Abstract
By following peroxiredoxin I (Prx I)-dependent NADPH oxidation spectrophotometrically, we observed that Prx I activity decreased gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by two-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Cys(51)-SH to Cys(51)-SO(2)H. Thus, Cys(51)-SOH generated as an intermediate during catalysis appeared to undergo occasional further oxidation to Cys(51)-SO(2)H, which cannot be reversed by thioredoxin. The presence of H(2)O(2) alone was not sufficient to cause oxidation of Cys(51) to Cys(51)-SO(2)H. Rather, the presence of complete catalytic components (H(2)O(2), thioredoxin, thioredoxin reductase, and NADPH) was necessary, indicating that such hyperoxidation occurs only when Prx I is engaged in the catalytic cycle. Likewise, hyperoxidation of Cys(172)/Ser(172) mutant Prx I required not only H(2)O(2), but also a catalysis-supporting thiol (dithiothreitol). Kinetic analysis of Prx I inactivation in the presence of a low steady-state level (<1 microm) of H(2)O(2) indicated that Prx I was hyperoxidized at a rate of 0.072% per turnover at 30 degrees C. Hyperoxidation of Prx I was also detected in HeLa cells treated with H(2)O(2).
Highlights
All Prx proteins contain a conserved Cys residue, which corresponds to Cys51 in mammalian peroxiredoxin I (Prx I), in the N-terminal portion of the molecule [10, 11]
The crystal structures of 2-Cys and 1-Cys Prx enzymes reveal that the catalytic cysteine is located in a small pocket formed by the N- and C-terminal domains of the two subunits amidopropyl)dimethylammonio]-1-propanesulfonic acid; High Performance Liquid Chromatography (HPLC), high performance liquid chromatography; Electrospray Ionization Mass Spectrometry (ESI-MS), electrospray ionization mass spectrometry; MS/MS, tandem mass spectrometry; MALDI-TOFMS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Prx I Inactivation in Vitro—The peroxidase activity of human Prx I was monitored by following the decrease in A340 attributable to the oxidation of NADPH in a reaction mixture containing NADPH, Trx, thioredoxin reductase (TrxR), and varying concentrations of H2O2
Summary
All Prx proteins contain a conserved Cys residue, which corresponds to Cys in mammalian Prx I, in the N-terminal portion of the molecule [10, 11]. In 1-Cys Prx enzymes, including mammalian Prx VI, the conserved cysteine is the site of oxidation, but remains in a sulfenic acid state upon oxidation because there is no nearby partner cysteine to form a disulfide bond [18, 19]. Trx cannot reduce this sulfenic acid-containing intermediate [18, 20]. In H2O2-treated cells, a number of proteins (including Prx I and Prx II) demonstrated altered migration consistent with decreased pI, suggesting that such oxidative inactivation might occur in cells These acidic Prx enzymes were not characterized in detail. We observed that only those Prx molecules actively engaged in the catalytic cycle are vulnerable to oxidative inactivation
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