E - Z Isomerization Mechanism of the Green Fluorescent Protein Chromophore: Remote Regulation by Proton Dissociation of the Phenol Group.

Dronpa, a GFP (green fluorescent protein)-like fluorescent protein, allows its fluorescent and nonfluorescent states to be switched to each other reversibly by light or heat through E - Z isomerization of the GFP chromophore. In this article, a GFP chromophore ( p -HBDI) in water is used as a model to explore this E - Z isomerization mechanism. Based on the experimental solvent isotope effect ( k H2 O / k D2 O = 2.30), the E - Z isomerization of p -HBDI in water is suggested to go through the remote-proton-dissociation-regulated direct mechanism with a proton transfer in the rate-determining step. The fractionation factor (ϕ) of the water-associated phenol proton of p -HBDI in the transition state is found to be 0.43, which is exactly in the range of 0.1-0.6 for the fractionation factor (ϕ) of the transferring proton in the transition state of R2 O··· H ···O+ H2 in water. This means that the phenol proton of E - p -HBDI in the transition state is on the way to the associated water oxygen during the E - Z isomerization. The proton dissociation from the phenol group of p -HBDI remotely regulates its E - Z isomerization. Less proton dissociation from the phenol group (p K a = 8.0) at pH = 1-4 results in a modest reduction in the E - Z isomerization rate of p -HBDI, while complete proton dissociation from the phenol group at pH = 11-12 also reduces its E - Z isomerization rate by one order of magnitude because of the larger charge separation in the transition state of the p -HBDI anion. All of these results are consistent with the remote-proton-dissociation-regulated direct mechanism but against the water-assisted addition/elimination mechanism.