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

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.

PsbQ-Like Protein 3 Functions as an Assembly Factor for the Chloroplast NADH Dehydrogenase-Like Complex in Arabidopsis.

Angiosperms have three PsbQ-like (PQL) proteins in addition to the PsbQ subunit of the oxygen-evolving complex of photosystem II. Previous studies have shown that two PQL proteins, PnsL2 and PnsL3, are subunits of the chloroplast NADH dehydrogenase-like (NDH) complex involved in the photosystem I (PSI) cyclic electron flow. In addition, another PsbQ homolog, PQL3, is required for the NDH activity; however, the molecular function of PQL3 has not been elucidated. Here, we show that PQL3 is an assembly factor, particularly for the accumulation of subcomplex B (SubB) of the chloroplast NDH. In the pql3 mutant of Arabidopsis thaliana, the amounts of NDH subunits in SubB, PnsB1 and PsnB4, were decreased, causing a severe reduction in the NDH-PSI supercomplex. Analysis using blue native polyacrylamide gel electrophoresis suggested that the incorporation of PnsL3 into SubB was affected in the pql3 mutant. Unlike other PsbQ homologs, PQL3 was weakly associated with thylakoid membranes and was only partially protected from thermolysin digestion. Consistent with the function as an assembly factor, PQL3 accumulated independently in other NDH mutants, such as pnsl1-3. Furthermore, PQL3 accumulated in young leaves in a manner similar to the accumulation of CRR3, an assembly factor for SubB. These results suggest that PQL3 has developed a distinct function as an assembly factor for the NDH complex during evolution of the PsbQ protein family in angiosperms.

Antimycin A inhibits cytochrome b559-mediated cyclic electron flow within photosystem II.

The light reactions of photosynthesis are known to comprise both linear and cyclic electron flow in order to convert light energy into chemical energy in the form of NADPH and ATP. Antimycin A (AA) has been proposed as an inhibitor of ferredoxin-dependent cyclic electron flow around photosystem I (CEF-PSI) in photosynthesis research. However, its precise inhibitory mechanism and target site had not been elucidated yet. Here we show that AA inhibits the cyclic (alternative) electron flow via cytochrome b559 (Cyt b559) within photosystem II (CEF-PSII). When AA was applied to thylakoid membranes isolated from spinach leaves, the high potential form of Cyt b559, which was reduced in the dark, was transformed into the lower potential forms and readily oxidized by molecular oxygen. In the absence of AA, the reduced Cyt b559 was oxidized by P680+ upon light illumination and re-reduced in the dark, mainly by the electron from the QB site on the acceptor side of PSII. In contrast, AA suppressed the oxidation of Cyt b559 and induced its reduction under the illumination. This inhibition of Cyt b559 oxidation by AA enhanced photoinhibition of PSII. Based on the above results, we propose caution regarding the use of AA for evaluating CEF-PSI per se and concurrently propose that AA provides for new insights into, and interpretations of, the physiological importance of Cyt b559, rather than that of CEF-PSI in photosynthetic organisms.

Boosting Effect of Amphiphilic Random Copolymers for Bicontinuous Phases.

Amphiphilic random copolymers, poly(oxyethylene)/poly(oxypropylene) butyl ethers (C4EmPn), have been used as raw materials for cosmetics. This paper reports on the influence of amphiphilic random copolymers on mixtures of n-decane, water, and a nonionic surfactant, hexa(oxyethylene) dodecyl ether (C12E6). Bicontinuous phases are formed from decane/water/C12E6 mixtures at high C12E6 weight fractions (> 70 wt%). Adding C4EmPn to decane/water/C12E6 mixtures brings about the formation of bicontinuous phases and a decrease in the amount of the surfactant required for their formation, indicating efficiency boosting. The bicontinuous phase formation region in the phase diagram of the decane/water/C12E6+C4E5P5 system is largest at a specific C4E5P5 weight fraction in the C12E6/C4E5P5 mixture. When a hydrophobic polymer, in which the poly(ethylene oxide) group in C4EmPn is absent, is added to decane/water/C12E6 mixtures, no efficiency boosting is observed. These results suggest that the adjustment of the hydrophilicity-hydrophobicity balance of C12E6/C4EmPn mixture causes the efficiency boosting.

Location of the extrinsic subunit PsbP in photosystem II studied by pulsed electron-electron double resonance.

The binding site of the extrinsic protein PsbP in plant photosystem II was mapped by pulsed electron-electron double resonance, using mutant spinach PsbP (Pro20Cys, Ser82Cys, Ala111Cys, and Ala186Cys) labeled with 4-maleimido-TEMPO (MSL) spin label. The distances between the spin label and the Tyr160 neutral radical (YD) in PsbD, the D2 subunit of plant photosystem II, were 50.8 ±â€¯3.5 Å, 54.9 ±â€¯4.0 Å, 57.8 ±â€¯4.9 Å, and 58.4 ±â€¯14.1 Å, respectively. The geometry inferred from these distances was fitted to the PsbP crystal structure (PDB: 4RTI) to obtain the coordinates of YD relative to PsbP. These coordinates were then fitted under boundary conditions to the structure of cyanobacterial photosystem II (PDB: 4UB6), by rotating on Euler angles centered at fixed YD coordinates. The result proposed two models which show possible acidic amino acid residues in CP43, CP47 and D2 that can bind the basic amino acids Arg48, Lys143, and Lys160 in PsbP.

In vivo system for analyzing the function of the PsbP protein using Chlamydomonas reinhardtii.

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically developed in green-plant species including land plants and green algae. The protein-protein interactions involving PsbP and its effect on oxygen evolution have been investigated in vitro using isolated PSII membranes. However, the importance of those interactions needs to be examined at the cellular level. To this end, we developed a system expressing exogenous PsbP in the background of the Chlamydomonas BF25 mutant lacking native PsbP. Expression of His-tagged PsbP successfully restored the oxygen-evolving activity and photoautotrophic growth of the mutant, while PsbP-∆15 lacking the N-terminal 15 residues, which are crucial for the oxygen-evolving activity of spinach PSII in vitro, only partially did. This demonstrated the importance of N-terminal sequence of PsbP for the photosynthetic activity in vivo. Furthermore, the PSII-LHCII supercomplex can be specifically purified from the Chlamydomonas cells having His-tagged PsbP using a metal affinity chromatography. This study provides a platform not only for the functional analysis of PsbP in vivo but also for structural analysis of the PSII-LHCII supercomplex from green algae.

The N-terminal sequence of the extrinsic PsbP protein modulates the redox potential of Cyt b559 in photosystem II.

The PsbP protein, an extrinsic subunit of photosystem II (PSII) in green plants, is known to induce a conformational change around the catalytic Mn4CaO5 cluster securing the binding of Ca(2+) and Cl(-) in PSII. PsbP has multiple interactions with the membrane subunits of PSII, but how these affect the structure and function of PSII requires clarification. Here, we focus on the interactions between the N-terminal residues of PsbP and the α subunit of Cytochrome (Cyt) b559 (PsbE). A key observation was that a peptide fragment formed of the first N-terminal 15 residues of PsbP, 'pN15', was able to convert Cyt b559 into its HP form. Interestingly, addition of pN15 to NaCl-washed PSII membranes decreased PSII's oxygen-evolving activity, even in the presence of saturating Ca(2+) and Cl(-) ions. In fact, pN15 reversibly inhibited the S1 to S2 transition of the OEC in PSII. These data suggest that pN15 can modulate the redox property of Cyt b559 involved in the side-electron pathway in PSII. This potential change of Cyt b559, in the absence of the C-terminal domain of PsbP, however, would interfere with any electron donation from the Mn4CaO5 cluster, leading to the possibility that multiple interactions of PsbP, binding to PSII, have distinct roles in regulating electron transfer within PSII.

Cross-linking evidence for multiple interactions of the PsbP and PsbQ proteins in a higher plant photosystem II supercomplex.

The extrinsic subunits of membrane-bound photosystem II (PSII) maintain an essential role in optimizing the water-splitting reaction of the oxygen-evolving complex (OEC), even though they have undergone drastic change during the evolution of oxyphototrophs from symbiotic cyanobacteria to chloroplasts. Two specific extrinsic proteins, PsbP and PsbQ, bind to the lumenal surface of PSII in green plants and maintain OEC conformation and stabilize overall enzymatic function; however, their precise location has not been fully resolved. In this study, PSII-enriched membranes, isolated from spinach, were subjected to chemical cross-linking combined with release-reconstitution experiments. We observed direct interactions between PsbP and PsbE, as well as with PsbR. Intriguingly, PsbP and PsbQ were further linked to the CP26 and CP43 light-harvesting proteins. In addition, two cross-linked sites, between PsbP and PsbR, and that of PsbP and CP26, were identified by tandem mass spectrometry. These data were used to estimate the binding topology and location of PsbP, and the putative positioning of PsbQ and PsbR on the lumenal surface of the PSII. Our model gives new insights into the organization of PSII extrinsic subunits in higher plants and their function in stabilizing the OEC of the PSII supercomplex.

Identification of the basic amino acid residues on the PsbP protein involved in the electrostatic interaction with photosystem II.

The PsbP protein is an extrinsic subunit of photosystem II (PSII) that is essential for photoautotrophic growth in higher plants. Several crystal structures of PsbP have been reported, but the binding topology of PsbP in PSII has not yet been clarified. In this study, we report that the basic pocket of PsbP, which consists of conserved Arg48, Lys143, and Lys160, is important for the electrostatic interaction with the PSII complex. Our release-reconstitution experiment showed that the binding affinities of PsbP-R48A, -K143A, and -K160A mutated proteins to PSII were lower than that of PsbP-WT, and triple mutations of these residues greatly diminished the binding affinity to PSII. Even when maximum possible binding had occurred, the R48A, K143A, and K160A proteins showed a reduced ability to restore the rate of oxygen evolution at low chloride concentrations. Fourier transform infrared resonance (FTIR) difference spectroscopy results were consistent with the above finding, and suggested that these mutated proteins were not able to induce the normal conformational change around the Mn cluster during S1 to S2 transition. Finally, chemical cross-linking experiments suggested that the interaction between the N-terminus of PsbP with PsbE was inhibited by these mutations. These data suggest that the basic pocket of PsbP is important for proper association and interaction with PSII. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

The conserved His-144 in the PsbP protein is important for the interaction between the PsbP N-terminus and the Cyt b559 subunit of photosystem II.

The PsbP protein regulates the binding properties of Ca(2+) and Cl(-), and stabilizes the Mn cluster of photosystem II (PSII); however, the binding site and topology in PSII have yet to be clarified. Here we report that the structure around His-144 and Asp-165 in PsbP, which is suggested to be a metal binding site, has a crucial role for the functional interaction between PsbP and PSII. The mutated PsbP-H144A protein exhibits reduced ability to retain Cl(-) anions in PSII, whereas the D165V mutation does not affect PsbP function. Interestingly, H144A/D165V double mutation suppresses the effect of H144A mutation, suggesting that these residues have a role other than metal binding. FTIR difference spectroscopy suggests that H144A/D165V restores proper interaction with PSII and induces the conformational change around the Mn cluster during the S(1)/S(2) transition. Cross-linking experiments show that the H144A mutation affects the direct interaction between PsbP and the Cyt b(559) α subunit of PSII (the PsbE protein). However, this interaction is restored in the H144A/D165V mutant. In the PsbP structure, His-144 and Asp-165 form a salt bridge. H144A mutation is likely to disrupt this bridge and liberate Asp-165, inhibiting the proper PsbP-PSII interaction. Finally, mass spectrometric analysis has identified the cross-linked sites of PsbP and PsbE as Ala-1 and Glu-57, respectively. Therefore His-144, in the C-terminal domain of PsbP, plays a crucial role in maintaining proper N terminus interaction. These data provide important information about the binding characteristics of PsbP in green plant PSII.

The PsbQ protein stabilizes the functional binding of the PsbP protein to photosystem II in higher plants.

PsbP and PsbQ proteins are extrinsic subunits of photosystem II (PSII) and optimize the oxygen evolution reaction by regulating the binding properties of the essential cofactors Ca(2+) and Cl(-). PsbP induces conformational changes around the catalytic Mn cluster required for Ca(2+) and Cl(-) retention, and the N-terminal region of PsbP is essential for this reaction. It was reported that PsbQ partially restores the functional defect of N-terminal truncated PsbP [Ifuku and Sato (2002) Plant Cell Physiol. 43, 1244-1249]; however, the mechanism of this restoration is yet to be clarified. In this study, we demonstrate that PsbQ is able to restore the functional binding of mutated PsbPs. In the presence of PsbQ, ∆15-PsbP, a truncated PsbP lacking 15 N-terminal residues, was able to specifically bind to NaCl-washed spinach PSII membranes and significantly restore the oxygen evolving activity. Furthermore, PsbQ was also able to compensate for the impaired ion-retention of H144A-PsbP, in which a conserved histidine at position 144 in the C-terminal domain was substituted with an alanine. Fourier transform infrared (FTIR) difference spectroscopy showed that PsbQ restored the ability of ∆15- and H144A-PsbP to induce proper conformational changes during S(1) to S(2) transition. These data suggest that the major function of PsbQ is to stabilize PsbP binding, thereby contributing to the maintenance of the catalytic Mn cluster of the water oxidation machinery in higher plant PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.