Dark-State-Mediated Efficient Energy Trapping in a Model GFP Chromophore

TL;DR

Dark excited states can act as trapping states affecting photostability and energy harvesting in chromophores. This work analyzes meta-HBDI, a GFP chromophore model, and outlines a combined experimental–theoretical approach to access and characterize the optically dark first singlet state with charge-transfer character in the gas phase, using action-absorption, photoelectron spectroscopy, and high-level ab initio calculations. Action-absorption and photoelectron spectroscopy reveal a near-IR dark S1 state in the meta-HBDI anion with pronounced charge-transfer character. Ultrafast pump–probe measurements show S2 decays to S1 in ~100 fs, followed by trapping in S1 for ~94 ps, and ab initio calculations identify three MECIs among S2, S1, and S0 with a lowest S1/S0 barrier of ~0.41 eV that channels ground-state recovery and suppresses detachment; betaine complexation corroborates the strong CT character of S1. This work provides a mechanistic framework for photostability predicated on dark-state trapping and suggests design principles for tailoring chromophore lifetimes via environmental polarity, with implications for biomolecular photoprotection and the development of robust photoactive materials.

Abstract

The functional properties of photoactive proteins are governed by the interplay between bright and dark excited states. While the bright states are well-studied, the dark states, which are fundamental to photostability and light harvesting, are notoriously difficult to characterize. Here, we report the direct observation and full characterization of an optically dark, low-lying singlet excited state in the isolated anion of the meta green fluorescent protein (GFP) chromophore. Using a combination of ultrafast time-resolved action-absorption and photoelectron spectroscopy, we have captured the formation of this state in 100 fs and measured its remarkably long lifetime of 94 ps. We unambiguously assign its charge-transfer character and reveal the precise trapping mechanism through high-level ab initio calculations. Our findings uncover a photoprotective mechanism in biomolecular anions where ultrafast internal conversion quenches electron emission, stabilizing long-lived electronic excitation even when the energy exceeds the electron detachment threshold.

Figures (8)

• Figure 1: Action-absorption spectrum of meta-HBDI (black data points) in comparison to para-HBDI (green data points). Notice the logarithmic vertical axis. The vertical line shows the electron-detachment threshold for the meta chromophore. Between 350 and 500 nm prompt action is detected after single-photon absorption (photofragment yield increases linearly with the laser-pulse energy, presented here for 465 nm in the lower graph) and associated with excitation into S$_2$ and vibrationally resonant (VR) photodetachment out of the dipole-bound state. The weak absorption between 550 and 700 nm for meta-HBDI is attributed to excitation of the dark S$_1$ state. Sequential absorption of two photons is necessary to cause fragmentation (photofragment yield increases quadratically with the laser-pulse energy, presented here for 600 nm in the lower graph). Data in the region 550 and 700 nm is normalized according to the quadratic laser-power dependence.
• Figure 2: Prompt action spectrum of meta-HBDI (black data points) in comparison with meta-HBDI complexed with betaine (red data points). A strong blueshift of the S$_0$-S$_1$ transition energy in the meta-HBDI - betaine complex reflects a strong CT character. Calculations of the $\pi$ and $\pi$* orbitals, which are primarily involved in the excitation, show that the transition is connected to charge transfer from the phenolate ring into the imidazolinone ring. Only little blueshift for the transition into S$_2$ is observed.
• Figure 3: Photoelectron spectra of the meta-HBDI anion. (a) Two-dimensional photoelectron spectrum recorded with nanosecond laser pulses. (b) One-dimensional spectral cuts at specified excitation energies. For comparison, the spectrum at 3.10 eV was acquired with femtosecond pulses, contrasting with the 3.02 eV spectrum measured using ns pulses.
• Figure 4: Wavelength-dependent photodetachment (PD) mechanisms for the meta-HBDI anion. The energy-level diagram, calculated at the XMCQDPT2/SA(3)-CASSCF(16,14)/(aug)-cc-pVDZ level, shows pathways for different excitation energies: direct PD; resonant PD through the S$_3$ shape resonance; multiphoton (MP) indirect PD via the S$_1$ state after internal conversion (IC) from the S$_2$ Feshbach resonance; thermionic emission (TE) from S$_0$; and vibrational autodetachment (VAD) from S$_1$. Channels ( i) -- ( v) are identified in the experimental 2D photoelectron spectrum (Fig. \ref{['fig:ExPES']}). Energies (in eV) and oscillator strengths (in brackets) are provided for key transitions. Adiabatic and vertical energies are shown as solid and dashed lines, respectively, with colored areas representing the vibrational manifold. Blue arrows indicate the electron-detachment process, labeled with the resulting electron kinetic energy (eKE).
• Figure 5: Excited-state lifetime measured in pump-probe experiments at the ion storage ring SAPHIRA. The molecules were first excited into S$_2$ by a 400 nm pump pulse and then probed by a 800 nm probe pulse. The experimental setup enables the detection of prompt (within the first 10 $\mu$s after photoexcitation) as well as delayed (after 10 $\mu$s up to several ms after photoexcitation) photofragmentation. The upper graph (a) shows the prompt and delayed fragmentation as a function of the pump-probe delay on a linear timescale. While the lifetime of S$_2$ and S$_1$ can be probed by detecting prompt fragmentation, the delayed action shows the ground state recovery. In the lower graph (b), the signal of the prompt action is plotted on a logarithmic time scale and shows that the decay consists of a fast and a slow component. The fast decay of 100 fs $\pm$ 26 fs corresponds to the fast relaxation out of S$_2$ with subsequent trapping in S$_1$ for 94 ps $\pm$ 11 ps. The solid line represents a fit through the data points using a rate model indicated by the inserted schematic. The dashed lines show the calculated population of S$_2$, S$_1$ and S$_0$.