Photoactivation of photosystem II has been studied in the FUD 39 mutant of Chlamydomonas reinhardtii that lacks the 23 kDa extrinsic subunit of photosystem II. We have taken advantage of the slow photoactivation rate of FUD 39, earlier demonstrated in Rova, E. M., et al. [(1996) J. Biol. Chem. 271, 28918-28924], to study events in photosystem II during intermediate stages of the process. By measuring the EPR multiline signal, the decay of the variable fluorescence after single flashes, and electron transfer from water to the QB site, we found a good correlation between the building of a tetrameric Mn cluster, longer recombination times between QA- and the donor side of photosystem II, and the achievement of water splitting ability. An increased rate of electron transfer from QA- to the QB site on the acceptor side of photosystem II, mainly due to enhanced efficiency of binding of QB to its site, was found to precede the building of the Mn cluster. We also showed that TyrD was oxidized simultaneously with this increase in electron-transfer rate. Thus, it appears that photoactivation is sequential, with an increased rate of electron transfer on the acceptor side occurring together with the oxidation of TyrD in the first step, followed by the assembly of the Mn cluster. We suggest that a conformational change of photosystem II is induced early in the photoactivation process facilitating electron transfer from the primary donor to the acceptor side. As a consequence, TyrD, an auxiliary electron donor to P680+/TyrZ*, is oxidized. That this occurs before the Mn cluster is fully functional serves to protect photosystem II against donor side induced photodamage.