title: Experimentally Dissecting the Origins of Peroxiredoxin Catalysis.
abstract: Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases involved in oxidant defense and signal transduction. Despite much study, the precise roles of conserved residues remain poorly defined. In this study, we carried out extensive functional and structural characterization of 10 variants of such residues in a model decameric bacterial Prx.
  
  
  
  Three active site proximal mutations of Salmonella typhimurium AhpC, T43V, R119A, and E49Q, lowered catalytic efficiency with hydrogen peroxide by 4-5 orders of magnitude, but did not affect reactivity toward their reductant, AhpF. pKavalues of the peroxidatic cysteine were also shifted up by 1-1.3 pH units for these and a decamer disruption mutant, T77I. Except for the decamer-stabilizing T77V, all mutations destabilized decamers in the reduced form. In the oxidized form, three mutants-T77V, T43A, and T43S-exhibited stabilized decamers and were more efficiently reduced by AhpF than wild-type AhpC. Crystal structures of most mutants were solved and many showed alterations in stability of the fully folded active site loop.
  
  
  
  This is the first study of Prx mutants to comprehensively assess the effects of mutations on catalytic activities, the active site cysteine pKa, and the protein structure and oligomeric status.
  
  
  
  The Arg119 side chain must be properly situated for efficient catalysis, but for other debilitating variants, the functional defects could be explained by structural perturbations and/or associated decamer destabilization rather than direct effects. This underscores the importance of our comprehensive approach. A remarkable new finding was the preference of the reductant for decamers. Antioxid. Redox Signal. 28, 521-536.
authors.affiliation: 1 Department of Biochemistry, Wake Forest School of Medicine , Winston-Salem, North Carolina.|2 Center for Redox Biology and Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina.
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Nelson
authors.affiliation: 3 Department of Biochemistry and Biophysics, Oregon State University , Corvallis, Oregon.
authors.forename: Arden
authors.initials: A
authors.lastname: Perkins
authors.affiliation: 1 Department of Biochemistry, Wake Forest School of Medicine , Winston-Salem, North Carolina.|2 Center for Redox Biology and Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina.
authors.forename: Amanda E D
authors.initials: AED
authors.lastname: Van Swearingen
authors.affiliation: 3 Department of Biochemistry and Biophysics, Oregon State University , Corvallis, Oregon.
authors.forename: Steven
authors.initials: S
authors.lastname: Hartman
authors.affiliation: 3 Department of Biochemistry and Biophysics, Oregon State University , Corvallis, Oregon.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Brereton
authors.affiliation: 1 Department of Biochemistry, Wake Forest School of Medicine , Winston-Salem, North Carolina.|2 Center for Redox Biology and Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina.
authors.forename: Derek
authors.initials: D
authors.lastname: Parsonage
authors.affiliation: 2 Center for Redox Biology and Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina.|4 Department of Physics, Wake Forest University , Winston-Salem, North Carolina.
authors.forename: Freddie R
authors.initials: FR
authors.lastname: Salsbury
authors.affiliation: 3 Department of Biochemistry and Biophysics, Oregon State University , Corvallis, Oregon.
authors.forename: P Andrew
authors.initials: PA
authors.lastname: Karplus
authors.affiliation: 1 Department of Biochemistry, Wake Forest School of Medicine , Winston-Salem, North Carolina.|2 Center for Redox Biology and Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina.
authors.forename: Leslie B
authors.initials: LB
authors.lastname: Poole
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054464:Peroxiredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics
chemical_list: D006861:Hydrogen Peroxide; D010544:Peroxidases; D054464:Peroxiredoxins; D003545:Cysteine
keywords: AhpC; decamer; enzyme mechanism; peroxide signaling; regulation; thiol peroxidase