**2. Structure of the Mn4CaO5 cluster**

The structure of the manganese cluster has been the focus of intense study. Early x-ray structures obtained at low to medium resolution were known to be affected by radiation damage, which alters the valence state of the manganese and also potentially alters the ligand field (Grabolle et al., 2006). Nonetheless, these structures provided the first data on the positions of the protein subunits, chlorophylls and other cofactors. However, they did not allow for a highly refined, detailed structure of the manganese cluster. The manganese cluster models were proposed relying on interatomic distances derived from EXAFS experiments (Dau et al., 2008). The recent x-ray structure at 1.9 Å from *Thermosynechococcus* 

<sup>1</sup> Due to differences in initiation sites, the CP43 proteins from various cyanobacteria are of varying lengths. In this article, unless otherwise specified, we will follow the common practice of numbering the amino acids using the corresponding numbering in *Thermosynechococcus vulcanus*, the strain from which the latest x-ray structure was derived.

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

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).

within the context of the most current x-ray structure of PSII.

**2. Structure of the Mn4CaO5 cluster** 

the latest x-ray structure was derived.

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

The structure of the manganese cluster has been the focus of intense study. Early x-ray structures obtained at low to medium resolution were known to be affected by radiation damage, which alters the valence state of the manganese and also potentially alters the ligand field (Grabolle et al., 2006). Nonetheless, these structures provided the first data on the positions of the protein subunits, chlorophylls and other cofactors. However, they did not allow for a highly refined, detailed structure of the manganese cluster. The manganese cluster models were proposed relying on interatomic distances derived from EXAFS experiments (Dau et al., 2008). The recent x-ray structure at 1.9 Å from *Thermosynechococcus* 

1 Due to differences in initiation sites, the CP43 proteins from various cyanobacteria are of varying lengths. In this article, unless otherwise specified, we will follow the common practice of numbering the amino acids using the corresponding numbering in *Thermosynechococcus vulcanus*, the strain from which

(Ghanotakis et al., 1984).

*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 (Umena et al., 2011).

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 the backbone nitrogen of a glutamate residue.

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 may also play a role in stabilizing these channels.
