植物色素构成了一系列光感蛋白,被各种生物体用来调节几种生理过程。植物色素结合bilin颜料,在吸收红色或远红色光子时转换其异构状态,导致生物体感觉到的蛋白质构象变化。以前,通过时间分辨串行飞秒X射线衍射(TR-SFX)将细菌植物色素中的超快动力学解析为原子分辨率,在1皮秒延迟时间时显示其分子构象的广泛变化。然而,TR-SFX中使用的mJ/mm2的大激发注量质疑观察到的动力学的有效性。在这项工作中,我们提出了一个激发相关的超快瞬态吸收研究,以测试相关的细菌植物色素对激发注量的响应。我们观察到激励功率相关的亚皮秒动力学,通过共振增强的双光子吸收分配给高激发态Sn的种群,随后快速内部转换为低洼的S1状态。对高通量下的长寿命光谱的检查表明,除了主要的中间Lumi-R,观察到溶剂化电子和电离发色团自由基的光谱特征。在数值模拟的支持下,我们建议在几十μJ/mm2及更高的激发注量下,细菌植物色素部分从Sn状态经历光电离,并在300fs内内部转化为S1状态。我们建议广泛的结构变化相关,较短的细菌植物色素,缺少PHY域,从TR-SFX解析可能受到电离物质的影响。我们提出了通过调节激发光谱远离S1吸收或使用表现出最小化或偏移的S1吸收的植物色素来最小化双光子吸收过程的方法。
Phytochromes constitute a family of photosensory proteins that are utilized by various organisms to regulate several physiological processes. Phytochromes bind a bilin pigment that switches its isomeric state upon absorption of red or far-red photons, resulting in protein conformational changes that are sensed by the organism. Previously, the ultrafast dynamics in bacterial phytochrome was resolved to atomic resolution by time-resolved serial femtosecond X-ray diffraction (TR-SFX), showing extensive changes in its molecular conformation at 1 picosecond delay time. However, the large excitation fluence of mJ/mm2 used in TR-SFX questions the validity of the observed dynamics. In this work, we present an excitation-dependent ultrafast transient absorption study to test the response of a related bacterial phytochrome to excitation fluence. We observe excitation power-dependent sub-picosecond dynamics, assigned to the population of high-lying excited state Sn through resonantly enhanced two-photon absorption, followed by rapid internal conversion to the low-lying S1 state. Inspection of the long-lived spectrum under high fluence shows that in addition to the primary intermediate Lumi-R, spectroscopic signatures of solvated electrons and ionized chromophore radicals are observed. Supported by numerical modelling, we propose that under excitation fluences of tens of μJ/mm2 and higher, bacterial phytochrome partly undergoes photoionization from the Sn state in competition with internal conversion to the S1 state in 300 fs. We suggest that the extensive structural changes of related, shorter bacterial phytochrome, lacking the PHY domain, resolved from TR-SFX may have been affected by the ionized species. We propose approaches to minimize the two-photon absorption process by tuning the excitation spectrum away from the S1 absorption or using phytochromes exhibiting minimized or shifted S1 absorption.