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The work in this thesis investigates the initial events surrounding light absorption by phytochrome Cph1 from the cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803. The photocycles in Cph1 were investigated with a home built pump−dump/repump−probe spectroscopy apparatus which employed a visible probe. In order to do this light pulses, on the same time scales that the events occur are needed. Advances in laser physics have made the production of wavelength−tuneable femtosecond pulses normal procedure in many labs. Phytochromes are a family of light−sensitive proteins acting as photo−receptors for biological signalling. Typically they have reversible red (‘Pr’) and far red (‘Pfr’) absorbing forms. The phytochrome Cph1 displays similar spectroscopic transitions to those observed in plant phytochrome receptors. The Pr state absorption maximum is at 660 nm and the Pfr meta−stable intermediated maximum is at 710 nm. The Pfr intermediate is long lived, with thermal reversion occurring on a second time scale. Additionally Pfr can be photo−transformed by absorption of far red light back to the Pr ground state. It is widely accepted that the interconversion between the Pr and Pfr forms is triggered by photo−induced Z−E and E−Z photoisomerisations, respectively, around the C15=C16 double bond of the billin chromophore. Additional structural rearrangements during the photocycle occurring in the phycocyanobilin chromophore of Cph1, particularly at C5, have been implicated. In addition to the spectral similarity to plant phytochromes, Cph1 has a 10−15% quantum yield; this low yield is yet to be fully understood. Pump−probe spectroscopy, with dispersive detection, is used to investigate the two photocycles in Cph1. This is the first time the Pfr photocycle of Cph1 has been investigated with dispersive detection. From this experiment it has been revealed that the Pfr S1 state decays with a 240 fs time constant; faster than previously reported. The wavelength dependence of the Pr photo−reaction is systematically investigated between 620 and 680 nm for the first time. Pump−dump/repump−probe spectroscopy employing visible detection is used for the first time to investigate the two photocycles in the phytochrome Cph1. Pump−dump/repump−probe spectroscopy is an extension of the pump−probe technique, from which information on connectivies between states can be found. By employing this method, evidence for the presence of a ground state intermediate in both the Pr and Pfr photo pathways is found. The wavelength dependence of states present in the Pr photocycle has been systematically investigated between 620 and 680 nm for the first time. The wavelength dependence of the Pump−dump/repump−probe experiments supports the assignment of a ground state intermediate. Coherent coupling between the electric and vibrational dipoles is seen in the Pfr photocycle. This coupling is revealed by varying the timing of the dump pulse within the first 300 fs of the photo−reaction. From the work in this thesis, the understanding of both the phytochrome photochemistry and pump−dump/repump−probe spectroscopy techniques has been advanced. |
