Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a “bucket” consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pK_a, whereas they are directly hydrogen bonded in the β-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the “leaky bucket” structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.

蓝细菌色素 (CBCR) 是光敏色素超家族的胆碱结合光传感器，具有显着的光谱多样性。绿色/红色 CBCR 亚家族对于调节蓝藻中光合天线的色度驯化很重要，并应用于合成生物学中基因表达的光遗传学控制。有人建议，该亚科的吸收变化是由比林C15造成-Z / C15- Ë光异构化和胆素质子化状态的后续变化。然而，缺乏胆素质子化状态的结构信息和直接证据。在这里，我们报告了红色吸收光产物状态下色温传感器 RcaE 的胆素结合域的高分辨率 (1.63Å) 晶体结构。后胆色素埋入一个“桶”由疏水性残基，其中后胆色素结构/构象是C5-内Ž，顺式/ C10- Ž，顺式/ C15- ë，顺式与通过C环的共面A-和D 形环倾斜。A-到 C-环的三个吡咯氮覆盖在 α-面，带有 Leu249 的疏水盖，影响胆碱 pK _a，而它们在 β 面与 Glu217 的羧基直接氢键合。Glu217 进一步连接到一组水，在桶中形成一个洞，在分子动力学模拟中与溶剂水进行交换。我们建议“漏桶”结构在光转换时充当质子退出/流入途径。核磁共振分析表明，四个吡咯氮原子确实在红色吸收状态下完全质子化，但其中一个，最有可能是 B 环氮，在绿色吸收状态下被去质子化。这些发现加深了我们对 CBCR 中存在的各种光谱调谐机制的理解。