Conformer specific photophysical properties of an analog of the green fluorescent protein chromophore anion

We present a spectroscopic study of a cryogenically cooled GFP chromophore derivative with an additional OH group attached to the phenol ring at the <i>ortho</i> position. Using a depletion technique, we decomposed the spectrum and identified the presence of two conformers. The absorption maximum of one conformer, the <i>para</i>-<i>trans</i> form, which resembles the native GFP chromophore, has a band origin at 482.8 nm, slightly red-shifted compared to the native chromophore. In contrast, the other conformer, the <i>para</i>-<i>cis</i> form, features a structure where the two rings of the chromophore are locked, resulting in a significantly higher excitation energy and a corresponding 14.4 nm blue shift. Comparisons with <i>ab initio</i> TDDFT calculations reveal that low-energy modes in the excited state significantly influence the spectral absorption profiles. For the <i>para</i>-<i>trans</i> form, the addition of the OH group increases the intrinsic energy barrier for internal conversion in the S₁ state from 250 cm^-1 (as observed for the native chromophore) to 390 cm^-1. This leads to an exceptionally long excited-state lifetime, which potentially may yield a high fluorescence-quantum yield. In contrast, for the <i>para</i>-<i>cis</i> conformer, an excited-state lifetime of 430 fs is measured, with internal conversion remaining a competing decay channel across the spectral range examined. Importantly, the OH-N ring locking in the electronic ground state of the <i>para</i>-<i>cis</i> isomer does not prevent internal conversion from de-activating fluorescence in the S₁ excited state.