Because these reaction are 'triggered' by light pulses and many intrinsic 'sensors' can be used, photosystems are well-suited for studying several aspects of the physics of biological macromolecules.

During the recent years, we worked on questions concerning all four functional areas of photosystems. Keywords are: exciton equilibration in PS II antenna, assembly of pigment-protein complexes, function of chlorophylls absorbing in the long-wavelength region, influence of electric fields on energetics and kinetics of primary charge separation, substrate and inhibitor binding at the acceptor site, pH-induced formation of macro-aggregates, mechanisms protecting against photooxidative damage.

At present, work in our group focuses on water oxidation by the PS II manganese complex. Our efforts aim towards elucidation of the mechanism of water cleavage (or make significant contributions) by examination of structural changes of the manganese complex during its functional cycle with X-ray absorption spectroscopy. Recent progress has facilitated tracking of the manganese complex in its reaction cycle by time-resolved X-ray absorption spectroscopy after Laser-pulse excitation. The figure below summarizes central results of the time-resolved experiments. (Haumann, Liebisch, Müller, Grabolle,  Barra, and Dau (2005) SCIENCE 310, 1019-1021.)

 

 

 

 

About half of all proteins contain transition metals in their active site. Presently, there is increasing scientific interest in these metal centers, because many important biological processes are catalyzed by protein-bound transition metals. Furthermore, biomimetic approaches lead to interesting perspectives in (bio-)technology. Thus, a new interdisciplinary field of research developed, called bioinorganic chemistry.

In many cases, e.g. membrane proteins, crystallographic determination of the molecules structure cannot be performed with reasonable expenditure. This is especially true if, for elucidation of the catalytic mechanism, the structure of the active site has to be unraveled in many intermediate states of the functional cycle. By X-ray absorption spectroscopy (XAS), determination of the oxidation state of the X-ray absorbing metal atom (position of the absorption edge) and of the adjacent structures (EXAFS effect) becomes feasible. The EXAFS phenomenon (extended X-ray absorption fine structure, EXAFS) allows precise determination of distances between the absorbing metal atom and surrounding atoms of the first coordination spheres. X-ray absorption measurements are performed by us at the outstation of the European Molecular Biology Laboratories (EMBL) at the Deutsches Elektronen-Synchrotron (DESY) in Hamburg (in cooperation with Dr. W. Meyer-Klaucke), at  the European Synchrotron Radiation Facility (ESRF) in Grenoble (in cooperation with Dr. P. Glatzel) and at the Berliner Elektronensynchrotron (BESSY, in cooperation with Prof. A. Erko and Dr. F. Schäfers).

One of our 'specialties' is the orientation of protein particles through a combination of centrifugation techniques and partial dehydration. Such vectorially oriented samples render XALDS measurements feasible (X-ray absorption linear dichroism spectroscopy, XALDS; variation of the angle between the sample and the X-ray's electric field vector). In many cases the achievable resolution of EXAFS measurements is increased by XALDS, and the determination of the spatial orientationa of the metal complex becomes possible. Our aim is to develop this method (regarding preparation and measurement techniques, theory of the effect, evaluation of experimental data) in order to take full advantage of the potential of the XALDS technique.

 

Another promising approach is time-resolved XAS. The metalloprotein is driven through its catalytic cycle right at the beamline, thereby allowing 'on-line' measurement of changes in structure and oxidation state of the metal center. First experiments at the ESRF have been successful (Haumann et al., 2005, Science 310, 1019-1021).

Presently, we use the XALDS technique to investigate structural changes of the PS II manganese-complex. XAS measurements of the atomic structure of volcano-shaped manganese deposits in the cell wall of certain green algae have been performed in cooperation with PD C. Plieth (Kiel). Furthermore, we are investigating the catalytic mechanism of the vanadium containing bromoperoxidase by XAS of both the substrate (bromine) and the metal center (vanadium) in cooperation with Prof. D. Rehder (Hamburg), Zn-sites of methyltransferases with Prof. R. Thauer (Marburg), oxygen-binding to the dicopper site of hemocyanin with Prof. H. Decker (Mainz), and other interesting questions. Besides measurements on the biological systems, investigation of structural and functional model compounds is of major importance.

 

Catalysis by biological metal centers - application perspective:     Hydrogen as a fuel produced from water by using solar energy

The sensing, utilization and production of molecular hydrogen by hydrogenases is investigated in cooperation with Prof. B. Friedrich (HU Berlin). Hydrogenases are connected to the research on Photosystem II by a long-term perspective: Solar energy drives the splitting of water (water oxidation, PSII function). The obtained 'energized electrons' (reducing equivalents) are used to produce of dihydrogen (reduction of protons, hydrogenase function). Since dihydogen is likely to fuel future cars, after exhaustion of the oil resources in 30-40 years, the light-driven hydrogen production is of significant interest.

Respective biomimetic and biotechnological approaches are explored in the framework of a joint project of nine German research groups (funded by the BMBF, see BioH2 Website) as well as in the consortium of groups from nine European countries (funded by the European Community, see SOLAR-H2 Website). Biomimetic compounds are also characterized in cooperation with the Swedish Consortium of Artificial Photosynthesis (centered at the Uppsalla University, see Website).

PS II is the target protein of a large group of herbicides, which during the recent years also have emerged as pollutants in drinking-water resources (e.g. Diuron, Atrazine). These herbicides competitively inhibit electron flux in PS II by blocking the quinone binding site (Q_B binding site). Binding of an inhibitor to this site can be detected fluorometrically with high sensitivity. In principle, herbicide concentrations below the limiting value (0.1 mg/L) defined by the EU can be detected.

The development of a sensitive system for detection of herbicides in water is developed; stabilized PSII particles serve as a sensor. Work on this project also includes investigation of the binding energetics and kinetics of different subtrates and inhibitors at the Q_B binding site.

Development of the sensor sensor system was finished in 2001. This project has been pursued in cooperation with the company bbe Moldaenke GmbH (Kiel). We also cooperate with this company regarding other development projects (biotest procedures and fluorimetrical measurements for ecosystem research). A milestone has been the development of a submersible probe which allows species-group specific, quantitative determination of a vertical population profile of algae within minutes.

We are working as a team of physicists, chemists, and biologists. The spectrum of employed methods ranges from experimental biophysics and theoretical chemistry to biochemistry and physiology. An important educational aim is to allow insights into specific methods and concepts of each discipline to every Ph.D.-student working in our group.