Sulfiredoxin Translocation into Mitochondria Plays a Crucial Role in Reducing Hyperoxidized Peroxiredoxin III

Abstract

The mitochondria are the major intracellular source of reactive oxygen species (ROS), which are generated during cellular respiration. The role of peroxiredoxin (Prx) III, a 2-Cys Prx family member, in the scavenging of mitochondrial H 2 O 2 has recently been emphasized. While eliminating H 2 O 2 , Prx can become overoxidized and inactivated by modifying the active cysteine into cysteine sulfinic acid (Cys-SO 2 H). When 2-Cys Prxs are inactivated in vitro, sulfiredoxin (Srx) reduces the cysteine sulfinic acid to cysteines. However, whereas Srx is localized in the cytoplasm, Prx III is present exclusively in the mitochondria. Although Srx reduces sulfinic Prx III in vitro, it remains unclear whether the reduction of Prx III in cells is actually mediated by Srx. Our gain-and loss-of-function experiments show that Srx is responsible for reducing not only sulfinic cytosolic Prxs (I and II) but also sulfinic mitochondrial Prx III. We further demonstrate that Srx translocates from the cytosol to mitochondria in response to oxidative stress. Overexpression of mitochondrion-targeted Srx promotes the regeneration of sulfinic Prx III and results in cellular resistance to apoptosis, with enhanced elimination of mitochondrial H 2 O 2 and decreased rates of mitochondrial membrane potential collapse. These results indicate that Srx plays a crucial role in the reactivation of sulfinic mitochondrial Prx III and that its mitochondrial translocation is critical in maintaining the balance between mitochondrial H 2 O 2 production and elimination. Peroxiredoxins (Prxs)2 are a family of enzymes that catalyze the reduction of hydrogen peroxide (H 2 O 2 ) and hydroperoxides to water and alcohol, respectively (1-5). The six isoforms of mammalian Prx (Prx I to Prx VI) are classified into three subfamilies: 2-Cys, atypical 2-Cys, and 1-Cys (2, 6). Prx I to Prx IV, which belong to the 2-Cys Prx subfamily, exist as homodimers and contain two conserved cysteine residues. In the catalytic cycle of 2-Cys Prxs, the conserved N-terminal Cys-SH is first oxidized by peroxides to cysteine sulfenic acid (Cys-SOH), which then reacts with the conserved COOH-terminal Cys-SH of the other subunit in the homodimer to form a disulfide bond. This disulfide is specifically reduced by thioredoxin, whose oxidized form is then regenerated by thioredoxin reductase, using NADPH-reducing equivalents (2-5, 7). As a result of the slow conversion rate to a disulfide, the sulfenic intermediate is occasionally further oxidized to cysteine sulfinic acid (Cys-SO 2 H), which causes inactivation of peroxidase that cannot be reduced by thioredoxin (7-9). Studies on the fate of the sulfinylated Prx enzyme have shown that its sulfinylation is actually a reversible reaction in mammalian cells (10). The enzyme responsible for the reduction of sulfinylated Prx was subsequently identified in yeast and named sulfiredoxin (Srx) (11). Sulfiredoxin regenerates inactive 2-Cys Prxs, returning it to the catalytic cycle and preventing its permanent oxidative inactivation by strong oxidat...

The Dual-Targeted Plant Sulfiredoxin Retroreduces the Sulfinic Form of Atypical Mitochondrial Peroxiredoxin

“…The mammalian Srx is a cytosolic protein. However, Srx translocates from cytosol to mitochondria in response to oxidative stress to retroreduce sulfinic form of Prx III (Noh et al, 2009). Prx III is a typical 2-Cys Prx in mitochondria of mammalia.…”

“…However, (1) the conditional mitochondrial localization in mammalia in response to oxidative stress (Noh et al, 2009) and (2) the high probability of dual targeting to chloroplast and mitochondrion in pea predicted by our bioinformatic analysis, stimulated us to investigate the intracellular localization of Srx from pea and Arabidopsis in detail.…”

“…genevestigator.com). Our objective was to demonstrate the presence of Srx into plant mitochondria under physiological conditions, in contrast to human Srx (Noh et al, 2009). The immunolocalization was also addressed by western blots with isolated chloroplasts and mitochondria, reaching the conclusion that Srx has the same localization than its target Prx IIF (Finkemeier et al, 2005;Barranco-Medina et al, 2007).…”

“…Although mammal Srx is a cytosolic enzyme, Woo et al (2005) demonstrated that the sulfinic form of mitochondrial hPrx III could be reduced in vitro by hSrx. More recently Noh et al (2009) reported the hSrx translocation from cytosol to mitochondria under oxidative stress conditions to reduce overoxidized hPrx III.…”

“…The presence of Srx in mammalian mitochondria in response to oxidative stress (Noh et al, 2009) and the importance of plant mitochondria as a significant site of reactive oxygen species (ROS) production encouraged us to investigate the presence of Srx in this organelle from plants and study the retroreduction of the atypical Prx IIF-SO 2 H. Moreover, it seemed to be a testable hypothesis that mitochondria contain their own constitutive pool of Srx for retroreduction of overoxidized Prx IIF, under physiological conditions, rather than depending on a slower translocation of Srx from the cytosol to the mitochondrion during oxidative stress.…”