Nanosecond reduction kinetics of photooxidized chlorophyll-aII (P-680) in single flashes as a probe for the electron pathway, H+-release and charge accumulation in the O2-evolving complex

(1) The re-reduction kinetics of chlorophyll a+II (P-680+) after the first, second, third etc. flash given to dark-adapted subchloroplasts have been monitored at 824 nm in the nanosecond range. After the first flash and, again, after the fifth flash, the re-reduction of chlorophyll a+II (Chl a+II) in the nanosecond range is nearly monophasic with t12 ≈ 23 ns. After the second and third flash, the re-reduction is significantly slower and biphasic; it can be well-adapted with t12 ≈ 50 ns and ≈260 ns. After the 4th flash, the re-reduction kinetics of Chl a+II are intermediate between the first/fifth and second/third flash. A similar dependence on flash number was obtained with a sample of oxygen-evolving Photosystem II particles from Synechococcus sp. (2) Considering the populations of the S-states of the O2-evolving complex before each flash, the following correlation of S-states to Chl a+II reduction kinetics and electron transfer times, respectively, is obtained: in state S0 as well as in state S1 Chl a+II is reduced with t12 ≈ 23 ns, whereas in state S2 as well as state S3 a biphasic reduction with t12 ≈ 50 ns and ≈260 ns (ratio of the amplitudes ≈1:1) occurs. (3) The observed multiphasic Chl a+II reduction under repetitive excitation is quantitatively explained by a superposition of the individual electron transfer times. (4) We suggest that the retardation of electron transfer to Chl a+II in states S2 and S3 as compared to S0 and S1 is caused by Coulomb attraction by one positive charge located in the O2-evolving complex. A positively charged O2-evolving complex in states S2 and S3 can be explained if the electron release pattern (1,1,1,1) is accompanied by a proton release pattern (1,0,1,2) for the transitions (S0 → S1, S1 → S2, S2 → S3, S3 → S0). (5) A kinetic model based on linear electron transfer from the O2-evolving complex (S) to Chl a+II via two carriers, D1 and D2, makes a quantitative description of the experimental results possible. (6) According to the kinetic model, the retardation of electron transfer to Chl a+II in states S2 and S3 is reflected by an increase in the change of standard free energy, ΔG0, of the reaction Chl a+IID1D2SChl aIID1+D2S from ΔG0 ≈ − 90 meV in states S0 and S1 to ΔG0 ≈ − 20 meV in states S2 and S3. (7) This increase by ≈ 70 meV can be quantitatively explained by the Coulomb potential of the positive charge in the O2-evolving complex, estimated by using the point charge approximation.