Prodhan Sayan, Mészáros Petra, Bódizs Szabolcs, Zhou Yalin, Maj Michał, Westenhoff Sebastian
Department of Chemistry - BMC, Biochemistry, Uppsala University, 75123 Uppsala, Sweden.
Department of Chemistry-Ångström Laboratory, Uppsala University, Uppsala, Sweden.
Phys Chem Chem Phys. 2025 Sep 10. doi: 10.1039/d5cp01831e.
Phytochromes are photosensor proteins found in plants, fungi, and bacteria. They photoswitch between red light absorbing (Pr) and far-red light absorbing (Pfr) states. Thermal reversion in the dark, however, is an equally important factor in controlling their signaling levels. Phytochromes are generally dimeric proteins, therefore, mixed PrPfr states are possible. These states have been implicated in dark reversion studies of plant phytochromes, but not in bacterial phytochromes. Here, we investigate the dark reversion kinetics of the 'bathy' phytochrome from (BphP) using UV-Vis absorption spectroscopy. A single set of time-resolved spectra does not conclusively reveal the presence of a mixed PrPfr state, as both a direct Pr → Pfr model or a sequential Pr → PrPfr → Pfr model fit the spectral kinetics. However, a systematic analysis of dark reversion kinetics with varying Pr/Pfr ratios can only be satisfactorily fit by the sequential model, which indicates the presence of an intermediate PrPfr state. A newly designed monomeric variant of BphP provides strong support for this interpretation. Temperature-dependent kinetics revealed similarly low activation energies for the dark reversion processes of both proteins, consistent with a previously proposed keto-enol tautomerization preceding dark reversion. Interestingly, our results suggest allosteric regulation of dark reversion across the dimer, which we propose to be a contributing factor in phytochrome signaling.
光敏色素是存在于植物、真菌和细菌中的光传感器蛋白。它们在吸收红光（Pr）和吸收远红光（Pfr）状态之间进行光转换。然而，黑暗中的热逆转是控制其信号水平的一个同样重要的因素。光敏色素通常是二聚体蛋白，因此，混合的PrPfr状态是可能的。这些状态在植物光敏色素的暗逆转研究中有所涉及，但在细菌光敏色素中并非如此。在这里，我们使用紫外-可见吸收光谱研究了来自（BphP）的“深海”光敏色素的暗逆转动力学。一组时间分辨光谱并不能确凿地揭示混合PrPfr状态的存在，因为直接的Pr→Pfr模型或顺序的Pr→PrPfr→Pfr模型都能拟合光谱动力学。然而，对不同Pr/Pfr比率的暗逆转动力学进行系统分析，只有顺序模型能令人满意地拟合，这表明存在中间的PrPfr状态。新设计的BphP单体变体为这一解释提供了有力支持。温度依赖性动力学显示，两种蛋白的暗逆转过程的活化能同样低，这与之前提出的暗逆转之前的酮-烯醇互变异构一致。有趣的是，我们的结果表明二聚体上暗逆转的变构调节，我们认为这是光敏色素信号传导的一个促成因素。
