Darkness inhibits autokinase activity of bacterial bathy phytochromes.

Bathy phytochromes are a subclass of bacterial biliprotein photoreceptors that carry a biliverdin IXα chromophore. In contrast to prototypical phytochromes that adopt a red-light-absorbing Pr ground state, the far-red light-absorbing Pfr-form is the thermally stable ground state of bathy phytochromes. Although the photobiology of bacterial phytochromes has been extensively studied since their discovery in the late 1990s, our understanding of the signal transduction process to the connected transmitter domains, which are often histidine kinases, remains insufficient. Initiated by the analysis of the bathy phytochrome PaBphP from Pseudomonas aeruginosa, we performed a systematic analysis of five different bathy phytochromes with the aim to derive a general statement on the correlation of photostate and autokinase output. While all proteins adopt different Pr/Pfr-fractions in response to red, blue, and far-red light, only darkness leads to a pure or highly enriched Pfr-form, directly correlated with the lowest level of autokinase activity. Using this information, we developed a method to quantitatively correlate the autokinase activity of phytochrome samples with well-defined stationary Pr/Pfr-fractions. We demonstrate that the off-state of the phytochromes is the Pfr-form and that different Pr/Pfr-fractions enable the organisms to fine-tune their kinase output in response to a certain light environment. Furthermore, the output response is regulated by the rate of dark reversion, which differs significantly from 5 s to 50 min half-life. Overall, our study indicates that bathy phytochromes function as sensors of light and darkness, rather than red and far-red light, as originally postulated.

A Photoswitchable Metallocycle Based on Azobenzene: Synthesis, Characterization, and Ultrafast Dynamics.

The novel photoswitchable ligand 3,3'-Azobenz(metPA)2 (1) is used to prepare a [Cu2(1)2](BF4)2 metallocycle (2), whose photoisomerization was characterized using static and time-resolved spectroscopic methods. Optical studies demonstrate the highly quantitative and reproducible photoinduced cyclic E/Z switching without decay of the complex. Accordingly and best to our knowledge, [Cu2(1)2](BF4)2 constitutes the first reversibly photoswitchable (3d)-metallocycle based on azobenzene. The photoinduced multiexponential dynamics in the sub-picosecond to few picosecond time domain of 1 and 2 have been assessed. These ultrafast dynamics as well as the yield of the respective photostationary state (PSSZ = 65 %) resemble the behavior of archetypical azobenzene. Also, the innovative pump-probe laser technique of gas phase transient photodissociation (τ-PD) in a mass spectrometric ion trap was used to determine the intrinsic relaxation dynamics for the isolated complex. These results are consistent with the results from femtosecond UV/Vis transient absorption (fs-TA) in solution, emphasizing the azobenzene-like dynamics of 2. This unique combination of fs-TA and τ-PD enables valuable insights into the prevailing interplay of dynamics and solvation. Both analyses (in solution and gas phase) and quantum chemical calculations reveal a negligible effect of the metal coordination on the switching mechanism and electronic pathway, which suggests a non-cooperative isomerization process.

Time-Resolved Spectroscopy and Electronic Structure of Mono-and Dinuclear Pyridyl-Triazole/DPEPhos-Based Cu(I) Complexes.

Chemical and spectroscopic characterization of the mononuclear photosensitizers [(DPEPhos)Cu(I)(MPyrT)]0/+ (CuL, CuLH) and their dinuclear analogues (Cu2 L', Cu2 L'H2 ), backed by (TD)DFT and high-level GW-Bethe-Salpeter equation calculations, exemplifies the complex influence of charge, nuclearity and structural flexibility on UV-induced photophysical pathways. Ultrafast transient absorption and step-scan FTIR spectroscopy reveal flattening distortion in the triplet state of CuLH as controlled by charge, which also appears to have a large impact on the symmetry of the long-lived triplet states in Cu2 L' and Cu2 L'H2 . Time-resolved luminescence spectroscopy (solid state), supported by transient photodissociation spectroscopy (gas phase), confirm a lifetime of some tens of µs for the respective triplet states, as well as the energetics of thermally activated delayed luminescence, both being essential parameters for application of these materials based on earth-abundant copper in photocatalysis and luminescent devices.

Metal-to-Metal Distance Modulated Au(I)/Ru(II) Cyclophanyl Complexes: Cooperative Effects in Photoredox Catalysis.

The modular synthesis of Au(I)/Ru(II) decorated mono- and heterobimetallic complexes with π-conjugated [2.2]paracyclophane is described. [2.2]Paracyclophane serves as a rigid spacer which holds the metal centers in precise spatial orientations and allows metal-to-metal distance modulation. A broad set of architectural arrangements of pseudo -geminal, -ortho, -meta, and -para substitution patterns were employed. Metal-to-metal distance modulation of Au(I)/Ru(II) heterobimetallic complexes and the innate transannular π-communication of the cyclophanyl scaffold provides a promising platform for the investigations of structure-activity relationship and cooperative effects. The Au(I)/Ru(II) heterobimetallic cyclophanyl complexes are stable, easily accessible, and exhibit promising catalytic activity in the visible-light promoted arylative Meyer-Schuster rearrangement.

Ultrafast proton release reaction and primary photochemistry of phycocyanobilin in solution observed with fs-time-resolved mid-IR and UV/Vis spectroscopy.

Deactivation processes of photoexcited (λex = 580 nm) phycocyanobilin (PCB) in methanol were investigated by means of UV/Vis and mid-IR femtosecond (fs) transient absorption (TA) as well as static fluorescence spectroscopy, supported by density-functional-theory calculations of three relevant ground state conformers, PCBA, PCBB and PCBC, their relative electronic state energies and normal mode vibrational analysis. UV/Vis fs-TA reveals time constants of 2.0, 18 and 67 ps, describing decay of PCBB*, of PCBA* and thermal re-equilibration of PCBA, PCBB and PCBC, respectively, in line with the model by Dietzek et al. (Chem Phys Lett 515:163, 2011) and predecessors. Significant substantiation and extension of this model is achieved first via mid-IR fs-TA, i.e. identification of molecular structures and their dynamics, with time constants of 2.6, 21 and 40 ps, respectively. Second, transient IR continuum absorption (CA) is observed in the region above 1755 cm-1 (CA1) and between 1550 and 1450 cm-1 (CA2), indicative for the IR absorption of highly polarizable protons in hydrogen bonding networks (X-H…Y). This allows to characterize chromophore protonation/deprotonation processes, associated with the electronic and structural dynamics, on a molecular level. The PCB photocycle is suggested to be closed via a long living (> 1 ns), PCBC-like (i.e. deprotonated), fluorescent species.

Real-time observation of molecular flattening and intersystem crossing in [(DPEPhos)Cu(i)(PyrTet)] via ultrafast UV/Vis- and mid-IR spectroscopy on solution and solid samples.

The primary photo-induced processes in the mononuclear, heteroleptic Cu(i) complex [(DPEPhos)Cu(PyrTet)] (1), relevant for OLED applications, were investigated in various solvents and in solid state samples via femtosecond (fs) time resolved UV/Vis and fs time resolved mid-IR transient absorption spectroscopy (TA) with MLCT excitation around 340 nm. UV/Vis fs-TA on 1 in solution reveals (i) a severe blue-shift of excited state absorption on the time scale of a few picoseconds (τ2) that is not observed in solids, and (ii), on the time scale of several tens of picoseconds (τ3), a process with very similar dynamics in all samples. Mid-IR fs-TA in solution indicates structural changes with τ2. Transient absorption anisotropy experiments temporally resolve a viscosity-dependent change of the excited state transition dipole moment orientation with τ2, as quantitatively predicted for the relaxed S1-state via TD-DFT. The results strongly suggest flattening distortion (FD) and structural rearrangement of the PyrTet-moiety to occur on the time scale of τ2 in liquid phase, and to be suppressed in solid phase. Moreover, intersystem crossing (ISC) is assigned to the process described by τ3, in line with its direct observation via time-resolved luminescence spectroscopy on 1 by Bergmann et al. (Sci. Adv., 2016, 2, e1500889). Overall, the use of structure-sensitive methods and the direct comparison of different preparations of 1 (i.e. solution vs. solid state), are a unique basis for a clear assignment of spectro-temporal characteristics to fundamental deactivation processes such as FD and ISC.

Exploring the Vibrational Side of Spin-Phonon Coupling in Single-Molecule Magnets via 161 Dy Nuclear Resonance Vibrational Spectroscopy.

Synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) using the Mössbauer isotope 161 Dy has been employed for the first time to study the vibrational properties of a single-molecule magnet (SMM) incorporating DyIII , namely [Dy(Cy3 PO)2 (H2 O)5 ]Br3 ⋅2 (Cy3 PO)⋅2 H2 O ⋅2 EtOH. The experimental partial phonon density of states (pDOS), which includes all vibrational modes involving a displacement of the DyIII ion, was reproduced by means of simulations using density functional theory (DFT), enabling the assignment of all intramolecular vibrational modes. This study proves that 161 Dy NRVS is a powerful experimental tool with significant potential to help to clarify the role of phonons in SMMs.

Photoinitiated Charge Transfer in a Triangular Silver(I) Hydride Complex and Its Oxophilicity.

The photoexcitation of a triangular silver(I) hydride complex, [Ag3 (µ3 -H)(µ2 -dcpm)3 ](PF6 )2 ([P](PF6 )2 , dcpm=bis(dicyclohexylphosphino)methane), designed with "UV-silent" bis-phosphine ligands, provokes hydride-to-Ag3 single and double electron transfer. The nature of the electronic transitions has been authenticated by absorption and photodissociation spectroscopy in parallel with high-level quantum-chemical computations utilizing the GW method and Bethe-Salpeter equation (GW-BSE). Specific photofragments of mass-selected [P]2+ ions testify to charge transfer and competing pathways resulting from the unique [Ag3 (µ3 -H)]2+ scaffold. This structural motif of [P](PF6 )2 has been unequivocally verified by 1 H NMR spectroscopy in concert with DFT and X-ray diffraction structural analysis, which revealed short equilateral Ag-Ag distances (dAgAg =3.08 Å) within the range of argentophilic interactions. The reduced radical cation [P]. + exhibits strong oxophilicity, forming [P+O2 ].+ ,which is a model intermediate for silver oxidation catalysis.

Photodynamics and Luminescence of Mono- and Tri-Nuclear Lanthanide Complexes in the Gas Phase and in Solution.

Lanthanide ions (DyIII , EuIII ) are stabilized by coordination with two Schiff base ligands in compounds [Dy{H3 L}2 ](NO3 )(EtOH)(H2 O)8 (1) and [Eu{H3 L}2 ](NO3 )(H2 O)8 (3) (H4 L, 2,2'-{[(2-aminoethyl)imino]bis[2,1-ethanediyl-nitriloethylidyne]}bis-2-hydroxy-benzoic acid). The latter is reported here for the first time. Both luminescence and ultrafast photodynamics after photoexcitation via a ligand absorption band (∼400 nm) have been studied. In solution, only the [Eu{H3 L}2 ]+ ([3]+ ) complex displays the typical lanthanide emission lines, whereas in gas phase both, [Dy{H3 L}2 ]+ ([1]+ ) and [3]+ , show their corresponding transitions depending on excitation energy. The ultrafast excited state dynamics, obtained in gas phase and in solution, are assigned to excited state intramolecular proton transfer processes in the ligands. The antenna ligand moiety of these complexes provides pockets for stabilization of two MnII ions so that we additionally investigated the photophysical behavior of the corresponding tri-nuclear (NHEt3 )2 [Ln{MnL}2 ](ClO4 )(H2 O)2 (Ln=DyIII , EuIII ) compounds (2, 4). Interestingly, the related complexes do not show lanthanide emission, neither in solution nor in gas phase. Transient data in solution and gas phase suggests an efficient quenching of the ligand's electronically excited state by strong interaction with the MnII ions. This effect could possibly be developed further into a design principle for luminescence-based sensing devices for metal cations.

Spectroscopic, Structural, and Kinetic Investigation of the Ultrafast Spin Crossover in an Unusual Cobalt(II) Semiquinonate Radical Complex.

A comprehensive spectroscopic and structural investigation of [CoII (l-N4 tBu2 )(dbsq)][B(p-C6 H4 Cl)4 ] (1, l-N4 tBu2 =N,N'-di-tert-butyl-2,11-diaza[3.3](2,6)pyridinophane, dbsq1- =3,5-di-tert-butylsemiquinonate), the first known octahedral complex with a low-spin (ls) CoII semiquinonate ground state, is reported. Above 200 K, solids as well as solutions of 1 exhibit thermally induced spin-crossover (SCO) from the ls to the high-spin (hs) CoII semiquinonate state instead of the frequently observed valence tautomerism from ls CoIII catecholate to hs CoII semiquinonate. DFT calculations demonstrate that the (closed shell) CoIII catecholate suffers from a triplet instability leading to the ls CoII semiquinonate ground state. The thorough temperature-dependent spectroscopic study of the SCO enables a photophysical investigation. Thus, by selective photoexcitation of the ls fraction of 1 in solution at room temperature, ultrafast conversion to the hs state is observed using femtosecond electronic and IR-vibrational (infrared) transient absorption spectroscopy. The kinetics of the photocycle is described by a stretched exponential with τ=3.3-3.6 ps and ß=0.52-0.54, representing an upper limit for the hs-ls relaxation time. This is, to our knowledge, the fastest interconversion ever determined for a SCO complex, and is attributed to the special situation that in 1 a CoII complex is coordinated to a π-radical ligand allowing very efficient coupling between the ls and hs spin states.

Spectroscopic Investigation on the Primary Photoreaction of Bathy Phytochrome Agp2-Pr of Agrobacterium fabrum: Isomerization in a pH-dependent H-bond Network.

Bathy phytochrome Agp2 from Agrobacterium fabrum exhibits an unusually low pKa =7.6 in the Pr state in contrast to a pKa >11 in the Pfr state, indicating a pH-dependent charge distribution and H-bond network in the Pr chromophore binding pocket around neutral pH. Here, we report on ultrafast UV/Vis absorption spectroscopy of the primary Pr photoisomerization of Agp2 at pH 6 and pH 9 and upon H2 O/D2 O buffer exchange. The triexponential Pr kinetics slows down at increased pH and pronounced pH-dependent kinetic isotope effects are observed. The results on the Pr photoreaction suggest: 1) component-wise hindered dynamics on the chromophore excited-state potential energy surface at high pH and 2) proton translocation processes either via single-proton transfer or via significant reorganization of H-bond networks. Both effects reflect the interplay between the pH-dependent charge distribution in the Pr chromophore binding pocket on the one hand and chromophore excitation and its Z→E isomerization on the other hand.

Ultrafast deactivation of bilirubin: dark intermediates and two-photon isomerization.

Bilirubin is a neurotoxic product responsible for neonatal jaundice, which is generally treated by phototherapy. The photoreaction involves ultrafast internal conversion via an elusive intermediate and Z-E isomerization with minor yield (less than 3% in solution). The structure of the intermediate remains unclear. Here, the combination of UV-vis and mid-IR ultrafast transient absorption spectroscopy reports a comprehensive picture of the mechanism and provides essential structural information about the intermediate species. Thus, spectral dynamics during the earliest ps unveils a wavepacket travelling from the Franck-Condon region to the crossing point with a dark state. The latter shows a tighter molecular skeleton than the ground state and decays with 15 ps time constant. Remarkably, the relative contribution of a non-decaying component increases linearly with pump energy, suggesting that Z-E isomerization could also be triggered by two-photon excitation. Implications for the photochemistry of protein-bound open tetrapyrroles are discussed.

Unusual spectral properties of bacteriophytochrome Agp2 result from a deprotonation of the chromophore in the red-absorbing form Pr.

Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pKa is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pKa shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.

Femtosecond dynamics in the lactim tautomer of phycocyanobilin: a long-wavelength absorbing model compound for the phytochrome chromophore.

Transient UV/Vis absorption spectroscopy is used to study the primary dynamics of the ring-A methyl imino ether of phycocyanobilin (PCB-AIE), which was shown to mimic the far-red absorbance of the Pfr chromophore in phytochromes (R. Micura, K. Grubmayr, Bioorg. Med. Chem. Lett.- 1994, 4, 2517-2522). After excitation at 615 nm, the excited electronic state is found to decay with τ1 =0.4 ps followed by electronic ground-state relaxation with τ2 =1.2 and τ3 =6.7 ps. Compared with phycocyanobilin (PCB), the initial kinetics of PCB-AIE is much faster. Thus, the lactim structure of PCB-AIE seems to be a suitable model that could not only explain the bathochromic shift in the ground-state absorption but also the short reaction of the Pfr as compared to the Pr chromophore in phytochrome. In addition, the equivalence of ring-A and ring-D lactim tautomers with respect to a red-shifted absorbance relative to the lactam tautomers is demonstrated by semiempirical calculations.

ESIPT and photodissociation of 3-hydroxychromone in solution: photoinduced processes studied by static and time-resolved UV/Vis, fluorescence, and IR spectroscopy.

The spectral properties of fluorescence sensors such as 3-hydroxychromone (3-HC) and its derivatives are sensitive to interaction with the surrounding medium as well as to substitution. 3-HC is a prototype system for other derivatives because it is the basic unit of all flavonoides undergoing ESIPT and is not perturbed by a substituent. In this study, the elementary processes and intermediate states in the photocycle of 3-HC as well as its anion were identified and characterized by the use of static and femtosecond time-resolved spectroscopy in different solvents (methylcyclohexane, acetonitrile, ethanol, and water at different pH). Electronic absorption and fluorescence spectra and lifetimes of the intermediate states were obtained for the normal, tautomer and anionic excited state, while mid-IR vibrational spectra yielded structural information on ground and excited states of 3-HC. A high sensitivity on hydrogen-bonding perturbations was observed, leading to photoinduced anion formation in water, while in organic solvents, different processes are suggested, including slow picosecond ESIPT and contribution of the trans-structure excited state or a different stable solvation state with different direction of OH. The formation of the latter could be favored by the lack of a substituent increasing contact points for specific solute-solvent interactions at the hydroxyl group compared to substituted derivatives. The effect of substituents has to be considered for the design of future fluorescence sensors based on 3-HC.

Transient IR spectroscopy and ab initio calculations on ESIPT in 3-hydroxyflavone solvated in acetonitrile.

Femtosecond polarization resolved UV/Vis and mid-infrared spectroscopy was used to thoroughly identify and characterize the relevant elementary chemical and physical processes in the photocycle of 3-hydroxyflavone (3-HF) in solution. In one set of experiments with the polar aprotic solvent acetonitrile-d(3), for the first time excited state intramolecular proton transfer (ESIPT), vibrational cooling/relaxation and rotational diffusion could be separated, and furthermore mid IR vibrational spectra of 3-HF excited states in solution phase were obtained. UV/Vis transient absorption data yield the time constant τ(Rot) = 22 ps for rotational diffusion and the time constant τ(VR) = 8.5 ps for vibrational cooling/relaxation in the tautomer excited state (S(1)'). Biphasic ESIPT with τ < 120 fs and τ = 2.4 ps as well as slow ground state recovery with τ > 500 ps was found. The time resolved mid IR data yield a time constant of ≈3.4 ps for the slow ESIPT step as well as the vibrational frequencies of S(0,) S(1)' and, in particular those of the short lived excited state S(1). Via quantum chemical calculations, structural parameters of these states are obtained. Various models were used, namely for the isolated molecule, aggregates with solvent as well as a polarizable continuum, that allow us to correlate the two ESIPT components with two mechanisms. Results are compared to those from previously published gas-phase experiments and indicate that the observed slow ESIPT is mediated by solute-solvent interaction via a hydrogen bond with the hydroxyl group of 3-HF.

Vibrational properties of the polymeric spin crossover (SCO) Fe(II) complexes [{Fe(4-amino-1,2,4-triazole)3}X2]n: a nuclear inelastic scattering (NIS), Raman and DFT study.

The vibrational properties of the cationic spin crossover (SCO) coordination polymers [{Fe(4-amino-1,2,4-triazole)(3)}(+2)](n) containing the anions chlorine, methanosulfonate and 1-naphthalenesulfonate have been studied via nuclear inelastic scattering of synchrotron radiation (NIS) as well as by Raman spectroscopy. Although the different anions have a strong influence on the spin crossover temperature, they have little effect on the positions of the spin marker bands in the NIS and Raman spectra. By comparing the line positions of the NIS spin marker bands with those observed by Raman spectroscopy, it has been possible to distinguish vibrations symmetry (A(u) or A(g)) because modes of A(u) and A(g) symmetries are NIS active, but only the A(g) modes are Raman active. The normal mode analysis of charge compensated cationic pentameric and hexameric model structures which have been obtained by density functional calculations reproduces the experimentally observed mode frequencies and the geometry optimization reproduces iron-ligand distances reported for these and related SCO coordination complexes. The effect of charge compensation appears to be independent of the choice of the functional and the basis set which shows that DFT calculations using B3LYP in conjunction with the basis set CEP-31G are a time effective approach in order to study vibrational properties of Fe(II) SCO compounds.

Excited-state dynamics of protochlorophyllide revealed by subpicosecond infrared spectroscopy.

To gain a better understanding of the light-induced reduction of protochlorophyllide (PChlide) to chlorophyllide as a key regulatory step in chlorophyll synthesis, we performed transient infrared absorption measurements on PChlide in d4-methanol. Excitation in the Q-band at 630 nm initiates dynamics characterized by three time constants: τ1 = 3.6 ± 0.2, τ2 = 38 ± 2, and τ3 = 215 ± 8 ps. As indicated by the C13'=O carbonyl stretching mode in the electronic ground state at 1686 cm⁻¹, showing partial ground-state recovery, and in the excited electronic state at 1625 cm⁻¹, showing excited-state decay, τ2 describes the formation of a state with a strong change in electronic structure, and τ3 represents the partial recovery of the PChlide electronic ground state. Furthermore, τ1 corresponds with vibrational energy relaxation. The observed kinetics strongly suggest a branched reaction scheme with a branching ratio of 0.5 for the path leading to the PChlide ground state on the 200 ps timescale and the path leading to a long-lived state (>>700 ps). The results clearly support a branched reaction scheme, as proposed previously, featuring the formation of an intramolecular charge transfer state with ∼25 ps, its decay into the PChlide ground state with 200 ps, and a parallel reaction path to the long-lived PChlide triplet state.

Wavelength-specific optoacoustic-induced vibrations of the guinea pig tympanic membrane.

SIGNIFICANCE: Optoacoustic-induced vibrations of the hearing organ can potentially be used for a hearing device. To increase the efficiency of such a hearing device, the conversion of the light energy into vibration energy within each type of irradiated tissue has to be optimized. AIM: To analyze the wavelength-dependency of optoacoustic-induced vibrations within the tympanic membrane (TM), and to define the most efficient and best-suited optical stimulation parameters for a novel auditory prosthesis. APPROACH: Single nanosecond laser pulses, continuously tunable in a range of visible to near-infrared, were used to excite the guinea pig TM. The induced vibrations of the hearing organ were recorded at the malleus using a laser Doppler vibrometer. RESULTS: Our results indicate a strong wavelength-dependency of the vibration's amplitude correlating with the superposition of the absorption spectra of the different specific tissue components. CONCLUSIONS: We investigated the spectrum of the vibrations of the hearing organ that were induced optoacoustically within a biological membrane embedded in air, in an animal model. First applications for these results can be envisioned for the optical stimulation of the peripheral hearing organ as well as for research purposes.

Primary photoinduced protein response in bacteriorhodopsin and sensory rhodopsin II.

Essential for the biological function of the light-driven proton pump, bacteriorhodopsin (BR), and the light sensor, sensory rhodopsin II (SRII), is the coupling of the activated retinal chromophore to the hosting protein moiety. In order to explore the dynamics of this process we have performed ultrafast transient mid-infrared spectroscopy on isotopically labeled BR and SRII samples. These include SRII in D(2)O buffer, BR in H(2)(18)O medium, SRII with (15)N-labeled protein, and BR with (13)C(14)(13)C(15)-labeled retinal chromophore. Via observed shifts of infrared difference bands after photoexcitation and their kinetics we provide evidence for nonchromophore bands in the amide I and the amide II region of BR and SRII. A band around 1550 cm(-1) is very likely due to an amide II vibration. In the amide I region, contributions of modes involving exchangeable protons and modes not involving exchangeable protons can be discerned. Observed bands in the amide I region of BR are not due to bending vibrations of protein-bound water molecules. The observed protein bands appear in the amide I region within the system response of ca. 0.3 ps and in the amide II region within 3 ps, and decay partially in both regions on a slower time scale of 9-18 ps. Similar observations have been presented earlier for BR5.12, containing a nonisomerizable chromophore (R. Gross et al. J. Phys. Chem. B 2009, 113, 7851-7860). Thus, the results suggest a common mechanism for ultrafast protein response in the artificial and the native system besides isomerization, which could be induced by initial chromophore polarization.