Duction of ROS, particularly the short-lived OHN, by the donor side of PS II [37,38]. 1O2 produced by the reaction of molecular oxygen with 3P680 has also been observed [17,18,19]. Interestingly, no oxidative 14636-12-5 modifications in the vicinity of P680 have been observed [20]. Since the production of 1O2 by PS II is well established [39], the lack of observed modifications in theOxidized Amino Acids on the Reducing Side of PS IITable 1. Oxidatively Modified SMER 28 web Residues in the Vicinity of PheoD1 and QA.Protein DModified Spinach Residues130 a 133 aCorresponding Thermosynechococcus Residues130 133 135 239 241 242 237 238 241E + go L + go F + go F + go Qb + ca E + gam P + ca T + go E + gam Mb + goQ L Y F Q E P T E M135 a239 b242 bD238 b 239 b 242 bIndividual residues are listed along with the modifications observed. For a complete list of oxidative modification types, the amino acids targeted, and mass modifications searched for in this study, as well as structures arising from these oxidative modifications, see [50,51]. Key: ca, carbonyl addition (+14 Da); gam, Glu/Asp modification (decarboxylation and oxidation, 230 Da); go, general oxidation (+16 Da). Oxidative modification of these residues was originally reported in Frankel et al. [20]. a Buried residues not adjacent to apparent cavities/channels. b Surface-exposed residues. doi:10.1371/journal.pone.0058042.tvicinity of P680 could be a detection issue, as the peptides in the vicinity of P680 are highly hydrophobic and difficult to resolve by reversed-phase HPLC. It is also possible that 1O2 may be vectored away from P680 rapidly, giving rise to a low yield of oxidative modifications. Finally, oxidative modification of residues in the vicinity of P680 may trigger D1 turnover more effectively than oxidative modifications at other sites in vivo. We cannot distinguish between these and other possibilities at this time. ROS may also be produced on the reducing side of the photosystem by the partial reduction of molecular oxygen, yielding long-lived O2N2 and H2O2 and the very short-lived OHN. It should be stressed, however, that at this time we cannot discriminate between these or other mechanisms that produce the ROS responsible for the oxidative modifications that we observe. Using mass spectrometry, it is also very difficult, and in most cases impossible, to differentiate oxidative modifications of amino acidside chains produced by OHN, 1O2, O2N2 or other oxidative species [40,41]. The site of ROS production by PS II has been the subject of much discussion [16]. Earlier, we reported that CP43: 354E, 355T, 356 M and 357R, which are in close proximity to the Mn4O5Ca cluster, were oxidatively modified [20]. These results indicate that the manganese cluster itself appears to be a source of oxidizingside ROS. On the reducing side of the photosystem, PheoD1, QA, QB22 and low potential cytochrome b559 have all been suggested as sites of ROS production. In this communication we have reported the oxidative modification of residues 
in close proximity to PheoD1 and QA. These results support the hypothesis that both of these sites can produce ROS that lead to amino acid residue oxidative modification. Since these modifications were observed on PS II membranes isolated from market spinach, it appears that they can accumulate to detectable levels within the D1 proteinFigure 2. Oxidized Residues Identified on the Stromally Exposed Regions of the D1 and D2 Proteins in the Vicinity of QA and PheoD1. The T. vulcanu.Duction of ROS, particularly the short-lived OHN, by the donor side of PS II [37,38]. 1O2 produced by the reaction of molecular oxygen with 3P680 has also been observed [17,18,19]. Interestingly, no oxidative modifications in the vicinity of P680 have been observed [20]. Since the production of 1O2 by PS II is well established [39], the lack of observed modifications in theOxidized Amino Acids on the Reducing Side of PS IITable 1. Oxidatively Modified Residues in the Vicinity of PheoD1 and QA.Protein DModified Spinach Residues130 a 133 aCorresponding Thermosynechococcus Residues130 133 135 239 241 242 237 238 241E + go L + go F + go F + go Qb + ca E + gam P + ca T + go E + gam Mb + goQ L Y F Q E P T E M135 a239 b242 bD238 b 239 b 242 bIndividual residues are listed along with the modifications observed. For a complete list of oxidative modification types, the amino acids targeted, and mass modifications searched for in this study, as well as structures arising from these oxidative modifications, see [50,51]. Key: ca, carbonyl addition (+14 Da); gam, Glu/Asp modification (decarboxylation and oxidation, 230 Da); go, general oxidation (+16 Da). Oxidative modification of these residues was originally reported in Frankel et al. [20]. a Buried residues not adjacent to apparent cavities/channels. b Surface-exposed residues. doi:10.1371/journal.pone.0058042.tvicinity of P680 could be a detection issue, as the peptides in the vicinity of P680 are highly hydrophobic and difficult to resolve by reversed-phase HPLC. It is also possible that 1O2 may be vectored away from P680 rapidly, giving rise to a 
low yield of oxidative modifications. Finally, oxidative modification of residues in the vicinity of P680 may trigger D1 turnover more effectively than oxidative modifications at other sites in vivo. We cannot distinguish between these and other possibilities at this time. ROS may also be produced on the reducing side of the photosystem by the partial reduction of molecular oxygen, yielding long-lived O2N2 and H2O2 and the very short-lived OHN. It should be stressed, however, that at this time we cannot discriminate between these or other mechanisms that produce the ROS responsible for the oxidative modifications that we observe. Using mass spectrometry, it is also very difficult, and in most cases impossible, to differentiate oxidative modifications of amino acidside chains produced by OHN, 1O2, O2N2 or other oxidative species [40,41]. The site of ROS production by PS II has been the subject of much discussion [16]. Earlier, we reported that CP43: 354E, 355T, 356 M and 357R, which are in close proximity to the Mn4O5Ca cluster, were oxidatively modified [20]. These results indicate that the manganese cluster itself appears to be a source of oxidizingside ROS. On the reducing side of the photosystem, PheoD1, QA, QB22 and low potential cytochrome b559 have all been suggested as sites of ROS production. In this communication we have reported the oxidative modification of residues in close proximity to PheoD1 and QA. These results support the hypothesis that both of these sites can produce ROS that lead to amino acid residue oxidative modification. Since these modifications were observed on PS II membranes isolated from market spinach, it appears that they can accumulate to detectable levels within the D1 proteinFigure 2. Oxidized Residues Identified on the Stromally Exposed Regions of the D1 and D2 Proteins in the Vicinity of QA and PheoD1. The T. vulcanu.
