The photosensitive unit triggering the negative phototaxis in the haloarchaeum Natronomonas pharaonis consists of the receptor sensory rhodopsin II (NpSRII) and its cognate transducer (NpHtrII) in a 2:2 stoichiometry. Upon light excitation, a structural rearrangement in the receptor initiates a displacement/rotation of the transducer helix TM2, which can be considered as starting event for the signal transduction. This signal is further transmitted to the cytoplasmic signaling domain through the signal transduction unit comprising two HAMP domains.Structural information already exists for the transmembrane region of this complex (crystal structure) as well as for the rod shaped cytoplasmic part of NpHtrII due to its high homologies with chemoreceptors. Moreover, the solution NMR structure of the isolated HAMP domain from A. fulgidus recently obtained shows a homodimeric, four-helical, parallel coiled-coil with an unusual interhelical packing, that is thought to propagate a signal by virtue of concerted helix rotations. Here, an electron paramagnetic resonance (EPR) investigation of site-directed spin labeled transducers in the NpSRII/NpHtrII complex has been carried out for structural and functional elucidation of the N. pharaonis HAMP. For this purpose, cw as well as pulse EPR techniques have been used in terms of mobility, accessibility and intra-transducer dimer distance analyses. Conformational changes induced by environmental inputs, namely salt, temperature and pH, give insight into the two-state equilibrium existing between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation of this linker region.

Das haloalkaphile Archaeon Natronomas pharaonis nutzt den Lichtrezeptor, das Sensorische Rhodopsin II (NpSRII), im Komplex mit dem halobakteriellen Transducer (NpHtrII) zur photophoben Antwort auf schädliches grün-blaues Licht und entsprechender Steuerung des Flagellenmotors um optimale Umgebungen zum Überleben aufzusuchen. In einer Membran rekonstituiert bildet der Rezeptor/Transducer Komplex eine 2:2 Stöchiometrie aus, wobei ein Transducer-Dimer von zwei Rezeptor-Molekülen flankiert wird. Durch die Lichtanregung wird ein Photozyklus initiiert, dessen Intermediate sich aufgrund ihrer optischen und/oder strukturellen Eigenschaften unterscheiden. In dieser Studie sind die strukturellen Änderungen des Rezeptors und des Transducers während des Photozyklus mit Hilfe der Elektronenspinresonanz (ESR)-Spektroskopie in Kombination mit der ortsspezifischen Spinmarkierung aufgeklärt worden. Als Methoden wurden dabei die zeitaufgelöste ESR-Spektroskopie und Abstandsmessungen in unterschiedlichen Intermediaten mit Hilfe von cw- und Puls-ESR-Techniken genutzt. Der Signaltransfer nach Initiierung des Photozyklus im Rezeptor, die Weiterleitung des Signals zum Transducer durch die Auswärtsbewegung der Helix F und die damit verbundene Verschiebung des thermodynamischen Gleichgewichts in der HAMP-Domäne des Transducers konnten beobachtet und analysiert werden. Die Methode der ESR-Spektroskopie erweist sich als mächtige biophysikalische Technik, die eine direkte und zeitaufgelöste Analyse von strukturellen Konformationsänderungen in Membranproteinen und die strukturelle Aufklärung unterschiedlicher Intermediate erlaubt.

"Laser flash spectroscopy of photosystem II" Photosystem II (PS II) of plants and cyanobacteria oxidizes water in a light-powered reaction. Thereby, this protein is the ultimate source of the atmospheric oxygen. The capacity to oxidize water is owed to two properties of PS II: (i) The midpoint potential of the oxidizing chlorophyll moiety is increased by 0.6 V compared to photosystem I or photochemical reaction centers of anoxygenic bacteria, and (ii) the energy requirements of the four steps needed for the tetravalent oxidation of water are adapted to the energy of red light quanta. This thesis deals with two particular aspects, namely: 1. The coupling of the electron transfer from tyrosine Z (YZ) to the primary donor (P680+) to proton transfer, and an inquiry on the role of a positive charge on YZox (plus base cluster) in increasing the oxidizing potential at the catalytic site. 2. The localization of the electron hole, P680+, among the excitonically coupled four inner chlorophyll a molecules, and an estimation of the midpoint potential differences between them. Electron-proton-coupling by YZ This study was carried out with PS II core complexes from spinach or pea with a deactivated (removed) manganese cluster. The reduction of P680+ was investigated as a function of pH by detecting the laser flash induced absorption changes with nanosecond resolution. Two kinetic components were found with different pH-dependence and activation energies. The alteration of kinetic parameters by H/D isotope substitutions or by addition of divalent cations implied two different types of YZ-oxidation: At acidic pH the electron transfer was coupled with proton transfer, whereas in the alkaline region it was more rapid and no longer controlled by proton transfer. The conversion between both mechanisms occured at pH 7.4. This value corresponds either to the apparent pK of YZ itself (i.e. of the hydroxy group of the phenol ring) or to the pK of an acid-base-cluster, which includes YZ. Independent measurements of pH-transients by following the absorption changes of hydrophilic proton indicators corroborated this notion. The data were interpreted as indicating that the phenolic proton of YZ was released into the medium at acidic, but not at alkaline pH. The electron transfer and proton release characteristics of intact, oxygen-evolving PS II resembled those in deactivated samples kept at alkaline pH. We concluded that the electron transfer from YZ to P680+ in the native system was not coupled with proton transfer into the bulk. This has shed doubt on a popular hypothesis on the role of YZ as 'hydrogen abstractor' from bound water. On the other hand, the energetic constraints of water oxidation could be eased by the positive upcharging during oxidation of YZox plus its base cluster. On the localization of the electron hole of P680+ Photooxidation of PS II oxidizes the set of four innermost chlorophyll a molecules giving rise to the only spectroscopically defined species P680+. The deconvolution of difference spectra into bands of pigments is ambiguous. By using photoselective excitation of antennae, i.e. chl a molecules with site specific energies at the long wavelength border of the mean Qy-band, and by polarized detection, it was possible to tag P680+QA-/P680QA and 3P680/P680 difference spectra with a further parameter, the (wavelength-dependent) anisotropy r. Results obtained at liquid nitrogen temperature (77 K) can be clearly interpreted in terms of two chl a monomer bands. The two main components of the P680+QA-/P680QA difference spectrum were marked by two distinct values of the anisotropy and could be interpreted in a straightforward manner: the bleaching of a band at 675 nm belonging to the charged species (chl a+) and an electrochromic blue-shift of a nearby chl a from 684 to 682 nm. The main bleaching band of the 3P680/P680 spectrum (at 77 K) can be apparently attributed to a third (or several) chl a component(s). The analysis of the P680+QA-/P680QA spectrum at cryogenic temperature is compatible with monomeric chl a bands. On the other hand, one could assume a system of excitonically coupled core pigments, as it was recently introduced in the literature on the basis of energy transfer studies ('multimer model'). However, in view of the clear indications for an electrochromic band shift and the location of the bleaching band, which absorbs in a wavelength region of monomeric chl a, one assumption of the 'multimer model' should be questioned. Presumably, the excitonic couplings are rather weak, in particular between each of the two central chl a-molecules (PA/PB) and its respective accessory chl a (BA/BB), because of (i) the distances and (ii) different site energies of the monomeric chromophores. At room temperature, the absorption difference and anisotropy spectra of P680+QA-/P680QA were clearly altered. The anisotropy data indicated that the changes could no longer exclusively be ascribed to thermal broadening of individual bands. The localization of the positive charge on one pigment, analogous to the situation at 77 K, was now unlikely. Hence, the midpoint potential differences between the inner four chlorophyll a molecules were small and were estimated as approximately 15 meV.

The presented thesis includes investigations to fully characterize the electronic structure and magnetic properties ofselected manganese containing high-spin molecules by means of various X-ray spectroscopic, magnetic and theoretical methods. The investigations on the Mn4 star-shaped molecule havelead to a number of interesting results. Magneto-chemical studies exhibit very weak exchange coupling constantsbetween the four Mn(II) ions, leading to complicated low lying states in which the ground state is not well separated, resulting from a dominant weak ferromagnetic coupling and a giant moment of up to 20 µB/f.u. XMCD measurements revealed that almost the completemagnetic moment is located around the Mn(II) ions.This is in agreement with only a few charge transfer states foundwithin the detailed X-ray absorption spectroscopic study. The electronic structure and detailed magnetic properties of the star-shaped heteronuclear CrIIIMnII3 complex have been precisely investigated.With XPS the homovalency of Mn and Cr have been verified. The XA-spectra of the manganese and chromium L edges were measured and compared to earlier investigated Mn4 spectra.The combination high-magnetic field magnetic measurements and element selective XMCD of Mn and Cr L edges and quantum model calculations lead to a complete analysis of the magnetic structure of the CrMn3 magnetic core. The III valence state of the manganese ions in MnIII6O2Salox has been verified. From X-ray diffraction, typical Jahn-Teller distorted oxygen octahedra have been found for Mn(III) ions. Comparisons of XPS and XAS spectra of the complex to corresponding spectraof maganite and tetranuclear manganese(II) cluster it was definitely possible to identify MnIII6O2Salox as a pure Mn(III) compound.