Crystal structure of decameric 2-cys peroxiredoxin from human erythrocytes Ewald Schröder, Jennifer A. Littlechild, Andrey A. Lebedev & Michail N. Isupov Schools of Chemistry and Biological Sciences, University of Exeter, Stocker Rd, Exeter, EX4 4QD, UK; Email: eschrode@ex.ac.uk Peroxiredoxin (Prx) enzymes are preferentially expressed under conditions of stress induced by elevated levels of reactive oxygen species and reactive nitrogen species. Mammalian Prxs down- regulate a range of redox-sensitive processes including apoptosis, mitochondrial permeability, tyrosine kinase activity and the activity of transcription factors [1]. Thioredoxin peroxidase B (TPx- B) is a 2-cys type II Prx and catalyses the reduction of hydrogen peroxide and alkyl hydroperoxides to water or alcohol. The 2-cys Prx mechanism involves the activity of two conserved cysteines and uses the thioredoxin enzyme system as a source of hydrogen [2]. The crystal structure for TPx-B has been solved to 1.7 Å. Native data were collected at the EMBL X11 beamline [3], as reported in last year’s report. A self-rotation function was calculated by MOLREP [4] at 10-3 Å resolution with an integration radius of 20 Å and clearly indicated that the Prx molecule is a decamer with 52 point group symmetry. The structure was solved by molecular replacement using the dimeric structure of hORF06 [5] as a search model, a distantly-related 1-cys Prx. Upon the basis of the self-rotation function, 3600 possible decamers with 52 point group symmetry were constructed and used in the translation function search over a 10-4 Å resolution range. A solution was found with a correlation of 17.4% and R-factor of 55.5%. This enabled the subsequent refinement using REFMAC to give a 1.7 Å resolution structure with R cryst of 19.2% and R free (for 1.0 % total data) of 25.6%. The structure is a toroid constructed from five dimers linked end-on through hydrophobic interactions and in-direct hydrogen bonds (Fig. 1). The subunit of each dimer contains 197 residues and consists of an N-terminal thioredoxin-like domain, and a C-terminal arm. Each dimer contains two active sites with residues from both subunits (Fig. 2). Cys 51, the site of peroxide reduction [2], is irreversibly oxidised to sulphinic acid [Csd 51] and is buried in a hydrophobic pocket. Cys 172 lies ~10 Å away from Cys 51 and recycles the catalytic activity in active TPx-B in vivo by forming a disulphide-bond with Cys 51 [2], as seen in the 2-cys Prx HBP23 structure [6], followed by reduction by thioredoxin [2]. Comparisons with the dimeric structures of hORF06 [5] and HBP23 [6] reveals that oxidation of the active site Cys 51 to sulphinic acid in TPx-B is linked to the unwinding of the active site helix α1, to conformational changes within four loops and to the non- reversible formation of decamers which traps the enzyme into an inactive toroid structure. Cysteine sulphinic acids can readily form in vivo under conditions of oxidative stress [2]. The clearly defined C-terminal arm is tethered at the dimer-dimer interface in the decameric TPx-B, but appears disordered within the dimeric HBP23 structure. Decameric toroids of TPx-B have been observed asssociated with the plasma membrane of erythrocyte ghosts by electron microscopy [7].