Electron transfer in cyanobacterial photosystem I -: II.: Determination of forward electron transfer rates of site-directed mutants in a putative electron transfer pathway from A0 through A1 to Fx

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (Q(K)) and F-X binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A(1) (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximate to 300 ns while optical data resolve an additional minor kinetic component of tau approximate to 10 ns. Whole cells and PS I complexes from the W697F(PsaA) and S692C(PsaA) mutants show a significant slowing of the slow kinetic component, whereas the W677F(PsaB) and S672C(PsaB) mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is similar to3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P-700 (+)A(1)(-), in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P-700 F-+(X)- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from Q(K)-A to F-X and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.