Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid.
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).
Yang
Kap-Seok
KS
Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kang
Sang Won
SW
Woo
Hyun Ae
HA
Hwang
Sung Chul
SC
Chae
Ho Zoon
HZ
Kim
Kanghwa
K
Rhee
Sue Goo
SG
D000595:Amino Acid Sequence; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054464:Peroxiredoxins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
D000975:Antioxidants; D016877:Oxidants; D010455:Peptides; D011994:Recombinant Proteins; D013879:Thioredoxins; D009249:NADP; D006861:Hydrogen Peroxide; D010544:Peroxidases; D054464:Peroxiredoxins; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine