Photosystem II (PSII) of plants, algae, and cyanobacteria utilize solar energy to catalyze one of the most important and most thermodynamically demanding reactions in nature: the oxidation of water into protons and molecular oxygen. Oxygen produced by PSII is toxic byproduct, however it is essential for respiration, the ozone layer and the extracted electrons drive the fixation of atmospheric CO2 to create biomass. The mechanism of water splitting driven by the light-induced charge separation is relatively well studied and high-resolution crystal structures are available to reveal the molecular aspects of PSII complex, however considerably less is known about how the inorganic Mn4O5Ca cluster is assembled de novo.The photosynthetic apparatus continuously experiences damage due to high light intensity and this results in the loss of photosynthetic activity. The primary photodamage occurs within main functional PSII unit, the D1 protein. To perform a highly efficient and sustained photosynthetic activity, damaged D1 protein should be replaced, with consequent reassembly of PSII. The key step in obtaining functional PSII de novo is the assembly of Mn4CaO5 core, driven by series of photo-oxidative reactions with incorporation of Mn and Ca ions into the coordination environment of PSII. The initial rate-limiting steps of the assembly of the PSII Mn4CaO5 core requires at least two quanta of light with the rate-limiting dark rearrangement step between them. A sensitive polarographic technique was used to track the assembly process under flash illumination as a function of the constituent Mn2+ and Ca2+ ions in genetically engineered membranes of the cyanobacterium Synechocystis sp. PCC6803 to elucidate the action of Ca2+ and peripheral proteins. We show that the protein scaffolding that organizes this process is allosterically modulated by the assembly protein Psb27, which together with Ca2+, stabilizes photoactivation intermediates.Photoactivation experiments with site-directed mutants D1-E189K and D1-E189R identified the role of D1-E189 in the formation the high affinity site of PSII. We have concluded that D1-E189 ligand is crucial during initial steps of photoactivation since it supports photoactivation intermediates by coordinating Ca2+ at its effectors site, which prevents the formation of inappropriately bound high-valency Mn at the oxygen evolving complex site
