Indirect Evidence for a Very Fast Recovery Kinetics of Chlorophyll-a_II in Spinach Chloroplasts

Abstract

The 690 nm absorption change reflecting the turnover of the system-II-reaction center chlorophyll, Chl-a_II (often referred to as P 680), has been investigated under different experimental conditions in spinach chloroplasts. A comparison was made with oxygen evolution and with absorption changes of Chl-a_I measured at 703 nm, both indicating the number of electrons produced by system II. It was found:

1. The dependency on actinic flash intensity of the initial amplitudes of the measured 690 nm absorption change, ∆A₀ (Chl-an), markedly differs for normal and for Tris-washed chloroplasts, respectively.

2. The saturation curve of ∆A₀ (Chl-an) in Tris-washed chloroplasts is similar to that for the total amplitude of the 703 nm absorption change, ∆A₀ (Chl-a_I), in normal chloroplasts, and can be described by an exponential function. On the other hand, ∆A₀ (Chl-aII) in normal chloroplasts exhibits a more complex biphasic dependency and much higher flash intensities are required for saturation.

3. Under repetitive flash group excitation and in the presence of an A D R Y (= acceleration of the deactivation reactions of the water-splitting enzyme system Y)-reagent the initial amplitude of the 690 nm absorption change oscillates in the same characteristic pattern as the oxygen evolution.

4. The initial amplitude of the 690 nm absorption change, ∆A₀(Chl-a_II), in Tris-washed chloroplasts becomes significantly smaller (more than 50%) by the addition of system-II-electron donors (benzidine, p-phenylendiamine, tetraphenylboron), whereas the total amplitude of the 703 nm absorption change, ∆A₀(Chl-a_I) increases 3 -4-fold.

In order to explain these results, the existance of a very fast reduction kinetics of Chl-a_II⁺ is postulated, which is not detectable by our measuring equipment. The half time of this reaction is ≦ 1 μs. Reaction centers with the very fast “undetected” Chl-a_II⁺-reduction are photochemically transformed into slower one by double hit processes with a comparatively low quantum yield. Furthermore, it is inferred, that the dark recovery kinetics of Chl-a_II is dependent on the charge accumulation state of the watersplitting enzyme system Y. This phenomenon is shown to explain also the oscillation pattern of delayed fluorescence. On the basis of the present results two alternative reaction schemes for the functional organization of the electron transport on the donor side of system II are discussed.