PH dependence of oxygen evolution and reduction of exogenous electron acceptor 2,6-dichlorophenolindophenol (DCPIP) by spinach membrane preparations of photosystem II (PSII) was investigated. PSII membranes were incubated in the buffer with pH which is explored, then membranes were pelleted by centrifugation, suspended in buffer with pH 6.5 and light-induced O2 evolution and DCPIP reduction were measured. At room temperature the maximum of functional activity was observed at pH around 6.5. Small decrease of the DCPIP reduction rate (about 10%) was observed after incubation at pH 4.5 and 7.5. The rate of O2 evolution is inhibited rather significantly at pH 4.5 (residual activity is about 40%) and more weakly at pH 7.5 (residual activity is about 80%). Since after the treatment of PSII samples by buffer with pH which is explored the functional activity was measured in the buffer with optimal pH (6.5) observed changes of activity reflect only irreversible effects of pH on the membranes whereas during standard measurement of O2 evolving activity (in buffer which is explored) observed changes of activity are the result not only irreversible but also reversible pH-dependent changes. Standard measurement of pH dependence of O2 evolution demonstrates the bell shape form of curve. Comparison of these two pH dependences permit to suggest that inhibition of O2 evolution in the region of pH 4.5 is the result of irreversible changes, possibly, connected with dissociation of extrinsic proteins PsbQ and PsbP whereas inhibition of O2 evolution in the alkaline region of pH is determined mainly by reversible pH-dependent changes, possibly connected with protonation/deprotonation of amino acid (for example, histidine).
photosystem II, oxygen-evolving complex, pH, temperature, oxygen, electron transport
1. Yamane Y., Kashino Y., Koike H., Satoh K. Effects of high temperatures on the photosynthetic systems in spinach: oxygen-evolving activities, fluorescence characteristics and the denaturation process. Photosynth. Res., 1998, vol. 57, pp. 51-59.
2. Busheva M., Tzonova I., Stoitchkova K., Andreeva A. Heat-induced reorganization of the structure of photosystem II membranes. Role of oxygen evolving complex. J. Photochem. Photobiol. B: Biol., 2012, vol. 117, pp. 214-221.
3. Enami I., Kitamura M., Tomo T., Isokawa Y., Ohta H., Katoh S. Is the primary cause of thermal inactivation of oxygen evolution in spinach PS II membranes release of the extrinsic protein or of Mn? Biochim. Biophys. Acta, 1994, vol. 1186, pp. 52-58.
4. Pospisil P., Tyystjarvi E. Molecular mechanism of high-temperature induced inhibition of acceptor side of photosystem II. Photosynth. Res., 1999, vol. 62, pp. 55-66.
5. Ghanotakis D.F., Babcock G.T., Yocum C.F. Calcium reconstitutes high rates of oxygen evolution in polypeptide depleted photosystem II preparations. FEBS Lett., 1984, vol. 167, pp. 127-130.
6. Dunahay T.G., Staechelin L.A., Seibert M., Ogilvie P.D., Berg S.P. Structural biochemical and biophysical characterization of four oxygen-evolving Photosystem 2 preparations from spinach. Biochim. Biophys. Acta, 1984, vol. 764, pp. 179-193.
7. Armstrong J.M. The molar extinction coefficient of 2,6-dichlorophenolindophenol. Biochim. Biophys. Acta, 1964, vol. 86, pp. 194-197.
8. Vass I., Styring S. pH-Dependent charge equilibria between tyrosine-D and the S states in photosystem II. Estimation of relative midpoint redox potentials. Biochemistry, 1991, vol. 30, pp. 830-839.
9. Schiller H., Dau H. Preparation protocols for high-activity photosystem II membrane particles of green algae and higher plants, pH dependence of oxygen evolution and comparison of the S2-state multiline signal by X-band EPR spectroscopy J. Photochem. Photobiol. B: Biol., 2000, vol. 55, pp. 138-144.
10. Commet A., Boswell N., Yocum C.F., Popelka H. pH optimum of the Photosystem II H2O oxidation reaction: effects of PsbO, the manganese-stabilizing protein, Cl- retention, and deprotonation of a component required for O2 evolution activity. Biochemistry, 2012, vol. 51, pp. 3808-3818.
11. Semin B.K., Davletshina L.N., Ivanov I.I., Rubin A.B., Seibert M. Decoupling of the processes of molecular oxygen synthesis and electron transport in Ca2+-depleted PSII membranes. Photosynth. Res., 2008, vol. 98, pp. 235-249.
12. Semin B.K., Davletshina L.N., Timofeev K.N., Ivanov I.I., Rubin A.B., Seibert M. Production of reactive oxygen species in decoupled, Ca2+-depleted PSII and their use in assigning a function to chloride on both sides of PSII. Photosynth. Res., 2013, vol. 117, pp. 385-399.
13. Shen J.R., Inoue Y. Low pH-induced dissociation of three extrinsic proteins from O2-evolving Photosystem II. Plant Sell Physiol., 1991, vol. 32(3), pp. 453-457.
14. Umena Y., Kawakami K., Shen J.-R., Kamiya N. Crystal structure of oxygen evolving Photosystem II at a resolution of 1.9 Å. Nature, 2011, vol. 473, pp. 55-60.
15. Homann P.H. The chloride and calcium requirement of photosynthetic water oxidation: effects of pH. Biochim. Biophys. Acta, 1988, vol. 93, pp. 1-13.
16. Briantais J.M., Vernotte C., Lavergne J., Arntzen C.J. Identification of S2 as the sensitive state to alkaline photoinactivation of Photosystem II in chloroplasts. Biochim. Biophys. Acta, 1977, vol. 461, pp. 61-74.
17. Schlodder E., Meyer B. pH dependence of oxygen evolution and reduction kinetics of photooxidized chlorophyll aII (P-680) in Photosystem II particles from Synechococcus sp. Biochim. Biophys. Acta, 1987, vol. 890, pp. 23-31.
