Wavelength-dependent photochemistry of biological chromophores in gas-phase, solution, and protein environments

We have shown that the photophysics of the photoprotective pigments trans-p-coumaric acid and trans-urocanic acid exhibit wavelength-dependent photophysics in the gas phase due to the presence of two-close lying electronic excited states which have different properties with respect to cis/trans isomerization. The electronic structure of these molecules is similar to styrene and not. The S₁ and S₂ states experience an avoided crossing, which results in an energy barrier to excitation on the S₁ state. The height of this barrier can be affected by both the S₂-S₁ energy gap and the stabilization of the perpendicular state. Upon overcoming the small barrier in the gas phase, isomerization occurs. The evidence for isomerization includes the onset of a dual-emission above the barrier, a shortening of the fluorescence lifetime similar to stilbene above the barrier, and HPLC analysis of the photoproducts collected upon photoexcitation of the molecular beam, which showed a large peak corresponding to the cis photoproduct. The mechanism of isomerization likely involves a conical intersection between the S₁ and S ₀ states. Upon excitation to the S₂ electronic excited state, we observe that internal conversion into S₁ occurs and that the amount of cis photoproduct that forms is significantly smaller than upon direct excitation into S₁. The reasons for the wavelength-dependence in the gas phase are likely due to subtleties in the S₂/S₁ internal conversion process and the shapes of the S₁ and S ₂ potential energy surfaces. We have also found that the photoisomerization quantum yield of pCA in its protein environment in solution is wavelength-dependent. This was measured by observing the decrease in absorbance of the dark state of PYP as a function of excitation wavelength using both pulsed and actinic illumination. We observe an approximately 40% decrease in the isomerization quantum yield with increasing excitation energy. In addition the fluorescence emission maximum exhibits a shift to higher energies as the excitation energy is increased in urocanic acid, p-coumaric acid, and the GFP chromophore. This shift is not present in a ring-locked form of pCA where the chromophore cannot isomerize, or in GFP where the protein is thought to prevent chromophore isomerization.