Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase-Insights into the Catalytic Cycle of Bacterial Peroxidases

Abstract

Neisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 ± 0.1 µM and 41 ± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae.

Keywords: Neisseria gonorrhoeae; ROS detoxification; active state; azide-inhibited structure; bacterial peroxidase; catalytic cycle; diheme enzymes.

Figure 1

UV-visible spectra of mixed-valence NgBCCP at pH 7.5 in the presence of increasing concentrations of cyanide (A), azide (C) and imidazole (E). The arrows indicate the direction of changes in the spectra from the light gray (no inhibitor) to dark gray (maximum inhibitor concentration). The absorption difference at specific wavelengths was plotted as a function of inhibitor concentration in solution ((B), cyanide; (D), azide; (F), imidazole). The dashed line (B,D) in the binding of cyanide and azide was fitted with a single binding site equation, with a K_app of 4 µM and 26 mM, respectively.

Scheme 1

Kinetic diagram of NgBCCP activity with H₂O₂ as substrate and H₂O as final product (protons and electrons in this reaction were omitted for simplicity). In the presence of the inhibitor (I) there is a mixed inhibition, where K_i is the binding constant to the free enzyme and K_i’ the binding constant in the presence of substrate. These two constants are related by a constant α, with K_i’ = α.K_i, and K’_M = αKs (see Section 4).

Figure 2

Determination of the inhibition constants for (A,B) cyanide, (C,D) azide, and (E,F) imidazole at pH 7.5. K_i’ and K_i estimates are given by the intercept value in the plots of S/v₀ (Dixon: A,C,E) or 1/v₀ (Cornish–Bowden: B,D,F), respectively, against i at three different substrate concentrations (● 0.1 µM, ■ 25 µM, and ▲ 100 µM H₂O₂). The error bars represent the average of duplicate assays.

Figure 3

Structure of the mixed-valence NgBCCP homodimer. In (A) is shown the dimer backbone, which comprises the asymmetric unit of the crystal, and in (B) its electrostatic surface potential is represented from −5 to 5 KT/e (colored from negative red surface to positive blue surface). Panel (A) was prepared with BIOVIA Discovery Studio Visualizer 4.5 and Panel (B) with Chimera 1.10.2.

Figure 4

View of the mixed-valence NgBCCP structure and calcium binding site. In (A) is shown the calcium ion (green sphere) located between the two hemes, the His/Met 6-coordinated E heme and the P heme coordinated by a proximal histidine residue and a distal water molecule (small red sphere). Although the two hemes have a Fe-Fe distance of 20.9 Å, electron transfer is possible because of the tryptophan residue located between their propionate groups. In (B) is shown the calcium binding site. The calcium ion (green sphere) is coordinated by the oxygens of four water molecules (small red spheres) and three conserved residues (Asn83, Thr261 and Pro263). This view also shows that the calcium ion is near the carboxylate group of the E heme propionate A, which forms hydrogen bonds with two of the coordinating water molecules. Figures prepared with BIOVIA Discovery Studio Visualizer 4.5.

Figure 5

Structure changes that occur upon activation. Structure comparison of (A) NgBCCP mixed-valence (MV) state with (B) P. aeruginosa BCCP in the MV (PsaBCCP–MV) and (C) oxidized (PsaBCCP–Ox) state. In red are highlighted the structural differences between MV states, and in green the differences between oxidation states. Figures prepared with BIOVIA Discovery Studio Visualizer 4.5 using PDB ID 2VHD and 1EB7 for P. aeruginosa BCCP in the mixed-valence and oxidized state, respectively.

Figure 6

Structure of the P heme active site in chain A. (A) In the active site, there are two water molecules, w1 and w2 (small red spheres representing their oxygen atoms), at a short distance from the conserved Gln108 and Glu118 side chains. (B) The surface above the P heme pocket showing a channel of waters above w1 and w2 (here as blue spheres). The water molecules around this pocket are represented as red spheres occupying small pockets and groves around the entrance. Figures prepared with BIOVIA Discovery Studio Visualizer 4.5.

Figure 7

Structure comparison of the NgBCCP active (blue) and azide-inhibited (orange) forms. Overall, the protein fold is conserved (A), as well as the residues in the active site (B). In the inhibited form there is an azide molecule, H-bonded to the conserved Gln108 and Glu118 (C). Figures prepared with BIOVIA Discovery Studio Visualizer 4.5.

Scheme 2

Proposed catalytic mechanism of bacterial peroxidases. (1.) P heme is 5-coordinated. (2.) In the presence of hydrogen peroxide, there is formation of a peroxide-bound complex, and Glu118 forms a hydrogen bond with one of the oxygens (blue O). (3.) The species formed is named Compound 0. (4.) The cleavage of O-O bond releases one water, and forms Compound I. (5.) Compound I can receive protons from the solvent forming Compound II. (6.) Transfer of an additional proton releases a second water molecule.