The reduction of the oxygen-evolving system in chloroplasts by thylakoid components

Using thoroughly dark-adapted thylakoids and an unmodulated Joliot-type oxygen electrode, the following results were obtained. (i) At high flash frequency (4 Hz), the oxygen yield at the fourth flash (Y₄) is lower compared to Y₃ than at lower flash frequency. At 4 Hz, the calculated S₀ concentration after thorough dark adaptation is found to approach zero, whereas at 0.5 Hz the apparent S₀ (S₀ + S₁) ratio increases to about 0.2. This is explained by a relatively fast donation (t _{1 2} = 1.0-1.5 s) of one electron by an electron donor to S₂ and S₃ in 15-25% of the Photosystem II reaction chains. The one-electron donor to S₂ and S₃ appears to be rereduced very slowly, and may be identical to the component that, after oxidation, gives rise to ESR signal II_s. (ii) The probability for the fast one-electron donation to S₂ and S₃ has nearly been the same in triazine-resistant and triazine-susceptible thylakoids. However, most of the slow phase of the S₂ decay becomes 10-fold faster (t _{1 2} = 5-6 s) in the triazine-resistant ones. In a small part of the Photosystem II reaction chains, the S₂ decay was extremely slow. The S₃ decay in the triazine-resistant thylakoids was not significantly different from that in triazine-susceptible thylakoids. This supports the hypothesis that S₂ is reduced mainly by Q^-_A, whereas S₃ is not. (iii) In the absence of CO₂/HCO^-_A and in the presence of formate, the fast one-electron donation to S₂ and S₃ does not occur. Addition of HCO^-₃ restores the fast decay of part of S₂ and S₃ to almost the same extent as in control thylakoids. The slow phase of S₂ and S₃ decay is not influenced significantly by CO₂/HCO^-₃. The chlorophyll a fluorescence decay kinetics in the presence of DCMU, however, monitoring the Q^-_A oxidation without interference of Q_B, were 2.3-fold slower in the absence of CO₂/HCO^-₃ than in its presence. (iv) An almost 3-fold decrease in decay rate of S₂ is observed upon lowering the pH from 7.6 to 6.0. The kinetics of chlorophyll a fluorescence decay in the presence of DCMU are slightly accelerated by a pH change from 7.6 to 6.0. This indicates that the equilibrium Q^-_A concentration after one flash is decreased (by about a factor of 4) upon changing the pH from 7.6 to 6.0. When direct or indirect protonation of Q^-_B is responsible for this shift of equilibrium Q^-_A concentration, these data would suggest that the pK_a value for Q^-_B protonation is somewhat higher than 7.6, assuming that the protonated form of Q^-_B cannot reduce Q_A.