Properties of the chloride-depleted oxygen-evolving complex of photosystem II studied by electron paramagnetic resonance

The effects of different Cl- depletion treatments in photosystem II (PS-II)-enriched membranes have been investigated by electron paramagnetic resonance (EPR) spectroscopy and by measurements of oxygen-evolving activity. The results indicated that the oxygen-evolving complex of PS-II exhibits two distinct Cl-dependent properties. (1) After Cl-free washes at pH 6.3, a reversibly altered distribution of structural states of PS-II was observed, manifested as the appearance of a g = 4 EPR signal from the S-2 State in a significant fraction of centers (20-40%) at the expense of the S-2 multiline signal. In addition, small but significant changes in the shape of the S-2 multiline EPR signal were observed. Reconstitution of Cl- to Cl-free washed PS-II rapidly reversed the observed effects of the Cl-free washing. The anions, SO42- and F-, which are often used during C-l- depletion treatments, had no effect on the S-2 EPR properties of PS-II under these conditions in the absence or presence of Cl-. Flash experiments and measurements of oxygen evolution versus light intensity indicated that the two structural states observed after the removal of C-l- at pH 6.3 originated from oxygen-evolving centers exhibiting a lowered quantum yield of water oxidation. (2) Depletion of C-l- in PS-II by pH 10 treatment reversibly inhibited the oxygen-evolving activity to approximate to 15%. The pH 10 treatment depleted the Cl- from a site which is considered to be equivalent to that studied in most earlier work on Cl--depleted PS-II. The S-2 State in pH 10/Cl--depleted PS-II was reversibly modified to a state from which no S-2 multiline EPR signal was generated and which exhibited an intense S-2 g = 4 EPR signal corresponding to at least 40% of the centers but possibly to a much larger fraction of centers. The state responsible for the intense S-2 g = 4 signal generated under these conditions is unlike that observed after removal of Cl- from PS-II at pH 6.3, in that this state was more stable in the dark, showing a half-decay time of approximate to 1.5 h at 0 degrees C, and was unable to undergo further charge accumulation. Nevertheless, a fraction of centers, probably different from those exhibiting the S-2 g = 4 signal, was able to advance to the formal S-3 state, giving rise to a narrow EPR signal around g = 2. Addition of the anions SO42- or F- to pH 10/C-l--depleted PS-II affected the properties of PS-II, resulting in EPR properties of the S-2 State similar to those reported earlier following Cl- depletion treatment of PS-II in the presence of these anions. Surprisingly, after addition of F-, the g = 4 EPR signal showed a damped flash-dependent oscillation. In addition, a narrow signal around g = 2, corresponding to the formal S-3 state, also showed a damped flash-dependent oscillation pattern. The presence of oscillating EPR signals (albeit damped) in F--treated pH 10/Cl--depleted PS-II indicates functional enzyme turnover. This was confirmed by measurements of the oxygen-evolving activity versus light intensity which indicated that in similar to 45% of oxygen-evolving centers the enzyme turnover was slowed by a factor of 2. The distinct Cl- depletion effects in PS-II observed under the two different Cl- depletion treatments are considered to reflect the presence of two distinct Cl--binding sites in PS-II.