Multiflash experiments reveal a new kinetic phase of photosystem II manganese cluster assembly in Synechocystis sp PCC6803 in vivo

The assembly of Mn2+ ions into the H2O oxidation complex (WOC) of the photosystern II (PSII) reaction center is a light-driven process, termed photoactivation. According to the two-quantum model, photoactivation involves two light-driven charge separations coupled to the photooxidation of Mn2+ in order to form the first stable intermediate in a process that culminates in the oxidative assembly of four Mn2+ ions and one Ca2+ ion to form the active, higher valence (Mn-4-Ca) center of the WOC. To better define the kinetics of the dark rearrangement and to gain some understanding of the basis for the very low quantum yield of the overall process, photoactivation experiments, involving different flash patterns, were conducted with Synechocystis sp. PCC6803. It was found that even the so-called first stable intermediate is readily lost during protracted (1 - 10 s) dark periods during photoactivation of Synechocystis cells. Low concentrations of the electron acceptor, DCBQ, improved the stability of the dark intermediates. The unstable photoactivation intermediates formed early in the photoactivation process were not, however, stabilized by the addition of Ca2+, although the overall yield of photoactivation is enhanced by the additional Ca2+. Measurements of the kinetics of fluorescence yield verify that Q(A)(-) to Q(B) electron transfer rates change during the course of photoactivation as the high potential form Of Q(A)(-) is converted to the low potential form and show that DCBQ acts as an efficient electron acceptor from Q(A)(-) even while in its high potential form. In addition the similar to 150 ms phase corresponding to the originally described dark rearrangement of photoactivation, repetitive, double flash experiments, with a 10 s intervening dark period, reveals a faster, 15 ms phase that is accentuated by DCBQ.