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Biochemistry. 2012 Apr 10;51(14):3050-8. doi: 10.1021/bi300020u. Epub 2012 Mar 30.

Thiol-based photocycle of the blue and teal light-sensing cyanobacteriochrome Tlr1999.

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Department of Life Sciences (Biology), Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan.


Cyanobacteriochromes are a spectrally diverse photoreceptor family that binds a bilin chromophore. For some cyanobacteriochromes, in addition to the widely conserved cysteine to anchor the chromophore, its ligation with a second cysteine is responsible for a remarkable blue shift. Herein, we report a newly discovered cyanobacteriochrome Tlr1999 exhibiting reversible photoconversion between a blue-absorbing form at 418 nm (P418) and a teal-absorbing form at 498 nm (P498). Acidic denaturation suggests that P418 harbors C15-Z phycoviolobilin, whereas P498 harbors C15-E phycoviolobilin. When treated with iodoacetamide, which irreversibly modifies thiol groups, P418 is slowly converted to a green-absorbing photoinactive form denoted P552. The absorption spectrum of P498 appears to be unaffected by iodoacetamide, but when iodoacetamide modified, it is photoconverted to P552. These results suggest that a covalent bond exists between the second Cys and the phycoviolobilin in P418 but not in P498. Subsequent treatment with dithiothreitol converts P552 into P418, whereas dithiothreitol reduces P498 to yield P420, a photoinactive form. Site-directed mutagenesis shows that the second Cys is essential for assembly of the photoactive holoprotein and that the photoactivity of this inert mutant is partially rescued by β-mercaptoethanol. These results suggest that the covalent attachment and detachment of a thiol, although not necessarily that of the second Cys, is critical for the reversible spectral blue shift and the complete photocycle. We propose a thiol-based photocycle, in which the thiol-modified P552 and P420 are intermediate-like forms.

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