On the Driving Force of the Excited-State Proton Shuttle in the Green Fluorescent Protein: A Time-Dependent Density Functional Theory (TD-DFT) Study of the Intrinsic Reaction Path.

We simulated the intrinsic reaction path of the Green Fluorescent Protein (GFP) proton shuttle in both the ground state (S0) and first singlet excited state (S1), accounting for the main energetic and steric effects of the protein in a convenient model including the chromophore, the crystallographic water, and the residues directly involved in the proton transfer event. We adopted density functional theory (DFT) and time-dependent density functional theory (TD-DFT) levels to define the potential energy surfaces of the two electronic states, and we compared results obtained by the Damped Velocity Verlet and the Hessian-based Predictor-Corrector integrators of the intrinsic reaction coordinate, which gave a comparable and consistent picture of the mechanism. We show that, at S1, the GFP proton transfer becomes favored, with respect to S0, as suggested by the experimental evidence. As an important finding, this change is strictly related to the rearrangement of the hydrogen bond network composing the reaction path, which, in S1, relaxes to a tighter and planar configuration, as a consequence of the photoinduced relaxation in the GFP chromophore structure, thus prompting more effectively for the proton shuttle. Therefore, we give an unprecedented direct proof of the key role played by the photoinduced structural relaxation of the GFP on the chromophore photoacidity, validating, in particular, the hypothesis of Fang and co-workers [Nature 2009, 462, 200].