Events near the reaction center in O2 evolving PS II enriched thylakoid membranes: The presence of an electric field during the S2 state in a population of centers.

Flash-induced absorption changes at 515 nm observed as a function of flash number are examined in relation to the flash-induced fluorescence yields in inside-out thylakoids. After partial dissipation of the delocalized transmembrane electric field by adding gramicidin, the analysis of period 4 oscillations and of the kinetics in the 10 ms-1 s range suggest that the variation of the absorption changes at 515 nm as a function of flash number is the result of at least two processes:1) an electric field increase related to the S2 state and 2) the fact that the field generated by the water protons inside the membrane decreases when these protons are released outside the membrane. The former field correlates with the flash-induced fluorescence yield increase induced by the donor side of Photosystem II. Both measurements show similar oscillations as a function of flash number, with maxima on the 1st, 5th and 9th flash. These oscillations, after a shift of two flashes, appear to be different from those of the O2 yield observed under similar conditions. It is proposed that, in a population of centers the electric field during the S2 state reflects the presence of a stabilized positive equivalent in the protein close to the Mn complex.

Light intensity saturation properties of O2 yields in a sequence of flashes in Chlorella.

As a function of the light intensity of flash n in a sequence, the O2 yields Yn, Yn+1 and Yn+2 have been measured: n = 1, 2, 3 and 6 in the examples given. It is shown that: (1) No double hit exists in the first saturating flash in Chlorella. (2) the flash saturation curve of the O2 yield Yn+1 as a function of the intensity of flash n exhibits a small sigmoidal shape at weak light. (3) If Yn+1 is detected at different times after the flash n of variable intensity, a well developed lag distinguishes the saturation curve of the O2 yield measured a long time after flash n (200 ms) with respect to that measured at shorter time (300 microseconds). Nevertheless, a large amount of double hits with the transitions S1 leads to S3 cannot occur in each flash, because it would lead to a periodicity of three rather than four in the O2 yield pattern. The saturation curve of the transition S2* leads to S3 is different from the other S-state saturation curves which are close to an exponential function; even with a short flash (0.3 microseconds), this curve shows a small lag at low light intensity, and its saturation intensity is higher than that of the other transitions. The low quantum yield of the transition S2* leads to S3 at low flash light intensity is explained by a product, T, partially inhibiting the formation of S3; at higher intensity, the quantity of formed S2* being larger than that of available T, only a part of S2* is inhibited and the quantum yield is higher than at low intensity.

Inhibition by ammonium chloride of the oxygen yield of photosynthesis.

Flash O2 yield experiments are described in spinach chloroplasts and Chlorella after addition of NH4Cl. (1) The damping of the sequence is increased by NH4Cl. (2) The turnover times are accelerated but the reaction during which O2 is released (S3bv leads to S4 leads to S0) is slowed. (3) We observed between the end of the turnover kinetic and the beginning of deactivation a latency time tl during which the S2 and S3 states are perfectly stable. In the presence of NH4Cl, this latency time for S2 is shortened, diminishing from 1 s to less than 12 ms; whereas it is lengthened for S3 (up to 4 s, 5 s). (4) After this latency time, two phases are clearly distinguished in the S2 deactivation: the beginning of the deactivation is abrupt, varying like the square root of the time, i.e. as is characteristic of a diffusive process. During the second phase, S2 is stabilized. Our experiment shows that S2 deactivation is monitored by the release of some product F after the latency time from one particular locus, so that nearby Photosystem II reaction centers are rapidly deactivated by diffusion of the product, whereas centers far from this locus are very slowly deactivated. These results are qualitatively complementary to the luminescence experiments of Velthuys (Biochim. Biophys, Acta (1975) 396, 392-401) except for the latency time which is invisible in the Velthuys results. We propose a modified model in which the binding of NH3 on states S2 and S3 occurs during deactivation.