Structure-based insights into the catalytic power and conformational dexterity of peroxiredoxins

Abstract

Peroxiredoxins (Prxs), some of nature's dominant peroxidases, use a conserved Cys residue to reduce peroxides. They are highly expressed in organisms from all kingdoms, and in eukaryotes they participate in hydrogen peroxide signaling. Seventy-two Prx structures have been determined that cover much of the diversity of the family. We review here the current knowledge and show that Prxs can be effectively classified by a structural/evolutionary organization into six subfamilies followed by specification of a 1-Cys or 2-Cys mechanism, and for 2-Cys Prxs, the structural location of the resolving Cys. We visualize the varied catalytic structural transitions and highlight how they differ depending on the location of the resolving Cys. We also review new insights into the question of how Prxs are such effective catalysts: the enzyme activates not only the conserved Cys thiolate but also the peroxide substrate. Moreover, the hydrogen-bonding network created by the four residues conserved in all Prx active sites stabilizes the transition state of the peroxidatic S(N)2 displacement reaction. Strict conservation of the peroxidatic active site along with the variation in structural transitions provides a fascinating picture of how the diverse Prxs function to break down peroxide substrates rapidly.

FIG. 1.

The Prx catalytic cycle. Peroxide reduction by Prxs involves three main chemical steps of (1) peroxidation, (2) resolution, and (3) recycling. Two distinct protein conformations are involved in the cycle: FF (fully folded, active-site intact) and LU (locally unfolded, disulfide between the C_P and the C_R). The local unfolding event is required for disulfide bond formation in step 2, as is the local refolding event to reform the peroxide-binding active site after the disulfide is reduced in step 3. Oxidative regulation (gray, steps 4 and 5) is seen in sensitive, eukaryotic floodgate-type 2-Cys Prxs. Inactivation of the Prx by overoxidation of the C_P (step 4) is peroxide dependent. The inactivated form can be rescued through an ATP-dependent reaction catalyzed by sulfiredoxin (Srx) (step 5). The generic Prx is represented as a monomer, with S_P designating the sulfur atom of the C_P. The C_R (from R′ with S_R designating the sulfur atom) can be supplied by a different protein (1-Cys mechanism) or by a second Cys within the same Prx, either on the same chain or on the other subunit of a B-type dimer (2-Cys mechanism). Different proteins, including Trx and AhpF, have been identified as R″ in step 3.

FIG. 2.

The common-core secondary structural elements of Prxs. (A) A representative FF Prx showing the α-helices (pale cyan and pink) and β-strands (dark blue) common to all known Prxs. The C_P (ball-and-stick with sulfur atom colored yellow) is located in the first turn of α2 (pink). The structure shown is a monomeric Prx from the BCP subfamily (entry 63 in Table 1). (B) Helix α2 lies in a cradle with the base formed by β-strands β3 and β4 and the sides formed by the flanking helices α3 and α5. Compared with (A), the view shown is from the backside (i.e., ∼180 degrees rotated around the y-axis). Coloring as in (A); figure prepared using Pymol (20). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 3.

The peroxidatic active site. (A) Stereoview of the FF Prx active site with a bound H₂O₂ molecule. Shown are the highly conserved contiguous C_P-loop and the first turn of α2 plus the active-site Arg and an associated Glu/Gln/His supporting residue. The proximity to the C_P and the hydrogen-bonding interactions (green dotted lines) highlight the importance of the conserved Pro, Thr, and Arg in binding and activating the peroxide substrate (*) and in activating the C_P sulfhydryl for attack of the substrate oxygen atom (orange dashed line). The Glu/Gln/His residue, although not 100% conserved across all Prxs, is important as a hydrogen-bond acceptor positioning the conserved Arg. This figure was created by using ApTpx (entry 37 in Table 1), colored by atom (C, gray; N, blue; O, red; S, yellow). (B) Stereoview of an overlay of the H₂O₂-bound Prx from (A) with benzoate (cyan tones, entries 38 and 24 in Table 1), acetate (green tones, entries 35 and 56 in Table 1), ethanediol (light blue, entry 29 in Table 1) and glycerol (violet, entry 36 in Table 1), as seen bound in other Prx structures. Protein atoms are shown only for the Prx bound to H₂O₂, and protein coloring and hydrogen bonds to H₂O₂ (*) are as in (A). (C) Cartoon representation of the active-site transition-state conformation. The stabilizing interactions between key atoms from the backbone and the four conserved residues, and with the H₂O₂ substrate, are indicated. In the transition state, a bond is forming between the S atom of the C_P and the O_A of H₂O₂, and a bond is breaking between the O_A and O_B atoms of H₂O₂. The geometry of the active site is ideal for stabilizing the larger distance between the O_A and O_B atoms as the bond is broken. (A, B) were prepared by using Pymol (20). (C) is based on a figure from Hall et al. (29). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 4.

Variations in Prx sequences. (A) Structure-based sequence alignment of representative Prxs. Residues that have a common main-chain path among all Prxs are highlighted by a yellow background. Secondary structure elements are indicated above the alignment with the common-core Prx elements labeled as in Fig. 2, and other elements present in only some Prxs are shown but not labeled. The four residues conserved in all Prxs are colored red, and the C_R position of each 2-Cys Prx is highlighted by a purple, green, orange, or cyan background for a C_R placed in α2, α3, α5, or the C-terminus, respectively (B). *The YF-motif helix associated with some Prxs sensitive to overoxidation. Residues involved in backbone-mediated passing chain stabilization of the conserved Arg are given a blue background; in one case, Asp 163 of PfAOP (underlined residue in line 5 sequence) stabilizes the Arg via its side chain. Structures are referenced by index number from Table 1 and include a sensitive Prx1 (1), a robust Prx1 (21), a 1-Cys Prx6 (23), a 2-Cys Prx6 (29), a 1-Cys Prx5 (49), a 2-Cys Prx5 (38), a Tpx (50), a 1-Cys BCP (, monomeric), a 2-Cys BCP (, C_R in α2, dimeric), a 2-Cys BCP (, C_R in α3, monomeric), and an AhpE (71). The last residue of each line is numbered and dots below the alignment mark every 10 spaces. (B) The four prototypical locations for the C_R [colored as in (A) and labeled by location and the subfamily it is commonly associated with] are mapped onto a composite structure based on StAhpC (entry 21 in Table 1). The conserved C_P (red) is also shown. The two chains of the B-type dimer are colored in dark and light blue, and helix α2 is colored pink. (C) Pie charts based on ∼3,500 Prx sequences showing the frequency at which the C_R is in a given location for each subfamily. Wedges are colored by C_R position consistent with (A) and (B), by using the notation in (B): no C_R (gray), C-term′ (cyan), α5 (orange), α3 (green), α2 (purple), and uncertain (pale yellow). The exact positions are defined as follows: C-term′ aligns with residue 172 in HsPrxII (entry 1 in Table 1); α5 aligns with residue 151 (or −2 residues) in HsPrxV (entry 38 in Table 1); α3 aligns with residue 95 in EcTpx (entry 50 in Table 1); and α2 aligns with residue 112 in ScnTPx (entry 63 in Table 1). Sequences marked “uncertain” have additional Cys residues present, but none aligns exactly with one of the known locations. (B) was prepared by using Pymol (20). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 5.

Local unfolding changes the conformation of α2 and the C_P-loop. Comparison of the canonic FF structure (pink, entry 1 in Table 1) with LU representatives from each subfamily shows that the LU conformations of the C_P-loop and helix α2 vary by subfamily. Shown in stereo and viewed as in Fig. 2B are the LU conformations of a Prx1 (light blue, entry 17 and dark blue, entry 15 in Table 1), a Prx5 (orange, entry 44 in Table 1), a Tpx (dark green, entry 53 in Table 1), an α2-BCP (purple, entry 64 in Table 1), and an α3-BCP (pale green, entry 69 in Table 1). Labels indicate the location of the C_R (α2, α3, and α5, as in Fig. 4; C′ and C′-alt for the C_R near the C-terminus as in Fig. 4 with “alt” for the ∼8-degree shift from the canonic conformation, as described). No LU example is given from the Prx6 or AhpE subfamilies. The C_P residue in each structure is shown as ball-and-stick, with the sulfur atom colored yellow. The LU structures are all disulfide-bonded forms, even though the C_R is shown only for the α2-BCP example. Figure was prepared by using Pymol (20). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 6.

Conformational changes for disulfide formation are localized to the positions surrounding the C_P and the C_R. Stereoview of the interpolated structural changes colored by rainbow between the FF (blue) and LU (red) conformations for a representative from each major subfamily: (A) Prx1 (StAhpC, entries 17 and 18 in Table 1); (B) Prx5 (HsPrx5, entries 38 and 44 in Table 1); (C) Tpx (EcTpx, entries 50 and 52 in Table 1); (D) α2-BCP (ApBCP, entry 64 in Table 1); and (E) α3-BCP (entries 68 and 69 in Table 1). The interpolations show how most of the protein structure does not change during the local unfolding transition. In (A), the C-terminus is truncated at residue 165 because of disorder in the rest of the chain, although in the FF conformation, residues through 186 are ordered. In (E), residues 78–80 are omitted, as they are disordered in the LU conformation. The C_P and the C_R are shown as sticks with sulfur atoms colored yellow, and the calculated intermediate structures are partially transparent. Interpolations are calculated by using the Yale morph server (47) and visualized by using Pymol (20). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 7.

Quaternary structures of Prxs. For some Prxs, the basic monomeric structure shown in Fig. 2 can form (A) A-type dimers, interacting near α3, or (B) B-type dimers, interacting at the β-sheet to form an extended 10-stranded β-sheet. (C) Some members of the Prx1 and Prx6 subfamilies form decameric structures through the interaction of five B-type dimers via the A-type dimer interface. Subunit coloring for the A-type dimer (purple and dark blue) and the B-type dimer (dark blue and light blue) are used in the decamer to show how it is composed of these two types of interactions. (D) The oligomerization of the decamers is redox dependent. In the Prx1 and Prx6 subfamilies, reduced and overoxidized Prxs form decamers, with the A-type dimer interface stabilizing the FF active site. The structural change with disulfide formation destabilizes the A-type dimer interface, and the decamer falls apart to B-type dimers. Octamers and dodecamers have also been observed (see Table 1) and are thought to be functionally equivalent to the decamer. (A–C) were prepared by using Pymol (20). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 8.

The structural difference between robust and sensitive Prx1s. Comparison of a sensitive Prx1 (A and B, RnPrx1, entries 5 and 4 in Table 1) and a robust Prx1 (C and D, StAhpC, entries 21 and 17 in Table 1) in the FF (left panels) and LU (right panels) conformations reveals the structural feature causing sensitivity. The two regions with differences in sequence that correlate with sensitive versus robust Prxs are a loop with an inserted GGLG motif and a C-terminal extension that forms a helix with a “YF” motif; they are colored orange. The regions that undergo conformational change during local unfolding are colored green (except for the C-terminus, which is orange). In sensitive Prx1s, the conserved C-terminal helix containing the YF motif and the adjacent GGLG motif bury the N-terminal end of α2, stabilizing the FF conformation. This hinders local unfolding, slowing disulfide-bond formation and thus enhancing the competing overoxidation pathway. Comparison of (A) with (C) shows the structural differences that result from the absence of GGLG and YF motifs in robust Prx1s. The C_P and C_R are shown as ball-and-stick with sulfur atoms colored yellow. *The end of the ordered C-terminus in the LU conformations (with additional residues being disordered). Figure prepared by using Pymol (20). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).