Evidence for only one stable electron acceptor in the reaction center of Photosystem II in spinach chloroplasts

Photooxidation of chlorophyll a 11 (P680) and its re-reduction has been measured, monitoring absorption changes at 824 nm. Oxidation of Chl-a 11 coupled with the reduction of the electron acceptor, Q A (X320), was induced by 3 ns single turnover laser flashes. The changes at 824 nm were analyzed under excitation by a probe flash following a saturating pre-flash at different time intervals (60 ns to 500 ms). At a time interval of 60 ns after the pre-flash about 40% of the total Chl-a 11 + is re-reduced, whereas reoxidation of Q A - is negligible (<1%). The probe flash fired 60 ns after the pre-flash does not induce a longer-lived (t 1/2 > 5 ns)‘photooxidation of the re-reduced Chl-a 11 fraction (40%). From this we may conclude that there does not exist an acceptor arranged parallel to Q A which can stabilize an electron for more than a few ns under repetitive excitation and in the presence of ferricyanide. Varying the time interval between pre- and probe flash yields the result that the amount of Chl-a 11 photooxidation induced by the probe flash rises with a half-life time of approx. 400 μ s, corresponding to the reoxidation kinetics of Q A - . In dark adapted samples in the presence of DCMU, but without artificial acceptors, even 500 ms after the first pre-flash no longer-lived photooxidation of Chl-a 11 by the probe flash could be observed. If, however, the sample is incubated with ferricyanide prior to addition of DCMU, two electrons can be subsequently stabilized once at the acceptor side of PS II. Considering the signal to noise ratio, the precision of these results is approx. 15% with reference to all photosystem II centers.