Figure 5.

APX1 catalyzes the luminol-H₂O₂ reaction in vitro. A, HRP catalyzes the luminol-H₂O₂ reaction. In the presence of HRP, luminol is oxidized by H₂O₂ to create an excited-state product, 3-aminophthalate (3-APA*), whose decay to the ground state results in photon emission. B, APX1 catalyzed the chemiluminescence reaction of luminol. MBP-tagged APX1 recombinant proteins were purified from E. coli. Luminol (300 μM) and MBP, MBP-APX1, or HRP (0.04 mg mL⁻¹) were added first, and light signals were recorded immediately after the addition of H₂O₂ (10 mM). Values represent the total photon counts within 10 min. Data are shown as the mean ± SE (n = 4). Different letters above the bars indicate significant differences (P ≤ 0.05, one-way ANOVA). C, APX1 activity was corresponded with luminol concentrations below 15 mM. Data are shown as the mean ± SE (n = 5). D, APX1 activity corresponded with H₂O₂ concentrations below 10 mM. Data are represented as the mean ± SE (n = 6). E, Homology modeling of the structure of APX1 using the 3D coordination of the soybean cAPX1 (PDB-1V0H) as a template. The heme cofactor and substrate salicylhydroxamic acid locate in the middle. The 3 important residues, Cys32, Arg38 and Trp41, are marked F, Catalytic abilities of APX1 variants, APX1^R38H and APX1^W41F. Luminol (300 μM) and MBP, MBP-APX1, MBP-APX1^R38H, or MBP-APX1^W41F (0.09 mg mL⁻¹) were added first, and light signals were recorded immediately after the addition of H₂O₂ (1 mM). Values represent the total photon counts within 10 min. Data are shown as the mean ± SE (n = 8). Different letters above the bars indicate significant differences (P ≤ 0.05, one-way ANOVA). All experiments were repeated 3 times with similar results.