The Roles of Cytochrome b559 in Assembly and Photoprotection of Photosystem II Revealed by Site-Directed Mutagenesis Studies

Cytochrome b559 (Cyt b559) is one of the essential components of the Photosystem II reaction center (PSII). Despite recent accomplishments in understanding the structure and function of PSII, the exact physiological function of Cyt b559 remains unclear. Cyt b559 is not involved in the primary electron transfer pathway in PSII but may participate in secondary electron transfer pathways that protect PSII against photoinhibition. Site-directed mutagenesis studies combined with spectroscopic and functional analysis have been used to characterize Cyt b559 mutant strains and their mutant PSII complex in higher plants, green algae, and cyanobacteria. These integrated studies have provided important in vivo evidence for possible physiological roles of Cyt b559 in the assembly and stability of PSII, protecting PSII against photoinhibition, and modulating photosynthetic light harvesting. This mini-review presents an overview of recent important progress in site-directed mutagenesis studies of Cyt b559 and implications for revealing the physiological functions of Cyt b559 in PSII.


INTRODUCTION
Cytochrome b 559 is one of the essential components of Photosystem II in all oxygenic photosynthetic organisms (Whitmarsh and Pakrasi, 1996;Stewart and Brudvig, 1998;Guskov et al., 2009;Umena et al., 2011). Cyt b 559 is a heme-bridged heterodimer protein comprising one αand one βsubunit (encoded by the psbE and psbF genes) of 9 and 4 kDa, respectively (see Figure 1). Each subunit provides a His ligand (His-22 residue of the αor β-subunit of Cyt b 559 in Synechocystis sp. PCC 6803, corresponding to His-23 residue of αor His-24 residue of the β-subunit of Cyt b 559 in Thermosynechococcus elongatus) for the non-covalently bound heme, which is located near the stromal side of PSII. In addition, Cyt b 559 has different redox potential forms depending on the type of PSII preparations and treatments: a HP form with a midpoint redox potential of about +400 mV, an IP form of about +200 mV, and a LP form with a midpoint redox potential of about 0-80 mV (Stewart and Brudvig, 1998;Roncel et al., 2001 and references therein). In intact PSII preparations, Cyt b 559 is mostly in the reduced HP form under FIGURE 1 | Structural model of the cytochrome b 559 in Photosystem II. Amino acid residues of cytochrome b 559 targeted by recent site-directed mutagenesis work are labeled by using amino acid sequences of cytochrome b 559 from Thermosynechococcus elongatus. The figure was created using PyMol and the PDB file 4UB8. ambient conditions. In inactive or less intact PSII preparations, Cyt b 559 is typically in the LP or IP form and mostly oxidized (presumably by molecular oxygen) under ambient conditions (Barber and De Las Rivas, 1993;Poulson et al., 1995;Pospíšil et al., 2006).
Several studies have proposed that Cyt b 559 participates in secondary electron transfer pathways that protect PSII against photoinhibition (see Figure 2; Heber et al., 1979;Falkowski et al., 1986;Thompson and Brudvig, 1988;Barber and De Las Rivas, 1993;Poulson et al., 1995;Magnuson et al., 1999;Faller et al., 2001;Tracewell and Brudvig, 2008 and references therein). In these models, the HP form of Cyt b 559 is thought to donate its electron, via a β-carotene molecule (Car D2 ), to reduce highly oxidized chlorophyll radicals in PSII under donor-side photoinhibitory conditions (e.g., the oxygen-evolving complex is impaired or under assembly). Oxidized Cyt b 559 may accept an electron from the acceptor side of PSII (Q B − , or reduced PQH 2 from the pool), thus forming a cyclic pathway of electron transfer within PSII. On the other hand, when the electron transfer on the acceptor side of PSII is inhibited (e.g., under high-light conditions), the oxidized Cyt b 559 might accept an electron from the acceptor side of PSII to prevent the formation of damaging singlet oxygen species (Nedbal et al., 1992;Vass et al., 1992;Barber and De Las Rivas, 1993;Bondarava et al., 2010). In addition, several different enzymatic functions of Cyt b 559 have been proposed, such as superoxide dismutase (Ananyev et al., 1994) and PQH 2 oxidase in intact PSII Strzałka, 1999, Kruk andStrzalka, 2001;Bondarava et al., 2003Bondarava et al., , 2010 and superoxide oxidase and reductase in tris-washed PSII (Tiwari and Pospísil, 2009;Pospíšil, 2011). Moreover, a novel quinone-binding site (Q C ) was identified close (about 15 Å) to the heme of Cyt b 559 in the 2.9-Å PSII crystal structure from T. elongatus (Guskov et al., 2009). The occupancy of this Q C site with PQ (or PQH 2 ) has been proposed to modulate the redox equilibration between Cyt b 559 and the PQ pool (Kaminskaya et al., 2006(Kaminskaya et al., , 2007Kaminskaya and Shuvalov, 2013) or be involved in the exchange of PQ on the Q B site from the pool (Guskov et al., 2009). However, the Q C site was not detected in the more recent 1.9-Å PSII crystal structure (Umena et al., 2011). Despite the recent remarkable progress in understanding the structure and function of PSII, the exact function of Cyt b 559 in PSII remains unclear.
This mini-review gives an overview of important progress in recent site-directed mutagenesis studies performed to reveal the physiological function(s) of Cyt b 559 in PSII. More comprehensive reviews on the structure and functions of Cyt b 559 are available (Whitmarsh and Pakrasi, 1996;Stewart and Brudvig, 1998;Faller et al., 2005;Pospíšil, 2011;Shinopoulos and Brudvig, 2012).

FUNCTION IN ASSEMBLY AND STABILITY OF PSII REACTION CENTERS
Prior mutagenesis studies with Synechocystis sp. PCC 6803, Chlamydomonas reinhardtii or Nicotiana tabacum showed no FIGURE 2 | A simplified scheme for electron transfer pathways within Photosystem II reaction centers. Primary electron transfer pathway (bold solid lines with arrows) and possible secondary electron transfer pathways involving cytochrome b 559 (dotted line with arrows) are shown. The scheme is modified from Whitmarsh and Pakrasi (1996). stable PSII reaction centers assembled in the absence of either Cyt b 559 subunit (Pakrasi et al., 1988(Pakrasi et al., , 1989(Pakrasi et al., , 1990Morais et al., 1998;Swiatek et al., 2003;Suorsa et al., 2004). Several authors proposed that Cyt b 559 plays an important structural role, such as being a nucleating factor, during the early stage of PSII assembly (Pakrasi et al., 1988;Morais et al., 1998;Komenda et al., 2004).
In addition, mutagenesis studies with Synechocystis sp. PCC 6803 showed that substituting either of the heme axial ligands (His22 of the α-subunit or His22 of the β-subunit of Cyt b 559 in Synechocystis sp. PCC 6803) with Leu, Met, Glu, Gln, Tyr, Lys, Arg, or Cys abolished the photoautotrophic growth and severely diminished the assembly or stability of PSII in the mutant cells, except for H22Kα mutant cells, which were able to grow photoautotrophically and accumulated stable PSII reaction centers (∼81% as compared with wild-type cells; Pakrasi et al., 1991;Hung et al., 2007Hung et al., , 2010. Electron paramagnetic resonance results indicated the displacement of one of the two axial ligands to the heme of Cyt b 559 in H22Kα mutant reaction centers, at least in isolated reaction centers (Hung et al., 2010). In addition, H22Kα and Y18Sα (corresponding to Y19Sα in T. elongatus) in mutant PSII core complexes contained predominately the LP form of Cyt b 559 . The findings support the concept that the redox properties of Cyt b 559 are strongly influenced by the hydrophobicity and ligation environment of the heme (Krishtalik et al., 1993;Gadjieva et al., 1999;Roncel et al., 2001;Pospíšil and Tiwari, 2010).
Spectroscopic and functional characterizations of the cyanobacterium Synechocystis sp. PCC 6803 with mutation of charged residues on the cytoplasmic side of Cyt b 559 in PSII have been reported (Chiu et al., 2013). All mutant cells grew photoautotrophically and assembled stable PSII. However, R7Eα, R17Eα, and R17Lβ mutant cells grew significantly slower and were more susceptible to photoinhibition as compared with wild-type cells. In addition, the PSII core complexes from R7Eα and R17Lβ cells contained predominantly the LP form of Cyt b 559 . Electron paramagnetic resonance results indicated the displacement of one of the two axial ligands to the heme of Cyt b 559 in the reaction centers of the R7Eα and R17Lβ mutants. In recent PSII crystal structural models (Guskov et al., 2009;Umena et al., 2011), the side chains of these Arg residues of Cyt b 559 (corresponding to Arg8 and Arg18 residues of the α-subunit and Arg19 residue of the β-subunit of Cyt b 559 in T. elongatus) are in close contact with the heme propionates of Cyt b 559 (see Figure 1). Thus, the electrostatic interactions between these Arg residues and the heme propionates of Cyt b 559 may affect the ligation structure and redox properties of the heme in Cyt b 559 (Chiu et al., 2013).
Furthermore, mutagenesis studies of C. reinhardtii showed that the H23Yα, H23Mα, and H23Cα mutant cells were unable to grow photoautotrophically, were sensitive to photoinhibition, accumulated 10-20% of the PSII (compared to wild-type cells), and contained a disrupted heme pocket while still retaining significant O 2 evolution activity (Morais et al., 2001;Hamilton et al., 2014). Thus, the heme of Cyt b 559 was not required for photosynthetic water oxidation by PSII (Morais et al., 2001). A recent study also presented evidence to ascribe the photoinhibition phenotype of H23Cα mutant cells to a faster rate of photodamage and an impaired PSII repair cycle (Hamilton et al., 2014). Hence, Cyt b 559 may play important roles in the assembly, repair and maintenance of the PSII complex in vivo.
In the other recent mutant study of T. elongatus that took advantage of the robustness of the PSII variant with PsbA3 as the D1 subunit, the four constructed Cyt b 559 mutants (H23Aα, H23Mα, Y19Fα, and T26Pα) grew photoautotrophically (T. elongatus is an obligate photoautotroph; Sugiura et al., 2015). Although the H23Aα and H23Mα mutants assembled only an apo-Cyt b 559 , the steady-state level of active PSII was comparable to that in the wild-type control. The results suggest that the heme has no structural role in the assembly of PSII in the presence of αand β-subunits of Cyt b 559 . This finding is in strong contrast to the Synechocystis sp. PCC 6803 mutant showing that proper coordination of the heme cofactor in Cyt b 559 is important to the assembly or stability of PSII (Pakrasi et al., 1991;Hung et al., 2007). In addition, Cyt b 559 mutant cells of T. elongatus showed no correlation between the rate of photoinhibition and the redox potential of the heme. However, the recovery of the oxygen-evolving activity of PSII after photoinhibition was significantly slower in these mutant cells. PsbA3 is the D1 isoform expressed in T. elongatus under high-light conditions (Nakamura et al., 2002;Kós et al., 2008). The high-light D1 isoform in cyanobacteria has a Glu instead of a Gln residue (for the low-light D1 isoform) at position 130 in the D1 protein sequence (for a review, see Mulo et al., 2009) and this Glu residue forms hydrogen-bonding interactions with pheophytinD1 (Dorlet et al., 2001;Shibuya et al., 2010). Cyanobacterial PSIIs with the high-light D1 isoform showed increased photo-tolerance and accelerated non-radiative charge recombination (Tichy et al., 2003). This phototolerant property has been attributed to a photoprotection mechanism involving the redox potential of pheophytinD1, which enhances the probability for non-radiative recombination of the singlet radical pair and prevents the formation of potentially damaging 3 P680 and singlet oxygen species (Vass and Cser, 2009;Sugiura et al., 2014). Further investigation could determine whether PsbA3 may compensate the photoprotective function of Cyt b 559 in the assembly and stability of PSII in these Cyt b 559 mutant cells.

FUNCTION IN PROTECTING PSII AGAINST PHOTOINHIBITION
Numerous site-directed mutagenesis studies have investigated the role of Cyt b 559 in protecting PSII against photoinhibition under high light. In the photoprotective models, oxidized Cyt b 559 may accept an electron from the acceptor side of PSII (Q B − , Q C , or reduced PQH 2 from the pool). Previous mutant studies of dark-adapted leaves of the F26Sβ Cyt b 559 tobacco mutant showed a greatly reduced PQ pool, conversion of the redox-potential form of Cyt b 559 to the LP form, and photosynthetic activities sensitive to high light (Bondarava et al., 2003(Bondarava et al., , 2010. In addition, R7Eα and R17Lβ Cyt b 559 mutant cells of Synechocystis sp. PCC 6803 and R18Sα Cyt b 559 mutant cells of T. elongatus showed markedly reduced PQ pools, altered redox-potential forms of Cyt b 559 , and high susceptibility to light stress (Chiu et al., 2013;Guerrero et al., 2014). A defect in PQH 2 oxidase activity of Cyt b 559 due to altered redoxpotential forms of Cyt b 559 in these mutant strains could explain their high susceptibility to strong light and greatly reduced PQ pools. Therefore, Cyt b 559 may function as a PQH 2 oxidase to keep the PQ pool and the acceptor side of PSII oxidized in the dark, thereby preventing PSII from acceptor-side photoinhibition Strzałka, 1999, Kruk andStrzalka, 2001;Bondarava et al., 2003Bondarava et al., , 2010. However, one recent study reported no defect in PQH 2 oxidation in the dark in H23Cα mutant cells of C. reinhardtii, even though H23Cα mutant cells contained a disrupted heme-binding pocket of Cyt b 559 and were sensitive to photoinhibition (Hamilton et al., 2014). Further studies are required to clarify this discrepancy. In addition, study of the 2.9-Å resolution PSII crystal structure reported the binding of Q C at a hydrophobic cavity near Cyt b 559 (Guskov et al., 2009). Several spectroscopic studies have provided evidence that the occupancy of the Q C site by PQ (or PQH 2 ) may modulate the redox potential of Cyt b 559 and mediate the redox equilibration between Cyt b 559 and the PQ pool (Kaminskaya et al., 2006(Kaminskaya et al., , 2007Kaminskaya and Shuvalov, 2013). A recent study provided evidence of a possible one-electron oxidation of PQH 2 by Cyt b 559 at the Q C site involved in the formation of a superoxide anion radical (Yadav et al., 2014). The above results are consistent with Cyt b 559 possibly accepting an electron from PQH 2 via the Q C site in PSII. However, the Q C site was not present in the more recent 1.9-Å PSII crystal structure (Umena et al., 2011). Further investigations are needed to solve this important issue.
Spectroscopic and functional characterization of the H22Kα and Y18Sα Cyt b 559 mutant cells of Synechocystis sp. PCC 6803 showed that both mutants have functional PSII and exhibited the normal period-four oscillation in oxygen yield (Hung et al., 2010). However, both mutants were more susceptible to photoinhibition than the wild type under high-light conditions. In addition, PSII core complexes from the H22Kα and Y18Sα mutants predominantly contained the oxidized LP form of Cyt b 559 (∼79 and 86%, respectively). A defect in the photoprotective function of Cyt b 559 in H22Kα and Y18Sα mutants could explain their high susceptibility to strong light. Furthermore, H22Kα and Y18Sα Cyt b 559 mutants in a D1-D170A genetic background that prevented assembly of the Mn cluster showed almost completely abolished accumulation of PSII even under normal-growth-light conditions. The data support an important redox role of Cyt b 559 in protecting PSII under donor-side photoinhibition conditions (Hung et al., 2010).
Furthermore, under low light, the H23Cα Cyt b 559 mutant showed more rapid assembly of the Mn 4 CaO 5 cluster than the wild-type control in C. reinhardtii (Hamilton et al., 2014). However, the photoactivation of oxygen-evolving PSII in the H23Cα mutant was inhibited under high light. The results suggest that reduction of P680 + via cyclic electron flow within PSII (via Cyt b 559 and Car D2 , Figure 2) may compete with the photoactivation process and provides important in vivo evidence for a photoprotective role of Cyt b 559 in photo-assembly of the Mn 4 CaO 5 cluster in PSII (Hamilton et al., 2014).
A recent mutant study involving T. elongatus showed that the midpoint redox potential of the HP form of Cyt b 559 was significantly destabilized (converted to the IP form) in mutant PSII core complexes of Cyt b 559 mutant strains (I14Aα, I14Sα, R18Sα, I27Aα, I27Tα, and F32Yβ;Guerrero et al., 2014). When the oxygen-evolving complex was inactive, the yield of darkreduction of Cyt b 559 was lower and the kinetics was slower in the R18Sα mutant than in wild-type cells. The results support the concept that the HP form of Cyt b 559 may function as a PQH 2 oxidase to keep the PQ pool oxidized and also as an electron reservoir for the cyclic electron flow within PSII when the donor-side of PSII is impaired (Guerrero et al., 2014).
Moreover, a previous spectroscopic study showed that different spectral forms of Car were oxidized in PSII samples containing different redox forms of Cyt b 559 (Tracewell and Brudvig, 2008). The authors proposed that the quenching properties of PSII may be controlled by the redox form of Cyt b 559 by modulating the different type of oxidized Car species (radical cation or neutral radical) formed in PSII. Future study could investigate the quenching properties of PSII in the wild type versus Cyt b 559 mutant strains of cyanobacteria with different redox forms of Cyt b 559 to validate this proposal.

EFFECTS ON PHOTOSYNTHETIC LIGHT HARVESTING
Recent mutant studies revealed a novel role of Cyt b 559 in modulating photosynthetic light harvesting in PSII reaction centers. A spontaneously generated mutant from Synechocystis sp. PCC 6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 μM DCMU carried an Arg7 to Leu mutation on the alpha-subunit of Cyt b 559 in PSII (Chiu et al., 2009). Results of 77-K fluorescence and roomtemperature chlorophyll a fluorescence spectra indicated that the energy transfer from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. In addition, the cytoplasmic side of Cyt b 559 is located within the predicted contact sites in PSII for the APC core complex of the phycobilisome (Barber et al., 2003). The Arg7 to Leu mutation of Cyt b 559 may alter the interaction between the APC core complex and PSII reaction centers, thereby reducing energy delivery from the antenna to the reaction center and protecting mutant cells against DCMU-induced photo-oxidative stress (Rutherford and Krieger-Liszkay, 2001).
Many cyanobacteria including Synechocystis sp. PCC 6803 have a novel blue-green light-induced NPQ mechanism to protect PSII reaction centers against photodamage under highlight stress (Kirilovsky and Kerfeld, 2012). Under high-light conditions, a soluble orange carotenoid protein is able to absorb blue-green light and undergoes photo-conversion into the active red form, which interacts with the APC core of the phycobilisome and dissipates excess excitation energy from the phycobilisome as heat. Interestingly, several Synechocystis sp. PCC 6803 mutant cells (e.g., R7Lα and R17Lβ) with mutations on the cytoplasmic side of Cyt b 559 in PSII showed significant inhibition of the effects of blue-green light-induced NPQ and apparent acceleration on its recovery (Chiu et al., 2013). These results are consistent with the proposal that the mutations on Cyt b 559 may alter the interaction between the phycobilisome and PSII reaction centers, thereby affecting the regulation of photosynthetic light harvesting in Synechocystis sp. PCC 6803.

CONCLUSION AND PERSPECTIVES
Site-directed mutagenesis studies combined with spectroscopic and functional characterization have revealed multiple roles of Cyt b 559 in the assembly and photoprotection of PSII reaction centers. The findings provide convincing evidence for the physiological role(s) of Cyt b 559 in a photoprotective secondary electron transfer pathway within PSII reaction centers, as was suggested from earlier studies of isolated PSII complexes (reviews in Whitmarsh and Pakrasi, 1996;Stewart and Brudvig, 1998;Shinopoulos and Brudvig, 2012). In the near future, site-directed mutagenesis studies combined with advanced high-resolution protein crystallography and spectroscopic and functional analysis will provide further new insights (e.g., structure and function relationships for different redox forms of Cyt b 559 ) and possibly the final proof of the molecular mechanisms of Cyt b 559 in PSII.

AUTHOR CONTRIBUTIONS
H-AC wrote the major part of the manuscript. Y-FC wrote the minor part of the manuscript, contributed the