Histidine-assisted reduction of arylnitrenes upon photo-activation of phenyl azide chromophores in GFP-like fluorescent proteins.

The photochemically active sites of the proteins sfGFP ^66azF and Venus ^66azF , members of the green fluorescent protein (GFP) family, contain a non-canonical amino acid residue p -azidophenylalanine (azF) instead of Tyr66. The light-induced decomposition of azF at these sites leads to the formation of reactive arylnitrene (nF) intermediates followed by the formation of phenylamine-containing chromophores. We report the first study of the reaction mechanism of the reduction of the arylnitrene intermediates in sfGFP ^66nF and Venus ^66nF using molecular modeling methods. The Gibbs energy profiles for the elementary steps of the chemical reaction in sfGFP ^66nF are computed using molecular dynamics simulations with quantum mechanics/molecular mechanics (QM/MM) potentials. Structures and energies along the reaction pathway in Venus ^66nF are evaluated using a QM/MM approach. According to the results of the simulations, arylnitrene reduction is coupled with oxidation of the histidine side chain on the His148 residue located near the chromophore.