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Peroxiredoxin 5 is a thioredoxin peroxidase ubiquitously expressed in mammalian tissues. Peroxiredoxin 5 can be addressed intracellularly to mitochondria, peroxisomes, the cytosol and the nucleus. Here, we show that mitochondrial human peroxiredoxin 5 protects mitochondrial DNA (mtDNA) from oxidative attacks. In an acellular assay, recombinant peroxiredoxin 5 was shown to protect plasmid DNA from damages induced by metal-catalyzed generation of reactive oxygen species. In Chinese hamster ovary cells, overexpression of mitochondrial peroxiredoxin 5 significantly decreased mtDNA damages caused by exogenously added hydrogen peroxide. Altogether our results suggest that mitochondrial peroxiredoxin 5 may play an important role in mitochondrial genome stability.

Peroxiredoxin 5 is the last discovered mammalian member of an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Mammalian peroxiredoxin 5 has been recently classified as an atypical 2-Cys peroxiredoxin due to the presence of a conserved peroxidatic N-terminal cysteine (Cys47) and an unconserved resolving C-terminal cysteine residue (Cys151) forming an intramolecular disulfide intermediate in the oxidized enzyme. We have recently reported the crystal structure of human peroxiredoxin 5 in its reduced form. Here, a new crystal form of human peroxiredoxin 5 is described at 2.0 Ǻ resolution. The asymmetric unit contains three polypeptide chains. Surprisingly, beside two reduced chains, the third one is oxidized although the enzyme was crystallized under initial reducing conditions in the presence of 1 mM 1,4-dithio-DL-threitol. The oxidized polypeptide chain forms an homo-dimer with a symmetry-related one through intermolecular disulfide bonds between Cys47 and Cys151. The formation of these disulfide bonds is accompanied by the partial unwinding of the N-terminal parts of the α2 helix, which, in the reduced form, contains the peroxidatic Cys47 and the α6 helix, which is sequentially close to the resolving residue Cys151. In each monomer of the oxidized chain, the C-terminal part including the α6 helix is completely reorganized and is isolated from the rest of the protein on an extended arm. In the oxidized dimer, the arm belonging to the first monomer now appears at the surface of the second subunit and vice versa.Keywords: antioxidant enzyme; peroxiredoxin; thioredoxin fold; thioredoxin peroxidase; crystal structure Abbreviations used: PRDX, human peroxiredoxin; Prdx, mouse and rat peroxiredoxin; 1hd2, RCSB Protein Data Bank code of the tetragonal form of human PRDX5; ROS, reactive oxygen species; RNS, reactive nitrogen species; MALDI, matrix-assisted laser desorption/ ionization.

Mammalian thioredoxin 2 is a mitochondrial isoform of highly evolutionary conserved thioredoxins. Thioredoxins are small ubiquitous protein-disulfide oxidoreductases implicated in a large variety of biological functions. In mammals, thioredoxin 2 is encoded by a nuclear gene and is targeted to mitochondria by a N-terminal mitochondrial presequence. Recently, mitochondrial thioredoxin 2 was shown to interact with components of the mitochondrial respiratory chain and to play a role in the control of mitochondrial membrane potential, regulating mitochondrial apoptosis signaling pathway. Here we report the first crystal structures of a mammalian mitochondrial thioredoxin 2. Crystal forms of reduced and oxidized human thioredoxin 2 are described at 2.0 and 1.8 Å resolution. Though the folding is rather similar to that of human cytosolic/nuclear thioredoxin 1, important differences are observed during the transition between the oxidized and the reduced states of human thioredoxin 2, compared with human thioredoxin 1. In spite of the absence of the Cys residue implicated in dimer formation in human thioredoxin 1, dimerization still occurs in the crystal structure of human thioredoxin 2, mainly mediated by hydrophobic contacts, and the dimers are associated to form two-dimensional polymers. Interestingly, the structure of human thioredoxin 2 reveals possible interaction domains with human peroxiredoxin 5, a substrate protein of human thioredoxin 2 in mitochondria.

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