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.