Additive effect of mutations affecting the rate of phylloquinone reoxidation and directionality of electron transfer within photosystem i

Optical pump-probe spectroscopy in the nanosecond-microsecond timescale has been used to study the electron transfer reactions taking place within the Photosystem I reaction center of intact Chlamydomonas reinhardtii cells. The biphasic kinetics of phylloquinone (PhQ) reoxidation were investigated in double mutants that combine a mutation (PsaA-Y696F) near the primary acceptor chlorophyll, ec3_A, with those near PhQ_A (PsaA-S692A, PsaA-W697F). The PsaA-S692A and PsaA-W697F mutations selectively lengthened the 200 ns lifetime component observed in the wild-type (WT). The ∼20 ns component was unaltered in the single mutant, both in terms of lifetime and relative amplitude. However, both double mutants possessed a ∼20 ns component (PhQ_B^- reoxidation) with increased amplitude compared with the WT and the individual PhQ_A mutants. The component assigned to PhQ_A^- reoxidation was slowed, like the individual PhQ_A mutants, and of lower amplitude, as observed in the single ec3_A mutant. Hence, the effects of these mutations are almost entirely additive, providing strong support for the previously proposed bidirectional electron transfer model, which attributes the ∼20 and ∼200 ns phases to reoxidation of PhQ_B or PhQ_A, respectively. Moreover, in all the mutants investigated, it was also possible to observe an intermediate (∼180 ns) component, as previously reported for mutants of the PhQ_A binding pocket (Biochim. Biophys. Acta [2006] 1757, 1529-1538), which we have tentatively attributed to forward electron transfer between the iron-sulfur clusters F_X and F_A/B.