The Effect of Conjugation on the Competition between Internal Conversion and Electron Detachment: A Comparison between Green Fluorescent and Red Kaede Protein Chromophores

Abstract

Kaede, an analogue of green fluorescent protein (GFP), is a green-to-red photoconvertible fluorescent protein used as an in vivo “optical highlighter” in bioimaging. The fluorescence quantum yield of the red Kaede protein is lower than that of GFP, suggesting that increasing the conjugation modifies the electronic relaxation pathway. Using a combination of anion photoelectron spectroscopy and electronic structure calculations, we find that the isolated red Kaede protein chromophore in the gas phase is deprotonated at the imidazole ring, unlike the GFP chromophore that is deprotonated at the phenol ring. We find evidence of an efficient electronic relaxation pathway from higher-lying electronically excited states to the S1 state of the red Kaede chromophore that is not accessible in the GFP chromophore. Rapid autodetachment from high-lying vibrational states of S1 is found to compete efficiently with internal conversion to the ground electronic state.

Photoelectron Imaging and Quantum Chemistry Study of Phenolate, Difluorophenolate, and Dimethoxyphenolate Anions

“…Broadening of photoelectron spectra following photoexcitation of resonances in the continuum has been observed in similar sized molecular anions by many other groups 14,30,31 including our own. 11,[32][33][34][35][36][37][38][39][40][41][42] This resonance in PhO − has been observed before 12,14 and assigned to indirect detachment from the S 1 (ππ * ) state of PhO − . The S 0 -S 1 (ππ * ) transition corresponds to promotion of an electron from the HOMO shown in Table 1 to the first antibonding orbital on the phenyl ring system (the LUMO of PhO − ) and therefore has shape resonance character with respect to the D 0 continuum.…”

Photoelectron spectroscopy of isolated luciferin and infraluciferin anions in vacuo: competing photodetachment, photofragmentation and internal conversion

“…To support this proposed relaxation mechanism, it is worth noting that our group observed a similar continuum of low eKE electrons in photoelectron spectra of the deprotonated red Kaede anion and complementary high-level quantum chemistry calculations allowed us to assign this feature to internal conversion through a series of conical intersections to the lowest lying excited electronic state which subsequently decayed by electron detachment. 43 The observation that there is an efficient electronic relaxation pathway from higher lying electronic states to the emissive S 1 (1pp*) state, that is also coupled strongly to the electron detachment continuum, is significant because it suggests that isolated LH À and iLH À may function as UV photoactivated chromophores for bioimaging or have applications in monitoring redox processes. The calculated D 0 value for the phenolate 1 isomer of LH À (3.13 eV) ( Table 1) is in good agreement with the position of the lowest energy peaks in the experimental LH À photoelectron spectra, whereas the calculated D 1 value (5.25 eV) is a little higher than the highest energy peak in the 230 nm photoelectron spectrum.…”

“…The electrospray anion photodetachment spectroscopy method was pioneered by LS Wang 31 and used by XB Wang 32 and photoelectron imaging spectroscopy of anions is employed by a number of groups [33][34][35][36][37] as well as our own. [38][39][40][41][42][43][44][45] The design of our spectrometer is described in an earlier publication. 46 For the negative ion electrospray, 1 mM solutions of luciferin and infraluciferin in dry methanol were treated with a few drops of ammonia as a base to deprotonate the acidic groups.…”

“…Our group has made similar observations in green fluorescent protein (GFP), red Kaede protein and photoactive yellow protein (PYP) chromophore anions and attributed the broadening to resonant excitation of higher lying electronically excited states that are coupled strongly to the detachment continuum, [38][39][40][41][42]44 or resonant excitation of higher lying electronic states, that are only weakly coupled to the detachment continuum but undergo rapid internal conversion to a lower lying electronically excited state that is coupled strongly to the detachment continuum. 43 In addition to the peak around 3.2 eV attributed to direct detachment, the 230 nm photoelectron spectrum has a weak feature around 4.5 eV and a more intense peak with a maximum just below 5 eV. There are two possible deprotonation sites in LH (phenol and carboxylic acid groups) so these higher eBE features may be attributed to detachment from different deprotonated forms of the anions or detachment to higher lying electronically excited states of the neutral radicals associated with the different deprotonated forms of the anions.…”

Anion photoelectron spectroscopy of protein chromophores

“…The resulting small droplets then evaporate further, eventually leading to the production of isolated gas-phase ions. ESI has been combined very successfully with photoelectron spectroscopy for studying the electronic structure of a wide range of deprotonated biomolecules, including protein chromophores [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. There are several techniques for measuring photoelectron spectra, the most popular of which are based on time-of-flight (TOF) or velocity map imaging (VMI) methods.…”