Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate

Peroxidases of the peroxiredoxin (Prx) family contain a Cys residue that is preceded by a conserved sequence in the NH2-terminal region. A new type of mammalian Prx, designated PrxV, has now been identified as the result of a data base search with this conserved Cys-containing sequence. The 162-amin o acid PrxV shares only similar to 10% sequence identity with previously id entified mammalian Prx enzymes and contains Cys residues at positions 73 an d 152 in addition to that (Cys(48)) corresponding to the conserved Cys. Ana lysis of mutant human PrxV proteins in which each of these three Cys residu es was individually replaced with serine suggested that the sulfhydryl grou p of Cys(48) is the site of oxidation by peroxides and that oxidized Cys(48 ) reacts with the sulfhydryl group of Cys(152) to form an intramolecular di sulfide linkage. The oxidized intermediate of PrxV is thus distinct from th ose of other Prx enzymes, which form either an intermolecular disulfide or a sulfenic acid intermediate. The disulfide formed by PrxV is reduced by th ioredoxin but not by glutaredoxin or glutathione. Thus, PrxV mutants lackin g Cys(48) or Cys(152) showed no detectable thioredoxin-dependent peroxidase activity, whereas mutation of Cys(73) had no effect on activity. Immunoblo t analysis revealed that PrxV is widely expressed in rat tissues and cultur ed mammalian cells and is localized intracellularly to cytosol, mitochondri a, and peroxisomes. The peroxidase function of PrxV in vivo was demonstrate d by the observations that transient expression of the wild-type protein, b ut not that of the Cys(48) mutant, in NIH 3T3 cells inhibited H2O2 accumula tion and activation of c-Jun NH2-terminal kinase induced by tumor necrosis factor-alpha.