Ultrafast protein response in the Pfr state of Cph1 phytochrome.

Photoisomerization is a fundamental process in several classes of photoreceptors. Phytochromes sense red and far-red light in their Pr and Pfr states, respectively. Upon light absorption, these states react via individual photoreactions to the other state. Cph1 phytochrome shows a photoisomerization of its phycocyanobilin (PCB) chromophore in the Pfr state with a time constant of 0.7 ps. The dynamics of the PCB chromophore has been described, but whether or not the apoprotein exhibits an ultrafast response too, is not known. Here, we compare the photoreaction of 13C/15N labeled apoprotein with unlabeled apoprotein to unravel ultrafast apoprotein dynamics in Cph1. In the spectral range from 1750 to 1620 cm-1 we assigned several signals due to ultrafast apoprotein dynamics. A bleaching signal at 1724 cm-1 is tentatively assigned to deprotonation of a carboxylic acid, probably Asp207, and signals around 1670 cm-1 are assigned to amide I vibrations of the capping helix close to the chromophore. These signals remain after photoisomerization. The apoprotein dynamics appear upon photoexcitation or concomitant with chromophore isomerization. Thus, apoprotein dynamics occur prior to and after photoisomerization on an ultrafast time-scale. We discuss the origin of the ultrafast apoprotein response with the 'Coulomb hammer' mechanism, i.e. an impulsive change of electric field and Coulombic force around the chromophore upon excitation.

Intramolecular Proton Transfer Controls Protein Structural Changes in Phytochrome.

Phytochromes are biological photoswitches that interconvert between two parent states (Pr and Pfr). The transformation is initiated by photoisomerization of the tetrapyrrole chromophore, followed by a sequence of chromophore and protein structural changes. In the last step, a phytochrome-specific peptide segment (tongue) undergoes a secondary structure change, which in prokaryotic phytochromes is associated with the (de)activation of the output module. The focus of this work is the Pfr-to-Pr photoconversion of the bathy bacteriophytochrome Agp2 in which Pfr is the thermodynamically stable state. Using spectroscopic techniques, we studied the structural and functional consequences of substituting Arg211, Tyr165, His278, and Phe192 close to the biliverdin (BV) chromophore. In Pfr, substitutions of these residues do not affect the BV structure. The characteristic Pfr properties of bathy phytochromes, including the protonated propionic side chain of ring C (propC) of BV, are preserved. However, replacing Arg211 or Tyr165 blocks the photoconversion in the Meta-F state, prior to the secondary structure transition of the tongue and without deprotonation of propC. The Meta-F state of these variants displays low photochemical activity, but electronic excitation causes ultrafast alterations of the hydrogen bond network surrounding the chromophore. In all variants studied here, thermal back conversion from the photoproducts to Pfr is decelerated but substitution of His278 or Phe192 is not critical for the Pfr-to-Pr photoconversion. These variants do not impair deprotonation of propC or the α-helix/ß-sheet transformation of the tongue during the Meta-F-to-Pr decay. Thus, we conclude that propC deprotonation is essential for restructuring of the tongue.

Ultrafast Backbone Protonation in Channelrhodopsin-1 Captured by Polarization Resolved Fs Vis-pump-IR-Probe Spectroscopy and Computational Methods.

Channelrhodopsins (ChR) are light-gated ion-channels heavily used in optogenetics. Upon light excitation an ultrafast all-trans to 13-cis isomerization of the retinal chromophore takes place. It is still uncertain by what means this reaction leads to further protein changes and channel conductivity. Channelrhodopsin-1 in Chlamydomonas augustae exhibits a 100 fs photoisomerization and a protonated counterion complex. By polarization resolved ultrafast spectroscopy in the mid-IR we show that the initial reaction of the retinal is accompanied by changes in the protein backbone and ultrafast protonation changes at the counterion complex comprising Asp299 and Glu169. In combination with homology modelling and quantum mechanics/molecular mechanics (QM/MM) geometry optimization we assign the protonation dynamics to ultrafast deprotonation of Glu169, and transient protonation of the Glu169 backbone, followed by a proton transfer from the backbone to the carboxylate group of Asp299 on a timescale of tens of picoseconds. The second proton transfer is not related to retinal dynamics and reflects pure protein changes in the first photoproduct. We assume these protein dynamics to be the first steps in a cascade of protein-wide changes resulting in channel conductivity.

Ultrafast Dynamics of Sb-Corroles: A Combined Vis-Pump Supercontinuum Probe and Broadband Fluorescence Up-Conversion Study.

Corroles are a developing class of tetrapyrrole-based molecules with significant chemical potential and relatively unexplored photophysical properties. We combined femtosecond broadband fluorescence up-conversion and fs broadband Vis-pump Vis-probe spectroscopy to comprehensively characterize the photoreaction of 5,10,15-tris-pentafluorophenyl-corrolato-antimony(V)-trans-difluoride (Sb-tpfc-F2). Upon fs Soret band excitation at ~400 nm, the energy relaxed almost completely to Q band electronic excited states with a time constant of 500 ± 100 fs; this is evident from the decay of Soret band fluorescence at around 430 nm and the rise time of Q band fluorescence, as well as from Q band stimulated emission signals at 600 and 650 nm with the same time constant. Relaxation processes on a time scale of 10 and 20 ps were observed in the fluorescence and absorption signals. Triplet formation showed a time constant of 400 ps, with an intersystem crossing yield from the Q band to the triplet manifold of between 95% and 99%. This efficient triplet formation is due to the spin-orbit coupling of the antimony ion.

Femtosecond anisotropy excitation spectroscopy to disentangle the Q x and Q y absorption in chlorophyll a.

Chlorophyll a (Chl a) belongs to the most important and most investigated molecules in the field of photosynthesis. The Q-band absorption is central for energy transfer in photosystems and the relative orientation of the Q y transitions of interacting chlorophylls governs the energy transfer. Chl a was well investigated, but a quantitative separation of Q x and Q y contributions to the Q-band of the Chl a absorption spectrum is still missing. We use femtosecond Vis-pump - IR-probe anisotropy excitation spectroscopy to disentangle the overlapping electronic Q x and Q y contributions quantitatively. In an anisotropy excitation spectrum we trace the dichroic ratio of a single vibration, i.e. the keto C[double bond, length as m-dash]O stretching vibration at 1690 cm-1, as a function of excitation wavelength. The change in dichroic ratio reflects altering Q y and Q x contributions. We identified Q x00 (0-0 transition of Q x ) and Q x01 transition at (636 ± 1) nm and (607 ± 2) nm, respectively, and the Q y01 and Q y02 at (650 ± 6) nm, and (619 ± 3) nm, respectively. We find that Q x absorption, contributes to 50% to 72% at 636 nm and 49% to 71% at 606 nm to the Chl a absorption at room temperature. The Q band was well modelled by a single vibronic progression for the Q x and Q y transition of (700 ± 100) cm-1, and the energy gap between Q x00 and Q y00 was found to be (820 ± 60) cm-1. This precise description of the hexa-coordinated Chl a absorption spectrum will foster more accurate calculations on energy transfer processes in photosystems, and advance the detailed understanding of the intricate interaction of chlorophyll molecules with the solvent.

Ultrafast proton-coupled isomerization in the phototransformation of phytochrome.

The biological function of phytochromes is triggered by an ultrafast photoisomerization of the tetrapyrrole chromophore biliverdin between two rings denoted C and D. The mechanism by which this process induces extended structural changes of the protein is unclear. Here we report ultrafast proton-coupled photoisomerization upon excitation of the parent state (Pfr) of bacteriophytochrome Agp2. Transient deprotonation of the chromophore's pyrrole ring D or ring C into a hydrogen-bonded water cluster, revealed by a broad continuum infrared band, is triggered by electronic excitation, coherent oscillations and the sudden electric-field change in the excited state. Subsequently, a dominant fraction of the excited population relaxes back to the Pfr state, while ~35% follows the forward reaction to the photoproduct. A combination of quantum mechanics/molecular mechanics calculations and ultrafast visible and infrared spectroscopies demonstrates how proton-coupled dynamics in the excited state of Pfr leads to a restructured hydrogen-bond environment of early Lumi-F, which is interpreted as a trigger for downstream protein structural changes.

Ultrafast Electron Transfer in a Self-Assembling Sulfonated Aluminum Corrole-Methylviologen Complex.

Photoinduced electron transfer systems can mimic certain features of natural photosynthetic reaction centers, which are crucial for solar energy production. Among other tetra-pyrroles, the versatile chemical and photophysical properties of corroles make them very promising donors applicable in donor-acceptor complexes. Here, we present a first comprehensive study of ultrafast photoinduced electron transfer in a self-assembling sulfonated aluminum corrole-methylviologen complex combining visible and mid-IR transient absorption spectroscopy. The noncovalent D-A association of the corrole-methylviologen complex has the great advantage that photoinduced charge separation becomes possible even though the back electron transfer (BET) rate is large. Initial forward electron transfer from corrole to methylviologen is observed on an ∼130 fs time scale. Subsequent back electron transfer takes place with τBET = (1.8 ± 0.5) ps, revealing very complex relaxation dynamics. Direct probing in the mid-IR allows us to unravel the back electron transfer and cooling dynamics/electronic reorganization. Upon tracing the dynamics of the methylviologen-radical marker band at 1640 cm-1 and the C═C stretching of corrole at around 1500 cm-1, we observe that large amounts of excess energy survive the back transfer, leading to the formation of hot ground state absorption. A closer examination of the signal after 300 ps, surviving the back transfer, exhibits a charge-separation yield of 10-15%.

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.

Acceleration of a ground-state reaction by selective femtosecond-infrared-laser-pulse excitation.

Infrared (IR) excitation of vibrations that participate in the reaction coordinate of an otherwise thermally driven chemical reaction are believed to lead to its acceleration. Attempts at the practical realization of this concept have been hampered so far by competing processes leading to sample heating. Here we demonstrate, using femtosecond IR-pump IR-probe experiments, the acceleration of urethane and polyurethane formation due to vibrational excitation of the reactants for 1:1 mixtures of phenylisocyanate and cyclohexanol, and toluene-2,4-diisocyanate and 2,2,2-trichloroethane-1,1-diol, respectively. We measured reaction rate changes upon selective vibrational excitation with negligible heating of the sample and observed an increase of the reaction rate up to 24%. The observation is rationalized using reactant and transition-state structures obtained from quantum chemical calculations. We subsequently used IR-driven reaction acceleration to write a polyurethane square on sample windows using a femtosecond IR pulse.

Influence of Heterogeneity on the Ultrafast Photoisomerization Dynamics of Pfr in Cph1 Phytochrome.

Photoisomerization of a protein-bound chromophore is the basis of light sensing and signaling in many photoreceptors. Phytochrome photoreceptors can be photoconverted reversibly between the Pr and Pfr states through photoisomerization of the methine bridge between rings C and D. Ground-state heterogeneity of the chromophore has been reported for both Pr and Pfr. Here, we report ultrafast visible (Vis) pump-probe and femtosecond polarization-resolved Vis pump-infrared (IR) probe studies of the Pfr photoreaction in native and 13 C/15 N-labeled Cph1 phytochrome with unlabeled PCB chromophore, demonstrating different S0 substates, Pfr-I and Pfr-II, with distinct IR absorptions, orientations and dynamics of the carbonyl vibration of ring D. We derived time constants of 0.24 ps, 0.7 ps and 6 ps, describing the complete initial photoreaction. We identified an isomerizing pathway with 0.7 ps for Pfr-I, and silent dynamics with 6 ps for Pfr-II. We discuss different origins of the Pfr substates, and favor different facial orientations of ring D. The model provides a quantum yield for Pfr-I of 38%, in line with ~35% ring D rotation in the electronic excited state. We tentatively assign the silent form Pfr-II to a dark-adapted state that can convert to Pfr-I upon light absorption.

The primary photoreaction of channelrhodopsin-1: wavelength dependent photoreactions induced by ground-state heterogeneity.

The primary photodynamics of channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) was investigated by VIS-pump supercontinuum probe experiments from femtoseconds to 100 picoseconds. In contrast to reported experiments on channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), we found a clear dependence of the photoreaction dynamics on varying the excitation wavelength. Upon excitation at 500 and at 550 nm we detected different bleaching bands, and spectrally distinct photoproduct absorptions in the first picoseconds. We assign the former to the ground-state heterogeneity of a mixture of 13-cis and all-trans retinal maximally absorbing around 480 and 540 nm, respectively. At 550 nm, all-trans retinal of the ground state is almost exclusively excited. Here, we found a fast all-trans to 13-cis isomerization process to a hot and spectrally broad P1 photoproduct with a time constant of (100 ± 50) fs, followed by photoproduct relaxation with time constants of (500 ± 100) fs and (5 ± 1) ps. The remaining fraction relaxes back to the parent ground state with time constants of (500 ± 100) fs and (5 ± 1) ps. Upon excitation at 500 nm a mixture of both chromophore conformations is excited, resulting in overlapping reaction dynamics with additional time constants of <300 fs, (1.8 ± 0.3) ps and (90 ± 25) ps. A new photoproduct Q is formed absorbing at around 600 nm. Strong coherent oscillatory signals were found pertaining up to several picoseconds. We determined low frequency modes around 200 cm(-1), similar to those reported for bacteriorhodopsin.