Articles Crystal Structure and Solution NMR Dynamics of a D (Type II) Peroxiredoxin Glutaredoxin and Thioredoxin Dependent: A New Insight into the Peroxiredoxin Oligomerism ² Aude Echalier, ‡,§ Xavier Trivelli, §,| Catherine Corbier, ‡ Nicolas Rouhier, ⊥ Olivier Walker, | Pascale Tsan, | Jean-Pierre Jacquot, ⊥ Andre ´ Aubry, ‡ Isabelle Krimm,* ,| and Jean-Marc Lancelin* ,| LCM3B, Groupe Biocristallographie, UniVersite ´ Henri Poincare ´ sNancy1, UMR CNRS 7036, 54506 VandoeuVre, France, RMN Biomole ´ culaire, UniVersite ´ Claude BernardsLyon1, UMR CNRS 5180 Sciences Analytiques, ESCPE-Lyon, 69622 Villeurbanne, France, and UMR Interaction Arbres Microorganismes, UniVersite ´ Henri Poincare ´ sNancy1, UMR INRA 1136, 54506 VandoeuVre, France ReceiVed August 17, 2004; ReVised Manuscript ReceiVed October 22, 2004 ABSTRACT: Peroxiredoxins (Prxs) constitute a family of thiol peroxidases that reduce hydrogen peroxide, peroxinitrite, and hydroperoxides using a strictly conserved cysteine. Very abundant in all organisms, Prxs are produced as diverse isoforms characterized by different catalytic mechanisms and various thiol- containing reducing agents. The oligomeric state of Prxs and the link with their functionality is a subject of intensive research. We present here a combined X-ray and nuclear magnetic resonance (NMR) study of a plant Prx that belongs to the D-Prx (type II) subfamily. The Populus trichocarpa Prx is the first Prx shown to be regenerated in Vitro by both the glutaredoxin and thioredoxin systems. The crystal structure and solution NMR provide evidence that the reduced protein is a specific noncovalent homodimer both in the crystal and in solution. The dimer interface is roughly perpendicular to the plane of the central  sheet and differs from the interface of A- and B-Prx dimers, where proteins associate in the plane parallel to the  sheet. The homodimer interface involves residues strongly conserved in the D (type II) Prxs, suggesting that all Prxs of this family can homodimerize. The study provides a new insight into the Prx oligomerism and the basis for protein-protein and enzyme-substrate interaction studies by NMR. Peroxiredoxins (Prxs) 1 are thiol-specific antioxidant pro- teins that catalyze the reduction of hydrogen peroxide, peroxinitrite, and hydroperoxides, some of the reactive oxygen species responsible for oxidation and degradation of lipids, nucleic acids, and proteins. The first Prx was discovered in 1988 as a yeast antioxidant protein devoid of a prosthetic group and selenium (1). Since then, the number of identified Prxs has increased steadily, and heterologues have been found in bacteria to humans with several isoforms ² This work was supported by grants from CNRS (GDR “Thiore ´dox” 2477) and financial support from the French Ministe `re de l’Education Nationale, de la Recherche et des Technologies to A.E., X.T., and N.R. * To whom correspondence should be addressed: RMN Bio- mole ´culaire, Universite ´ Claude BernardsLyon1, Domaine Scientifique de la Doua, ESCPE-Lyon, F-69622 Villeurbanne Cedex, France. Telephone/Fax: (+33) 4 72 43 13 95. E-mail: isabelle@hikari.cpe.fr (I.K.); Telephone/Fax: (+33) 4 72 43 13 95. E-mail: lancelin@hikari.cpe.fr (J.-M.L.). ‡ LCM3B, Groupe Biocristallographie. § A.E. and X.T. have contributed equally. | UMR CNRS 5180 Sciences Analytiques. ⊥ UMR Interaction Arbres Microorganismes. 1 Abbreviations: AhpD, alkyl hydroperoxide reductase D; AhpF, alkyl hydroperoxide reductase flavoprotein; BMRB, BioMagResBank; Grx, glutaredoxin; MW, molecular weight; NMR, nuclear magnetic reso- nance; NOE, nuclear Overhauser effect; PDB, Protein Data Bank; PEG, poly(ethylene glycol); Prx, peroxiredoxin; R 1, longitudinal relaxation rate; R2, transversal relaxation rate; Trx, thioredoxin. © Copyright 2005 by the American Chemical Society Volume 44, Number 6 February 15, 2005 10.1021/bi048226s CCC: $30.25 © 2005 American Chemical Society Published on Web 01/15/2005