9-Aminoacridine and dibucaine exhibit competitive interactions and complicated inhibitory effects that interfere with measurements of ΔpH and xanthophyll cycle-dependent Photosystem II energy dissipation

Abstract

This study concerns measurements and interpretations of the trans-thylakoid membrane pH gradient, ΔpH, and xanthophyll cycle-dependent energy dissipation in Photosystem II (PS II). Compared and contrasted are the concentration-dependent inhibitory effects and interactions between two lipophilic tertiary amines, namely, 9-aminoacridine the ΔpH indicator and dibucaine a local anesthetic reported to inhibit both the ΔpH and xanthophyll cycle deepoxidation. Chlorophyll a fluorescence monitored both electron transport efficiency and xanthophyll cycle-dependent energy dissipation, high-performance liquid chromatography monitored deepoxidase and chloroplast ATPase activities and steady-state fluorescence monitored various activities of the amines in solution. Low concentrations (up to 2 μM) of both 9-aminoacridine and dibucaine showed similar fluorescence properties and ΔpH-dependent uptake into thylakoids. Importantly both amines exhibited mutually competitive inhibitory effects with respect to this ΔpH-dependent uptake and fluorescence quenching. The fluorescence yields of both compounds in aqueous solution were strongly quenched by sodium ascorbate, a necessary cofactor for in vitro deepoxidation. Both compounds similarly inhibited several light induced activities including deepoxidation, photosynthetic electron transport and PS II energy dissipation. However, for all these activities 9-aminoacridine was 2 to 5 times more potent. Importantly, 9-aminoacridine inhibited deepoxidation with an I_50≈1 μM, a concentration far below that which inhibits the ΔpH, ATP synthesis/hydrolysis or electron transport. The inhibitory effects of both compounds on PS II energy dissipation were exerted at 3 to 5 times lower concentration if added before as opposed to after a saturating level of deepoxidation. This result confirms the important role for deepoxidation in mediating PS II energy dissipation. Compared to 9-aminoacridine and in contrast to similar effects on the light-induced activities, dibucaine exhibited significantly different inhibitory effects on ATPase activity and ATPase mediated PS II energy dissipation. However, we conclude from the more potent inhibition by 9-aminoacridine and the similar inhibitory patterns of all the light-induced activities that neither 9-aminoacridine nor dibucaine possess unique capacities to neutralize the light-mediated ΔpH. DCMU–3-(3,4-dichlorophenyl)-1,1-dimethylurea; DTT–dithiothreitol; f_x–fractional intensity of fluorescence lifetime component x; F(′)_m–maximal PS II Chl a fluorescence intensity with all Q_A reduced in the absence (presence) of thylakoid membrane energization; F_o–minimal PS II Chl a fluorescence intensity with all Q_A oxi dized; F_s–steady state PS II Chl a fluorescence; HPLC–high performance liquid chromatography; I_(o)–intensity of fluorescence in the presence (absence) of quencher; K_a–association constant between Z (and A) and protonated PS II units; LA–local anesthetic; NaAsc–sodium ascorbate; NR–neutral red; PAM–pulse-amplitude modulation fluorometer; PFD–photon-flux density, μmols photons m^-2 s^-1; PS I–Photosystem I; PS II–Photosystem II; [PS II^-+]–concentration of PS II units with inactive/deprotonated (active/protonated ) xanthophyll binding sites; [PS II_tot]–total concentration of PS II units; [PS II⁺-Z]–concentration of PS II units with Z or A bound; Q–fraction of fluorescence intensity that is quenched; Q_max–fraction of fluorescence intensity that is quenched under control conditions; Q_A–primary quinone electron acceptor of PS II; V–violaxanthin; Z–zeaxanthin; 9AA–9-aminoacridine; ΔpH–trans-thylakoid membrane proton gradient; τ_f–lifetime of Chl a fluorescence