Fig. 4: H2O2 inhibits recombinant AtPCO enzymes.
From: H2O2 repurposes plant O2 sensing to regulate post-hypoxia responses
a, Oxidation of RAP22–15 by 50 µM H2O2- and 50 µM TBHP-treated or non-treated recombinant AtPCO4 enzyme. Non-enzymatic RAP22–15 oxidation by 1 mM H2O2 was included for comparison with oxidation of RAP22–15 by AtPCO4 (n = 3). b, H2O2-mediated inhibition of (10 µM) AtPCOs 1–5; statistical differences were evaluated using two-way ANOVA followed by Tukey’s HSD test (P < 0.05). Lines show the mean, with error bars representing the s.d. (n = 4). c, H2O2 dose-dependent effect on (2 µM) PCO4 enzyme activity (n = 3). d, Active site view of crystal structure of AtPCO4 (PDB 6S7E); the Fe cofactor (orange sphere) is bound by a triad of His residues (His98, His100 and His164, yellow sticks), and Cys residues found to be oxidized by H2O2 are shown in cyan. e, X-band CW-EPR spectra of (i) AtPCO4 with H2O2, (ii) AtPCO4 only and (iii) FeSO4(aqueous) with H2O2. The spectrum in (i) shows Fe(III) signal intensity at 150 mT, similar to that seen in (iii) but with signal splitting at geff = 4.29, g′eff = 6.4 and g″eff = 5.7, first as typical rhombic coordination of Fe(III) with two axially symmetric species. f, Activity restoration test of 10 µM AtPCO4 enzyme treated with 100 µM H2O2, using known cellular reductants glutathione (GSH, 1 mM) and ascorbate (Asc, 1 mM), as well as DTT (1 mM) (n = 3).
