The sensitive balance between the fully folded and locally unfolded conformations of a model peroxiredoxin

Biochemistry. 2013 Dec 3;52(48):8708-21. doi: 10.1021/bi4011573. Epub 2013 Nov 20.


To reduce peroxides, peroxiredoxins (Prxs) require a key "peroxidatic" Cys that, in a substrate-ready fully folded (FF) conformation, is oxidized to sulfenic acid and then, after a local unfolding (LU) of the active site, forms a disulfide bond with a second "resolving" Cys. For Salmonella typhimurium alkyl hydroperoxide reductase C (StAhpC) and some other Prxs, the FF structure is only known for a peroxidatic Cys→Ser variant, which may not accurately represent the wild-type enzyme. Here, we obtain the structure of authentic reduced wild-type StAhpC by dithiothreitol treatment of disulfide form crystals that fortuitously accommodate both the LU and FF conformations. The unique environment of one molecule in the crystal reveals a thermodynamic linkage between the folding of the active site loop and C-terminal regions, and comparisons with the Ser variant show structural and mobility differences from which we infer that the Cys→Ser mutation stabilizes the FF active site. A structure for the C165A variant (a resolving Cys to Ala mutant) in the same crystal form reveals that this mutation destabilizes the folding of the C-terminal region. These structures prove that subtle modifications to Prx structures can substantially influence enzymatic properties. We also present a simple thermodynamic framework for understanding the various mixtures of FF and LU conformations seen in these structures. On the basis of this framework, we rationalize how physiologically relevant regulatory post-translational modifications may modulate activity, and we propose a nonconventional strategy for designing selective Prx inhibitors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Catalytic Domain
  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Cysteine / genetics
  • Models, Molecular
  • Peroxiredoxins / chemistry*
  • Peroxiredoxins / genetics
  • Protein Folding*
  • Protein Structure, Tertiary / physiology
  • Salmonella typhimurium / enzymology
  • Salmonella typhimurium / genetics
  • Serine / chemistry
  • Serine / genetics
  • Structure-Activity Relationship
  • Thermodynamics


  • Serine
  • Peroxiredoxins
  • Cysteine