The Mn cluster of Photosystem II (PSII) from Synechococcus elongatus was studied using EPR. A signal with features between g = 5 and g = 9 is reported from the S2-state. The signal is attributed to the manganese cluster in a state with a spin 5/2 state. Spectral simulations of the signal indicate zero field splitting parameters where the |E/D| was 0.13. The new signal is formed by irradiating PSII samples which contain the spin = 1/2 S2-state using 813 nm light below 200 K. This effect is attributed to a spin-state change in the manganese cluster due to absorption of the IR light by the Mn-cluster itself. The signal is similar to that reported recently in PSII of plants [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. In plant PSII the comparable signal is formed at a lower temperature (optimally below 77 K), and gradual warming of the sample in the dark leads to the formation of the state responsible for the well-known g = 4.1 signal prior to formation of the spin 1/2 multiline signal. In the present work using cyanobacterial PSII, warming of the sample in the dark leads to the formation of the spin 1/2 multiline signal without formation of the g = 4 type signal as an intermediate. These observations provide a partial explanation for the long-standing "mystery of the missing g = 4 state" in cyanobacterial PSII. The observations are rationalized in terms of three possible states which can exist for S2: (i) the spin 1/2 multiline signal, (ii) the state responsible for the g = 4.1 signal, and (iii) the new spin 5/2 state. The relative stability of these states differs between plants and cyanobacteria.

A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the Q B binding site. The oxygen-evolving activity, 5000 -6000 mol of O 2 /(h⅐mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50°C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 Å. The space group was determined to be P2 1 2 1 2 1 with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization. Photosystem II (PS II)1 functions as a water/plastoquinone oxidoreductase in the thylakoid membrane of chloroplasts and cyanobacteria yielding oxygen, protons, and reduced plastoquinone by the splitting of water. Powered by sunlight, this is the key reaction that created and still sustains an oxygenic atmosphere on our planet. As a pigment-protein complex, PS II consists of more than 20 different subunits (1), most of them being integral membrane proteins. The PS II reaction center (RC) itself is composed of the D1 and D2 proteins, which bind the cofactors for the light-driven electron transfer processes (2). Upon light absorption, an electron is transferred from the primary electron donor P680 to a nearby pheophytin molecule. This charge separation is stabilized by rapid electron transfer to a tightly bound plastoquinone molecule, Q A , and finally a mobile plastoquinone, Q B , on the stromal/cytoplasmic side of the membrane. After double reduction and protonation, the reduced Q B is released from its binding site on the D1 protein and replaced by an oxidized plastoquinone molecule. On the donor side, P680ϩ is reduced by a tyrosine residue of the D1 protein, tyrZ (3-5), which in turn is reduced by a cluster of four manganese atoms located on the luminal side of PS II. This manganese cluster is the place where water splitting occurs. In four successive charge separation steps, four positive charge equivalents are accumulated that yield, finally, one molecule of oxygen out of two molecules of water. Accordingly, the manganese cluster can assume five different oxidation states, S 0 -S 4 , with S 4 being the state in which oxygen is released. Because water is a very stable molecule, its oxidation requires a potential of ϩ1 V. Therefore, PS II is the most oxidizing enzyme in nature.Closely associated with the reaction center is cytochrome b 559 consisting of two subunits, ␣ and ␤. Each subunit of the cytochrome provides one histidine side chain for heme binding. The function of cytochrome b 559 is still not completely understood (fo...