Structural and biochemical characterization of a mitochondrial peroxiredoxin from Plasmodium falciparum

Abstract

Plasmodium falciparum possesses a single mitochondrion with a functional electron transport chain. During respiration, reactive oxygen species are generated that need to be removed to protect the organelle from oxidative damage. In the absence of catalase and glutathione peroxidase, the parasites rely primarily on peroxiredoxin-linked systems for protection. We have analysed the biochemical and structural features of the mitochondrial peroxiredoxin and thioredoxin of P. falciparum. The mitochondrial localization of both proteins was confirmed by expressing green fluorescent protein fusions in parasite erythrocytic stages. Recombinant protein was kinetically characterized using the cytosolic and the mitochondrial thioredoxin (PfTrx1 and PfTrx2 respectively). The peroxiredoxin clearly preferred PfTrx2 to PfTrx1 as a reducing partner, reflected by the KM values of 11.6 microM and 130.4 microM respectively. Substitution of the two dyads asparagine-62/tyrosine-63 and phenylalanine-139/alanine-140 residues by aspartate-phenylalaine and valine-serine, respectively, reduced the KM for Trx1 but had no effect on the KM of Trx2 suggesting some role for these residues in the discrimination between the two substrates. Solution studies suggest that the protein exists primarily in a homodecameric form. The crystal structure of the mitochondrial peroxiredoxin reveals a fold typical of the 2-Cys class peroxiredoxins and a dimeric form with an intermolecular disulphide bridge between Cys67 and Cys187. These results show that the mitochondrial peroxiredoxin of P. falciparum occurs in both dimeric and decameric forms when purified under non-reducing conditions.

Fig. 1

Localization of PfTrx-Px2 and PfTrx2 in P. falciparum erythrocytic stages. P. falciparum erythrocytic stages were transfected with construct pHH2-Px2-GFP, pHH2-Trx2-GFP leading to the expression of the peroxiredoxin or thioredoxin2 C-terminally fused to green fluorescent protein (GFP). A. The localization of the peroxiredoxin-GFP fusion protein in parasites previously treated with MitoTracker CMX-Ros was analysed by fluorescence light microscopy. Phase, phase contrast of parasitized erythrocytes infected with P. falciparum trophozoites; PfTrx-Px2-GFP, parasitized erythrocytes expressing the peroxiredoxin-GFP fusion protein analysed using the FITC channel; MitoTracker, parasitized erythrocytes expressing the peroxiredoxin-GFP fusion protein analysed using the rhodamine channel; PfTrx-Px2-Mito; merge of FITC and rhodamine channels; merge, merge of all images. The images show that the peroxiredoxin-GFP fusion protein is colocalizing with the mitochondrion (stained by MitoTracker). B. The expression of pHH2-PfTrx2 results in the localization of the fusion protein the mitochondrion. Phase, phase contrast of parasitized erythrocytes infected with P. falciparum trophozoites; PfTrx2-GFP, parasitized erythrocytes expressing the Trx2-GFP fusion protein analysed using the FITC channel; MitoTracker, parasitized erythrocytes expressing the Trx2-GFP fusion protein analysed using the rhodamine channel; PfTrx2-Mito; merge of FITC and rhodamine channels; merge, merge of all images.

Fig. 2

A. Ribbon diagram of the PfTrx-Px2 monomer colour ramped from its N- (orange) to C-terminus (purple) and with elements of secondary structure labelled. The side-chains of the active cysteine residues 67 and 187 (which has a dual conformation) are drawn together with the two pairs of residues (Asn62-Tyr63 and Phe139-Ala140) that were substituted by mutagenesis. B. Amino acid sequence alignment of selected Plasmodium 2-Cys peroxiredoxins. Strictly conserved residues are highlighted with red boxes, conservative substitutions are also boxed. Secondary structure elements are shown above the alignment, with TT denoting turns. The active site cysteines are labelled with green inverted triangles. The residues chosen for mutation are indicated by black triangles. This figure was generated using ESPript (Gouet et al., 1999). Pf, P. falciparum; Pk, Plasmodium knowlesi; Pv, Plasmodium vivax.

Fig. 3

A. The PfTrx-Px2 dimer with subunits coloured in purple and red. This figure was made using the program ccp4mg (Potterton et al., 2004). B. Orthogonal view to A to show the intersubunit disulphide bridges. C. Electron density in the vicinity of the intermolecular cystine. The 2F_obs − F_calc, α_calc map is contoured at the 1 σ level. The chains are coloured in cyan and green and the dual conformation of the resolving cysteine from chain B is evident. D. Ribbon diagram showing the active site disulphide in the context of the Cp loop (red) and the C-terminal tail (blue).

Fig. 4

Analytical ultracentrifugation of PfTrx-Px2. A sample of PfTrx-Px2 at an initial concentration of approximately 0.2 mg ml⁻¹ was centrifuged at 28 000 r.p.m. A plot of relative concentration versus sedimentation coefficient is shown revealing the presence of peaks at 3.2 S, 9.4 S and 14.2 S. The increases at the limits of the plot indicate there may be some additional aggregates and some very-low-molecular-weight material.