PMID- 24003226 OWN - NLM STAT- MEDLINE DCOM- 20131211 LR - 20211021 IS - 1083-351X (Electronic) IS - 0021-9258 (Print) IS - 0021-9258 (Linking) VI - 288 IP - 41 DP - 2013 Oct 11 TI - Molecular basis for the resistance of human mitochondrial 2-Cys peroxiredoxin 3 to hyperoxidation. PG - 29714-23 LID - 10.1074/jbc.M113.473470 [doi] AB - Peroxiredoxins (Prxs) detoxify peroxides and modulate H2O2-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1-4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H2O2 to form Cys sulfinic acid (Cys-SO2H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H2O2 and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation. FAU - Haynes, Alexina C AU - Haynes AC AD - From the Center for Structural Biology and Department of Biochemistry. FAU - Qian, Jiang AU - Qian J FAU - Reisz, Julie A AU - Reisz JA FAU - Furdui, Cristina M AU - Furdui CM FAU - Lowther, W Todd AU - Lowther WT LA - eng GR - R01 CA136810/CA/NCI NIH HHS/United States GR - R01 GM072866/GM/NIGMS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20130903 PL - United States TA - J Biol Chem JT - The Journal of biological chemistry JID - 2985121R RN - 0 (Disulfides) RN - 0 (Oxidants) RN - 0 (Sulfinic Acids) RN - BBX060AN9V (Hydrogen Peroxide) RN - EC 1.11.1.15 (2-cys peroxiredoxin, human) RN - EC 1.11.1.15 (Peroxiredoxin III) RN - EC 1.11.1.15 (Peroxiredoxins) SB - IM MH - Amino Acid Sequence MH - Biocatalysis MH - Disulfides/chemistry/metabolism MH - Enzyme Activation/drug effects MH - Humans MH - Hydrogen Peroxide/chemistry/*metabolism/pharmacology MH - Kinetics MH - Mitochondria/*metabolism MH - Molecular Sequence Data MH - Mutation MH - Oxidants/chemistry/metabolism/pharmacology MH - Oxidation-Reduction/drug effects MH - Peroxiredoxin III/chemistry/genetics/metabolism MH - Peroxiredoxins/chemistry/genetics/*metabolism MH - Sequence Homology, Amino Acid MH - Spectrometry, Mass, Electrospray Ionization MH - Sulfinic Acids/chemistry/metabolism PMC - PMC3795269 OTO - NOTNLM OT - Mass Spectrometry (MS) OT - Mitochondria OT - Peroxiredoxin OT - Redox OT - Thiol EDAT- 2013/09/05 06:00 MHDA- 2013/12/16 06:00 PMCR- 2014/10/11 CRDT- 2013/09/05 06:00 PHST- 2013/09/05 06:00 [entrez] PHST- 2013/09/05 06:00 [pubmed] PHST- 2013/12/16 06:00 [medline] PHST- 2014/10/11 00:00 [pmc-release] AID - S0021-9258(20)48790-3 [pii] AID - M113.473470 [pii] AID - 10.1074/jbc.M113.473470 [doi] PST - ppublish SO - J Biol Chem. 2013 Oct 11;288(41):29714-23. doi: 10.1074/jbc.M113.473470. Epub 2013 Sep 3.