Phytochromes are widely occurring red/far-red photoreceptors that utilize a linear tetrapyrrole (bilin) chromophore covalently bound within a knotted PAS-GAF domain pair. Cyanobacteria also contain more distant relatives of phytochromes that lack this knot, such as the phytochrome-related cyanobacteriochromes implicated to function as blue/green switchable photoreceptors. In this study, we characterize the cyanobacteriochrome Tlr0924 from the thermophilic cyanobacterium Thermosynechococcus elongatus. Full-length Tlr0924 exhibits blue/green photoconversion across a broad range of temperatures, including physiologically relevant temperatures for this organism. Spectroscopic characterization of Tlr0924 demonstrates that its green-absorbing state is in equilibrium with a labile, spectrally distinct blue-absorbing species. The photochemically generated blue-absorbing state is in equilibrium with another species absorbing at longer wavelengths, giving a total of 4 states. Cys499 is essential for this behavior, because mutagenesis of this residue results in red-absorbing mutant biliproteins. Characterization of the C 499 D mutant protein by absorbance and CD spectroscopy supports the conclusion that its bilin chromophore adopts a similar conformation to the red-light-absorbing P r form of phytochrome. We propose a model photocycle in which Z/E photoisomerization of the 15/16 bond modulates formation of a reversible thioether linkage between Cys499 and C10 of the chromophore, providing the basis for the blue/green switching of cyanobacteriochromes.Photosynthetic organisms face the need to coordinate their metabolic responses to their light environment, so that photosynthesis and redox balance are properly maintained for growth. This is accomplished by a wide range of photosensory proteins (1,2). The first such proteins to be discovered were the phytochromes, which are red/far-red photosensors initially described in plants and later shown to be widespread in both photosynthetic and nonphotosynthetic organisms (3,4). Upon excitation with red light, phytochromes photoconvert from the redabsorbing P r state 1 , which is usually thermally stable, to the far-red absorbing P fr state (5). This reversible interconversion is the result of light-driven Z/E isomerization of the 15/16 double bond of the protein-bound bilin chromophore ( Fig. 1), which is covalently attached to a Cys residue in the conserved photosensory core of phytochromes. This photosensory core is generally found N-terminal to putative output domains implicated in signal transduction, such as the histidine kinase domain of the cyanobacterial phytochrome Cph1 (6). Since both P r and NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 July 8. Published in final edited form as:Biochemistry. 2008 July 8; 47(27): 7304-7316. doi:10.1021/bi800088t. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript P fr states can generate signaling outputs (4,7), light modulates the signaling activity of phytochrome...