The 2-cysteine peroxiredoxins (2-Cys Prx) constitute an ancient family of peroxide detoxifying enzymes and have acquired a plant-specific function in the oxygenic environment of the chloroplast. Immunocytochemical analysis and work with isolated intact chloroplasts revealed a reversible binding of the oligomeric form of 2-Cys Prx to the thylakoid membrane. The oligomeric form of the enzyme was enhanced under stress. The 2-Cys Prx has a broad substrate specificity with activity toward hydrogen peroxides and complex alkyl hydroperoxides. During the peroxide reduction reaction, 2-Cys Prx is alternatively oxidized and reduced as it catalyzes an electron flow from an electron donor to peroxide. Escherichia coli thioredoxin, but also spinach thioredoxin f and m were able to reduce oxidized 2-Cys Prx. The midpoint redox potential of ؊315 mV places 2-Cys Prx reduction after Calvin cycle activation and before switching the malate valve for export of excess reduction equivalents to the cytosol. Thus the 2-Cys Prx has a defined and preferential place in the hierarchy of photosynthetic electron transport. The activity of 2-Cys Prx also is linked to chloroplastic NAD(P)H metabolism as indicated by the presence of the reduced form of the enzyme after feeding dihydroxyacetone phosphate to intact chloroplasts. The function of the 2-Cys Prx is therefore not confined to its role in the water-water cycle pathway for energy dissipation in photosynthesis but also mediates peroxide detoxification in the plastids during the dark phase.

Peroxiredoxins (prxs) are peroxidases with broad substrate specificity. The seven prx genes expressed in Arabidopsis shoots were analyzed for their expressional response to changing photon fluence rates, oxidative stress, and ascorbate application. The results reveal a highly variable and gene-specific response to reducing and oxidizing conditions. The steady-state transcript amounts of the chloroplast-targeted prxs, namely the two-cysteine (2-Cys) prxs, prx Q and prx II E, decreased upon application of ascorbate. prx Q also responded to peroxides and diamide treatment. prx II B was induced by tertiary butylhydroperoxide, but rather unaffected by ascorbate. The strongest responses were observed for prx II C, which was induced with all treatments. The two Arabidopsis 2-Cys Prxs and four Prx II proteins were expressed heterologously in Escherichia coli. In an in vitro test system, they all showed peroxidase activity, but could be distinguished by their ability to accept dithiothreitol and thioredoxin as electron donor in the regeneration reaction. The midpoint redox potentials (E m Ј) of Prx II B, Prx II C, and Prx II E were around Ϫ290 mV and, thus, less negative than E m Ј of Prx II F, 2-Cys Prx A, and 2-Cys Prx B (Ϫ307 to Ϫ322 mV). The data characterize expression and function of the mitochondrial Prx II F and the chloroplast Prx II E for the first time, to our knowledge. Antibodies directed against 2-Cys Prx and Prx II C showed a slight up-regulation of Prx II protein in strong light and of 2-Cys Prx upon transfer both to high and low light. The results are discussed in context with the subcellular localization of the Prx gene products.Peroxiredoxins (Prxs) are enzymes that reduce hydrogen peroxide (H 2 O 2 ) and alkyl hydroperoxides. They are grouped in four classes: (a) 2-Cys Prx; (b) Prx Q; (c) Prx II, which all contain two catalytic Cys residues in distinct sequence environment; and (d) 1-Cys Prx with one conserved Cys residue only (Dietz, 2003). A phylogenetic distance analysis suggests that 2-Cys Prx, Prx Q, and 1-Cys Prx are related proteins, whereas the group of Prx II is likely to have evolved independently (Verdoucq et al., 1999;Horling et al., 2002). The catalytic Cys residues undergo oxidation during the peroxide reduction reaction and need to be reduced by electron donors such as glutaredoxins, thioredoxins, or cyclophilins before the next catalytic cycle (Lee et al., 2001;Rouhier et al., 2001;Kö nig et al., 2002). For the bacterial and animal homologs, a broad substrate specificity has been described (Nogoceke et al., 1997; Bryk et al., 2000; Hillas et al., 2000). In in vitro tests, these Prx proteins reduced H 2 O 2 , lipid peroxides, such as butyl hydroperoxide, phospholipid peroxides and cumene hydroperoxide, and peroxynitrite. For plant Prxs, the catalytic properties have only poorly been investigated.The Arabidopsis genome encodes 10 open reading frames (ORFs) for peroxiredoxins. Based on sequence similarities, they can be assigned to the four subgroups of peroxiredoxins: two ORFs code for 2...

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