Bioluminescence is a fascinating natural phenomenon, wherein organisms produce light through specific biochemical reactions. Among these organisms, Renilla luciferase (RLuc) derived from the sea pansy Renilla reniformis is notable for its blue light emission and has potential applications in bioluminescent tagging. Our study focuses on RLuc8, a variant of RLuc with eight amino acid substitutions. Recent studies have shown that the luminescent emitter coelenteramide can adopt multiple protonation states, which may be influenced by nearby residues at the enzyme’s active site, demonstrating a complex interplay between protein structure and bioluminescence. Herein, using the quantum mechanical consistent force field method and the semimacroscopic protein dipole-Langevin dipole method with linear response approximation, we show that the phenolate state of coelenteramide in RLuc8 is the primary light-emitting species in agreement with experimental results. Our calculations also suggest that the proton transfer (PT) from neutral coelenteramide to Asp162 plays a crucial role in the bioluminescence process. Additionally, we reproduced the observed emission maximum for the amide anion in RLuc8-D120A and the pyrazine anion in the presence of a Na⁺ counterion in RLuc8-D162A, suggesting that these are the primary emitters. Furthermore, our calculations on the neutral emitter in the engineered AncFT-D160A enzyme, structurally akin to RLuc8-D162A but with a considerably blue-shifted emission peak, aligned with the observed data, possibly explaining the variance in emission peaks. Overall, this study demonstrates an effective approach to investigate chromophores’ bimolecular states while incorporating the PT process in emission spectra calculations, contributing valuable insights for future studies of PT in photoproteins.

中文翻译：

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通过有效的 QM/MM 方法探索海肾荧光素酶中的发光剂


生物发光是一种令人着迷的自然现象，生物体通过特定的生化反应产生光。在这些生物体中，源自海三色海肾 (Renilla reniformis) 的海肾荧光素酶 (RLuc) 以其蓝光发射而闻名，在生物发光标记方面具有潜在的应用。我们的研究重点是 RLuc8，它是具有 8 个氨基酸取代的 RLuc 变体。最近的研究表明，发光发射体腔肠酰胺可以采用多种质子化状态，这可能会受到酶活性位点附近残基的影响，证明蛋白质结构和生物发光之间存在复杂的相互作用。在此，我们利用量子力学一致力场法和线性响应近似的半宏观蛋白质偶极子-朗之万偶极子法，证明RLuc8中腔肠酰胺的酚盐态是主要的发光物质，与实验结果一致。我们的计算还表明，从中性腔肠酰胺到 Asp162 的质子转移 (PT) 在生物发光过程中起着至关重要的作用。此外，我们在 RLuc8-D162A 中重现了在 Na ⁺ 反离子存在下观察到的 RLuc8-D120A 中酰胺阴离子和吡嗪阴离子的发射最大值，表明这些是主要发射器。此外，我们对工程化 AncFT-D160A 酶中的中性发射体进行了计算，该酶在结构上类似于 RLuc8-D162A，但具有相当大的蓝移发射峰，与观察到的数据一致，这可能解释了发射峰的变化。 总的来说，这项研究展示了一种研究发色团双分子态的有效方法，同时将 PT 过程纳入发射光谱计算中，为发光蛋白中 PT 的未来研究提供了宝贵的见解。