The active site for water oxidation in photosystem II goes through five sequential oxidation states (S 0 to S 4 ) before O 2 is evolved. It consists of a Mn 4 Ca cluster close to a redox-active tyrosine residue (Tyr Z ). Cl ؊ is also required for enzyme activity. To study the role of Ca 2؉ and Cl ؊ in PSII, these ions were biosynthetically substituted by Sr 2؉ and Br ؊ , respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O 2 polarography, and thermoluminescence spectroscopy. The effect of the Ca 2؉ /Sr 2؉ and Cl ؊ /Br ؊ exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O 2 release was similar to that of the S 3 Tyr Z ⅐ to S 0 Tyr Z transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being ≈6 -7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S 3 state as manifest by thermoluminescence. These observations indicate that Cl ؊ is involved in the water oxidation mechanism. The possibility that Cl ؊ is close to the active site is discussed in terms of recent structural models.Light-driven water oxidation by the photosystem II (PSII) 3 enzyme is responsible for the O 2 on Earth and is at the origin of the production of most of the biomass. Refined three-dimensional x-ray structures at 3.5 and 3.0 Å resolution have been obtained by using PSII isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus (1, 2). PSII is made up of more than 20 membrane protein subunits, 35-36 chlorophyll molecules, more than 10 carotenoid molecules, several lipids, two hemes, and the cofactors involved in the electron transfer reactions (1, 2). Absorption of a photon by chlorophyll is followed by the formation of a radical pair in which the pheophytin molecule, Pheo D1 , is reduced and the accessory chlorophyll molecule, Chl D1 , is oxidized (3-5). The cation is then stabilized on P 680 , a weakly coupled chlorophyll dimer (see e.g. Refs. 6 and 7 for energetic considerations). The pheophytin anion transfers the electron to a quinone, Q A , which in turn reduces a second quinone, Q B . P 680 .ϩ oxidizes a tyrosine residue of the D1 polypeptide, Tyr Z , which in turn oxidizes the Mn 4 Ca cluster.The Mn 4 Ca cluster acts as a device for accumulating oxidizing equivalents and as the active site for water oxidation. During the enzyme cycle, the oxidizing side of PSII goes through five sequential redox states, denoted as S n , where n varies from 0 to 4 upon the absorption of four photons (8). Upon formation of the S 4 state, two molecules of water are rapidly oxidized, the S 0 state is r...