Structural evidence that peroxiredoxin catalytic power is based on transition-state stabilization

Abstract

Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cell-wide peroxide reduction, but the origins of their high activity have been mysterious. We present here a novel structure of human PrxV at 1.45 A resolution that has a dithiothreitol bound in the active site with its diol moiety mimicking the two oxygens of a peroxide substrate. This suggests diols and similar di-oxygen compounds as a novel class of competitive inhibitors for the Prxs. Common features of this and other structures containing peroxide, peroxide-mimicking ligands, or peroxide-mimicking water molecules reveal hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen track along which the peroxide oxygens move as the reaction proceeds. Key insights include how the active-site microenvironment activates both the peroxidatic cysteine side chain and the peroxide substrate and how it is exquisitely well suited to stabilize the transition state of the in-line S(N)2 substitution reaction that is peroxidation.

Figure 1

Electron density for bound DTT in the active site. Shown is the electron density in the active site (A) before including DTT in the model and (B) after the final round of refinement of the model containing DTT (HsPrxV·DTT). A peptide segment including the C_P and the two residues prior to it is shown in sticks and colored by atom (oxygen = red, nitrogen = blue, sulfur = yellow), and electron density is shown as mesh: 2F_o-F_c density is contoured at 1.5 ρ_rms (pale blue) and 6.5 ρ_rms (dark blue), and F_o-F_c density is contoured at 4 ρ_rms (green). Panel B inset: An overlay of the final HsPrx·DTT structure (dark grey carbons) with the published HsPrx·BEZ structure (light grey carbons; PDB entry 1HD2) in the same orientation as the main panel.

Figure 2

The HsPrx·DTT structure. The protein backbone ribbon is shown with α-helices (green), β-strands (purple), coils/turns (grey), and the C_P residue (yellow) highlighted. The bound DTT molecule (colored by atom, oxygen = red, sulfur = yellow) is shown as sticks, and the surface of the active site pocket into which it binds is shown. Secondary structures are labeled according to the common fold of all Prxs; the short helix between α4 and α5 is not universally conserved in the Prx fold and is labeled simply as “α”.

Figure 3

Stereoview of the HsPrx·DTT active site with bound DTT. Shown are the C_P-loop and the first turn of α2 plus the conserved Arg colored by atom as in Figure 1. Hydrogen bonding (cyan dotted lines) important for DTT binding and the S_P-O_A interaction (orange dashed line) are indicated. The four residues conserved in all Prxs and the His that interacts with the active site Arg are labeled.

Figure 4

Two distinct positions for the conserved Arg in the Prx active site. (A) The overlaid active sites from all ligand-bound Prx structures (Table 1) are shown with side chains (shown as sticks) for the C_P, the conserved active site Arg, the supporting Glu/Gln/His residue and for those that contain it, a second Arg (indicated by an asterisk) of the Arg-Glu-Arg hydrogen bonding network. The carbon atoms are colored according to the position of the Arg, light grey for position I and purple for position II; oxygen (red), nitrogen (blue) and sulfur (yellow) are colored by atom. The distinct positioning of the conserved active site Arg correlates with the presence of the second Arg residue. (B) Same as panel A but including all FF Prx structures. The overlay includes 179 chains from 32 pdb entries (3A2V, 3A2W, 1QMV, 2Z9S, 2PN8, 1UUL, 2I81, 2V2G, 2V32, 2V41, 1XCC, 2CV4, 1X0R, 3A5W, 1HD2, 1H4O, 1OC3, 1URM, 2VL2, 1TP9, 2VL3, 1NM3, 1XIY, 3HVV, 1Y25, 1PSQ, 2YZH, 2CX4, 1XXU, 3MNG, 3DRN, 3GKM).

Figure 5

Overlays of ligands that bind in the Prx active site. In all panels, a peptide segment containing the C_P-loop is shown as a ribbon in the same orientation as in Figure 3. Side chains are shown for the conserved Pro, Thr, C_P and Arg, and ligands are shown as sticks with coloring as in Figure 4. (A) H₂O₂-bound structure with O_A, O_B and S_P atoms identified. In the remaining panels, a bound H₂O₂ molecule (transparent pale green) is shown for reference. Panels B through I feature the following ligands: (B) DTT and ethanediol; (C) glycerol; (D) citrate; (E) acetate; (F) benzoate; (G) sulfate and formate; (H) ethanediol binding as only O_A; (I) glycerol binding as only O_A. The structures included in each panel are listed in Table 1.

Figure 6

Summary of peroxide-mimics binding in the Prx active site. (A) The oxygen atoms (red spheres) mimicking O_A and O_B from all ligands shown in Figure 5. (B) Active site water molecules (red spheres) from unliganded FF structures. In both panels, the C_P-loop and conserved Arg are shown as sticks in the same orientation as Figure 3 and colored by atom type. As in Figure 5, a reference H₂O₂ molecule (pale green) is included for reference. In this figure the C_P-loop is extended by one residue to emphasize it is the first turn of helix α2 by showing the first hydrogen bond of the helix (cyan). Waters shown are from pdb codes 1X0R, 1QMV, 2PN8, 1XXU, 1PSQ, 1XIY, 3A5W, 1CXX and 1N8J.

Figure 7

Interactions in the Prx active site Michaelis complex. Shown is a schematic drawing of the peroxide-bound active site emphasizing the hydrogen bonding interactions present. The in-line geometry for S_P to attack O_A for the peroxidatic S_N2 reaction (bold dashed line) and key hydrogen bonding interactions (pale dashed, dotted, and solid lines) are indicated. The view is as in Figures 3, 5, 6 and 8. Backbone atoms in the C_P-loop are identified by their residue position relative to C_P. The negative charge on the C_P-thiolate is indicated.

Figure 8

Reaction path for the peroxidatic S_N2 reaction. (A) The Michaelis complex structure as seen in ApTpx·H₂O₂ chain F (PDB entry 3A2V20) shows how the active site aligns the peroxide substrate for an in-line attack from the C_P-thiolate (orange dashed line). (B) Simplistically-derived model for the transition state (see text) showing the partial S_P-O_A (orange dashed line) and O_A-O_B (red dashed line) bonds. The S_P-O_A and O_A-O_B distances are 2.6 and 2.4 Å, respectively. (C) The product complex as seen at ~25% occupancy in the rerefined ApTpx·H₂O₂ chain F (see text). The S_PO_AH and O_B hydroxide leaving group (red sphere) are shown. Stabilizing active site hydrogen bonds (cyan dotted lines) are shown in all panels.