The O-2 release of the oxygen-evolving complex of the photosystem II (PSII) is one of the essential processes responsible for the highly efficient O-2 production. Despite its importance, the detailed molecular mechanism is still unsolved. In the present study, we show that the O-2 release is directly coupled with water insertion into the Mn cluster based on the quantum mechanics/molecular mechanics (QM/MM) calculations. In this mechanism, the O-2 molecule first dissociates from the Mn sites in order, that is, the O atom coordinating to the Mn3 (O5a) first dissociates, then the other O atom coordinating to the Mn1 (O5d) dissociates in the next step in the late S-4 state (1 -> 2). Next, the O-2 migrates to a space surrounded by the Val185 and His332 side chains as one water molecule coordinating to the Ca2+ ion (W3) comes into the O-2 bonded site (2 -> 3). Finally, a pre-S-0 state (4) is formed after a proton transfer from the inserted water to the other proton acceptor site (W2) (3 -> 4). The highest activation barrier during these reactions was found at the O-2 release step (2 -> 3) that only requires E-double dagger = 12.7 kcal mol(-1) (G(double dagger) = 10.4 kcal mol(-1)). A series of the reactions (2 -> 3) look like a chain crash of billiard balls because the W3 is inserted into the catalytic center from the water-abundant side (Ca2+ ion side), and then the O-2 moiety is pushed out to the opposite side (Val185 side). The hydrophobic residue of Val185 covers the active O5 site and forms an O-2-specific permeation tunnel. The present sequential reactions clearly demonstrate the efficient removal of the toxic O-2 from the catalytic center and implications of the essential roles of Val185, Ca2+ ions and water molecules, which are all present in the active site of PSII as the indispensable constituents.