Electron transfer through the quinone acceptor complex of Photosystem II after one or two actinic flashes in bicarbonate-depleted spinach thylakoid membranes

Abstract

We report here the pH dependence of the kinetics of the decay of variable chlorophyll a fluorescence after one or two actinic flashes in the absence or the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethyl-urea) in HCO 2 −-depleted or anion-inhibited spinach thylakoid membranes. All the reported effects of HCO 3 − removal are reversed by the addition of 5 mM HCO 3 −. The initial first-order component for the oxidation of Q A − (the reduced primary plastoquinone acceptor of Photosystem II (PS II) by Q B (the secondary plastoquinone acceptor) was reversibly inhibited in a pH-dependent manner in HCO 3 −-depleted membranes. After a single actinic flash, the half-time of Q A − decay was 630 μs (amplitude, 29%) at pH 6.5 which changed to a value of 320 μs (amplitude, 66%) at pH 7.75. The rate and amplitude at pH 7.75 were approximately the same as found in the restored and control membranes which were pH independent over the same pH range. A similar observation was made after the second actinic flash. Thus, at alkaline pH HCO 3 −-depleted membranes behave as control membranes with respect to electron flow from Q A to Q B or to Q B −. The time ( t 50) at which the [Q A −] is 50% of the maximum [Q A −] during the back reaction between Q A − and the S 2 state of the oxygen-evolving complex, in the presence of 5 μM DCMU, was increased from 1.3 s in control and restored samples to 5.3 s in HCO 3 −-depleted samples below pH 7.0, but was unaffected above pH 7.5 (2.3–2.9 s in all cases). Furthermore, a new pathway of Q A − with a half-time of less than 100 μs was present at pH 8.0 in the presence of DCMU, in approx. one-third of the PS II centers in HCO 3 −-depleted membranes. The apparent equilibrium for the sharing of an electron between Q A and Q B is estimated to decrease by a factor of 4 at pH 6.0 in treated membranes ( K app ≈ 16) as compared to the restored or control membranes ( K app ≈ 62); there was no difference in K app at pH 7.75. Estimates of the operating redox potential for the Q B/Q B − couple from the results presented here indicated that the pH dependence of this parameter is greatly reduced in treated membranes (−60 mV at pH 6.0 to −72 mV at pH 7.75) as compared to restored or control membranes (−25 mV at pH 6.0 to −72 mV at pH 7.75). We iscuss our results in the context of a model that envisages HCO 3 − to act as a proton donor to the protein dissociable group believed to participate in the protonation of Q B −. Finally, the possibility of HCO 3 − being a ligand to Fe 2+ in the Q A-Fe-Q B complex of the PS II reaction center is also discussed.