Fundamental differences between period-4 oscillations of the oxygen and fluorescence yield induced by flash excitation in inside-out thylakoids

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The period-4 oscillation pattern of the chlorophyll a fluorescence induced by flash excitation of dark-adapted inside-out thylakoids was compared to the oxygen-yield pattern obtained under the same conditions of flash energy and adaptation to dark. For a quantitative comparison, a least-square best fitting method was used. In the Kok model, besides the miss and the double hit parameters α and β, it has been necessary to introduce a new variable, z, which describes a decrease of the apparent number of active centers by a factor z at each flash of a series. We demonstrate that under similar flash conditions, the oscillation pattern of fluorescence yield (measured 80 ms after each of a series of flashes) fundamentally differs from that of the O 2 yield. After a 3 h dark-adaptation period, the oscillation pattern of the oxygen yield presents a change of phase as a function of flash number, associated with the damping coming from misses ( α ⩾ 0.12). In contrast, under the same conditions, the fluorescence oscillation showed no phase change. The same oscillation pattern was repeated every four flashes with a decreasing amplitude. The numerical fitting of the oscillation pattern of fluorescence yields no misses, no double hits, but a progressive vanishing of fluorescent centers in a proportion 1 − z F. The higher the flask energy the faster the decrease of the oscillating part of fluorescence. The product of flash energy, I, by the flash number necessary to decrease the fluorescence oscillation by half, N 1 2 , was found to be independent of the exciting flash energy. The initial amplitude of the fluorescence oscillation was not light saturated in our experimental conditions, though the flash energy used was at least 5-times higher than necessary to saturate the transitions S i of O 2 evolution (except S 2 → S 3). All these results show that the fluorescence oscillation with period 4 is not related to the S i states of most O 2-evolving centers. The existence of a 4-step reaction series (the S′ i states), functioning in parallel to the S i states, is postulated. The S′ i states seen by fluorescence are controlled by the limited amount of a component stored in the dark, which is exhausted after each flash of a series in a quantity proportional to flash energy.