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L. Flohe and J. R. Harris (eds.), Peroxiredoxin Systems, 61-81. © 2007 Springer.

this family grew (Chae et al., 1994), it became clear that, in addition to or as a part of the various cellular functions that were ascribed to these proteins, the Prxs have in common their ability to enzymatically reduce peroxides, including (for broad specificity Prxs, which seems to include most) hydrogen peroxide, organic hydroperoxides and peroxynitrite (Hofmann et al., 2002; Wood et al., 2003b). In Escherichia coli, the major Prx in the cytosol, AhpC, is the primary reducing system for endogenously-generated hydrogen peroxide, with catalase becoming the primary scavenger only at relatively high levels of peroxides (> 5 ^M), because the capacity to regenerate reduced AhpC becomes limiting (Seaver and Imlay, 2001). The increasing availability of structural and kinetic information for the widespread and in some ways diverse group of Prx proteins has given us insight into many aspects of the structure-function relationships within them. As described in further detail in this chapter, a coalition of structural, dynamic and chemical features are all intimately involved in imparting to Prxs their modulated catalytic and cellular functions.

2. CATALYSIS OF THE PEROXIDE REDUCTION STEP 2.1. Reaction at the Critical Cysteine Residue

With one notable exception of a selenocysteine-containing Prx (Söhling et al., 2001), the only redox center in Prxs, completely conserved among all members, is the Cys residue at the active site (Cys51 in Prxll numbering), termed the peroxidatic Cys (Cp or Sp symbols used for the peroxidatic Cys or sulfur atom, respectively, Fig. 1). Accordingly, any mutations of this residue completely abrogate activity (Chae et al., 1994; Ellis and Poole, 1997; Montemartini et al, 1999; Chen et al., 2000; Flohe et al., 2002; König et al., 2003; Deponte and Becker, 2005). A second Cys residue, designated as the resolving Cys (CR or SR) can and often does participate in catalysis in later steps (see below and Fig. 2), but this residue is both unnecessary and peripheral (in a structural and chemical sense) to the peroxidatic active site and mechanism. Interestingly, although the Prxs have diverged into separate classes based on their sequences, oligomerization properties and recycling mechanisms, they exhibit very highly conserved structures and sequences around the active site. The peroxidatic Cys is located within the first turn of an a-helix following a flexible loop region, in a location that parallels that of the second Cys of the CxxC motif in thioredoxin (Trx); Trx is a distant homologue which defines the core structural fold of Prxs and is prototypical of a superfamily of redox enzymes built on related motifs (T/S/CxxC and CxxT/S/C) (Fomenko and Gladyshev, 2003; Wood et al., 2003b ; Copley et al., 2004). In Prxs, this Cys residue sits at the base of the active site pocket and is activated for catalysis by interactions with several conserved residues surrounding it (see below), in part acting to lower the pKa of this group for nucleophilic attack on the terminal oxygen of the hydroperoxide (ROOH) substrate (Fig. 3). The need for deprotonation as well as additional activation by other features of the active site microenvironment is clear, given that protonated

Figure 1. Conserved structure of Prx active sites. Shown in red are the conserved Pro, Thr, and Cys in the PxxxTxxC active site motif, as well as the conserved Arg residue contributed by another part of the polypeptide (from human PrxV, PDB code 1hd2). Dotted lines indicate the conserved hydrogen bonding network. Blue indicates the loop-helix region around the active site that undergoes local unfolding following oxidation to form a disulfide bond in typical and atypical 2-Cys Prxs. This figure was reprinted from Trends in Biochemical Sciences, Vol. 28, article by Wood, Z.A., Schröder, E., Harris, J.R., and Poole, L.B., Structure, mechanism and regulation of peroxiredoxins, pages 32-40, Copyright 2003, with permission from Elsevier

Figure 1. Conserved structure of Prx active sites. Shown in red are the conserved Pro, Thr, and Cys in the PxxxTxxC active site motif, as well as the conserved Arg residue contributed by another part of the polypeptide (from human PrxV, PDB code 1hd2). Dotted lines indicate the conserved hydrogen bonding network. Blue indicates the loop-helix region around the active site that undergoes local unfolding following oxidation to form a disulfide bond in typical and atypical 2-Cys Prxs. This figure was reprinted from Trends in Biochemical Sciences, Vol. 28, article by Wood, Z.A., Schröder, E., Harris, J.R., and Poole, L.B., Structure, mechanism and regulation of peroxiredoxins, pages 32-40, Copyright 2003, with permission from Elsevier

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