Liquid-microjet photoelectron spectroscopy of the green fluorescent protein chromophore.

Green fluorescent protein (GFP), the most widely used fluorescent protein for in vivo monitoring of biological processes, is known to undergo photooxidation reactions. However, the most fundamental property underpinning photooxidation, the electron detachment energy, has only been measured for the deprotonated GFP chromophore in the gas phase. Here, we use multiphoton ultraviolet photoelectron spectroscopy in a liquid-microjet and high-level quantum chemistry calculations to determine the electron detachment energy of the GFP chromophore in aqueous solution. The aqueous environment is found to raise the detachment energy by around 4 eV compared to the gas phase, similar to calculations of the chromophore in its native protein environment. In most cases, electron detachment is found to occur resonantly through electronically excited states of the chromophore, highlighting their importance in photo-induced electron transfer processes in the condensed phase. Our results suggest that the photooxidation properties of the GFP chromophore in an aqueous environment will be similar to those in the protein.

A photoelectron imaging study of the deprotonated GFP chromophore anion and RNA fluorescent tags.

Green fluorescent protein (GFP), together with its family of variants, is the most widely used fluorescent protein for in vivo imaging. Numerous spectroscopic studies of the isolated GFP chromophore have been aimed at understanding the electronic properties of GFP. Here, we build on earlier work [A. V. Bochenkova, C. Mooney, M. A. Parkes, J. Woodhouse, L. Zhang, R. Lewin, J. M. Ward, H. Hailes, L. H. Andersen and H. H. Fielding, Chem. Sci., 2017, 8, 3154] investigating the impact of fluorine and methoxy substituents that have been employed to tune the electronic structure of the GFP chromophore for use as fluorescent RNA tags. We present photoelectron spectra following photoexcitation over a broad range of wavelengths (364-230 nm) together with photoelectron angular distributions following photoexcitation at 364 nm, which are interpreted with the aid of quantum chemistry calculations. The results support the earlier high-level quantum chemistry calculations that predicted how fluorine and methoxy substituents tune the electronic structure and we find evidence to suggest that the methoxy substituents enhance internal conversion, most likely from the 2ππ* state which has predominantly Feshbach resonance character, to the 1ππ* state.

Shining light on the electronic structure and relaxation dynamics of the isolated oxyluciferin anion.

Firefly bioluminescence is exploited widely in imaging in the biochemical and biomedical sciences; however, our fundamental understanding of the electronic structure and relaxation processes of the oxyluciferin that emits the light is still rudimentary. Here, we employ photoelectron spectroscopy and quantum chemistry calculations to investigate the electronic structure and relaxation of a series of model oxyluciferin anions. We find that changing the deprotonation site has a dramatic influence on the relaxation pathway following photoexcitation of higher lying electronically excited states. The keto form of the oxyluciferin anion is found to undergo internal conversion to the fluorescent S1 state, whereas we find evidence to suggest that the enol and enolate forms undergo internal conversion to a dipole bound state, possibly via the fluorescent S1 state. Partially resolved vibrational structure points towards the involvement of out-of-plane torsional motions in internal conversion to the dipole bound state, emphasising the combined electronic and structural role that the microenvironment plays in controlling the electronic relaxation pathway in the enzyme.

An experimental and computational study of the effect of aqueous solution on the multiphoton ionisation photoelectron spectrum of phenol.

We revisit the photoelectron spectroscopy of aqueous phenol in an effort to improve our understanding of the impact of inhomogeneous broadening and inelastic scattering on solution-phase photoelectron spectra. Following resonance-enhanced multiphoton ionisation via the 11ππ* and 11πσ* states of phenol, we observe 11ππ*-D0/D1 ionisation and competing direct S0-D0/D1 ionisation. Following resonance-enhanced multiphoton ionisation via the 21ππ* state, we observe the signature of solvated electrons. By comparing the photoelectron spectra of aqueous phenol with those of gas-phase phenol, we find that inelastic scattering results in peak shifts with similar values to those that have been observed in photoelectron spectra of solvated electrons, highlighting the need for a robust way of deconvoluting the effect of inelastic scattering from liquid-phase photoelectron spectra. We also present a computational strategy for calculating vertical ionisation energies using a quantum-mechanics/effective fragmentation potential (QM/EFP) approach, in which we find that optimising the configurations obtained from molecular dynamics simulations and using the [phenol·(H2O)5]QM[(H2O)n≥250]EFP (B3LYP/aug-cc-pvdz) method gives good agreement with experiment.

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

Phenolates and their substituted analogues are important molecular motifs in many biological molecules, including the family of fluorescent proteins based on green fluorescent protein. We have used a combination of anion photoelectron velocity-map imaging measurements and quantum chemistry calculations to probe the electronic structure of the phenolate anion and difluoro- and dimethoxy-substituted analogues. We report vertical detachment energies (VDEs) and quantify the photoelectron angular distributions. The VDEs for phenolate (2.26 ± 0.03 eV, 3.22 ± 0.02 eV) are in agreement with high-resolution measurements, whereas the values for the substituted analogues (2.61 ± 0.03 eV for difluorophenolate; ∼2.35 eV for dimethoxyphenolate) are new measurements. We also report adiabatic excitation energies (AEEs) of anion resonances and discuss their contributions to the overall photoelectron angular distributions. The AEE of the lowest lying resonance in phenolate (∼3.36 eV) is consistent with previous measurements, whereas the value for the next resonance (∼3.7 eV) is a new measurement. The AEEs of the resonances in the substituted analogues (∼3.74 eV for difluorophenolate; ∼3.4 and 3.74 eV for dimethoxyphenolate) are new measurements.

Role of Photoisomerization on the Photodetachment of the Photoactive Yellow Protein Chromophore.

The photocycle of photoactive yellow protein (PYP) is initiated by a photoinduced trans-cis isomerization around a C═C bond in the chromophore that lies at the heart of the protein; however, in addition to the desired photochemical pathway, the chromophore can undergo competing electronic relaxation processes. Here we combine gas-phase anion photoelectron spectroscopy and quantum chemistry calculations to investigate how locking the C═C bond in the chromophore controls the competition between these electronic relaxation processes following photoexcitation in the range 400-310 nm. We find evidence to suggest that preventing trans-cis isomerization effectively turns off internal conversion to the ground electronic state and enhances electron emission from the first electronically excited state.

A photoelectron imaging and quantum chemistry study of the deprotonated indole anion.

Indole is an important molecular motif in many biological molecules and exists in its deprotonated anionic form in the cyan fluorescent protein, an analogue of green fluorescent protein. However, the electronic structure of the deprotonated indole anion has been relatively unexplored. Here, we use a combination of anion photoelectron velocity-map imaging measurements and quantum chemistry calculations to probe the electronic structure of the deprotonated indole anion. We report vertical detachment energies (VDEs) of 2.45 ± 0.05 eV and 3.20 ± 0.05 eV, respectively. The value for D0 is in agreement with recent high-resolution measurements whereas the value for D1 is a new measurement. We find that the first electronically excited singlet state of the anion, S1(ππ*), lies above the VDE and has shape resonance character with respect to the D0 detachment continuum and Feshbach resonance character with respect to the D1 continuum.

Enhancement of CO2 Uptake and Selectivity in a Metal-Organic Framework by the Incorporation of Thiophene Functionality.

The complex [Zn2(tdc)2dabco] (H2tdc = thiophene-2,5-dicarboxylic acid; dabco = 1,4-diazabicyclooctane) shows a remarkable increase in carbon dioxide (CO2) uptake and CO2/dinitrogen (N2) selectivity compared to the nonthiophene analogue [Zn2(bdc)2dabco] (H2bdc = benzene-1,4-dicarboxylic acid; terephthalic acid). CO2 adsorption at 1 bar for [Zn2(tdc)2dabco] is 67.4 cm3·g-1 (13.2 wt %) at 298 K and 153 cm3·g-1 (30.0 wt %) at 273 K. For [Zn2(bdc)2dabco], the equivalent values are 46 cm3·g-1 (9.0 wt %) and 122 cm3·g-1 (23.9 wt %), respectively. The isosteric heat of adsorption for CO2 in [Zn2(tdc)2dabco] at zero coverage is low (23.65 kJ·mol-1), ensuring facile regeneration of the porous material. Enhancement by the thiophene group on the separation of CO2/N2 gas mixtures has been confirmed by both ideal adsorbate solution theory calculations and dynamic breakthrough experiments. The preferred binding sites of adsorbed CO2 in [Zn2(tdc)2dabco] have been unambiguously determined by in situ single-crystal diffraction studies on CO2-loaded [Zn2(tdc)2dabco], coupled with quantum-chemical calculations. These studies unveil the role of the thiophene moieties in the specific CO2 binding via an induced dipole interaction between CO2 and the sulfur center, confirming that an enhanced CO2 capacity in [Zn2(tdc)2dabco] is achieved without the presence of open metal sites. The experimental data and theoretical insight suggest a viable strategy for improvement of the adsorption properties of already known materials through the incorporation of sulfur-based heterocycles within their porous structures.

Electronic structure and dynamics of torsion-locked photoactive yellow protein chromophores.

The photocycle of photoactive yellow protein (PYP) begins with small-scale torsional motions of the chromophore leading to large-scale movements of the protein scaffold triggering a biological response. The role of single-bond torsional molecular motions of the chromophore in the initial steps of the PYP photocycle are not fully understood. Here, we employ anion photoelectron spectroscopy measurements and quantum chemistry calculations to investigate the electronic relaxation dynamics following photoexcitation of four model chromophores, para-coumaric acid, its methyl ester, and two analogues with aliphatic bridges hindering torsional motions around the single bonds adjacent to the alkene group. Following direct photoexcitation of S1 at 400 nm, we find that both single bond rotations play a role in steering the PYP chromophore through the S1/S0 conical intersection but that rotation around the single bond between the alkene moiety and the phenoxide group is particularly important. Following photoexcitation of higher lying electronic states in the range 346-310 nm, we find that rotation around the single bond between the alkene and phenoxide groups also plays a key role in the electronic relaxation from higher lying states to the S1 state. These results have potential applications in tuning the photoresponse of photoactive proteins and materials with chromophores based on PYP.

Effective Binding of Methane Using a Weak Hydrogen Bond.

The weak hydrogen bond is an important type of noncovalent interaction, which has been shown to contribute to stability and conformation of proteins and large biochemical membranes, stereoselectivity, crystal packing, and effective gas storage in porous materials. In this work, we systematically explore the interaction of methane with a series of functionalized organic molecules specifically selected to exhibit a weak hydrogen bond with methane molecules. To enhance the strength of hydrogen bond interactions, the functional groups include electron-enriched sites to allow sufficient polarization of the C-H bond of methane. The binding between nine functionalized benzene molecules and methane has been studied using the second order Møller-Plesset perturbation theory to reveal that benzenesulfonic acid (C6H5-SO3H) and phenylphosphonic acid (C6H5-PO3H2) have the greatest potential for efficient methane capture through hydrogen bonding interactions. Both acids exhibit efficient binding potential with up to three methane molecules. For additional insight, the atomic charge distribution associated with each binding site is presented.