Exploring the interdependence of calcium and chloride activation of O2 evolution in photosystem II

Abstract

Calcium and chloride are activators of oxygen evolution in photosystem II (PSII), the light-absorbing water oxidase of higher plants, algae, and cyanobacteria. Calcium is an essential part of the catalytic Mn₄CaO₅ cluster that carries out water oxidation and chloride has two nearby binding sites, one of which is associated with a major water channel. The co-activation of oxygen evolution by the two ions is examined in higher plant PSII lacking the extrinsic PsbP and PsbQ subunits using a bisubstrate enzyme kinetics approach. Analysis of three different preparations at pH 6.3 indicates that the Michaelis constant, K_M, for each ion is less than the dissociation constant, K_S, and that the affinity of PSII for Ca²⁺ is about ten-fold greater than for Cl^-, in agreement with previous studies. Results are consistent with a sequential binding model in which either ion can bind first and each promotes the activation by the second ion. At pH 5.5, similar results are found, except with a higher affinity for Cl^- and lower affinity for Ca²⁺. Observation of the slow-decaying Tyr Z radical, Y_Z•, at 77 K and the coupled S₂Y_Z• radical at 10 K, which are both associated with Ca²⁺ depletion, shows that Cl^- is necessary for their observation. Given the order of electron and proton transfer events, this indicates that chloride is required to reach the S₃ state preceding Ca²⁺ loss and possibly for stabilization of Y_Z• after it forms. Interdependence through hydrogen bonding is considered in the context of the water environment that intervenes between Cl^- at the Cl-1 site and the Ca²⁺/Tyr Z region.

Keywords: Calcium; Chloride; Electron paramagnetic resonance; Oxygen evolution; Photosystem II; Water oxidation.

Scheme 1

Bisubstrate binding models for enzyme kinetics analyses

Fig. 1

Dependence of O₂ evolution activity on Ca²⁺ in the presence of various concentrations of Cl⁻ at pH 6.3: red circles, 12.0 mM Cl⁻; green squares, 5.0 mM Cl⁻; yellow diamonds, 2.0 mM Cl⁻; and blue triangles, 1.0 mM Cl⁻. Solid lines show the fits to the data sets. PSII lacking PsbP/PsbQ was depleted of Ca²⁺ as described. Assays were carried out in the presence of 1 mM EDTA; the Ca²⁺ concentrations given were corrected for that complexed with EDTA

Fig. 2

Secondary plots of Cl⁻ dependence of Ca²⁺-activated O₂ evolution in PSII lacking PsbP/PsbQ: A, Lineweaver–Burk slopes vs. 1/[Cl⁻]; B, Lineweaver–Burk intercepts vs. 1/[Cl⁻]. Data points correspond to the fits shown in Fig. 1, except with the omission of the inhibitory effect observed at high Cl⁻ concentrations as described in the text. Error bars were propagated from the data in Table 1

Fig. 3

Dependence of O₂ evolution activity on Cl⁻ concentration in the presence of various concentrations of Ca²⁺ at pH 6.3 using PSII lacking PsbP/PsbQ with: A, no further treatment; B, with Ca²⁺ depletion treatment. Ca²⁺ concentrations were: red circles, 5.0 mM Ca²⁺; green squares, 3.0 mM Ca²⁺; yellow diamonds, 1.0 mM Ca²⁺; blue triangles, 0.50 mM Ca²⁺; pink inverted triangles, 0.25 mM Ca²⁺; and cyan hexagons, 0.10 mM Ca²⁺. Solid lines show direct fits to the data sets

Fig. 4

Dependence of O₂ evolution activity on Cl⁻ concentration in the presence of various concentrations of Ca²⁺ at pH 5.5 using PSII lacking PsbP/PsbQ. Ca²⁺ concentrations were: red circles, 4.0 mM Ca²⁺; green squares, 1.0 mM Ca²⁺; and yellow diamonds, 0.5 mM Ca²⁺. Solid lines show direct fits to the data sets

Fig. 5

EPR spectra of tyrosine radicals in dark-adapted (dashed line) and illuminated (solid line) PSII lacking PsbP/PsbQ in the presence of: A bottom, 5 mM Ca²⁺ and 25 mM Cl⁻ (sample 1); A top, 25 mM Cl⁻ (sample 2); B bottom, 5 mM Ca²⁺ (sample 3); and B top, no Cl⁻ or Ca²⁺ (sample 4). Samples without Ca²⁺ were prepared with 1 mM EDTA to ensure absence of Ca²⁺, as described in Materials and Methods. EPR spectra were taken at 77 K using 1 mW power and 3 G modulation amplitude

Fig. 6

EPR spectra of PSII lacking PsbP/PsbQ that had been dark-adapted (A), followed by illumination at 195 K (B), and finally by illumination at 273 K (C). Panels B and C show difference spectra resulting from subtraction of the dark-adapted spectra of Panel A. Samples were prepared as described in Materials and Methods with: top, 25 mM Cl⁻; second, 6 mM Ca²⁺ and 25 mM Cl⁻; third, no Cl⁻ or Ca²⁺; and bottom, 6 mM Ca²⁺. Samples also contained 0.1 mM EDTA. EPR spectra were taken at 10 K using 20 mW power and 18 G modulation amplitude. For comparison, the intensity scales in Panels B and C are 0.63 and 5.2 relative to that of Panel A. (The top spectrum in panel A was published previously in (Haddy and Ore 2010).)