Keywords

Summary

Photosystem II (PS II) catalyzes photosynthetic water oxidation and is composed of more than 20 subunits, including membrane-intrinsic and extrinsic proteins. PS II extrinsic proteins shield the catalytic Mn4CaO5 cluster from the outside bulk solution and stabilize the binding of inorganic cofactors, such as Ca2+ and Cl−, in the oxygen-evolving center of PS II. Among the PS II extrinsic proteins, PsbO is commonly found in all oxygenic organisms, while PsbP and PsbQ are specific to green plants, including vascular plants and green algae, and PsbU, PsbV, CyanoQ, and CyanoP exist in cyanobacteria. Additionally, red algae and diatoms have unique PS II extrinsic proteins, such as PsbQ′ and Psb31. Recent structural studies revealed the structure and binding manner of each extrinsic subunit in PS II from different species. Furthermore, various functions of PsbP- and PsbQ-homologs in photosynthetic electron transfer have been identified, indicating that gene duplication and successive functional diversification occurred during the evolution of PS II extrinsic proteins. This chapter focuses on recent results on the structural and functional studies of PS II extrinsic proteins, and discusses their evolutionary changes during the development of PS II.

I.Introduction

Photosystem II (PS II) converts light energy into the electrochemical potential energy required to split water into H+, electrons, and molecular oxygen (Dau et al. 2012; Cox and Messinger 2013; Vinyard et al. 2013). The PS II core complex is composed of more than 20 subunits, including CP47, CP43, D1, D2, Cyt b559 α- and β-subunits, and PsbI, with numerous small subunits that stabilize the reaction (Pagliano et al. 2013; Shen 2015). X-ray structural analysis of the PS II dimeric complex from cyanobacteria at atomic resolution revealed the location of most subunits, pigments, and redox cofactors (Ferreira et al. 2004; Guskov et al. 2009; Umena et al. 2011; Suga et al. 2015) (Chap. 1). Light excitation of the primary donor P680, the special pair chlorophylls in PS II, results in electron transfer to a nearby pheophytin followed by electron transfer to the acceptor quinones (QA and QB). The resulting cation radical of P680+ receives electrons from the Mn4CaO5 cluster via a redox-active tyrosine of D1, YZ. The Mn4CaO5 cluster converts two water molecules into one molecular oxygen and four protons through a light-driven cycle consisting of five intermediates known as the Si states (i = 0–4). Among them, the S1 state is the most dark-stable, and flash illumination advances each Si state (i = 0–3) to the next Si + 1 state. Molecular oxygen is released during the S3–S4–S0 transition after the transient S4 state. Recent studies using a femtosecond X-ray free laser reported the transient structure of intermediate S-states, revealing the nearly complete mechanism of water oxidation in PS II (Suga et al. 2017, 2019; Kern et al. 2018) (Chap. 1).

The mechanism of water oxidation and the basic subunit structure of the PS II core are mostly conserved across oxygenic photosynthetic organisms ranging from cyanobacteria to vascular plants, whereas several peripheral PS II subunits differ (Nelson and Yocum 2006). Particularly, the composition of the extrinsic subunits of PS II surrounding the catalytic Mn4CaO5 cluster has undergone a large evolutionary change (Enami et al. 2008). Green eukaryotes, such as vascular plants and green algae, contain a set of three extrinsic proteins, PsbO, PsbP, and PsbQ, which bind to the lumenal surface of PS II (Kuwabara and Murata 1982; Ghanotakis et al. 1984a). In cyanobacterial PS II, PsbV and PsbU are present rather than PsbP and PsbQ (Shen et al. 1992; Shen and Inoue 1993). Furthermore, cyanobacteria contain PsbP and PsbQ homologs designated as CyanoP and CyanoQ, respectively (Kashino et al. 2002; Thornton et al. 2004). Additionally, red algae and diatoms possess PsbQ′, a 20-kDa homolog of CyanoQ, bound to PS II as an extrinsic subunit in addition to PsbO, PsbU, and PsbV (Ohta et al. 2003; Nagao et al. 2010a). Diatoms further possess Psb31 as an additional, specific extrinsic subunit (Okumura et al. 2008). High-resolution structures of individual PS II extrinsic subunits from eukaryotes have been reported (Calderone et al. 2003; Ifuku et al. 2004; Balsera et al. 2005; Kopecky et al. 2012; Nagao et al. 2013; Michoux et al. 2014; Cao et al. 2015); however, their binding sites and topologies were unclear until recently because crystallographic information derived from prokaryotic cyanobacterial PS II cannot be fully applied to eukaryotic PS II.

The X-ray structure of red algal PS II and cryo-electron microscopy (cryo-EM) structure of PS II supercomplex from vascular plants were recently reported (Ago et al. 2016; Wei et al. 2016; Su et al. 2017). It is now possible to describe the structural basis of the binding and function of extrinsic proteins in eukaryotic PS II. Extensive reviews have been published on the structure and interaction of each PS II extrinsic proteins in PS II (Enami et al. 2008; Ifuku et al. 2008, 2011; Bricker et al. 2012; Ifuku 2015; Ifuku and Noguchi 2016; Roose et al. 2016). Therefore, this chapter focus on their evolutional aspects.

II.Localization of Extrinsic Subunits in Photosystem II Structures

Figure 16.1 shows the X-ray and cryo-EM structures of the PS II dimer from cyanobacteria, red algae, and vascular (green) plants, viewed horizontally from the thylakoid lumenal side. The positions of PsbO, PsbU, and PsbV are similar between cyanobacterial and red algal PS II, except that PsbQ′ binds to one of the PS II monomers to interact with the outer surface of CP43 in red algal PS II (Ago et al. 2016). Binding of CyanoQ is not included in the current X-ray structure of cyanobacterial PS II, whereas its individual structure has been reported (Jackson et al. 2010). The study using a chemical crosslinker suggested that CyanoQ forms a dimer at the interface between monomers in the dimeric PS II complex (Liu et al. 2014). Binding of multiple copies of CyanoQ to the PS II assembly intermediates has also been reported (Liu et al. 2015). Unlike eukaryotic PsbQ (PsbQ′), CyanoQ is a lipoprotein with a lipid modification on its N-terminus (Juneau et al. 2016). Therefore, the manner of interaction with PS II may differ between PsbQ and CyanoQ.

Fig. 16.1.
figure 1

Binding sites of extrinsic subunits in the PS II structure from cyanobacteria (Thermosynechococcus vulcanus, PDB ID: 3WU2), red algae (Cyanidium caldarium, 4YUU), and green plants (Spinacia oleracea, 3JCU). PsbO, red; PsbP, green; PsbQ′ and PsbQ, pink; PsbV, blue; PsbU, yellow

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In the cryo-EM structure of green plant PS II, PsbP specifically replaces the binding site of PsbV in the structure of cyanobacterial and red algal PS II (Wei et al. 2016; Su et al. 2017). This is consistent with previous in vitro and in vivo studies indicating that both PsbP and PsbV are required to retain Ca2+ and Cl− ions in the oxygen-evolving center of PS II (Shen et al. 1992, 1995; Shen and Inoue 1993). The binding site of PsbQ is similar to that of PsbQ′ in red algal PS II, suggesting functional conservation between the green and red lineages (Ago et al. 2016). In fact, phylogenetic analysis indicated that PsbQ in green algae and Euglena gracilis is more closely related to PsbQ′ in red algae and diatoms (Yabuta et al. 2010). In this chapter, we use the term “PsbQ′” in red algal and diatom PSII; however, the term “PsbQ” may be more appropriate to avoid confusion in the future.

The above structural comparison suggests that the most drastic change in PS II at the branching point of the red and green lineages was the development of PsbP from CyanoP in cyanobacteria (Ifuku 2015). CyanoP is absent from the current X-ray structures of cyanobacterial PS II, whereas its individual structure has been reported (Michoux et al. 2010; Jackson et al. 2012). Recent studies reported that CyanoP interacts with premature PS II subcomplexes during the early stage of its assembly (Cormann et al. 2014; Knoppová et al. 2016). This suggests that CyanoP may function as an assembly factor in cyanobacterial PS II, and PsbP, which functions as an essential extrinsic subunit of PS II in green plants, developed from such an assembly factor for PS II. The molecular evolution of PsbP and PsbQ family proteins is discussed in a later section.

III.Functions of Each Extrinsic Subunit

A.PsbO

In all oxyphototrophs, PsbO, also known as OEC33 because of its apparent molecular mass of 33 kDa, or manganese-stabilizing protein, plays essential roles in stabilizing the Mn4CaO5 cluster and preventing destructive reduction of the cluster from exogenous reductants (Ghanotakis et al. 1984b; Kuwabara et al. 1985). PsbO along with other extrinsic subunits interacts with the extensive lumenal domains of D1 (PsbA), D2 (PsbD), CP43 (PsbC), and CP47 (PsbB) to optimize the structure for the water-oxidizing reaction, conceptually known as the oxygen-evolving complex, or oxygen-evolving center (OEC), or water-oxidizing center. The structure, interactions, and functions of PsbO have been extensively summarized in previous reviews (Enami et al. 2008; Bricker et al. 2012).

Fourier transform infrared (FTIR) spectroscopy marked a turning point in the functional analyses of extrinsic subunits in PS II (Ifuku and Noguchi 2016). Nagao et al. (2015) reported the FTIR measurements of S2-minus-S1 difference spectra using PS II core complexes from Thermosynechococcus elongatus reconstituted with its extrinsic proteins, PsbO, PsbV, and PsbU. Under a low-CaCl2 condition, PsbO is essential for inducing normal spectral changes during the S1→S2 transition, indicating its significant role in stabilizing the Mn4CaO5 cluster. Even at a high-CaCl2 concentration, protein conformational changes in the OEC are induced by removal of all extrinsic proteins, and these changes are largely recovered by the binding of PsbO, with further recovery observed by PsbV and then PsbU in a stepwise manner. Therefore, binding of PsbO mainly supports the conformation of the OEC in cyanobacterial PS II. The recoveries of the OEC protein conformation were consistent with those of oxygen-evolving activity recovered by the binding of respective extrinsic proteins (Shen and Inoue 1993), confirming structural coupling between the activity and protein conformation of the OEC.

The X-ray structures and theoretical calculations suggest that extrinsic proteins play another important role by forming access channels for substrate water to the Mn4CaO5 cluster and exit channels for the products (oxygen and protons) (Bondar and Dau 2012; Vassiliev et al. 2013; Linke and Ho 2014). These channels involve numerous water molecules forming hydrogen-bond networks, connecting the OEC towards the protein bulk surface at the lumenal side. As shown in Fig. 16.2, four channels have been proposed (Nagao et al. 2017c), and PsbO participates in the “Cl-1 path” starting from D1-Asp61 to PsbO through the Cl-1 site and the D1-Glu65/D2-Glu312/D1-Arg334 triad. Molecular dynamics (MD) simulation suggested that the absence of PsbO is correlated with the rapid loss of Cl-1 coordinated by D2-Lys317 (Guerra et al. 2018). At the end of the extended hydrogen-bond network, PsbO-Asp224 is part of a cluster of carboxylate groups, which may accept protons on the protein surface (Shutova et al. 2007; Lorch et al. 2015; Bommer et al. 2016). This Cl-1 path is thought to be a major pathway for protons or substrate water and is conserved in PS II-light-harvesting complex II (LHCII) structures from vascular plants, recently resolved by cryo-EM (Wei et al. 2016; Su et al. 2017). These results support the general importance of PsbO in the water-oxidizing reaction in oxyphototrophs.

Fig. 16.2.
figure 2

Hydrogen-bond networks around the OEC. The picture was drawn using a high-resolution PS II core structure (PDB ID: 3ARC). Wheat and cyan colored spheres represent water molecules and chloride ions, respectively. Arrows indicate putative proton exit channels, which were designated “Cl-1 path”, “O4 path”, “O1 path”, and “YZ-N298 path” in this work

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B.PsbV

PsbV was found in PS II core complexes of the cyanobacterium Thermosynechococcus vulcanus (Shen et al. 1992; Shen and Inoue 1993) and was subsequently observed in functional PS II complexes in various red-lineage oxyphototrophs but not in the green lineages (Enami et al. 2008). PsbV is a c-type cytochrome (Cyt), known as Cyt c550, with a molecular weight of approximately 15 kDa. The basic function of PsbV is to maintain Ca2+ and Cl− retention (Shen et al. 1992, 1995; Shen and Inoue 1993); this function is conserved in red algal and diatom PsbV (Enami et al. 1998; Nagao et al. 2010a). The redox function for Cyt c550 in PS II has been proposed, but has not been demonstrated experimentally (Guerrero et al. 2011). The physiological functions and structural organization of PsbV are summarized in previous reviews (Enami et al. 2008; Bricker et al. 2012).

As described above, the binding of PsbV together with PsbO to extrinsic protein-depleted PS II induces the recovery of protein conformations around the OEC in the S1→S2 transition of the Mn4CaO5 cluster (Nagao et al. 2015). PsbV induced the recovery of specific amide I bands, suggesting that it affects different regions of polypeptide chains from those affected by PsbO. In the high-resolution PS II structure (Umena et al. 2011), PsbV is associated with the C-terminal helix of the D2 protein including D2-Lys317, which is a ligand of Cl-1 ion, and with the C-terminal helix near D1-His332 and C-terminal loop between D1-His337 and D1-Asp342 of the D1 protein. Eventually, PsbV binding to the PS II cores appears to stabilize the structure of hydrogen-bond networks involved in the Cl-1 path (Fig. 16.2).

PsbV has also been suggested to be connected to the hydrogen-bond network near YZ. YZ forms a hydrogen-bonded triad with D1-His190 and D1-Asn298, which is connected to a hydrogen-bond network leading to PsbV through D1-Asn322 (YZ-N298 path) (Nagao et al. 2017c) (Fig. 16.2). MD simulations suggest that the tight hydrogen-bond network of this pathway is advantageous for proton transfer (Sakashita et al. 2017b; Ogata et al. 2013). Furthermore, a large water cluster interacting with the YZ/D1-His190/D1-Asn298 triad also forms another hydrogen-bond network through O1 and D1-Glu329, leading to PsbV (O1 path). MD simulation showed that this channel contains numerous mobile water molecules (Sakashita et al. 2017b). FTIR analysis of the D1-N298A mutant suggested that the YZ-N298 path functions as the proton-exit pathway in S2→S3 and S3→S0 transitions (Nagao et al. 2017c). These observations indicate that the protein conformational changes of the OEC upon association of PsbV with the cores induce a better structural arrangement for the proton-release events of S2→S3 and S3→S0 transitions. Thermoluminescence analysis suggested large contributions of PsbV to the S2→S3 and S3→S0 transitions (Shen and Inoue 1993). The further recovery of oxygen-evolving activity observed by PsbV binding plus PsbO compared to the activity by the sole binding of PsbO (Shen and Inoue 1993) may be attributed to the fine-tuning of the OEC structures, particularly the Cl-1 and YZ-N298 proton-exit channels. Based on these observations, together with the similar function and location of PsbV in the PS II cores of a primitive red alga Cyanidium caldarium (Enami et al. 1995, 1998; Uno et al. 2013; Ago et al. 2016), the contribution of PsbV to the OEC structure has been largely conserved in the red lineages. Notably, red algal PsbV plays a major role in the recovery of the secondary structure in the OEC to a larger extent than the cyanobacterial PsbV as revealed by FTIR measurements (Uno et al. 2013). Differences in the contribution to OEC recovery between cyanobacteria and red algae PsbV is an interesting point indicating the evolutional diversity of PS II.

C.PsbU

PsbU has a molecular weight of approximately 12 kDa and was first found in PS II core complexes isolated from T. vulcanus (Shen et al. 1992) followed by that in the red-lineage oxyphototrophs (Enami et al. 1995, 2008). The basic function of PsbU is similar to that of PsbV, i.e., PsbU binding maintains Ca2+ and Cl− retention in PS II of cyanobacteria, red algae, and diatoms (Shen and Inoue 1993; Enami et al. 1998; Nagao et al. 2010a). The physiological functions and structural organization of PsbU have been summarized in previous reviews (Enami et al. 2008; Bricker et al. 2012).

Similar to the functional examination of PsbV, the role of PsbU in the OEC structures was also investigated by FTIR measurements (Nagao et al. 2015). The binding of PsbU together with PsbO and PsbV to extrinsic protein-depleted PS II recovers the secondary structures of the polypeptide main chains in the OEC (Nagao et al. 2015). In the PS II structure, PsbU contacts with the C-terminal loop between D1-His337 and D1-Asp342 of the D1 protein, suggesting that PsbU also stabilizes the Cl-1 hydrogen-bond network. Additionally, the map of hydrogen-bond networks suggests that a distinct water chain in the O4 path is linked to PsbU (Takaoka et al. 2016) (Fig. 16.2). No studies have been conducted on the spectroscopic analyses of S-state transitions using site-directed mutants of amino acid residues along the O4 path; therefore, it is difficult to experimentally assess the contribution of the O4 path to the S-state transitions. However, theoretical calculation proposed that the O4 path functions as a proton-exit pathway in the S0→S1 transition (Saito et al. 2015; Sakashita et al. 2017b). These observations indicate that the proton-release event in the S0→S1 transition is promoted by protein conformational changes of the OEC induced by PsbU binding. The maximum recovery of oxygen-evolving activity by PsbU plus PsbO/V binding compared to the activity of PsbO/V binding (Shen and Inoue 1993) may be related with the fine-tuning of OEC structures, particularly the Cl-1 and O4 proton-exit channels. Given the similar function and location of PsbU in the PS II cores of a primitive red alga C. caldarium (Enami et al. 1995, 1998; Uno et al. 2013; Ago et al. 2016) with that of the cyanobacterial PS II, the contribution of PsbU to the OEC structure is largely conserved in the red lineages.

D.PsbP

PsbP, also known as OEC23 or OEC24 because of its apparent molecular mass of 23–24 kDa, is specifically found in green plant PS II (Åkerlund et al. 1982). Like PsbV and PsbU in cyanobacterial PS II, PsbP maintains the required levels of Ca2+ and Cl− in PS II of green lineage organisms (Ghanotakis et al. 1984a; Miyao and Murata 1986). A lack of PsbP largely diminishes photoautotrophy in Arabidopsis thaliana (Yi et al. 2007) and Nicotiana tabacum (Ifuku et al. 2005b). Therefore, the development of the PsbP protein as an extrinsic subunit is a crucial event in PS II function during evolution from the red lineage to green lineage organisms. The structure and function of PsbP have been intensively reviewed previously (Ifuku et al. 2008, 2011; Ifuku and Noguchi 2016; Roose et al. 2016).

The importance of PsbP in structural coupling with the OEC has been investigated by in vitro reconstitution (Ifuku and Sato 2001; Ifuku et al. 2005a) and FTIR analysis: PS II membranes depleted of PsbP and PsbQ by NaCl washing showed clear changes in amide I bands in S2-minus-S1 FTIR difference spectra, reflecting conformational changes in polypeptide main chains (Tomita et al. 2009). Unlike cyanobacterial PS II, further depletion of PsbO did not induce additional changes, and the original amide I features were recovered by rebinding of PsbP to NaCl-washed PS II membranes. These results indicate that the PsbP protein mainly contributes to the protein conformation during the S1→S2 transition in green plant PS II. Various mutations or truncations have been introduced in the PsbP structure and their effects were examined by FTIR (Tomita et al. 2009; Kakiuchi et al. 2012; Ido et al. 2012; Nishimura et al. 2014; Ifuku and Noguchi 2016), and the results obtained revealed the manner of interactions of PsbP with PS II core subunits. Subsequent FTIR measurement of NO3−-substituted PS II membranes indicated that PsbP binding perturbs the protein conformation around Cl− ion(s) in the OEC (Kondo and Noguchi 2018). Chemical cross-linking data and cryo-EM structures suggested that PsbP forms multiple interactions with the D1, D2, and CP43 subunits, indicating that both the Cl-1 and Cl-2 sites are affected by PsbP binding (Ido et al. 2012, 2014). Notably, the O1 path and the O4-PsbU path in cyanobacterial PS II appear to be structurally conserved as the channels proceeding along PsbP toward the protein bulk surface in the cryo-EM structure of plant PS II (Sakashita et al. 2017a). Therefore, binding of PsbP may play a crucial role in maintaining the hydrogen-bond network both for H+ export and water uptake in the OEC.

In addition to optimizing the water-oxidizing reaction in PS II, it has been reported that a lack of PsbP perturbs electron transport at the reducing side of PS II (Ido et al. 2009). Removal of PsbP and PsbQ with 2 M NaCl significantly slowed the rate of electron transfer from QA− to QB (Roose et al. 2010). However, the mechanism of this trans-membrane effect remains unknown. Related to this, Nishimura et al. (2016) reported that N-terminal amino-acid residues of PsbP interacting with PsbE of Cyt b559 modulate the redox potential of the heme of Cyt b559 located at the stroma side. Even a synthetic pN15 peptide consisting of the 15 N-terminal residues of PsbP altered the structure of Cyt b559 in a transmembrane manner and triggered the redox potential change of the heme in Cyt b559, converting it into its high-potential form (Nishimura et al. 2016). The interaction of PsbP with Cyt b559 was confirmed by the recently determined cryo-EM structure (Wei et al. 2016) (Fig. 16.3). Cyt b559 is known to be involved in alternative or secondary electron flow to suppress photoinhibition: its high potential form donates electrons to P680+, whereas its low potential form oxidizes the reducing side of PS II (Takagi et al. 2019). Interestingly, the pN15 peptide alone reduces the oxygen-evolving activity of PS II and inhibits the S1→S2 transition in the OEC as suggested by thermoluminescence and FTIR analyses (Nishimura et al. 2016). These data suggest that PsbP forms multiple interactions with PS II and each has a distinct role in regulating electron transfer within PS II. Therefore, binding of PsbP is important for balancing the oxidizing and reducing reactions of PS II in a trans-membrane manner.

Fig. 16.3.
figure 3

Interactions of PsbP with PS II core subunits in spinach PS II structure (PDB ID: 3JCU). CP43, cyan; CP47, pink; PsbE, red; PsbF, orange; PsbP, green

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E.PsbQ

PsbQ, previously referred to as OEC16 or OEC18 because of its apparent molecular mass of 16–18 kDa, was originally found in green plant PS II (Åkerlund et al. 1982). Release-reconstitution experiments showed that a lack of PsbQ reduces the oxygen-evolving activity of PS II membranes under low Cl− conditions (Akabori et al. 1984; Miyao and Murata 1985), whereas PsbQ is not essential for photoautotrophic growth in both A. thaliana and N. tabacum (Ifuku et al. 2005b; Yi et al. 2006). Cryo-EM structures suggested that PsbQ is located at the interface between CP43 and the monomer-type light-harvesting protein CP26 (Wei et al. 2016; Su et al. 2017). This location is consistent with the fact that PsbQ is required for stabilizing the PS II-LHCII supercomplex (Yi et al. 2006; Allahverdiyeva et al. 2013). FTIR analysis did not suggest a specific function for PsbQ; however, binding of PsbQ partly restored the function of the ∆15-PsbP protein, an N-terminal truncated mutant of PsbP lacking the function to induce conformational changes and activate oxygen evolution of OEC (Kakiuchi et al. 2012). Therefore, PsbQ may play an auxiliary role in supporting PsbP binding and function in vascular plants. The interaction between PsbP and PsbQ was confirmed in the cryo-EM structure of the plant PS II-LHCII supercomplex (Wei et al. 2016). Modification of PsbP binding in the absence of PsbQ was also suggested by pulsed electron-electron double resonance analysis using spin-labeled proteins (Asada et al. 2018).

PsbQ′ was found in the PS II core complexes isolated from the primitive red alga C. caldarium (Enami et al. 1995, 1998) and showed low sequence similarity to PsbQ in vascular plants with a molecular weight of approximately 20 kDa (Ohta et al. 2003). This protein was found in highly purified red algal PS II cores (Adachi et al. 2009) and its structure was revealed by the X-ray crystal structure of the red algal PS II core complexes (Ago et al. 2016). The structure of PsbQ′ is composed of a four-helix bundle domain, which is similar to the structure of plant PsbQ (Calderone et al. 2003; Balsera et al. 2005). PsbQ′ is located near CP43, similar to the position of plant PsbQ (Wei et al. 2016; Su et al. 2017). Diatom PS II core also possesses PsbQ′ with a low sequence similarity (Nagao et al. 2007; Okumura et al. 2008; Nagao et al. 2010b), indicating that the diatom PsbQ′ is also located near CP43. Release-reconstitution experiments in red algal PS II showed that rebinding of only PsbQ′ without other extrinsic subunits showed no recovery of oxygen-evolving activity, whereas PsbQ′ significantly contributed to the full binding of PsbV and PsbU (Enami et al. 1998). Similarly, the complete association of diatom PsbV and PsbU also requires the binding of PsbQ′ (Nagao et al. 2010a). Interestingly, conformational recovery of the OEC in the S1→S2 transition of the Mn4CaO5 cluster was not observed in FTIR measurements of the binding of PsbQ′ to PS II (Uno et al. 2013). This is consistent with the binding site of PsbQ′, which is rather far from the Mn4CaO5 cluster in the PS II structure compared to those of PsbO, PsbV, and PsbU (Ago et al. 2016).

Genome editing has enabled deletion of PsbQ′ genes in a primitive red alga Cyanidioschyzon merolae (Zienkiewicz et al. 2018). There are two PsbQ′ genes encoded in the genome of C. merolae, and each ΔpsbQ′1 and ΔpsbQ′2 mutant showed a lower growth rate and oxygen-evolving activity compared to wild-type cells. This is likely because of the partial lack of PsbV from isolated PS II cores; therefore, impaired photosynthetic ability may be an indirect effect of PsbQ′ on oxygen-evolving reactions, consistent with the in vitro release-reconstitution experiments (Enami et al. 1998; Uno et al. 2013). The main role of PsbQ′ in the oxygen-evolving reactions appears to maintain the protein environments for full binding of PsbV and PsbU. This function of PsbQ′ in red algae is analogous to that of PsbQ for PsbP in vascular plants (Kakiuchi et al. 2012). Notably, CyanoQ also stabilizes the binding of PsbV in cyanobacterial PS II (Summerfield et al. 2005; Kashino et al. 2006), although a different binding manner has been proposed for CyanoQ (Liu et al. 2014, 2015).

Enami and coworkers examined the functional preservations of extrinsic subunits by comparing the recovery of oxygen-evolving activity using reconstituted PS II samples, particularly in cross-reconstitution experiments, in which, red-algal and plant PsbO were bound to spinach and red-algal PS II, respectively (Enami et al. 2000, 2003; Suzuki et al. 2005). Their results suggested that PsbQ′ is exchangeable with PsbQ in plant PS II, whereas PsbQ is not exchangeable with PsbQ′ in red algal PS II (Enami et al. 2000). Therefore, binding and function of PsbQ and PsbQ′ are preserved to some extent in eukaryotic oxyphototrophs. It has also been reported that red-algal PsbQ′ regulates the redox-potential of QA in cyanobacterial PS II (Yamada et al. 2018). Because such a transmembrane effect has not been reported for PsbQ binding, there may be some functional differences between these proteins.

F.Psb31

Psb31 was identified in PS II complexes isolated from the marine diatom Chaetoceros gracilis (Nagao et al. 2007, 2010b). This protein is encoded in the nuclear genome with a molecular weight of approximately 13 kDa and has a homologue in numerous chromophyte algae and red algae, but not in green-lineage oxyphototrophs (Okumura et al. 2008). The electrostatic interactions of Psb31 with PS II cores, independent of the other extrinsic subunits, were observed by cross-linking and release-reconstitution experiments (Nagao et al. 2010a). Cross-linking studies further suggested that the binding partners of Psb31 are PsbE and/or PsbH according to mass spectrometry and immunological detection (Okumura et al. 2008) (Fig. 16.4). The structure of Psb31 is composed of a four-helix bundle domain including the N-terminus, which is similar to the structures of PsbQ and PsbQ′, and a flexible C-terminal domain as revealed by X-ray crystallography (Nagao et al. 2013). Chemical modification analysis showed that the positively charged amino acids on the Psb31 surface, but not the negative charges, contribute to electrostatic interactions with the cores (Nagao et al. 2017b). Because Lys residues are mainly changed to non-charged Lys by chemical modifications, each of the 10 Lys residues in Psb31 was replaced with Ala (Nagao et al. 2017a, b). The study revealed that involvement of the 10 Lys in the electrostatic interactions can be classified into three groups: Lys39/Lys54/Lys57 associate strongly with the PS II core, Lys33/Lys56/Lys69 associate moderately with the core, and Lys24/Lys76/Lys80/Lys117 do not interact with the core directly. The important Lys residues for PS II binding are well-conserved among diatom species but not among other chromophytes, suggesting high conservation of the Lys residues at the binding sites only in the diatom species.

Fig. 16.4.
figure 4

Putative model of diatom PS II core complexes. The model was built using red algal PS II core (PDB ID: 4YUU) and diatom Psb31 (PDB ID: 4K7B). PsbO, red; PsbQ′, pink; PsbV, blue; PsbU, yellow; PsbH and PsbE, black; and Psb31, orange

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Psb31 plays a unique role in oxygen-evolving reactions. Release-reconstitution experiments showed that binding of Psb31 alone induces the recovery of oxygen-evolving activity (Nagao et al. 2010a). This recovery is a unique feature of diatom PS II, as the recovery of oxygen-evolutoin was not observed in extrinsic subunit-reconstituted PS II samples without PsbO from cyanobacterial, red algae, green algae, and plants (Enami et al. 2008). In chemical modification and site-directed mutagenesis studies, we found that the flexible C-terminal domain of Psb31 may be elongated near the OEC, resulting in the regulation of oxygen-evolving reactions (Nagao et al. 2017a, b). The structure of diatom PS II in complex with its light-harvesting antenna, fucoxanthin Chl a/c-binding proteins (FCPII), has been analyzed by cryo-EM recently (Nagao et al. 2019); however, Psb31 was not visible in this structure. Detailed function of Psb31 in the oxygen-evolving reaction remains unclear, and thus further structural, FTIR, and genetic studies of Psb31 are needed.

IV.Molecular Evolution of PsbP and PsbQ Family Proteins

During the acquisition of PsbP and PsbQ in PS II of the green lineage organisms, gene duplication likely occurred in parallel, as multiple homologs for PsbP and PsbQ are found in the genome of green plants. Two PsbP-like proteins (PPL1, 2), seven PsbP-domain proteins (PPD1–7), and three PsbQ-like proteins (PQL1–3) have been found in A. thaliana and Oriza sativa (Ifuku et al. 2010). Their structures and functions were summarized elsewhere (Ifuku et al. 2008, 2010, 2011; Bricker et al. 2013; Ifuku 2014). Briefly, PPL1 is most closely related to CyanoP, while PsbP and the other PsbP homologs are paralogs of PPL1. PPL and PPD proteins should have distinct functions from PsbP, as no PPL and PPD proteins can compensate for the loss of PsbP (Ifuku et al. 2005b; Yi et al. 2009). Genetic studies using A. thaliana mutants reported that PPL1 functions in efficient PS II repair, while PPL2 is a subunit of the chloroplast NADH dehydrogenase-like (NDH) complex involved in photosystem I (PSI) cyclic electron transport and chlororespiration (Ishihara et al. 2007). PPD1 protein is required for PSI accumulation (Liu et al. 2012; Roose et al. 2014). PPD2 is involved in singlet oxygen-dependent signaling in Chlamydomonas reinhartdii (Brzezowski et al. 2012). PPD5 was linked to plant development via the plant hormone strigolactone (Roose et al. 2011). PPD6 was identified as a putative lumenal target of thioredoxin, but its function is currently unknown (Hall et al. 2012). All three PQL proteins are required for the activity of the chloroplast NDH complex in A. thaliana (Yabuta et al. 2010). These facts suggest that functional diversification also occurred during evolution of the PsbP and PsbQ family.

Phylogenetic analysis based on structural modeling suggested that PsbP homologs in green plants are classified into eight families from Family A to H (Sato 2010). Family A contains PsbP in PS II, Family B – G contains PPD proteins, and Family H consists of subfamilies H1 and H2, including PPL proteins in green plants and CyanoP-type protein in red algae and cyanobacteria, respectively. The H1 and H2 families are very similar in structure and are the simplest of all PsbP families, suggesting that the PsbP superfamily originated from CyanoP and that the PPL protein likely emerged first. Functional and sequence similarity suggest that PPL1, but not PPL2, is a direct descendant of CyanoP, although PPL1 appears to have developed its specific function in the PS II assembly (Che et al. 2020). After branching of PPL1, various families including PsbP, PPL2, and PPD diverged. Red algae and diatom contain genes for several PsbP family proteins in addition to H2-type PsbP (Sato 2010). This suggests that if we do not consider horizontal gene transfer, a small degree of diversification of the PsbP family likely also occurred in the red lineage; however, the A-type PsbP for PS II is developed at a later stage.

Unlike the PsbP protein family, phylogenetic analysis indicated that PsbQ and PsbQ′ are direct descendants of CyanoQ (De Las Rivas et al. 2004; De Las Rivas and Roman 2005). As described above, green algal PsbQ is more closely related to red algal PsbQ′ than to vascular plant PsbQ, suggesting that functional differentiation of PsbQ homologs continued after the red and green lineages had branched. Although sequence conservation among PsbQ family proteins is very low, the four-helices bundled structure was largely preserved. The dispensability of PsbQ family proteins for photoautotrophy may facilitate a high evolutionary rate, resulting in lower sequence similarity among the PsbQ protein family and subsequent diversification of the PQL protein in the chloroplast NDH complex (Yabuta et al. 2010). Interestingly, PPL2 and PQLs are found specifically in angiosperms that have chloroplast NDH activity but not in mosses and liverworts with other NDH genes (Ueda et al. 2012). Therefore, diversification of NDH-type PPL2 and PQLs likely occurred rather recently in evolution.

V.Concluding Remarks

Figure 16.5 shows changes in PS II extrinsic proteins occurred during the evolution of oxyphototrophs. As described in this chapter, structural, biochemical, biophysical, and genetic studies are needed to reconsider historical concepts regarding the evolution of the extrinsic subunits of PS II. PsbO clearly exerts the important function of stabilizing the Mn4CaO5 cluster in all species; however, its function has not been fully conserved during evolution. Particularly, FTIR studies suggested that the function of regulating the OEC conformation was transferred to PsbP and PsbV in the green and red lineage organisms, respectively. The cryo-EM structure of plant PS II-LHCII complexes indicates that PsbP replaced the function of PsbV and PsbU to maintain the hydrogen-bond network both for H+ export and water uptake in the OEC. The binding of PsbP also regulates electron transfer at the reducing side of PS II from the thylakoid lumenal side in a long distance, trans-membrane manner. The function and manner of interaction of PsbQ (PsbQ′) with the PS II core appear to be largely conserved between the green and red lineage organisms. Therefore, the development of PsbP is a crucial event at the branching point of the green and red lineages, involving gene duplication and functional diversification. Unlike other extrinsic subunits, only the gene for PsbV is encoded in the plastid genome in the red algae and diatom, indicating the difficulty in replacing it. A recent study reported that most isolates of the marine cyanobacterium Prochlorococcus naturally do not contain PsbV and PsbU, but they contain PsbO and evolve oxygen (Partensky et al. 2018); however, the mechanism compensating for their absence remains unknown. One possibility is that CyanoP, commonly found in marine picocyanobacteria, functions as a major extrinsic subunit in these species, which may be relevant to the exchange process of PS II extrinsic subunits. Alternatively, some Prochlorococcus-specific proteins may replace PsbU and PsbV. In fact, diatom has developed Psb31 as a unique PS II extrinsic subunit, which partially substitutes for the function of PsbO. It is expected that issues will be solved in the future studies.

Fig. 16.5.
figure 5

Evolutionary changes in extrinsic proteins of PS II. Only PsbO (O) has been retained in all oxyphototrophs. In the green lineage, PsbV (V) and PsbU (U) were lost and replaced with PsbP (P) which evolved from CyanoP (cP). In red algae, PsbQ′ (Q′) developed from CyanoQ. PsbQ′ is also used in green algae and subsequently evolved to PsbQ in vascular plants. CyanoP/Q are reported to function as assembly factors for PS II. During the molecular evolution of PsbP and PsbQ, the number of PsbP and PsbQ homologs, such as PsbP-like (PPL), PsbP-domain (PPD), and PsbQ-like (PQL) proteins, were developed by gene duplication and functional diversification. Psb31 (31) has arisen in diatoms. These models are designed to show differences in the PS II extrinsic subunit but do not show the exact location and interaction of these extrinsic subunits within PS II

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Acknowledgements

The authors gratefully acknowledge the financial support from MEXT KAKENHI 16H06554 and JSPS KAKENHI 16K07690 to KI.