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Abstract

The BLUF domain of the transcriptional anti−repressor protein AppA from the non−sulfur anoxyphototrophic purple bacterium Rhodobacter sphaeroides was characterized by absorption and emission spectroscopy. The BLUF domain constructs AppA148 (consisting of amino−acid residues 1−148) and AppA126 (amino−acid residues 1−126) are investigated. The cofactor of the investigated domains is found to consist of a mixture of the flavins riboflavin, FMN, and FAD. The dark−adapted domains exist in two different active receptor conformations (receptor states) with different sub−nanosecond fluorescence lifetimes (BLUFr,f and BLUFr,sl) and a small non−interacting conformation (BLUFnc). The active receptor conformations are transformed to putative signalling states (BLUFs,f and BLUFs,sl) of low fluorescence efficiency and picosecond fluorescence lifetime by blue−light excitation (light−adapted domains). In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 17 min. A quantum yield of signalling state formation of about 25% was determined by intensity dependent transmission measurements. A photo−cycle scheme is presented including photo−induced charge transfer complex formation, charge recombination, and protein binding pocket reorganisation