Uncoupling effect of lipid peroxidation in spinach thylakoids exposed to peroxyl radicals generated by 2,2′-azobis (2-amidinopropane) dihydrochloride

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In this study, we characterized how lipid peroxidation alters the functionality of spinach thylakoids exposed to peroxyl radicals generated by the azo compound 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH). Incubation of thylakoids in the presence of different concentrations (0 to 200 mmol·L–1) of AAPH inhibited the formation of ΔpH (IC50 ≈ 1.5 mmol·L–1) estimated by the quenching of 9-aminoacridine fluorescence (Q9-AA). The Q9-AA inhibition was correlated (R2 = 0.98) to the extent of lipid peroxidation determined by the accumulation of thiobarbituric acid reactive substances. Much higher AAPH concentrations were required to inhibit the maximum (Fv/Fm) and effective (ΔF/Fm′) photochemical efficiencies of photosystem II (IC50 ≈ 120 mmol·L–1 and 50 mmol·L–1, respectively), indicating that moderate lipid peroxidation caused the uncoupling of spinach thylakoids. This was confirmed by the 62% stimulation of the O2 uptake rates measured without the artificial uncoupler NH4Cl when the AAPH concentrations increased from 0 to 20 mmol·L–1, reaching similar values to the rates measured in the presence of NH4Cl. Above 20 mmol·L–1 AAPH, the O2 uptake rates measured with and without NH4Cl declined similarly to the decrease of ΔF/Fm′. These results suggest that the increased H+-leakiness of thylakoid membranes could be one of the primary effects of oxidative stress.