View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 589 (2015) 779–786 journal homepage: www.FEBSLetters.org Experimental evidence suggesting that H2O2 is produced within the thylakoid membrane in a reaction between plastoquinol and singlet oxygen ⇑ Sergey A. Khorobrykh a, Maarit Karonen b, Esa Tyystjärvi a, a Department of Biochemistry/Molecular Plant Biology, University of Turku, FI-20014 Turku, Finland b Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, FI-20014 Turku, Finland article info abstract Article history: Plastoquinol (PQH2-9) and plastoquinone (PQ-9) mediate photosynthetic electron transfer. We iso- Received 17 December 2014 lated PQH2-9 from thylakoid membranes, purified it with HPLC, subjected the purified PQH2-9 to sin- Revised 30 January 2015 1 1 glet oxygen ( O2) and analyzed the products. The main reaction of O2 with PQH2-9 in methanol was Accepted 10 February 2015 found to result in formation of PQ-9 and H2O2, and the amount of H2O2 produced was essentially the Available online 18 February 2015 1 same as the amount of oxidized PQH2-9. Formation of H2O2 in the reaction between O2 and PQH2-9 Edited by Miguel De la Rosa may be an important source of H2O2 within the lipophilic thylakoid membrane. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Singlet oxygen Plastoquinone Hydrogen peroxide Thylakoid membrane Photosystem II 1. Introduction from singlet excited chlorophyll (1Chl⁄) [7], but transfer of excita- tion energy to the reaction center shortens the lifetime of 1Chl⁄, 1 Reactive oxygen species (ROS) like singlet oxygen ( O2), super- thereby lowering the probability of intersystem crossing. Triplet- ÅÀ 3 ⁄ oxide anion radical (O2 ) and hydrogen peroxide (H2O2) are ubiqui- triplet energy transfer from Chl to carotenoids lowers the prob- 1 tously formed in plants. Besides causing direct damage to ability of O2 formation in the antenna complexes of both photo- 1 biomolecules, ROS also play important roles in plant signaling systems [8], and the reaction center of PSI does not produce O2 1 [1–3]. ROS are formed in the apoplast, mitochondria and chloro- [9]. Therefore, the main source of O2 appears to be the reaction 3 plasts [4–6], and formation of ROS in chloroplasts depends on center of PSII, where the triplet state of the primary donor, P680, + absorption of light by the photosynthetic apparatus. is formed by charge recombination reactions between P680 and 1 3 À O2 is mainly formed via interaction of oxygen ( O2) with the QA, the primary quinone acceptor of PSII [8,10]. Furthermore, triplet excited state of chlorophyll (3Chl⁄), which occurs in the time-dependent formation of a virtual triplet state of the primary + À light-harvesting antenna complexes and in the reaction centers radical pair P680 Pheo and subsequent recombination may pro- 3 of photosystems II and I (PSII and PSI, respectively). In light-har- duce P680 [11], although the short lifetime of the primary pair ren- vesting complexes, 3Chl⁄ can be formed via intersystem crossing ders this pathway inefficient. The rate constants and concentrations of molecules that poten- 1 tially function as O2 quenchers suggest that carotenoids are the Abbreviations: Chl, chlorophyll; DNP-INT, 2-iodo-6-isopropyl-3-methyl-20,4,40- main quenchers of 1O in the thylakoid membrane. The second- trinitrodiphenyl ether; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 2 1 Å 1 ÅÀ order rate constants of the reaction of O with thylakoid carote- acid; HO2 , hydroperoxyl radical; MeOH, methanol; O2, singlet oxygen; O2 , 2 9 10 À1 À1 superoxide anion radical; P680, primary electron donor of PSII; Pheo, pheophytin a— noids are in the range of 7 Â 10 to 1.4 Â 10 M s [12], much primary electron acceptor of PSII; PQHÅ-9 and PQÅÀ-9, plastosemiquinone radical 1 higher than the rate constants of tocopherols with O2 [13]. Caro- and plastosemiquinone anion radical, respectively; PQH2-9 and PQ-9, plastoquinol 1 tenoids can prevent the formation of O2 via direct quenching of and plastoquinone, respectively; PSI and PSII, photosystems I and II, respectively; 3 ⁄ ROS, reactive oxygen species; UQH -9, ubiquinol-9 Chl . However, the two ß-carotene molecules of the reaction cen- 2 3 ⇑ Corresponding author. ter of PS II are too far away from P680 to quench P680 [14,15], and 1 E-mail address: esatyy@utu.fi (E. Tyystjärvi). may therefore mainly function as O2 quenchers. http://dx.doi.org/10.1016/j.febslet.2015.02.011 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 780 S.A. Khorobrykh et al. / FEBS Letters 589 (2015) 779–786 ÅÀ O2 and H2O2 are produced via one-electron reduction of oxygen system (methanol:hexane, 34:2). The flow rate was 0.75 ml/min, at the acceptor side of photosystem I (PSI). This reaction is known the temperature of the column was maintained at 25 °C, and the as Mehler’s reaction, and H2O2 is produced by a subsequent dismu- injection volume was 20 ll. PQ-9 was detected with absorption ÅÀ tation reaction from O2 . The terminal acceptors FX,FA, and FB likely at 255 nm, and PQH2-9 was detected with fluorescence (kex = 290 - function as electron donors to oxygen [16]. It has also been sug- nm, kem = 330 nm). Pure PQH2-9 was collected according to the gested that phylloquinone A1, a secondary electron acceptor in PS retention times of the corresponding peaks on the chromatogram I, might donate an electron to oxygen within the thylakoid mem- using Analyt-FC (Agilent Technologies, Palo Alto, CA) collector. ÅÀ brane [17,18].O2 can also be generated on the acceptor side of The same HPLC-based method, except that the isocratic solvent ÅÀ PS II [19,20]. However, the acceptor side of PSII produces O2 at a system (methanol:hexane, 34:2) was replaced with methanol, much lower rate than Mehler’s reaction [21]. was applied to detection of PQ-derived compounds formed in the 1 1 Mehler’s reaction can be fully suppressed with the herbicide 3- reaction of O2 with PQH2-9. Reaction of O2 with PQH2-9 was car- (3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of electron ried out in MeOH containing 10 lM Rose Bengal in a glass cuvette 1 transfer from PSII to the plastoquinone pool. However, significant under continuous stirring. Generation of O2 was triggered by illu- formation of H2O2 was shown to occur in isolated thylakoid mem- minating the samples with white light. Visible light between 400 branes when the oxidation of PQH2-9 by the cytochrome b6/f com- and 700 nm was quantified with a Li-Cor quantum sensor (LiCor, plex was inhibited with 2,4-dinitrophenylether of iodonitrothymol Lincoln, NE, USA). (DNP-INT) [21]. In this case, H2O2 was suggested to be formed from HPLC-MS analyses were carried out by an HPLC/DAD-ESI-MS superoxide produced by reduction of oxygen by the plas- system consisting of an Agilent HPLC 1200 Series equipped with ÅÀ tosemiquinone radical (PQ -9). The production of H2O2 was found an absorbance detector (Agilent Technologies, Waldbronn, Ger- to occur mainly within the thylakoid membrane, not via dismuta- many) and a micrOTOFQ mass spectrometer (Bruker Daltonics, Bre- ÅÀ tion of O2 in the soluble phase [22], and it was suggested that a men, Germany). Chromatographic separations were performed ÅÀ reaction between O2 and PQH2-9 produces H2O2 [21,23]. Thus, using a LiChroCART C18 reversed-phase column (Superspher 100 ÅÀ PQH2-9 may convert O2 to H2O2. RP-18, 125 Â 4 mm, 5 lM; Merck KGaA, Darmstadt, Germany) 1 The PQ pool has also been suggested to scavenge O2 [24–26]. with isocratic elution with methanol. The flow rate was 0.7 ml/ The suggestion is supported by the high concentration of PQ in min and it was reduced to approximately 0.3 ml/min by splitting the chloroplast together with a modest rate constant of the reac- before the introduction into the ion source. The injection volume 1 8 À1 À1 tion of O2 with PQH2-9, 0.97 ± 0.08 Â 10 M s in acetonitrile was 20 ll. The HPLC system was controlled by Hystar software 1 [27]. O2 can also efficiently react with thylakoid lipids, which (version 3.2., Bruker BioSpin). The mass spectrometer was con- leads to lipid peroxidation [28]. Thus, the reaction of PQH2-9 with trolled by Bruker Compass micrOTOF control software and operat- 1 1 O2 also slows down the oxidation of lipids by O2. The oxidized ed in the negative ion mode. The capillary voltage was maintained 1 form (PQ-9) can also quench O2 [27]. Earlier work has shown that at +4000 V with the end plate offset at À500V. The pressure for 1 oxidation of PQH2-9 by O2 produces PQ-9 and hydroxy derivatives nebulizer gas was set at 1.6 bar and the drying gas flow was of PQ [27]. The results of the present work indicate that the reac- 8.0 l/min and the drying gas temperature 200 °C. The full scan 1 tion between PQH2-9 and O2 is accompanied with formation of mass ranged from m/z 50 up to m/z 3000. Calibration with 5 mM H2O2. Oxidation of PQH2-9 to PQ-9 and H2O2 was found to be the sodium formate injected via a six-port-valve was used at the end 1 main reaction when O2 reacts with PQH2-9, whereas the PQ-hy- of the HPLC-MS experiment in order to provide high-accuracy droperoxides are minor products.