Evidence for the involvement of loosely bound plastosemiquinones in superoxide anion radical production in photosystem II

Abstract

Recent evidence has indicated the presence of novel plastoquinone-binding sites, QC and QD, in photosystem II (PSII). Here, we investigated the potential involvement of loosely bound plastosemiquinones in superoxide anion radical (O2-) formation in spinach PSII membranes using electron paramagnetic resonance (EPR) spin-trapping spectroscopy. Illumination of PSII membranes in the presence of the spin trap EMPO (5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide) resulted in the formation of O2-, which was monitored by the appearance of EMPO-OOH adduct EPR signal. Addition of exogenous short-chain plastoquinone to PSII membranes markedly enhanced the EMPO-OOH adduct EPR signal. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, the EMPO-OOH adduct EPR signal was suppressed by 50% when the urea-type herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) was bound at the QB site. However, the EMPO-OOH adduct EPR signal was enhanced by binding of the phenolic-type herbicide dinoseb (2,4-dinitro-6-sec-butylphenol) at the QD site. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, DCMU and dinoseb inhibited photoreduction of the high-potential form of cytochrome b559 (cyt b559). Based on these results, we propose that O2- is formed via the reduction of molecular oxygen by plastosemiquinones formed through one-electron reduction of plastoquinone at the QB site and one-electron oxidation of plastoquinol by cyt b559 at the QC site. On the contrary, the involvement of a plastosemiquinone formed via the one-electron oxidation of plastoquinol by cyt b559 at the QD site seems to be ambiguous. In spite of the fact that the existence of QC and QD sites is not generally accepted yet, the present study provided more spectroscopic data on the potential functional role of these new plastoquinone-binding sites.

Fig 1. Light-induced EMPO-OOH adduct EPR spectra measured using unsupplemented and PQ-supplemented PSII membranes.

EMPO-OOH adduct EPR spectra were obtained after illumination of PSII membranes (150 μg Chl ml^-1) with white light (1000 μmol photons m^-2 s^-1) in the absence [A, B] and presence of exogenous PQ-1 [C, D] and in the presence of 25 mM EMPO, 100 μM Desferal and 40 mM MES (pH 6.5). Figures B and D shows mean ± SD, where n = 3. 30 μM PQ-1 was added to PSII membranes prior to illumination.

Fig 2. The effects of DCMU and dinoseb on EMPO-OOH adduct EPR spectra measured using unsupplemented and PQ-supplemented PSII membranes.

EMPO-OOH adduct EPR spectra were measured using unsupplemented [A] and PQ-supplemented PSII membranes [B] in the presence of DCMU and dinoseb. Prior to illumination, DCMU (20 μM) and dinoseb (200 μM) were added to the membranes. [C] The relative intensity (mean ± SD, n = 3) of the light-induced EMPO-OOH adduct EPR signal measured using unsupplemented and PQ-supplemented PSII membranes. The other experimental conditions were the same as described in Fig. 1.

Fig 3. Differences in redox spectra and cyt b ₅₅₉ photoreduction measured using unsupplemented and PQ-supplemented PSII membranes.

Differences in the redox spectra of cyt b ₅₅₉ measured in the dark using unsupplemented [A] and PQ-supplemented PSII membranes [B]. 100 μM PQ-1 was added to the PSII membranes prior to the experiments. To measure cyt b ₅₅₉ photoreduction, unsupplemented [C] and PQ-supplemented PSII membranes [D] were illuminated for 180 s at high light intensity (1000 μmol photons m^-2 s^-1). The spectra represent the difference in the light minus ferricyanide-oxidized spectra [the photoreduced HP form of cyt b ₅₅₉, (PH)], hydroquinone-reduced minus ferricyanide-oxidized or hydroquinone-reduced minus light spectra [HP form of cyt b ₅₅₉, (HP)], ascorbate-reduced minus hydroquinone-reduced spectra [IP form of cyt b ₅₅₉, (IP)] and dithionite-reduced minus ascorbate-reduced spectra [LP form of cyt b ₅₅₉, (LP)].

Fig 4. The effects of DCMU and dinoseb on cyt b ₅₅₉ photoreduction measured using unsupplemented and PQ-supplemented PSII membranes.

Cyt b ₅₅₉ photoreduction was measured using unsupplemented [A,C] and PQ-supplemented [B,D] PSII membranes in the presence of DCMU [A, B] and dinoseb [C, D]. The other experimental conditions were the same as described in Fig. 3.

Fig 5. Proposed mechanism for the involvement of loosely bound plastosemiquinone at the Q_B and Q_C sites in O₂ ^•– formation in PSII.

Superoxide anion radicals are produced via one-electron reduction of molecular oxygen by plastosemiquinones, which are formed via one-electron reduction of plastoquinone at the Q_B sites and one-electron oxidation of plastoquinol at the Q_C site but unlikely at the Q_D site.