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)(.) to S(0)Tyr(Z) transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being approximately 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.
Highlights
The Mn4Ca cluster acts as a device for accumulating oxidizing equivalents and as the active site for water oxidation
(22), we have shown that growing the thermophilic cyanobacterium T. elongatus in the presence of Sr2ϩ instead of Ca2ϩ resulted in the exchange of Ca2ϩ by Sr2ϩ, and this produced a significant slowdown of the oxygen evolution rate
An alternative, abnormally stable form appears to be induced in which the Mn4 cluster is in the same redox state as it was in S2 state but in magnetic interaction with a radical [38], likely TyrZ1⁄7 [39, 40], giving rise to a characteristic EPR signal that is known as the split signal
Summary
Culture of the Cells and Purification of Thylakoids and PSII— Thylakoids, and thence PSII, were purified from a T. elongatus strain that had a His tag on the CP43 subunit [58] and in which the psbA1 and psbA2 genes were deleted (WT*) [59]. Thylakoids and PSII were stored in liquid nitrogen at a concentration of about 1.5–2 mg Chl/ml in a medium containing 10% glycerol, 1 M betaine, 15 mM CaX2, 15 mM MgX2, and 40 mM MES, pH 6.5 (pH adjusted with NaOH), until they were used. UV-visible Absorption Change Spectroscopy—Absorption changes were measured with a laboratory-built spectrophotometer where the absorption changes were sampled at discrete times by short flashes [62] These flashes were provided by a neodymium-yttrium aluminum garnet (Nd:YAG) pumped (355 nm) optical parametric oscillator, which produces monochromatic flashes (1 nm full-width at half-maximum) with a duration of 6 ns. The samples were in some cases synchronized in the S1 state with one pre-flash [66] After another dark period of 1 h at room temperature, 0.5 mM PPBQ was added (the final concentration of dimethyl sulfoxide was Ϸ2%). Analysis of the data was done using Excel (Microsoft), Mathcad 14 (Parametric Technology Corp.), and Origin 7.5 (OriginLab Corp.)
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