Londi Giacomo, Salvadori Giacomo, Mazzeo Patrizia, Cupellini Lorenzo, Mennucci Benedetta
Department of Chemistry and Industrial Chemistry, University of Pisa, 56124 Pisa, Italy.
Institute for Computational Biomedicine (INM-9), Forschungszentrum Jülich, 52428 Jülich, Germany.
JACS Au. 2024 Dec 18;5(1):158-168. doi: 10.1021/jacsau.4c00853. eCollection 2025 Jan 27.
Naturally occurring photoenzymes are rare in nature, but among them, fatty acid photodecarboxylases derived from (FAPs) have emerged as promising photobiocatalysts capable of performing the redox-neutral, light-induced decarboxylation of free fatty acids (FAs) into C1-shortened alka(e)nes. Using a hybrid QM/MM approach combined with a polarizable embedding scheme, we identify the structural changes of the active site and determine the energetic landscape of the forward electron transfer (fET) from the FA substrate to the excited flavin adenine dinucleotide. We obtain a charge-transfer diradical structure where a water molecule rearranges spontaneously to form a H-bond interaction with the excited flavin, while the FA's carboxylate group twists and migrates away from it. Together, these structural modifications provide the driving force necessary for the fET to proceed in a downhill direction. Moreover, by examining the R451K mutant where the FA substrate is farther from the flavin core, we show that the marked reduction of the electronic coupling is counterbalanced by an increased driving force, resulting in a fET lifetime similar to the WT, thereby suggesting a resilience of the process to this mutation. Finally, through QM/MM molecular dynamic simulations, we reveal that, following fET, the decarboxylation of the FA radical occurs within tens of picoseconds, overcoming an energy barrier of ∼0.1 eV. Overall, by providing an atomistic characterization of the photoactivation of FAP, this work can be used for future protein engineering.
天然存在的光酶在自然界中很罕见，但在它们之中，源自（FAPs）的脂肪酸光脱羧酶已成为有前途的光生物催化剂，能够将游离脂肪酸（FAs）进行氧化还原中性的光诱导脱羧反应，生成碳链缩短一个碳原子的烷烃（烯烃）。使用结合了可极化嵌入方案的混合量子力学/分子力学（QM/MM）方法，我们确定了活性位点的结构变化，并确定了从FA底物到激发态黄素腺嘌呤二核苷酸的正向电子转移（fET）的能量景观。我们得到了一种电荷转移双自由基结构，其中一个水分子自发重排，与激发态黄素形成氢键相互作用，而FA的羧基扭曲并远离它。这些结构修饰共同提供了fET向下进行所需的驱动力。此外，通过研究FA底物离黄素核心更远的R451K突变体，我们表明电子耦合的显著降低被增加的驱动力所抵消，导致fET寿命与野生型相似，从而表明该过程对这种突变具有弹性。最后，通过QM/MM分子动力学模拟，我们揭示了在fET之后，FA自由基的脱羧反应在几十皮秒内发生，克服了约0.1 eV的能垒。总体而言，通过提供FAP光激活的原子水平表征，这项工作可用于未来的蛋白质工程。
