Kinetische, theoretische und strukturelle Charakterisierung des Cytochrom c-Photosystem I-Komplexes

Photosystem I (PSI) from the thermophilic cyanobacterium Thermosynechococcus elongatus is a membrane-bound, multipigment protein supercomplex. It converts light to electrochemical energy with a quantum efficiency of almost 100 %. It reduces theluminal proteins plastocyanin and cytochrome c6 (Cyt c6) to oxidize the stromalprotein Ferredoxin. The structure of PSI has been solved in 2001 at a resolution of 2,5 ˚A (Jordan et al. 2001). PSI can be assembled on an electrode surface to produce photocurrents and the generated electrons can be used for the production of biofuels.The mitochondrial cytochrome c from horse heart (Cyt cHH) binds strongly to both, PSI and the electrode surface, and can therefore be applied to improve the electrical coupling. Due to the practical use of the PSI-Cyt c complex, the aim of my thesis isto characterize the interaction of PSI with Cyt c6 and the analog Cyt cHH. To this end, the binding of both cytochromes to PSI was analyzed by kinetic, calorimetric, theory-based and structural methods.Cyt c6 binds to PSI while being reduced and decreases its affinity after transferring its electron. In contrast, Cyt cHH binds to PSI in both oxidation states, reduced and oxidized, with identical affinity. By means of kinetic measurements, I identified condi-tions in which PSI forms a stable complex with either of the two cytochromes. The posi-tions of the cytochrome binding sites at PSI were calculated by a rigid-body docking.For the calculation with Cyt c6, the majority of the potential binding sites are located at the luminal side of PSI, close to P700. The theoretic properties of one of these bindingsites are in good agreement with my own kinetic measurements and literature data.The position and orientation of Cyt c6 in this theoretic binding site is almost iden-tical to the localization of Cyt c2 in cocrystals with the bacterial reaction center from Rhodobacter sphaeroides. The potential Cyt cHH binding sites are uniformly distri-buted over the luminal surface of PSI. Still, cocrystals consisting of PSI and Cyt cHH in a 1 : 1 ratio were produced. The crystal structure was solved to a resolution of 3,4 ˚A,however, no electron density corresponding to Cyt cHH was found. To verify the PSI-Cyt c6 complex, the two proteins were crosslinked and the structure was solved by cryo electron microscopy (cryo-EM) to a resolution of 2,9 ˚A. No density correspondingto Cyt c6 is present in the cryo-EM reconstruction, but masked classification revealed3D classes containing additional density at the luminal surface of PSI. The cryo-EM structure reveals density of five additional cofactors and the flexible subunit PsaK. For the analysis of electron transport processes, serial femtosecond crystallography (SFX) was used. Datasets were measured for PSI crystals without cytochromes at resolutions of 2,75 ˚A (oxidized), 3,4 ˚A (reduced, dark adapted) and 3,4 ˚A (100 ns after light flash).Additionally, a protocol for the production of PSI-Cyt cHH cocrystals, suitable for the measurement by the SFX-method, was designed. With the crystal structure, the cryo-EM-structure and the SFX-structure, three high resolution structures from the same PSI-protein are now available. The differences between these structures are discussed in respect to their influence on the function of PSI