Exploring the Light-Emitting Agents in Renilla Luciferases by an Effective QM/MM Approach

Bioluminescence is a remarkable natural process in which living organisms produce light via specific biochemical reactions. Among these organisms, Renilla luciferase (RLuc), derived from the sea pansy Renilla reniformis, is notable for its blue light emission, making it one of the promising candidates for bioluminescent tagging. Our study focuses on RLuc8, a modified variant of RLuc characterized by eight amino acid substitutions. Recent findings have illuminated that its luminescent emitter, coelenteramide, is capable of existing in multiple protonation states. These states may significantly be influenced by adjacent proton acceptor residues at the enzyme's active site, highlighting the complex interplay between the protein structure and its bioluminescent activity. Herein employing the Quantum Mechanical Consistent Force Field (QCFF/PI) method and the semi-macroscopic Protein Dipole-Langevin Dipole method with Linear Response Approximation (PDLD/S-LRA), we show that the phenolate state of coelenteramide in RLuc8 is the predominant light-emitting species, corroborating experimental results. Our calculations also demonstrate that proton transfer from neutral coelenteramide to Asp162 is integral to the bioluminescence mechanism. Furthermore, our calculations reproduce the observed emission maximum for the amide anion in RLuc8-D120A mutant. In the case of RLuc8-D162A, we predicted that the pyrazine anion, existing in the presence of a Na+ counterion, has an emission maximum consistent with experimental data, suggesting its primary role as potential emitter. Additionally, our calculations on the engineered AncFT-D160A enzyme, structurally similar to RLuc8-D162A but with a significantly blue-shifted emission peak, show that the emission peak of its neutral form of the emitter agrees well with observed data. This agreement may explain the variations in observed emission peaks. This study not only showcases an effective way for investigating the bimolecular states of chromophores in light emission but also introduces an efficient approach that integrates the proton transfer process into the calculations of the emission spectra, proving vital for further research of proton transfer and light emission in photoproteins.