【JACS】惊人发现！455nm蓝光下 photoenzyme 发生不可逆光失活，核心辅助因子释放与降解机制被首次揭示#

Figure 1. Native mass spectra of FAP (200 mM NH4CH3COO, pH 7.4) in the dark and following 455 nm irradiation, and photofragment identification. (A) Native mass spectra of FAP dark (bottom panel) and under irradiation (top panel). Each spectrum was acquired for 5 min; the insert in the top spectrum shows the appearance of FAD under blue light illumination; the corresponding region in the dark shows very little signal of FAD (Figure S6C); major forms of the FAP protein are depicted as cyan star: holo-FAP bound to two fatty acid substrates, yellow square: holo-FAP bound to natively present SA, green triangle: substrate-free FAP bound to FAD and blue circle: apo-FAP. (B) Summed selected ion chromatograms from the signal of each of the major forms of FAP [M + nH]n+ where n = 14–16 for a total acquisition time of 800 s, where the sample is irradiated after 200 s. (C) Deconvoluted mass and assignment of FAP forms. The experimental mass differences between each neighboring proteoform are also tabulated. (D) FAD photofragmentation sites, corresponding photoproduct masses, and quantitative classification of their photodegradation reaction time scales. (E) Schematic workflow and multidimensional outputs of light footprinting IM-MS. The pipeline spans from sample preparation to multidimensional outputs in the dark and under 455 nm irradiation, featuring m/z distributions forming the native mass spectra and arrival time distributions (ATD) that we convert to CCS distributions; aIMS heatmaps and fragmentation data; permitting subsequent multivariate statistical results (PCA).#

Figure 2. Traveling wave ion mobility and activated ion mobility of apo-FAP and holo-FAP, and representative structure of compact and extended conformers of FAP from gas phase MD simulations. (A) CCS distributions of 14+, 15+, and 16+ charge states of FAP with FAD in the dark. (B) CCS distributions of 14+, 15+, and 16+ charge states of apo-FAP ions produced during irradiation. (C) CCS distributions of 14+, 15+, and 16+ charge state ions of irradiated FAP. (D–F) Representative structures produced by gas-phase MD simulations. (D) Compact geometry of apo-FAP, theoretical CCSN2 of 4459 Å2. (E) Compact geometry of holo-FAP, theoretical CCSN2 of 4515 Å2. FAD shown in yellow; residues with structural differences from the apo form shown in red. (F) Extended geometry of FAP, theoretical CCSN2 of 4782 Å2. (G, H) Comparison of the unfolding pathways of apo- and holo-FAP forms, as well as holo-FAP under dark and blue light conditions, using aIMS. (G) aIMS heat maps and difference heat map for 16+ apo-FAP intermediate and 16+ FAP in the dark. (H) aIMS heat maps and difference heat map for 16+ FAP under dark and 16+ FAP under blue light.#

然而，一旦暴露在 455纳米的蓝光高功率 LED 光源下，质谱信号发生了戏剧性的重构。随着光照时间的延长，所有原本结合有底物和 FAD 辅因子的蛋白质复合物信号开始协同衰减，与此同时，一个全新的不含任何配体的 apo-FAP 蛋白质形态信号迅速涌现并随后达到动态平衡。这一动态变化直接揭示了溶液中同时并存的两个激烈竞争的反应路径，即正常的底物光脱羧催化路径与光诱导的 FAD 辅因子释放失活路径。质谱的高分辨监测进一步捕捉到了游离 FAD 分子的大量出现以及伴随而来的多条动力学断裂碎片线索。研究人员根据响应速度将其划分为百秒内即饱和的快速光解阶段与更长周期的慢速降解阶段，且结合态与游离态 FAD 迥异的碎片图谱证实，FAP 内部独特的微环境在早期对 FAD 的光化学行为具有强烈的控制作用。

Figure 3. CD spectra of FAP under dark and irradiated conditions. (A, C) Far-UV (205–260 nm) CD at temperatures over the 20 to 94 °C range. (B, D) fitting of thermodynamic transitions using three thermodynamic ensembles. (A, B) dark state of FAP. (C, D) FAP after 60 s of irradiation at 1200 μmol m–2 s–1. (E) Far-UV (180–260 nm) CD spectra of FAP in the dark and irradiated states. Far-UV CD spectra indicate a subtle loss of secondary structure due to 455 nm light exposure. (F) UV–vis CD spectra of the FAP dark state (black line), the FAP after 60 s of irradiation (blue line), and FAD in aqueous solution (yellow line).#

为了从三维结构层面解析这一失活过程，离子迁移质谱技术发挥了核心作用。实验表明，无论是处于黑暗还是光照状态，FAP 在气相中均展现出分别集中在特定碰撞截面积的“闭合”紧凑构象与“开放”延伸构象。在蓝光照射下，整个碰撞截面积分布显著变宽，表明光照引发了蛋白质全局构象动态变动性的剧烈增加。时间分辨的构象转化动力学数据表明，紧凑构象的蛋白质在光照下转化为 apo 形态的速度显著快于延伸构象，说明紧凑状态对光表现出更高的敏感性。结合分子动力学模拟结果，研究团队发现从 holo 转化为 apo 形态时光释放导致的局部结构翻转相对温和，而紧凑与延伸构象之间的转换则涉及巨大的全局结构重排。

Figure 4. Multivariate analysis of FAP from top-down MS and IM-MS data in both dark and light. (A) PCA scores plot of FAP using aIMS data, which yields peptide fragments from 16+ apo-FAP and holo-FAP ions. (B) PCA loading plot of the total ATDs from all ions (1st PC), where the most contributing features correspond to the ATD of the 16+ charge state of FAP (apo and holo) (∼85 ms) as well as FAD photofragments. (C) Confusion matrix trained on IM arrival time data. (D) PCA scores plot of +16 holo-FAP from top-down CID data. (E) Fragments that distinguish dark and blue light irradiation groups (eight significant features) mapped onto the FAP sequence, b and internal fragment ions are underlined in red and blue, respectively. Residues on the sequence proximal to FAD, stearic acid, and palmitic acid binding sites from pdb 6zh77 are labeled with green, yellow, and cyan, respectively.#

随后开展的碰撞激活离子迁移质谱（aIMS） 实验则进一步明确了这种稳定性差异。在逐级提高的碰撞电压下，脱去 FAD 的 apo-FAP 在更低的激活能量下便引发了多步解折叠过渡，且最终达到了更为松散舒展的终态，直接证明了 FAD 辅因子的流失会导致 FAP 全局折叠结构的严重去稳定化。同样地，历经蓝光照射后的 holo-FAP 较之黑暗对照组，其解折叠起始电压也明显降低。在溶液状态下进行的温度依赖性远紫外圆二色光谱实验完美印证了这一气相结论，黑暗状态下的 FAP 表现出高度协同的蛋白质协作折叠熔解曲线，而光照后的样本其热变性过渡变得极为平缓，表明光化学损伤导致了天然折叠协同性的丧失。

Figure 5. Fluorescence spectra of FAP under varying light intensities and with and without additional palmitic acid (PA). (A, B) Fluorescence spectrum of FAP under continuous pulse laser irradiation. (A) Without PA, (B) with 300 μM PA. (C) Time-dependent fluorescence emission with 455 nm excitation under light intensities between 100 and 1600 μmol m–2 s–1, in the presence (solid traces) or absence of PA (dashed traces). (D) Concentrations of FAD in solution in the dark and after irradiation, deduced from absorbance spectra of filtrates. Investigation of the effect of blue light intensity and substrate on the release of FAD from FAP. (E, F) Rate coefficients (fluorescence units/s) of initial fluorescence growth as a function of light intensity, along with power law and linear fits. (E) FAP without additional PA. (F) FAP with 500 μM PA.#

有趣的是，通过定量计算不同形态蛋白质的光失活速率常数，研究人员发现了一条非常关键的保护机制。无底物结合的空载 FAP 复合物其信号衰减速率最快，而当其结合有一个或两个底物分子时，时光失活速率大幅度慢化。这表明脂肪酸底物的结合对光酶结构具有显著的物理保护效应，底物的存在介导了高效的光氧化还原脱羧，从而动态抑制了低效且具破坏性的光失活旁路。溶液荧光光谱动力学实验进一步证实，在不添加外源底物棕榈酸时，光照会导致 isoalloxazine 荧光由于 FAD 释放而呈指数级飙升；而当提供饱和浓度的底物时，这种荧光的增长被极大地淬灭和压制。

最后，科研团队通过对光化学动力学规律进行幂律公式拟合，深入挖掘了光强对失活机制的调控规律。在缺乏底物的情况下，初始荧光增长速率对光强的依赖性表现出高达 1.37 的非线性多光子过程特征，这意味着空载的光酶内部处于激发态的强氧化性 FAD 1* 无法将电子从未结合底物的活性位点移走，转而低效地夺取周围关键氨基酸残基的电子，引发了诸如过氧化等链式蛋白质光化学损伤及骨架降解。而在充足底物存在下，反应则退化为标准的单光子线性依赖过程。基于多元统计学（PCA 和 PLS-DA）对拓扑质谱碎片谱及迁移时间数据的深度挖掘，研究团队成功将黑暗与光照组进行了百分之百准确度的分类判别，并精准定位了八个远离活性中心、由于光损伤而非热裂解产生特异性肽段断裂位点，全景式地勾勒出了 FAP 结构受损的分子图谱。