Biophysical studies of Photosystem II and related model systems

Photosystem II (PSII) utilizes the energy of sunlight to split water into molecular oxygen, protons and chemically bound electrons that are ultimately used in the Calvin cycle for the reduction of carbon dioxide to carbohydrates. From geological records it is known that the origin of PSII dates back over 2.2 billion years. Despite this long time span and the enormous evolutionary advantage of being able to derive reducing equivalents from the ubiquitous water, PSII is the only known biological complex that is able to perform this complicated reaction, which changed life on earth fundamentally by producing the oxygen that formed the aerobic atmosphere of our planet. PSII can be found in the thylakoid membranes of several organism such as higher plants; green, red and brown algae; cyanobacteria, and diatoms. It is remarkable that to our current knowledge all PSII complexes from these different organisms are essentially identical, with the exception of some peripheral proteins and the light harvesting apparatus.

PSII consists of close to 30 protein subunits from which only two bind all the electron transfer components. These two proteins, known as D1 and D2, and their cofactors show similarities to the reaction centers of simple photosynthetic bacteria. The primary charge separation occurs between a Chla containing unit known as P680 and the pheophytin molecule (Pheo) of the active branch. The reduced Pheo transfers the electron via a specially bound plastoquinone molecule, the one-electron acceptor Q_A, to a second plastoquinone Q_B, which after double reduction and protonation is replaced by a ‘fresh’ plastoquinone from the thylakoid membrane. In contrast to the bacterial reaction center the cation radical P680˙⁺ is reduced by a functional unit of PSII known as the ‘oxygen evolving complex’ (OEC) or the ‘water oxidizing complex’ (WOC). This unit couples the one electron photochemistry of primary charge separation with the four-electron chemistry of water oxidation to molecular oxygen. Therefore, the OEC cycles through five different oxidation states (S₀, S₁, S₂, S₃, S₄). The S₄ state contains four oxidizing equivalents and decomposes immediately to the S₀ state under the release of triplet O₂.

What makes PSII so unique? Firstly, the high oxidizing potential of almost +1.3 V of the redox couple P680˙⁺/P680. Therefore, many studies try to explore which factors tune this potential to a value much higher than that of the primary donor in the bacterial reaction centers. Secondly, the unique OEC, which contains the redox-active tyrosine Y_Z and a catalytic complex consisting of four Mn and one Ca²⁺ ions connected by several μ-oxo bridges (Mn₄O_xCa cluster) and surrounded by a special protein matrix. In addition Cl⁻ and bicarbonate are discussed as cofactors for water oxidation in PSII. The roles of these two anions are not fully understood and diverging opinions exist.

The Mn₄O_xCa cluster is the storage unit for the oxidizing equivalents and also templates the O–O bond formation by binding at least one of the two substrate water molecules. Y_Z is the immediate electron donor to P680˙⁺ and is in turn reduced by the Mn₄O_xCa cluster. Many recent studies show that the coupling of electron and proton transfer plays a pivotal role in the process of water splitting. In this regard the functions of Y_Z and the protein matrix are still under investigation, a direct participation of Y_Z in the water oxidation chemistry has been discussed. Interestingly, PSII contains a second redox active tyrosine (Y_D) that, in contrast to the D1-residue Y_Z, is located in the D2 protein. Y_D is not part of the normal electron transfer pathway leading to O₂ evolution, but can reduce the S₂ and S₃ states to the S₁ and S₂ states, respectively. Its oxidized form (denoted as Y_D^ox or Y_D˙) is very stable and oxidizes in a slow dark-reaction (half-time of about 30 min) the S₀ state to the dark stable S₁ state. The properties of Y_D/Y_D^ox have been studied extensively in the past in comparison to Y_Z/Y_Z^ox signals of systems inhibited in O₂ evolution. Recently it has become possible to also obtain EPR signals from Y_Z^ox in intact PSII samples by various illuminations at cryogenic temperatures.

Many different biophysical techniques have been used to study the various processes within PSII that range from the ultra fast reactions during light capture, energy transfer and charge separation (fs to ps time ranges), over oxygen release and water exchange kinetics (ms to second time scales) to the lifetimes of the S₀, S₂ and S₃ states that range between seconds to hours. Examples for these techniques are: ultrafast optical spectroscopy, FTIR/Raman, cw and pulse EPR/ENDOR, NMR, X-ray absorption and fluorescence spectroscopy (e.g. XANES, EXAFS, Kβ XES, RIXS), polarography, time resolved mass spectrometry, electron microscopy and X-ray crystallography. These studies are supplemented by theoretical calculations. Furthermore, the efforts of coordination chemists in synthesizing relevant model complexes are crucial for guiding the interpretation of the complex PSII data. Similarly, specific mutants have become available in different PSII containing organisms; these are very important for elucidating the structure–function relationships within PSII.

The first crystal structure of PSII was published in 2001 and had a resolution of 3.8 Å (ref. 1). In this issue of PCCP the latest crystal structure of the same group with now 3.2 Å resolution is presented. Earlier this year a detailed model for PSII was developed on the basis of data with a resolution of 3.5 Å (ref. 2). As can be seen from many articles in this issue this model was immensely inspiring to the researchers in this field. However, due to the limited resolution and problems with radiation damage all crystal structures can provide only limited information on the precise structure of the Mn₄O_xCa cluster. Therefore, presently the combination of crystallography and X-ray spectroscopy appears to be the most promising way to solve the geometrical structure of this multi-nuclear metal binding site.

The basis for this special issue was laid by the 314th WE-Heraeus seminar on “Water Oxidation in Photosynthesis”. It was held in Bad Honnef, Germany, November 22–25, 2003. Most of the authors of this special issue participated actively in this seminar, which also included a very memorable celebration of Prof. G. Renger’s 66th birthday and retirement (see Plate 1 with Prof. G. Renger in front left position). All manuscripts of this special issue have been reviewed according to the normal guidelines of PCCP. We are glad to have obtained such an excellent set of papers covering almost all relevant aspects concerning the function of PSII and the mechanism of water oxidation. Currently a large body of data that has been obtained over many years of intense research is being pieced together. We hope that the work presented in this special issue will further stimulate the field that has clearly gained fresh impetus over the last few years.

Johannes Messinger

Wolfgang Lubitz

Max-Planck-Institute for Bioinorganic Chemistry

Plate1 Participants of the 314th WE-Heraeus Seminar on “Water Oxidation in Photosynthesis” held in the Physikzentrum Bad Honnef, Germany, 22–25 November 2003. Photo: WE-Heraeus Foundation (with kind permission).

References

1. A. Zouni, H.-T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger and P. Orth, Nature, 2001, 409, 739–743.
2. K. N. Ferreira, T. M. Iverson, K. Maghlaoui, J. Barber and S. Iwata, Science, 2004, 303, 1831–1838.

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