Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.
Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state (  Pb) and orange-absorbing 15E state (  Po). pH titration analysis revealed that  Pb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy,  Po harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state (  Pg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in  Pb and bilin protonation in  Po, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated  Pg and protonated  Pr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed  Pg formation as an intermediate in the  Pr-to-  Pg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP-to-15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blue-absorbing CBCR subfamilies.
['Sato|Teppei|T|', 'Kikukawa|Takashi|T|', 'Miyoshi|Risako|R|', 'Kajimoto|Kousuke|K|', 'Yonekawa|Chinatsu|C|', 'Fujisawa|Tomotsumi|T|', 'Unno|Masashi|M|', 'Eki|Toshihiko|T|', 'Hirose|Yuu|Y|']
D001654:Bile Pigments D003545:Cysteine D006863:Hydrogen-Ion Concentration D054300:Oscillatoria D010834:Phytochrome
cyanobacteriochrome bilin Raman spectroscopy spectroscopy time-resolved spectroscopy cyanobacteria photobiology photoreceptor phytochrome