Ultrafast Hydrogen-Bonding Dynamics in the Electronic Excited State of Photoactive Yellow Protein Revealed by Femtosecond Stimulated Raman Spectroscopy

Abstract

The ultrafast structural dynamics in the electronic excited state of photoactive yellow protein (PYP) is studied by femtosecond stimulated Raman spectroscopy. Stimulated Raman spectra in the electronic excited state, S(1), can be obtained by using a Raman pump pulse in resonance with the S(1)-S(0) transition. This is confirmed by comparing the experimental results with numerical calculations based on the density matrix treatment. We also investigate the hydrogen-bonding network surrounding the wild-type (WT)-PYP chromophore in the ground and excited states by comparing its stimulated Raman spectra with those of the E46Q-PYP mutant. We focus on the relative intensity of the Raman band at 1555 cm(-1), which includes both vinyl bond C═C stretching and ring vibrations and is sensitive to the hydrogen-bonding network around the phenolic oxygen of the chromophore. The relative intensity for the WT-PYP decreases after actinic excitation within the 150 fs time resolution and reaches a similar intensity to that for E46Q-PYP. These observations indicate that the WT-PYP hydrogen-bonding network is immediately rearranged in the electronic excited state to form a structure similar to that of E46Q-PYP.

Quantum mechanical/molecular mechanical/continuum style solvation model: Time-dependent density functional theory

“…In addition, we found that the initial geometry relaxation on the S 1 potential energy surface does not alter the Tyr42 and Glu46 hydrogen bond strengths, implying that the weakening of these hydrogen bonds can be observed immediately after the excitation and probably for a short period of time. This is consistent with Nakamura et al 109 observation that the pCA hydrogen bond rearrangements occurs within ∼150 fs upon excitation.…”

“…In addition, we found that the initial geometry relaxation on the S 1 potential energy surface does not appear to alter the Tyr42 and Glu46 hydrogen bond strengths (Table III). Therefore, our current result does not support the main conclusion (preferential strengthening of the pCA-Tyr42 hydrogen bond upon excitation) in the paper of Mizuno et al 108 Based on the analyses of femtosecond stimulated Raman spectroscopy spectra of PYP, Nakamura et al 109 found that the 1555 cm −1 mode of the wild type PYP has a reduced intensity that is similar to the same mode of Glu46Gln-PYP, implying that the pCA-Glu46 hydrogen bond is weakened upon excitation. They observed that the weakening process is accomplished within ∼150 fs after the excitation.…”

Femtosecond Stimulated Raman Spectroscopy

“…After this initial investigation of the isolated chromophore, several groups extended their efforts to study the chromophore in its proper protein environment. Thereby, they unraveled ultrafast hydrogen‐bonding dynamics and could even follow the photocycle through its intermediates for the first 300 ps …”

Femtosecond stimulated Raman line shapes: Dependence on resonance conditions of pump and probe pulses

“…In an electronic excited state, transient vibrational population complicates the Raman line shapes to a greater extent. The resonance with excited-state absorption (ESA) and stimulated emission (SE) bands provides more channels for "dynamic" resonance Raman during the study of a broad range of photoactive systems [20,35,39,[50][51][52][53][54]. Some theoretical work has been performed for the ES Raman line shapes [18,20,46,50].…”