| Abstract |
Photosystem II is a membrane multiprotein complex, which catalyzes water splitting, via a unique mechanism. The significant functional properties of this complex are attributed to its unique structural characteristics. Indirect biochemical studies have yielded information on the location of the PSII proteins. Recently, electron microscopy studies have led to models concerning the relative assembly and subunit topology in PSII. However, the only available information on the PSII structure on a molecular level derive from purple photosynthetic bacteria, whose structure has been determined by X-ray crystallography. To date, a three-dimensional model, which would reveal the unique functional properties of PSII, is absent. In the present work the aim has been the structural study of PSII on a molecular level, as well as the level of PSII assembly. The study of the molecular structure of PSII was based on the production of three dimensional crystals and the analysis of the structure with X-ray crystallography. The analysis of the tertiary structure of PSII was based on electron microscopy studies of the reaction center. Concerning the crystallization of PSII, a new purification protocol was established, which enabled quantitative isolation of CP 47 and the reaction center complex, in a stable state. This protocol is based on the selective extraction of membrane proteins, using the appropriate combination of detergents and the enrichment of the subcomplexes with ultracentrifugation, using a linear sucrose gradient. The crystallization of a PSII membrane protein was accomplished for the first time. In order to improve the CP 47 crystals a thorough screening of the parameters, which influence membrane protein crystallization, was carried out. The low resolution of the crystals was attributed to a degradation of the CP 47 protein. This degradation takes place after the protein's dissociation from the rest of the complex and continues in the dark. This sensitivity of the protein could be the consequence of structural changes, induced by its dissociation from the core complex. The structural study of the PSII reaction center complex yielded information on the oligomerization state and the location of the membrane proteins in the PSII-core. The reaction center complex was isolated in a monomeric form and its molecular weight was estimated by a combination of two independent techniques. Analysis of the images, which were obtained by electron microscopy, led to a protein density map of the D1 and D2 subunits. A comparison of the reaction center map to the map of the whole PSII-core complex revealed the relative positions of the major membrane proteins. These results supported a central location for the D1/D2 heterodimer and a peripheral location for CP 47 and CP 43, giving the complex a pseudo twofold symmetry. In addition, in the present thesis a study on HPO lyase was carried out. This enzyme is widely spread in plants and its products are involved in plant defense. HPO lyase belongs to a new category of enzymes (CYP 74), which are considered to be a subclass of the cytochrome P450 family. CYP 74 enzymes have unique functional features, since they do not need a reductant and oxygen for their function and exhibit an extremely high turnover rate, in contrast to P450. In the present study HPO lyase was purified from E. coli, which contained the enzyme's recombinant DNA from green bell pepper. E. coli expression of HPO lyase yielded sufficient amounts of enzyme, which enabled its biochemical and spectroscopical characterization. This work has focused on the structural study of the heme environment of the metal center using UV-Vis spectroscopy and EPR. The spectroscopic study of the enzyme was based on the interaction of exogenous ligands with the heme iron and the comparison of the resulted spectroscopical characteristics to the ones of heme proteins with a known structure. These studies revealed that the heme iron is five-coordinated and that it exists in a high spin state. For the first time, spectroscopic data were obtained which supported that the proximal site is occupied by cystein and that HPO lyase is indeed a cytochrome P450.
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