Theoretical Study on the Excited State Transformation of Oxyluciferin-Luciferase Complex with Free Energy Simulation

Abstract

Firefly bioluminescence has been widely studied due to its high quantum yield and its broad range of color modulation, but debates on the origin of its color modulation and the exact chemical form of the emitter are still lingering. While the experimental observation that the spectrum can be decomposed into multiple peaks cannot be easily explained by the color shifts of a single chemical form, computational studies have questioned the possibility of having multiple chemical forms of oxyluciferin. Here, this issue is revisited with different pathway and free energy simulation. Free energy calculation including dynamic contribution may provide different results from those based on the minimum energy path. In order to obtain a free energy information, it is important to sample a wide range of conformational space. High level of quantum mechanical methods are also required for accurate description, but their computations are often too demanding from the view point of conformational sampling. As a remedy, level correction schemes that allow calculating high level free energies based on conformations from lower level simulations have been developed. In this dissertation, two topics are discussed: 1) the development of free energy level correction scheme and 2) the free energy study on the keto-enolate transformation of the oxyluciferin-luciferase complex. In the first part, I present a Monte Carlo (MC) resampling scheme associated with weighted histogram analysis method (WHAM). It is shown that MC resampled WHAM displays potentials of mean force (PMFs) that converge to the exact one with an enough number of sampled configurations and generates smoother PMF morphology than conventional WHAM correction scheme with a reduced sample size. Also, I show that MC resampling can provide a guide for checking the uncertainty of the level-corrected surface and a mathematically defined criterion for deciding the extent of smoothing on the free energy surface for its visual improvement. These features demonstrate that the MC resampled WHAM scheme can be a useful tool for producing free energy surfaces of realistic systems. In the second part, the investigation on the keto–enolate transformation of the oxyluciferin–luciferase complex with quantum mechanics / molecular mechanics (QM/MM) free energy simulations is presented. Although the free energy barrier for enolate formation presents much lower than that from previous studies, the overall transform is still unlikely. However, I additionally show that the energetics of the transformation can be heavily modulated with the protein electrostatics. This suggests that carefully modeling the protein–ligand interaction is an important key to understanding the reaction mechanism of the firefly bioluminescence. Thus, I conclude that it is not yet appropriate to completely rule out the possibility of having different form participation.