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, derived from the sea pansy Renilla reniformis, is notable for its blue light emission, making it an ideal candidate for bioluminescent tagging. Our focus lies at RLuc8, a variant with eight amino acid substitutions, recent research has shown that its emitter, coelenteramide, can exist in different protonation states, significantly influenced by proximal proton acceptor residues at the active site. 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 accurately reproduce the observed emission maximum for the amide anion in RLuc8-D120A, which is responsible for chemiluminescence. In the case of RLuc8-D162A, we observed 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. 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 spectrum, proving vital for further research of proton transfer and light emission in photoproteins.