Model for the Exceptional Reactivity of Peroxiredoxins 2 and 3 with Hydrogen Peroxide A KINETIC AND COMPUTATIONAL STUDY * □ S Received for publication, February 17, 2011, and in revised form, March 2, 2011 Published, JBC Papers in Press, March 8, 2011, DOI 10.1074/jbc.M111.232355 Pe ´ ter Nagy ‡1 , Amir Karton § , Andrea Betz ‡ , Alexander V. Peskin ‡ , Paul Pace ‡ , Robert J. O’Reilly § , Mark B. Hampton ‡ , Leo Radom § , and Christine C. Winterbourn ‡ From the ‡ Department of Pathology and National Research Centre for Growth and Development, University of Otago Christchurch, P.O. Box 4345, Christchurch, New Zealand and the § School of Chemistry and ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, University of Sydney, Sydney, New South Wales 2006, Australia Peroxiredoxins (Prx) are thiol peroxidases that exhibit excep- tionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are 3– 4 A ˚ or 6 –7 A ˚ away from the active site peroxidative cys- teine (C p ), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooper- ative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure sug- gest strong catalytic effects of H-bond-donating functional groups to the C p sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cat- ion in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bond- ing to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H 2 O 2 and may have wider implications for protein thiol reactivity toward peroxides. Peroxiredoxins (Prx) 2 are a family of ubiquitous proteins that are important for antioxidant defense and redox signaling (1, 2). Prx reduce H 2 O 2 extremely rapidly (3–5). They are also highly reactive against peroxynitrite and other peroxides (4, 6, 7), but less so with other typical thiol-reactive reagents such as chloramines or alkylating electrophiles (5, 8). There are six subfamilies (Prx1, Prx6, AhpE (one-cysteine peroxiredoxin from Mycobacterium tuberculosis), Prx5, Tpx, and bacterioferritin comigratory protein), categorized by amino acid sequence, which share a similar catalytic cycle. A number of highly conserved amino acid residues promote similar structures around their active site cysteine (peroxidatic cysteine; C p ) (9). Based on mech- anistic considerations they are further subcategorized into 1-Cys and 2-Cys Prx. Prx2 and -3, the focus of this manuscript, are 2-Cys Prx that belong to the Prx1 subfamily. The reactivity of the C p thiol toward H 2 O 2 is many orders of magnitude larger than that of free cysteine (e.g. the second- order rate constants for the reaction of human Prx2 and Prx3 with H 2 O 2 are  10 7 M -1 s -1 (3, 5), compared with 1 M -1 s -1 for free cysteine or GSH (10)). Due to the low pK a  6 of the C p sulfhydryl group, it is in its more nucleophilic thiolate form at physiological pH (5, 11, 12). However, this is not sufficient to explain the high reactivity of Prx with peroxides and further lowering of the pK a below 6 would decrease reactivity due to the linear free energy relationship (13). It is likely that conserved amino acid residues at the active site influence the rate of reac- tion via H-bonding interactions with the C p sulfur as well as with H 2 O 2 . Based on structural considerations, Hall et al. (9, 14) proposed that a highly conserved arginine residue (ArgI) near the C p (Fig. 1, Prx3 Arg-123) activates the peroxide at the active site of Prx. There is another arginine (ArgII; Fig. 1a, Prx3 Arg- 146) close to the active site (6 –7 Å from the C p sulfur, Fig. 1b) present in all members of the Prx1, Prx6, and AhpE subfamilies (see “Experimental Procedures”), which we hypothesize to have a role in the catalytic activity of these proteins. Although an activator role for ArgI on H 2 O 2 has been pro- posed, only one study has tested the role of these arginine res- idues (along with other conserved amino acids) using site-di- rected mutagenesis. This showed that barley Prx ArgI and ArgII are important for its peroxidatic activity (15). However, activity was measured using a catalytic assay where the rate-determin- ing step is reduction of the disulfide-linked Prx dimer, so it was not possible to assess the extent to which the reactivity of C p with H 2 O 2 was decreased. The effects of ArgI and ArgII on the kinetics of the reaction of C p with H 2 O 2 have not been investi- gated. In a combined crystallographic and quantum chemical * This work was supported by the Marsden Fund, the Health Research Council of New Zealand, and the Australian Research Council. □ S The on-line version of this article (available at http://www.jbc.org) con- tains supplemental “Methods,” Tables S1–S5, Figs. S1–S4, and addi- tional references. 1 To whom correspondence should be addressed: The National Institute of Oncology, Department of Molecular Immunology and Toxicology, 1122 Budapest, Rath Gyorgy 7-9, Hungary. Tel.: 36-1-224-8787; Fax: 36-1-224- 8620; E-mail: peter.nagy@oncol.hu. 2 The abbreviations used are: Prx, peroxiredoxin(s); Tpx, thiol peroxidase; ApTPx, archeal peroxiredoxin; C p , peroxidative cysteine; O a , electrophilic oxygen of H 2 O 2 (the one being attacked by HS - ); O b , leaving hydroxide group of H 2 O 2 ; G4, Gaussian-4; TS, transition structures; H ‡ , activation enthalpy; G ‡ , activation free energy; H ‡ =H ‡ (uncat,aq) -H ‡ (cat,enz) ; , dielectric constant; CPCM-G4//B3-LYP/6 –31+G(2df,p), conductor-like polarizable continuum model solvation correction on top of a gas phase G4//B3-LYP/6 –31+G(2df,p) enthalpy; uncat, uncatalised. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 20, pp. 18048 –18055, May 20, 2011 © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. 18048 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 20 • MAY 20, 2011 at University of Pennsylvania Library, on May 23, 2011 www.jbc.org Downloaded from http://www.jbc.org/content/suppl/2011/03/08/M111.232355.DC1.html Supplemental Material can be found at: