalent. The present work demonstrates that the heme sites of TDO are not equivalent. Quantitative interpretation of EPR and Mössbauer spectroscopic data indicates the presence of two dominant inequivalent heme species in reduced and oxidized states of the enzyme, which is consistent with a dimer of dimer protein quaternary structure that now extends to the electronic properties of the hemes. The electronic properties of the hemes in the reduced state of TDO change significantly upon L-Trp addition, which is attributed to a change in the protonation state of the proximal histidine to the hemes. The binding of O(2) surrogates NO or CO shows two inequivalent heme sites. The heme-NO complexes are 5- and 6-coordinate without L-Trp, and both 6-coordinate with L-Trp. NO can be selectively photodissociated from only one of the heme-NO sites and only in the presence of L-Trp. Cryoreduction of TDO produces a novel diamagnetic heme species, tentatively assigned as a reduced heme-OH complex. This work presents a new description of the heme interactions with the protein, and with the proximal His, which must be considered during the general interpretation of physical data as it relates to kinetics, mechanism, and function of TDO.

Figure 1

EPR spectra of 220 μM oxidized TDO, pH 7.4. (A) As-isolated, (B) simulation of (A), (C) with 10 equiv of L-Trp, (D) with 50 equiv of L-Trp, (E) with 110 equiv of L-Trp, and (F) simulation of (E). Experimental conditions: microwaves, 0.2 mW at 9.65 GHz; temperature, 11 K. See text and Table 1 for simulation parameters.

Figure 2

EPR spectra of 115 μM oxidized TDO. (A) In 50 mM MES pH 6.0, 10% glycerol; (B) pH 6.0 with 50 equiv of L-Trp; (C) after buffer exchange to 50 mM Tris-HCl pH 7.4, 10% glycerol. Experimental conditions are the same as in Figure 1.

Figure 3

Mössbauer spectra of 1 mM oxidized TDO, pH 7.4. (A) Oxidized TDO in a magnetic field of 8 T, (B) oxidized TDO, (C) with L-Trp, (D) difference spectrum of (C) – 0.4 (B). Experimental conditions: temperature, 4.2 K; magnetic field of 45 mT (except (A)) parallel to γ-ray direction. The solid lines are least-squares fits with parameters given in the text.

Figure 4

Mössbauer spectrum of 1 mM TDO, pH 7.4. (A) Reduced, (B) reduced with 10 equiv of L-Trp, (C) difference spectrum (B)–(A) for equal areas of (A) and (B). All spectra recorded at 4.2 K in a magnetic field of 45 mT parallel to γ-ray direction. The solid lines overlaid on the data are least-squares fits using the parameters given in Table 2. The additional lines in (A) show the doublets of the individual species.

Figure 5

Mössbauer spectrum after cryoreduction of (A) 1.5 mM oxidized TDO, pH 7.4. The difference spectra (B–E) after subtracting the spectrum of oxidized TDO are: (B) 45 mT; (C) 8 T; (D) after annealing; (E) after thawing, including an overlay (solid line) of the reduced TDO spectrum. All spectra are recorded at 4.2 K and magnetic field of 45 mT parallel to γ-ray direction, except (C). The solid lines in (B) and (D) are least-squares fits for species: (B) o1^red-OH (OH), o2^red (2); and (D) o1^red-anneal (1), o2^red-anneal (2). The solid line in (C) is a simulation at 8 T for the S = 0 species o1^red-OH. See Table 2 for parameter listings of species.

Figure 6

Mössbauer spectrum of (A) 1.5 mM ferrous TDO, pH 7.4, with 10 equiv of L-Trp and excess CO; (B) Fourier transform of spectrum A with removal of intrinsic ⁵⁷Fe source line width. All spectra recorded at 4.2 K in a magnetic field of 45 mT parallel to γ-ray direction. The solid lines are least-squares fits using the parameters given in Table 2 for r_a-CO and r_b-CO.

Figure 7

(A) X- and (E) Q-band EPR spectra of the NO adduct of 0.3 mM reduced TDO without L-Trp. (B) Simulation sum (C) + (D) of the two sites, (C) simulation of r1-NO, (D) simulation of r2-NO, and (F) simulation sum of the two sites of (E). Experimental conditions: microwave frequencies as listed; power, 0.02 mW (X), 0.005 mW (Q); temperature, 20 K (X), 27 K (Q). The simulation parameters are given in Table 1, and σ_g1 = (0.0015, 0.0032, 0.0044), σ_g2 = (0.0074, 0.0089, 0.0107). The weak signal near g = 1.96 is due to a minor amount of free NO in solution.

Figure 8

(A) X- and (E) Q-band EPR spectra of the NO adduct of 0.3 mM reduced TDO with 10 equiv of L-Trp. (B) Simulation sum (C) + (D) for the two sites, (C) simulation of r1-Trp-NO, (D) simulation of r2-Trp-NO, and (F) simulation sum for the two sites of (E). Experimental conditions: microwave frequencies as listed; power, 0.02 mW (X), 0.05 mW (Q); temperature, 20 K (X), 27 K (Q).

Figure 9

Photodissociation of the NO adduct from r2-Trp-NO. X- (top part) and Q-band (lower part) EPR spectra of the NO adduct of 0.3 mM reduced TDO with 10 equiv of L-Trp. (A) Before light, (B) after exposure to light at 77 K, (C) simulation of r1-Trp-NO, (D) difference (A)–(B), (E) simulation of r2-Trp-NO, (F) after anaerobic thawing in dark, (G) before light, (H) after exposure to light at 77 K, and (I) simulation of r1-Trp-NO. The experimental conditions and simulation parameters of the species are the same as in Figure 8.

Figure 10

Mössbauer spectra of (A) 2 mM reduced TDO, pH 7.4, with 10 equiv of L-Trp and excess NO, and (B) after exposure to light for 1 h at 77 K. All spectra were recorded at 4.2 K in a magnetic field of 45 mT parallel to γ-ray direction. The solid line is a simulation with parameters given in the text.

Figure 11

Spectroscopic states of reduced TDO with NO, highlighting 5- to 6-coordinate conversion of r1-NO with L-Trp, and specific conversion of r2-Trp-NO to r2-Trp after illumination.

Figure 12

Spectroscopic states observed for oxidized TDO.

Figure 13

Heme states observed from the cryoreduction of oxidized TDO with spin states as shown. The numbers in parentheses are the values of ΔE_Q (in mm/s) for the respective states. Red indicates a change from the previous state.