**3. Role of chloride in oxygen evolution**

Chloride is required for both the assembly and the stability of the oxygen-evolving complex and is sequestered at the active site by the PSII extrinsic proteins. Chloride depletion of isolated PSII preparations has profound effects on PSII function. Such preparations show large decreases in steady state oxygen evolution rates, and S-state defects including stabilization of the S2 state lifetimes, and slowing of the S2 → S3 transition. Chloride is necessary for S-state advancement in both the S2 → S3, and S3 → S4→ S0 transitions (Wincencjusz, et al., 1997). Removal of the extrinsic proteins from PSII results in a requirement for high, non-physiological concentrations of both calcium and chloride to maintain some oxygen-evolving activity (Bricker, et al., 2011). In PSII membranes depleted of PsbO, and also in *Synechocystis* mutants lacking the gene encoding PsbO, two manganese atoms were lost at chloride concentrations less than 100 mM (Kuwabara et al., 1985; Burnap et al., 1994). In both the higher plant and cyanobacterial systems, removal of the PsbO protein resulted in increases in the S2 and/or S3 lifetimes (Miyao et al., 1987; Burnap et al., 1992; Liu et al., 2007) and also in a slowing of the S3 → S4→ S0 transition (Ono & Inoue, 1985; Burnap et al., 1992; Liu et al., 2007). Removal of the PsbP and PsbQ extrinsic proteins in higher plant PSII also results in a large decrease in oxygen-evolving activity, which can be

Mutations in the CP43 Protein of Photosystem II

**4. Structure and function of CP43** 

structure (Umena et. al., 2011).

large extrinsic loop.

PsbU, as well as the manganese cluster.

Affect PSII Function and Cytochrome C550 Binding 57

The product of the *psb*C gene, the CP43 mature protein from *Synechocystis* 6803 consists of 460 amino acid residues and is well conserved. The nucleotide sequence of *psb*C has been determined from more than 50 species. The predicted plant apoproteins show approximately 95% homology with each other, and 85% and 77% homology with *Chlamydomonas* and cyanobacterial CP43, respectively. Early crystallographic studies confirmed that CP43 contains six transmembrane alpha helices (Zouni et al., 2001; Kamiya and Shen, 2003; Ferreira et al., 2004). The transmembrane helices of CP43 contain a number of conserved histidyl residues that function as chlorophyll-*a* ligands. Replacement of these histidyl residues with either tyrosyl or arginyl residues (Shen et al., 1993) results in mutants showing decreased PSII stability and defects in light-harvesting efficiency. Thirteen chlorophyll-*a* molecules associated with CP43 were assigned in the most current crystal

In addition to the six transmembrane alpha helices, CP43 contains five hydrophilic loops that connect the membrane-spanning domains. The positions of these loops are as follows (*Synechocystis* 6803 numbering): Loop A, Glu58-Phe98; Loop B, Gly123-Thr145; Loop C, Gly170-Asp219; Loop D, Lys242-Tyr258 and Loop E, Asn280-Arg410. Loop E, with 130 amino acids, is quite large and contains approximately 30% of the amino acids in this protein. Loops A, C and the large extrinsic Loop E are exposed to the lumenal side of the thylakoid membrane. The N- (Val1-Lys36) and C-termini (Arg448-Asp460), in addition to Loops B and D, face the stromal surface of the membrane (von Heijne and Gavel, 1988; Sayre and Wrobelboerner, 1994;). The CP47 protein has a similar structure and also possesses a

All crystal structures of dimeric PSII show that CP43 is positioned on the D1 side of the D1- D2 heterodimer and CP47 on the D2 side. The two PSII monomers are related by a local-C2 rotation axis oriented perpendicular to the plane of the membrane. CP47 lies at the interface of the two monomers while CP43 is located at the periphery. This location is speculated to be necessary for the removal and replacement of damaged D1 during photoinhibition. In both CP43 and CP47, the six transmembrane helices are arranged as a "trimer of dimers". The large extrinsic loop of CP47 lies adjacent to the PsbO protein, consistent with a large body of biochemical evidence (Bricker & Frankel, 2002), and is also close to the extrinsic PsbU protein. The large extrinsic loop of CP43 is located adjacent to cytochrome c550 and

Early mutagenesis and biochemical studies clearly outlined a role for CP43 in oxygen evolution and the stable assembly of the PSII complex. CP43 is required for optimal oxygenevolving activity from isolated PSII preparations (Bricker, 1990). *Chlamydomonas* strains incorporating mutations that affected either the synthesis or stability of CP43 were deficient in PSII activity (Rochaix et al., 1989). In these mutants, levels of the other PSII core proteins were severely reduced. *Synechocystis* mutants lacking the *psb*C gene as a result of deletion mutagenesis accumulated PSII core complexes (minus CP43) to only 10% of wild-type levels and could not grow photoautotrophically or evolve oxygen (Rogner et al., 1991; Carpenter et al., 1990). Interruption of the *psb*C gene by insertional mutagenesis also produced a mutant incapable of evolving oxygen, but which exhibited primary charge separation (Vermaas et al., 1988). A S*ynechocystis* mutant recovered by nitrosoguanidine mutagenesis was unable to evolve oxygen or support PSII electron transport from water to either dichlorobenzoquinone or methyl viologen (Dzelzkalns & Bogorad, 1988). Further characterization of this mutant

partially restored by high concentrations of calcium and chloride (Popelkova & Yocum, 2007). The absence of these proteins also makes the Mn4CaO5 cluster vulnerable to the effects of exogenous reductants (Ghanotakis et al., 1984).

Fig. 1. Coordination Environment of the Mn4CaO5 Cluster.

The amino acid residues in the first and second coordination sphere are labeled. Please note that D342:D1 is obscured in this view by the metal cluster. The manganese ions are shown in purple and are labeled. Calcium is shown in cyan and the oxygens are shown in red. The proximal and distal chlorides are shown in yellow and labeled Cl-1 and Cl-2, respectively. This figure was generated in Pymol from the crystal structure of *Thermosynechococcus vulcanus* (PDB: 3ARC; Umena et al., 2011).

In cyanobacteria, deletion of the PsbU protein results in a mutant capable of growth in normal media, but which shows a reduced growth rate in media depleted of chloride (Shen et al., 1997). This mutant has reduced rates of oxygen evolution, a more stable S2 state, and is more susceptible to damage to its PSII centers by both light and heat stress (Shen et al., 1997; Shen et al., 1998; Inoue-Kashino et al., 2005). Mutants constructed by deletion of the extrinsic cytochrome c550 protein, encoded by the *psb*V gene, do not grow well photoautotrophically, and exhibit no capacity for photoautotrophic growth in media depleted of either calcium or chloride (Shen et al., 1998; Katoh et al., 2001). The *psb*V deletion mutants also show decreased ability to evolve oxygen and reduced numbers of PSII centers in the thylakoids (Shen et al., 1995; Katoh et al., 2001). They also exhibit S-state cycling defects, including an increase in miss and double hit factors and a slowing of the S3 → S4→ S0 transition (Shen et al., 1998).

These observations are all supportive of a role of the extrinsic proteins in maintaining the proper ionic environment around the Mn4CaO5 cluster. How chloride is actually bound and transported to the active site is currently unknown. Interestingly, the current crystal structure identified a third chloride ion bound near the C-terminal amino acid of PsbU, which is located between PsbU and cytochrome c550 (Umena et al., 2011; Kawakami et al., 2011). This chloride is ligated by water molecules and lies close to the exit of a proposed hydrogen-bonding network leading from the Mn4CaO5 cluster to the lumen that could possibly serve to transport anions, water or protons (Gabdulkhakov et al., 2009; Vassiliev et al., 2010; Kawakami et al., 2011).
