Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome
Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome
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DOI：
10.1074/jbc.ra119.010384
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发表时间：
2019-12-06
影响因子：
4.8
通讯作者：
Hirose, Yuu
中科院分区：
文献类型：
--
作者：
Sato, Teppei;Kikukawa, Takashi;Hirose, Yuu
蓝细菌色素（CBCR）是光敏色素相关的光传感器，具有跨越整个可见光谱的不同光谱灵敏度。它们通过保守的半胱氨酸残基共价结合胆色素发色团，并在光照下发生15 Z/15 E胆色素光异构化。CBCR亚家族吸收紫蓝色光使用额外的半胱氨酸残基形成第二个胆色素？巯基加合物。然而，第二硫醇加合物形成的过程是不完全理解的，特别是参与的胆色素质子化状态。在这里，我们专注于Oscil6304_2705蛋白从蓝藻颤藻acuminata PCC 6304，它之间的蓝色吸收15 Z状态（铅-15Z）和橙色吸收15 E状态（Po-15 E）的光转换。pH滴定分析显示Pb-15 Z在宽pH范围内是稳定的，表明胆色素质子化通过第二硫醇加合物稳定。正如所揭示的共振拉曼光谱，Po-15 E窝藏质子化的胆色素在酸性和中性pH值，但很容易转化为去质子化的绿色吸收15 Z状态（（15 Z）Pg）在碱性pH值。定点突变显示，保守的Asp-71和His-102残基所需的第二个硫醇加合物形成Pb-15 Z和胆色素质子化Po-15 E，分别。缺乏第二个半胱氨酸残基Cys-73的Oscil6304_2705变体在去质子化（15 Z）Pg和质子化Pr-15 E之间光转化，类似于绿色/红色CBCR亚家族的原变色光循环。时间分辨光谱显示（15 Z）Pg形成的中间体在Pr-15 E？去哪（15 Z）Pg转换具有显着的溶剂-同位素效应，表明15 EP？去哪15 Z光异构化、去质子化和第二硫醇加合物形成。我们的研究结果揭示了紫蓝色的两个半胱氨酸光循环的原色吸收变化的细节？吸收CBCR亚家族。
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-15Z) and orange-absorbing 15E state (Po-15E). pH titration analysis revealed that Pb-15Z 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-15E harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state ((15Z)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-15Z and bilin protonation in Po-15E, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated (15Z)Pg and protonated Pr-15E, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed (15Z)Pg formation as an intermediate in the Pr-15E?to?(15Z)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.