The photoreceptor bacteriorhodopsin (HsBR) from Halobacterium salinarum is a model system for studying ultrafast photoinduced reactions in proteins. Recent time-resolved serial femtosecond crystallography (TR-SFX) experiments require high pump energies, raising concerns about nonlinear excitation and multi-photon effects. Here, we systematically investigate the influence of excitation energy, pulse duration and the sign of the chirp on the initial HsBR photo-reaction using femtosecond Vis-pump IR-probe spectroscopy in the retinal C=C stretching region. An acousto-optic programmable dispersive filter enabled independent control of pulse energy and chirp. Within the tested range, the retinal dynamics were independent of pulse duration and chirp, indicating that fluence alone does not fully describe excitation conditions. Increasing excitation energy leads to nonlinear saturation of the retinal signals and the appearance of an additional band near 1550 cm−1. However, this band rises linearly with the excitation energy. Hence, the additional band is not directly caused by non-resonant multi-photon absorption. Spectral decomposition reveals two components: a low-energy contribution consistent with the known retinal isomerization dynamics and a high-energy contribution attributed to a small population of photo-damaged HsBR likely formed via a resonant two-photon process. These findings clarify the role of excitation conditions in ultrafast HsBR spectroscopy and suggest that spectral changes at high pump energies mainly arise from damaged species upon resonant two-photon excitation.

Linked photosensitizer-catalyst dyads based on earth-abundant metal complexes are currently potential candidates for sustainable proton reduction systems. Fe(III) carbene complexes showing low-lying ligand-to-metal charge transfer (LMCT) states with lifetimes in the 0.2-0.3 ns range are promising candidates for the photosensitizer side. The latter must be linked to a proton reduction catalyst, usually of cationic character, like cobaloxime. We explore the feasibility of this approach with a model compound mimicking the inductive effect of the cobaloxime moiety on the electronic state structure of a Fe(III) photosensitizer. Thus, the homoleptic pyridine-substituted [Fe(ImPPy)2]+ (HImPPy = 1,1'-(5-(pyridin-4-yl)-1,3-phenylene)bis(3-methyl-1H-imidazol-3-ium)) complex was modified by attaching a methyl group at the pyridinyl nitrogen, to model the inductive effect of a positively charged catalytic centre on the Fe(III) photosensitizer. Reduction of ligand electron density leads to stabilization of metal-to-ligand (2MLCT) excited states, as predicted by TD-DFT calculations. Experimental support is provided by a combination of cyclic voltammetry and ultrafast spectroscopies. [Fe(ImPPyMe)2]3+ shows a characteristic lifetime of 80 ± 10 ps for the stabilized 2MLCT state. This constitutes a promising starting point for later use of [Fe(ImPPy)2]+ as a photosensitizer in bimetallic one-component dyads for photoinduced hydrogen production.

The orientation of the Qy transition dipole moment (tdm) in Chlorophyll a (Chl a) is a fundamental parameter for optimizing energy transfer in light harvesting systems. Here, we apply polarization resolved transient VIS-pump/IR-probe and 2D-IR spectroscopy in combination with density functional theory (DFT) calculations to determining the orientation of the Qy tdm for hexacoordinated Chl a. Analysis of the 2D-IR spectra provide a comprehensive study of the C═O vibrations, allowing us to refine the modeling of the vibrational tdm (vtdm) vectors. Polarization resolved transient IR spectra provide relative angles between the Qy tdm and different probed vtdms, which determine cones of possible orientations of the Qy tdm. Calculating the intersection of three cones from linearly independent vtdm vectors, we determine the orientation of the Qy tdm. We find that the Qy tdm for hexacoordinated Chl a is oriented along the y-axis of the macrocycle, exhibiting only a small angular deviation within -1° to 4°.

Photoreceptor proteins have a variety of functions and properties that are utilized in cells. The starting point is the absorption of light by a chromophore, triggering a cascade of specific reactions in the protein lasting from femtoseconds to seconds. To understand the individual early reaction steps, we need to trace the ultrafast structural dynamics. In this report, we present our approach towards improving the signal-to-noise ratio in polarization-resolved transient infrared (IR) spectroscopy, enabling such measurements. We outline our polarization-resolved ultrafast visible (Vis)-pump/IR-probe spectroscopy setup using IR referencing. The setup is tailored towards tracing small signals of photo-degrading biological samples (e.g. site-specific mutant proteins, that are available only in limited quantities). We provide a comprehensive overview on characterizing the excitation conditions for polarization resolved measurements. The obtained spectra allow for direct tracing of reaction dynamics and conformational changes of molecular groups within the chromophore, protein side chains and hydrogen-bond network.

Phytochromes are biliprotein photoreceptors widespread amongst microorganisms and ubiquitous in plants where they control developmental processes as diverse as germination, stem elongation and floral induction through the photoconversion of inactive Pr to the Pfr signalling state. Here we report crystal structures of the chromophore-binding module of soybean phytochrome A, including ~2.2 Å XFEL structures of Pr and Pfr at ambient temperature and high resolution cryogenic structures of Pr. In the Pfr structure, the chromophore is exposed to the medium, the D-ring remaining α-facial following the likely clockwise photoflip. The chromophore shifts within its pocket, while its propionate side chains, their partners as well as three neighbouring tyrosines shift radically. Helices near the chromophore show substantial shifts that might represent components of the light signal. These changes reflect those in bacteriophytochromes despite their quite different signalling mechanisms, implying that fundamental aspects of phytochrome photoactivation have been repurposed for photoregulation in the eukaryotic plant.

Phytochromes are red-light-sensitive biliprotein photoreceptors that control a variety of physiological processes in plants, fungi, and bacteria. Lately, greater attention has been paid to these photoreceptors due to their potential as fluorescent probes for deep-tissue microscopy. Such fluorescing phytochromes have been generated by multiple amino acid substitutions in weakly fluorescent wild-type (WT) proteins. Remarkably, the single substitution of conserved Tyr176 by His in cyanobacterial phytochrome Cph1 increases the fluorescence quantum yield from 2.4 to 14.5%. In this work, we studied this Y176H variant by crystallography, MAS NMR, resonance Raman spectroscopy, and ultrafast absorption spectroscopy complemented by theoretical methods. Two factors were identified to account for the strong fluorescence increase. First, the equilibrium between the photoactive and fluorescent substates of WT Cph1 was shown to shift entirely to the fluorescent substate in Y176H. Second, structural flexibility of the chromophore is drastically reduced and the photoisomerization barrier is raised, thereby increasing the excited-state lifetime. The most striking finding, however, is that Y176H includes the structural properties of both the dark-adapted Pr and the light-activated Pfr state. While the chromophore adopts the Pr-typical ZZZssa configuration, the tongue segment of the protein adopts a Pfr-typical α-helical structure. This implies that Tyr176 plays a key role in coupling chromophore photoisomerization to the sheet-to-helix transition of the tongue and the final Pfr structure. This conclusion extends to plant phytochromes, where the homologous substitution causes light-independent signaling activity akin to that of Pfr.

In a recent issue of Nature, Barends et al.1 studied the photodissociation of carboxymyoglobin with ultrafast laser pump-probe serial femtosecond crystallography experiments. They observed significant differences in heme protein structural dynamics for biologically relevant 1-photon excitation relative to high excitation leading to the absorption of several photons per heme.

Tuning the photophysical properties of iron-based transition-metal complexes is crucial for their employment as photosensitizers in solar energy conversion. For the optimization of these new complexes, a detailed understanding of the excited-state deactivation paths is necessary. Here, we report femtosecond transient mid-IR spectroscopy data on a recently developed octahedral ligand-field enhancing [Fe(dqp)(2)](2+) (C1) complex with dqp = 2,6-diquinolylpyridine and prototypical [Fe(bpy)(3)](2+) (C0). By combining mid-IR spectroscopy with quantum chemical DFT calculations, we propose a method for disentangling the (5)Q(1) and T-3(1) multiplicities of the long-lived metal-centered (MC) states, applicable to a variety of metal-organic iron complexes. Our results for C0 align well with the established assignment toward the (5)Q(1), validating our approach. For C1, we find that deactivation of the initially excited metal-to-ligand charge-transfer state leads to a population of a long-lived MC (5)Q(1) state. Analysis of transient changes in the mid-IR shows an ultrafast sub 200 fs rearrangement of ligand geometry for both complexes, accompanying the MLCT -> MC deactivation. This confirms that the flexibility in the ligand sphere supports the stabilization of high spin states and plays a crucial role in the MLCT lifetime of metal-organic iron complexes.

Abstract Supercontinuum generation is an enabling technology for a host of intriguing applications such as tuneable ultra-fast light sources, ultra-short pulse generation, etc. It is governed by self-phase modulation, in which new frequencies are created at the leading and trailing edge of the light pulse with lower and higher frequencies, respectively. This spectral broadening is accompanied by positive chirp, opening the possibility to compress the pulse to a shorter duration by adding negative chirp. However, in many cases – e.g. in the ultraviolet spectral range - it is very difficult to introduce well-controlled negative chirp. Here, we present a solution to this problem, the first negatively chirped supercontinuum generation driven by ghost pulses. Ghost pulses have properties of real light pulses and are imprinted on longer carrier pulses with inverted intensity. Flipping the intensity, enables negatively chirped supercontinuum in the normal dispersion regime. With support from simulations, we have experimentally created and characterized ghost pulses, and demonstrated creation of a negatively chirped supercontinuum. We present a first application by a self-compressing non-collinear optical parametric amplifier. Our approach adds a new item to the toolbox of ultrafast technology enabling pulse compression for difficult cases where classical chirp management is not a viable option.

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.

A quantum cascade laser-based sensing technique is presented which allows for in situ high-precision temperature and/or CO2 concentration measurements of gases in the room temperature regime with sampling rates up to about 40 kHz. The method is based on Boltzmann-like thermally populated fundamental and hot-band rovibrational transitions of CO2 with opposite temperature dependence. Single absorption spectra at about 2350 to 2352 cm−1 are recorded by a nanosecond frequency down chirped IR pulse of a pulsed distributed feedback quantum cascade laser (intrapulse mode). The statistical uncertainty (1σ) in the temperature measurement within one laser pulse is about 1 K and can be further reduced down to about 0.1 K by time averaging over 100 ms. Online temperature and CO2 concentration measurements on a breath simulator controlled gas flow were performed to demonstrate response-time and sensitivity for an application-driven test system.

Studies on photoreceptors provide a wealth of information on cofactor and protein dynamics on the microsecond to seconds time-scale. Up to now, ultrafast dynamics addresses mainly the cofactor or chromophore, but ultrafast protein dynamics are poorly understood. Increasing evidence show that protein responses can occur even faster than the cofactor dynamics. The causal reason for the ultrafast protein response cannot be explained by the localized cofactor excitation or its excited-state decay, alone. We propose a Coulomb interaction mechanism started by a shock wave and stabilized by a dipole moment change at least partially responsible for coherent oscillations in proteins, protonation changes, water dislocations, and protein changes prior to and beyond chromophore's excited-state decay. Photoexcitation changes the electron density distribution of the chromophore within a few femtoseconds: The Coulomb shock wave affects polar groups, hydrogen bonds, and protein bound water molecules. The process occurs on a time-scale even faster than excited-state decay of the chromophore. We discuss studies on selected photoreceptors in light of this mechanism and its impact on a detailed understanding of protein dynamics.

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.

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.

A non-invasive CO2 gas temperature sensing technique at or close to the room temperature range based on quantum cascade laser absorption spectroscopy is presented. The method probes thermally populated ground state and hot-band rotational-vibrational transitions of CO2 in the frequency range from 2349 to 2351 cm−1 from which the gas temperature is obtained from Boltzmann statistics. Transmission spectra are recorded by injection-current driven frequency-scans, the so-called inter pulse mode, of a pulsed distributed feedback quantum cascade laser. The statistical uncertainty (1σ) in temperature for single frequency scans with time resolution of 10 ms is 4 K and can be further reduced down to ∼50 mK by long-time averaging of about 1 min. The technique is evaluated with particular emphasis on implementation, data acquisition, data analysis and potential improvements.

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%.

Understanding the kinetics between the components of time‐resolved spectra is a crucial step in the study of photo‐activated processes. However, modeling the kinetics requires usually some a priori knowledge about the system. In our approach, we build a Markov State Model (MSM) from the spectral data, and obtain a Koopman transition matrix K (τ). With genPCCA, an invariant subspace projection, we project the process into its metastable components. The result of the application of gen‐PCCA is a transition matrix K c (τ), from which we can read the transition probability between the metastable components of the reaction. We discuss the application of this analysis method to the transient absorption spectrum of brominated Al‐corrole.

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.