| dc.description.abstracteng | Photosynthesis is the source of life on Earth. In oxygenic photosynthesis, water and carbon dioxide are converted into organic matter using of solar energy that results in the release of oxygen. PSII, a supra-molecular complex embedded in the thylakoid membrane containing various protein subunits and co-factors, is involved in the key reactions of this process. From the available structure of the PSII super complex so far, we could understand the basic water splitting mechanism and electron transport. Nevertheless, precise pathway of excitation energy transfer between peripheral and core complex, contribution of lipids in the reaction catalysis, and dimeric structure of the complex remains largely unknown. In this thesis, we developed a new method for purification of the PSII super complex dimer from Spinacia oleracea. Using cryo electron microscopy, we were able to reconstruct the dimer of PSII super complex to a resolution of 14Å. The monomers of the PSII super complex are aligned against each other at an angle of 30° as expected for the native and functionally active PSII.
Rubisco (Ribulose 1,5-bisphosphate carboxylase/oxygenase) is the key enzyme in the photosynthesis process fixing carbon dioxide in the biosphere. In a side reaction, Rubisco also catalyses the fixation of oxygen into phosphoglycolate which is further recycled in the photorespiration pathway. Unfortunately, the species-specific efficiency of the enzyme is invariably compromised vastly due to this side reaction. In spite of extensive structural and kinetic investigations, the key steps in the oxygenation reaction as well as the chemistry of the reaction is unknown. Therefore, acquiring high-resolution structures of Rubisco is necessary to better understand the mechanisms of oxygenation and carboxylation reactions. In this thesis, we designed an efficient method for purification and crystallization of Rubisco from Spinacia oleracea. I was able to crystallize the activated protein with substrate and product as well as the non-activated protein with substrate. This resulted in five different high-resolution structures at 1Å. Comparative analysis of these structures led to the identification of key residues involved in the enolization (Lys 177), and hydration (His 294) steps of the reaction which have so far been misinterpreted or remained unidentified, respectively.
Additionally, the two novel structural models in this thesis helped us to analyze the conformational changes of Rubisco at residue, as well as subunit levels during the transition from closed to open state and vice versa. Conformational changes of the conserved Glu 60, Asn 123 and Phe 127 residues cause the N-terminal domain of large subunit to move closer to the active site in the closed state. Flipping of the of Phe 211 and Gln 156 side chains cause the C-terminal domains of large and small subunits to move away from the active site. The novel conformational changes observed in this thesis indicate that Rubisco undergoes much more structural changes in the catalytic cycle than previously described. Additionally, we found alternate conformations for multiple residues indicating allosteric communication in Rubisco. In summary, the combination of biochemical methods, newly developed X-ray data collection, and data processing strategies enabled us to analyze the structural features of Rubisco protein in more detail and better understand the mechanism of the oxygenation reaction. | de |