D139N mutation of PsbP enhances the oxygen-evolving activity of photosystem II through stabilized binding of a chloride ion

Abstract

Photosystem II (PSII) is a multisubunit membrane protein complex that catalyzes light-driven oxidation of water to molecular oxygen. The chloride ion (Cl^-) has long been known as an essential cofactor for oxygen evolution by PSII, and two Cl^- ions (Cl-1 and Cl-2) have been found to specifically bind near the Mn₄CaO₅ cluster within the oxygen-evolving center (OEC). However, despite intensive studies on these Cl^- ions, little is known about the function of Cl-2, the Cl^- ion that is associated with the backbone nitrogens of D1-Asn338, D1-Phe339, and CP43-Glu354. In green plant PSII, the membrane extrinsic subunits-PsbP and PsbQ-are responsible for Cl^- retention within the OEC. The Loop 4 region of PsbP, consisting of highly conserved residues Thr135-Gly142, is inserted close to Cl-2, but its importance has not been examined to date. Here, we investigated the importance of PsbP-Loop 4 using spinach PSII membranes reconstituted with spinach PsbP proteins harboring mutations in this region. Mutations in PsbP-Loop 4 had remarkable effects on the rate of oxygen evolution by PSII. Moreover, we found that a specific mutation, PsbP-D139N, significantly enhances the oxygen-evolving activity in the absence of PsbQ, but not significantly in its presence. The D139N mutation increased the Cl^- retention ability of PsbP and induced a unique structural change in the OEC, as indicated by light-induced Fourier transform infrared (FTIR) difference spectroscopy and theoretical calculations. Our findings provide insight into the functional significance of Cl-2 in the water-oxidizing reaction of PSII.

Keywords: chloride ions; membrane-extrinsic proteins; oxygen evolution; photosynthesis.

Fig. 1.

Structures of water channels, hydrogen-bond networks, Cl⁻ binding sites, and PsbP-Loop 4 in the OEC of green plant PSII. (A) Structures of the O4, O1, and Cl-1 (E65/E312; E65/E313 in pea) channels and the Cl-2 and Y_Z-N298 networks in PSII from pea (PDB ID: 5XNL). The channel space was analyzed using Caver (108). The O4 channel was analyzed in spinach PSII and it is composed of D1, D2, CP43, and PsbP subunits (6). The components of the O4 channel in the pea PSII are identical to those in the spinach PSII. (B) The Cl⁻ binding sites and the location of the Loop 4 region of PsbP (PDB ID: 5XNL). D1, D2, and PsbP are shown in green, blue, and yellow cartoon view, respectively, and the Loop 4 region of PsbP is colored in red. The Cl⁻ ions are shown as pink spheres. The amino acid residues associated to the Cl⁻ ions are shown as stick models.

Fig. 2.

Effects of various mutations in PsbP-Loop 4 on the oxygen-evolving activity of PSII. (A) The oxygen-evolving activities of PSII membranes reconstituted with various PsbP mutant proteins were measured in buffer (25 mM MES-NaOH, 0.4 M sucrose, pH 6.5) with 0.4 mM DCBQ as an electron acceptor. The sample “−PsbP” is NaCl-washed PSII without reconstitution of PsbP. Oxygen-evolving activity of WT-reconstituted PSII (177 to 190 µmol O₂ mg Chl⁻¹ h⁻¹ in independent experiments) was set as 100%; error bars = SD (n = 3, technical replicates). (B) PSII membranes reconstituted with various PsbP mutant proteins were subjected to SDS-PAGE in order to confirm the binding of the recombinant PsbPs. Proteins equivalent to 3 µg chlorophyll were loaded onto each lane, and the gels were stained with Oriole stain (Bio-Rad). The arrow head indicates PsbP-6xHis bands.

Fig. 3.

Effect of the PsbP-D139N mutation on the oxygen-evolving activity of PSII and binding of PsbP to PSII. PSII membranes were reconstituted with various amounts of PsbP (WT (blue circles) or D139N (red squares)), and (A) the oxygen-evolving activity and (B) the extent of PsbP binding was measured. For (A), oxygen-evolving activity of PSII reconstituted with WT PsbP at a ratio of PsbP: PSII = 4:1 (157 to 167 µmol O₂ mg Chl⁻¹ h⁻¹ in independent experiments) was set as 100%; error bars = SD (n = 3, technical replicates), and for (B), the amount of WT PsbP bound to PSII when reconstituted with a ratio of PsbP: PSII = 4:1 was set as 100%; error bars = SD (n = 3, technical replicates).

Fig. 4.

Cl⁻ dependence of the oxygen-evolving activity of WT- and D139N-reconstituted PSII. The oxygen-evolving activity of WT- (blue circles) and D139N- (red squares) reconstituted PSII was measured in the presence of various concentrations of Cl⁻ at pH 6.5. Oxygen-evolving activity of WT-reconstituted PSII under 10.2 mM Cl⁻ (241 to 282 µmol O₂ mg Chl⁻¹ h⁻¹ in independent experiments) was set as 100%; error bars = SD (n = 3, technical replicates).

Fig. 5.

pH dependence and effect of Cl⁻ at pH 7.0 on the oxygen-evolving activity of WT- and D139N-reconstituted PSII. The oxygen-evolving activity of WT- (blue circles) and D139N- (red squares) reconstituted PSII was measured (A) at different pH (under 0.2 mM Cl⁻), (B) in the presence of various concentrations of Cl⁻ at pH 7.0. Oxygen-evolving activity of WT-reconstituted PSII (A) at pH 6.5 (142 µmol O₂ mg Chl⁻¹ h⁻¹), (B) under 10.2 mM Cl⁻ (225 to 239 µmol O₂ mg Chl⁻¹ h⁻¹ in independent experiments) was set as 100%; error bars = SD (n = 3, technical replicates).

Fig. 6.

Effect of the PsbP-D139N mutation on the S₂/S₁ FTIR difference spectrum of PSII membranes. (A) Light-induced S₂/S₁ FTIR difference spectra of untreated (a–c, black lines), NaCl-washed (a, red line), WT-reconstituted (b, red line), and D139N-reconstituted (c, red line) PSII membranes. (B) The amide I region of the untreated-minus-treated double-difference spectra of the S₂/S₁ FTIR difference spectra: (a) untreated-minus-NaCl-washed, (b) untreated-minus-WT-reconstituted, and (c) untreated-minus-D139N-reconstituted PSII.

Fig. 7.

Optimized protein structures of (A) WT PSII and (B) PsbP-D139N mutant PSII obtained by calculations based on the reported PSII structure (PDB ID: 5XNL). D1, D2, and PsbP are shown in green, blue, and yellow cartoon view, respectively. Dotted lines indicate interactions (hydrogen-bonds and salt-bridges) between the side chains of amino acid residues, and those that are formed/diminished by the D139N mutation are colored in pink, with the N–O distances [Å] shown. The hydrogen atoms are not shown for clarity. In the alternative conformation where the Nδ atom of PsbP-Asn139 is oriented toward the side chain of PsbP-Lys143 (Figure S5, Supplementary Material), the calculated energy was 16.1 kcal mol⁻¹ higher than that of the original conformation where the Nδ atom of PsbP-Asn139 is hydrogen-bonded with the backbone carboxyl group of D2-Leu353 as shown in (B).

Fig. 8.

Comparison of the structure of cyanobacterial PSII and green plant PSII. (A) Superposition of (B) the structure of green plant PSII (PDB ID: 5XNL) surrounding PsbP-Loop 4 and (C) the structure of cyanobacterial PSII (PDB ID: 3WU2) surrounding the C-terminus of PsbU. D1, D2, CP43, PsbP (in A and B), PsbU (in A and C), and PsbV (in A and C) are shown in green, blue, orange, yellow, dark gray, and light gray, respectively. The Cl⁻ are shown as pink spheres, and residues PsbP-Asp139 and PsbU-Tyr103 are shown as stick models. The glycerol molecules of the cyanobacterial PSII have been removed for clarity.