The effect of the protein environment on the electronic structure of the green fluorescent protein (GFP) chromophore is investigated by QM/MM (quantum mechanics/molecular mechanics) calculations. The protein has very small effect on the excitation energy of the bright absorbing and the lowest triplet states of the anionic GFP chromophore, deprotonated 4-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) anion, however, it increases vertical detachment energy from 2.5 eV (gas-phase deprotonated HBDI anion) to 5.0 eV (solvated protein). We also investigated possible existence of the charge-transfer-to-solvent (CTTS) states associated with the GFP chromophore. Although precursors of such states appear in cluster calculations, a tightly packed structure of the protein prevents the formation of the CTTS states in this system. Motivated by a recently discovered new type of photoconversion, oxidative redding, we characterized the redox properties of GFP. The computed standard reduction potential of the anionic form of GFP is 0.47 V (for the GFP(•) + 1e → GFP(-) reaction), and the reduction potential at physiological conditions (pH 7, T = 25 °C) is 0.06 V.

The proposed mechanisms of photoinduced reactions in the blue light using flavin chromophore photoreceptor proteins are primarily based on the results of X-ray crystallography and spectroscopy studies. Of particular value are the observed band shifts in optical and vibrational spectra upon formation of the signaling (light-induced) state. However, the same set of experimental data has given rise to contradictory interpretations suggesting different structures of the dark and signaling states. To verify the specific mechanism of light-induced changes involving the rotation/tautomerization transformations with the conserved Gln residue near the flavin chromophore, we performed accurate quantum chemical calculations of the equilibrium structures, vibrational and absorption bands of the model systems mimicking the BLUF domain of flavoprotein AppA. Geometry optimization and calculations of vibrational frequencies were carried out with the QM(B3LYP/cc-pVDZ)/MM(AMBER) approach starting from the representative molecular dynamics (MD) snapshots. The MD simulations were initiated from the available crystal structures of the AppA protein. Calculations of the vertical excitation energies were performed with the scaled opposite spin configuration interaction with single substitutions SOS-CIS(D) method that enables efficient treatment of excited states in large molecular systems. The computed molecular structures as well as the spectral shifts (the red shift by 12÷16 nm in absorption and the downshift by 25 cm(-1) for the C4═O flavin vibrational mode) are in excellent agreement with the experimental results, lending a strong support to the mechanism proposed by Domratcheva et al. (Biophys. J. 2008, 94, 3872).