Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate

Abstract

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp₂₃₃^•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

Keywords: Chagas disease; ascorbate; cytochrome c; heme; oxidants; peroxidase.

Figure 1

Crystal structures ofTcAPx-CcP and related heme peroxidases.A, TcAPx-CcP (PDB 7OPT). B, sAPX with ascorbate bound (1OAF). C, CcP (1ZBY). D, LmP (3RIV). Active site residues are shown as red sticks. Sodium atoms are shown as purple spheres, calcium as green spheres, and potassium as orange spheres. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase; PDB, Protein Data Bank.

Figure 2

The active site heme environment in TcAPx-CcP (in green) superimposed with CcP (cyan). Active site residues are shown as red sticks and labeled for TcAPx-CcP with the equivalent residue in CcP in parentheses. TcAPx-CcP features the C222 residue (in yellow), whereas T180 is found in CcP (see Fig. S2). An equivalent of C222 is also found in LmP (C197). CcP, cytochrome c peroxidase.

Figure 3

Low-temperature EPR spectra of TcAPx-CcP.A, 9-GHz CW EPR spectra of (i) ferric TcAPx-CcP, (ii) compound I of TcAPx-CcP formed by reaction of the ferric sample with H₂O₂ for 10 s, (iii) ferric W233F, (iv) ferric W233F mixed with H₂O₂ for 10 s, (v) ferric C222A, and (vi) C222A mixed with H₂O₂ for 10 s. Spectra were recorded at 4.3 K, 4 G modulation amplitude, 1 mW microwave power, 100 kHz modulation frequency, two scans. B, (i) The same sample of compound I of TcAPx-CcP as in (A)(ii), (ii) The same sample as in (A)(iv), (iii) The same sample as in (A)(vi). Spectra in (B) were recorded at 70 K, 1 G modulation amplitude, 0.2 mW microwave power, 100 kHz modulation frequency, 100 scans. (ii) and (iii) have been multiplied by a factor of 6 to allow comparison. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase.

Figure 4

Comparison of the structures of the γ-heme edge of TcAPx-CcP (green), sAPX (yellow), and yeast CcP (cyan). Ascorbate bound to sAPX is shown as yellow sticks. The important R172 ascorbate-binding residue in APX is shown superimposed with the equivalent residues (N226, N184) from TcAPx-CcP and CcP, respectively. The relevant sequence in this region is also shown, along with sequence logos obtained from Trypanosoma cruzi and Leishmania spp. alignments (see also Figs. S2 and S3). The extended loop which might interfere with ascorbate binding (22) is clearly visible in CcP but missing in TcAPx-CcP. Hydrogen bonds and their distances between R172 of sAPX and ascorbate are indicated with yellow dashes. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase.

Figure 5

Interaction of ascorbate at the active site of APX evaluated by MD simulation.A, superimposition of the conformations adopted by ascorbate at the active site of sAPX (blue cartoon, up) and TcAPx-CcP (black cartoon, down). B, ascorbate binding free energy (ΔG_bind, kcal/mol) estimation using the MM-GBSA formalism, along with a residue basis decomposition of the free energy. Only residues contributing more than 0.5 kcal/mol to the calculated binding free energy are shown. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase; MD, molecular dymamics.

Figure 6

Comparison of electron transfer pathways and surface electrostatics. A, alignment of the structures of TcAPx-CcP (faded green) and CcP (faded cyan). The residues involved in the delivery of electrons from cytochrome c in CcP (Trp191, Gly192, Ala193, and Ala194, in green) overlay well with an equivalent electron pathway in TcAPx-CcP (Trp233, Thr234, His235, and Asp236, in green). The heme group is shown for both proteins. The residues involved in binding of cytochrome c in CcP (Asp34, Glu35 (23)) overlay well in the TcAPx-CcP structure (Glu79 and Asp80, respectively). B, electrostatic surface representation of TcAPx-CcP (±5 kT), CcP, sAPX, and LmP, obtained using the APBS software (63). The predicted cyt c binding surface is represented in red where the overall charge is strongly electronegative. This electronegative area is substantially less prominent in sAPX; sAPX does not bind cyt c. The residues in TcAPx-CcP which are expected to be responsible for the electron transfer path are shown as dark blue sticks (W233, T234, H235, and D236). The equivalent residues (W191, G192, A193, and A194) in CcP are shown as cyan sticks in (B). See Fig. S2 for a sequence alignment highlighting these residues. APX, ascorbate peroxidase; CcP, cytochrome c peroxidase.