**1. Introduction**

52 Artificial Photosynthesis

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Synthetic Manganese-Calcium Oxides Mimic the Water-Oxidizing Complex of Photosynthesis Functionally and Structurally *Energy Environ Sci.,* DOI:

During the process of photosynthesis, light energy is converted to chemical energy that is utilized in the biosynthesis of carbohydrate. The initial events of photosynthesis in cyanobacteria, green algae and higher plants are the photoinduced transfer of electrons from water to plastoquinone. These reactions are catalyzed by Photosystem II (PSII), a large pigment-protein complex embedded within the thylakoid membrane (Renger, et al., 2008). The reactions catalyzed by PSII proceed as follows. First, light is trapped by pigments, predominately chlorophyll, associated with the thylakoid membrane (Glazer, 1983). Excitation energy is then transferred to the photochemically active chlorophyll species P680. Excited P680 then donates an electron (charge separation) to the primary PSII electron acceptor, a protein-bound pheophytin molecule (Klimov et al., 1980). Pheophytin is then oxidized by a tightly bound plastoquinone, QA, which in turn reduces a loosely bound plastoquinone, QB. A second light-induced charge separation results in the formation of plastoquinol (QBH2). Photooxidized P680 is reduced by the primary donor of PSII, a tyrosyl radical (YZ●) that is Tyr161 of the D1 protein of PSII (Debus et al., 1988a; Debus et al., 1988b). YZ then is reduced by an electron from the oxygen-evolving complex of PSII, located on the lumenal face of the thylakoid. The catalytic site of the oxygen-evolving complex consists of a metal ion cluster of four manganese atoms and one calcium atom bound via five -oxobridges (Mn4CaO5 cluster; Umena et al., 2011). Two chloride ions bound nearby are also required for activity. The oxygen-evolving complex functions to extract electrons and protons from water, ultimately resulting in the release of molecular oxygen. The manganese atoms cycle through a series of redox states, or S states (Joliot et al., 1969; Kok et al., 1970), from S0 to S4, with each S state representing a successively more oxidized form of the cluster. One molecule of O2 is evolved when four electrons and four protons are extracted by the Mn4CaO5 cluster.

PSII consists of both intrinsic thylakoid polypeptides and extrinsic polypeptides located within the thylakoid lumen. Together, these proteins ligate the Mn4CaO5 cluster, chlorophylls and other pigments, and the electron transport chain components. The intrinsic polypeptides necessary to form a PSII complex capable of evolving oxygen are CP47, CP43, D1, D2, the and subunits of cytochrome b559, and the 4kDa *psb*I gene product (Bricker et

Mutations in the CP43 Protein of Photosystem II

the backbone nitrogen of a glutamate residue.

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

Chloride may also play a role in stabilizing these channels.

(Umena et al., 2011).

Affect PSII Function and Cytochrome C550 Binding 55

*vulcanus* provided a well-resolved, detailed structure of the 4 manganese, calcium, and two chloride atoms, as well as revealing the positions of potential substrate water molecules, thus paving the way for an understanding of the mechanistic aspects of water oxidation

The Mn4CaO5 cluster has a distorted, chair-like structure in which three of the manganese (Mn1, Mn2, Mn3) and the calcium atom form a distorted cubane-like structure (see Fig. 1). The fourth manganese atom (Mn4), the so-called "dangler" manganese, lies outside of the cubane. Mn1, Mn2 and Mn3 and the calcium atom are linked via four oxygen atoms (oxo bridges). Mn4 is linked by a fifth oxygen to Mn1 and Mn3. In fact, each two adjacent manganese atoms are linked by a di--oxo bridge and the calcium atom is linked to all four manganese via oxo bridges. Four water molecules were observed in the x-ray structure in close proximity to the Mn4CaO5 cluster. Two of these appear to be associated with Mn4 and the other two with the calcium atom. It has been speculated that least one of these is a substrate for the water oxidizing reaction. Two chloride ions were also revealed in the structure. These flank the cluster and are not bound directly to the manganese or calcium atoms, but rather are ligated via two amino acids and two water molecules each. For each of these chloride ions, bound indirectly to the Mn4CaO5 cluster, one ligand is contributed by

In addition to the metal atoms and water, the protein ligands for the Mn4CaO5 cluster were identified. While most of the ligands are contributed by the D1 protein, CP43 contributes one bi-dentate ligand to the cluster, Glu354. Glu354 ligates both Mn2 and Mn3. What is additionally interesting is that this glutamate residue binds one of the chloride ions flanking the cluster. The other chloride ion is bound by Glu333 of the D1 protein. Glu333 is a bidentate ligand to Mn3 and Mn4. It has been proposed that the chloride ions help to maintain the structural integrity of the Mn4CaO5 cluster by maintaining the stable coordination of these glutamate residues to manganese (Umena et al., 2011; Kawakami et al., 2011). Also, the chloride ions lie at the beginning of two putative proton and/or water exit channels.

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

al., 2011). In higher plants, three extrinsic proteins, PsbO (previously termed the manganesestabilizing protein), PsbP and PsbQ, are required for maximal rates of oxygen evolution under physiological conditions (Bricker, 1992). In cyanobacteria, cytochrome c550 and a 12 kDa protein (PsbU) perform similar functions to PsbP and PsbQ. However, cyanobacteria also contain homologues of the higher plant PsbP and PsbQ proteins, though their roles are not well defined (Roose et al., 2007). In the absence of these extrinsic proteins, PSII complexes retain the ability to evolve oxygen, but at significantly reduced rates. This ability to evolve oxygen is dependent upon the presence of high, non-physiological, concentrations of calcium and chloride. The extrinsic proteins appear to act as a diffusional barrier that sequesters chloride and calcium in the vicinity of the oxygen-evolving complex (Bricker et al., 2011). They also act to protect the Mn4CaO5 cluster from exogenous reductants (Ghanotakis et al., 1984).

CP43 is an integral thylakoid protein. CP43 binds 13 chlorophyll molecules and is a component of the proximal antenna of PSII. X-ray analysis of PS II from *Thermosynechococcus elongatus* and *Thermosynechococcus vulcanus* shows that CP43 contains six transmembrane alpha helices and five hydrophilic loops that connect the membrane-spanning domains (Zouni et al., 2001; Kamiya & Shen, 2003; Ferreira et al., 2004; Umena et al., 2011). One of these loops, the large extrinsic loop E**,** spans amino acid residues 278Asn-410Trp and is located between the fifth and sixth membrane-spanning helices. The large extrinsic loop is exposed to the lumenal side of the thylakoid membrane and lies close to all three of the extrinsic PSII proteins as well as the Mn4CaO5 cluster. We previously constructed and extensively characterized the R320S (R305S in our original numbering scheme1) mutant in the large extrinsic loop E of CP43 in the cyanobacterium *Synechocystis* 6803, and showed that it has impaired PSII activity under chloride-limiting conditions (Knoepfle et al., 1999; Young et al., 2002). Isolated PSII from this mutant failed to bind the extrinsic PSII protein, cytochrome c550 (Bricker et al., 2002).

In this chapter, we will briefly review the role of both chloride and the CP43 large extrinsic loop E in the water-oxidizing process, and present new data on the characterization of additional mutations within the large extrinsic loop E that provide information about the nature of interaction of CP43 with the extrinsic PSII proteins. These data will be discussed within the context of the most current x-ray structure of PSII.
