Biosynthetic Exchange of Bromide for Chloride and Strontium for Calcium in the Photosystem II Oxygen-evolving Enzymes

Abstract

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...

Structure of Sr-substituted photosystem II at 2.1 Å resolution and its implications in the mechanism of water oxidation

“…Among various other cations, only Sr 2+ was shown to be able to partially replace the role of Ca 2+ , resulting in an OEC with approximately half of the activity compared with that having Ca 2+ -associated activity (15,(17)(18)(19)(20). Similar results were obtained with cyanobacteria, where Ca 2+ cannot be removed and replaced in isolated PSII so that it has to be replaced by Sr 2+ in culture medium (21,22). Substitution of Ca 2+ with Sr 2+ modifies the EPR properties of the S 2 -state (15,23) and alters several carboxylate-stretching modes of the S 2 -S 1 FTIR difference spectrum (24)(25)(26).…”

“…Substitution by Sr 2+ slows down S-state transition beyond S 2 and S 3 -states, leading to the formation of an S 2 -state with somewhat different properties in terms of its redox potential, stability, and the EPR multiline signal (17)(18)(19)(20)(22)(23)(24)(25)(26). However, because of the absence of detailed structural information, the exact role of Ca 2+ and the causes underlying the effects of substitution by Sr 2+ are not clear.…”

“…Although Ca 2+ in the Mn 4 CaO 5 -cluster can be replaced by Sr 2+ easily in purified PSII from higher plants, Ca 2+ in cyanobacterial PSII cannot be removed and replaced by Sr 2+ easily, and it has to be substituted by Sr 2+ biosynthetically. Thus, we substituted Ca 2+ with Sr 2+ in the culture medium of T. vulcanus, and purified Sr 2+ -substituted PSII according to the protocols reported previously (21,22). In agreement with the previous reports, the cells appeared to grow in the Sr 2+ -containing medium slightly slower than those in Ca 2+ -containing medium, and the oxygen-evolving activity of purified Sr 2+ -PSII dimer was lower by 40-50% than those of the Ca 2+ -PSII dimer (Table 1).…”

“…Extensive studies have been performed on the roles of Ca 2+ in the oxygen-evolving reaction, and perturbations of the S-state transition have been well documented upon replacement of Ca 2+ by Sr 2+ (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Substitution by Sr 2+ slows down S-state transition beyond S 2 and S 3 -states, leading to the formation of an S 2 -state with somewhat different properties in terms of its redox potential, stability, and the EPR multiline signal (17)(18)(19)(20)(22)(23)(24)(25)(26).…”

“…The growth was monitored by OD 730nm and photosynthetic activity. The thylakoid membranes and Sr-PSII core dimer complexes were purified with the method of Shen and Inoue (47), with modifications as previously described (21,22,48). The Sr-PSII dimer compositions, activity, and homogeneity were assessed before each crystallization experiment.…”