title: Kinetic analysis of structural influences on the susceptibility of peroxiredoxins 2 and 3 to hyperoxidation.
abstract: Mammalian 2-cysteine peroxiredoxins (Prxs) are susceptible to hyperoxidation by excess H2O2. The cytoplasmic family member Prx2 hyperoxidizes more readily than mitochondrial Prx3 due to slower dimerization of the sulfenic acid (SpOH) intermediate. Four variant amino acids near the C-terminus have been shown to contribute to this difference. We have performed kinetic analysis of the relationship between hyperoxidation and disulfide formation, using whole-protein MS and comparing wild-type (WT) Prx2 and Prx3 with tail-swap mutants in which the four amino acids were reversed. These changes make Prx3 more sensitive and Prx2 less sensitive to hyperoxidation and accounted for ∼70% of the difference between the two proteins. The tail swap mutant of Prx3 was also more susceptible when expressed in the mitochondria of HeLa cells. The hyperoxidized product at lower excesses of H2O2 was a semi-hyperoxidized dimer with one active site disulfide and the other a sulfinic acid. For Prx2, increasing the H2O2 concentration resulted in complete hyperoxidation. In contrast, only approximately half the Prx3 active sites underwent hyperoxidation and, even with high H2O2, the predominant product was the hyperoxidized dimer. Size exclusion chromatography (SEC) showed that the oligomeric forms of all redox states of Prx3 dissociated more readily into dimeric units than their Prx2 counterparts. Notably the species with one disulfide and one hyperoxidized active site was decameric for Prx2 and dimeric for Prx3. Reduction and re-oxidation of the hyperoxidized dimer of Prx3 produced hyperoxidized monomers, implying dissociation and rearrangement of the subunits of the functional homodimer.
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch 8011, New Zealand.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Poynton
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch 8011, New Zealand.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Peskin
authors.affiliation: Department of Biochemistry and Center for Structural Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157, U.S.A.
authors.forename: Alexina C
authors.initials: AC
authors.lastname: Haynes
authors.affiliation: Department of Biochemistry and Center for Structural Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157, U.S.A.
authors.forename: W Todd
authors.initials: WT
authors.lastname: Lowther
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch 8011, New Zealand.
authors.forename: Mark B
authors.initials: MB
authors.lastname: Hampton
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch 8011, New Zealand christine.winterbourn@otago.ac.nz.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Winterbourn
mesh_terms: D000595:Amino Acid Sequence; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D061105:Peroxiredoxin III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054464:Peroxiredoxins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D011994:Recombinant Proteins; D061105:Peroxiredoxin III; D054464:Peroxiredoxins
keywords: hydrogen peroxide; kinetics; peroxiredoxin inactivation; thiol antioxidants; thiol oxidation