Photosynthetic water oxidation is catalyzed by a Mn4CaO4 cluster bound to the protein subunits of photosystem II (PSII). We have solved the crystal structure of PSII from a thermophilic cyanobacterium at a resolution of 1.9 Å[1] using synchrotron radiation X-rays, and further solved the radiation damage-free structure using a femtosecond X-ray free electron laser (XFEL) by fixed-target serial femtosecond X-ray crystallography[2]. These structures showed that the Mn4CaO5-cluster is organized into a distorted chair in which, O5 is in a unique position with a rather longer distances to its nearby Mn ions, suggesting its possible involvement in the O=O bond formation. We further used a pump-probe approach by serial femtosecond crystallography (SFX) with XFEL to solve the structure of the S2 and S3 intermediate-states induced by 1 or 2-flash illumination[3,4], which showed the insertion of an oxygen atom O6 (water molecule) in the vicinity of O5 during S2-S3 transition. Accompanying the insertion of O6, a number of structural changes are found in the Mn4CaO5-cluster itself as well as in its environment. These structural changes are further investigated by the time-resolved pump-probe SFX approach in a time range of nsec to msec following 1 or 2 flashes, which revealed a number of fast structural changes that reflect the water inlet, proton egress, and O5-O6 coupling in the S2 and S3 states[5]. From these results, the precursor of O6 was found to bound to the Ca2+ ion immediately following 2F, and subsequently transported to its mature position close to O5.