Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical-quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.

Bacterial phytochromes are sensoric photoreceptors that transform light absorbed by the photosensor core module (PCM) to protein structural changes that eventually lead to the activation of the enzymatic output module. The underlying photoinduced reaction cascade in the PCM starts with the isomerization of the tetrapyrrole chromophore, followed by conformational relaxations, proton transfer steps, and a secondary structure transition of a peptide segment (tongue) that is essential for communicating the signal to the output module. In this work, we employed various static and time-resolved IR and resonance Raman spectroscopic techniques to study the structural and reaction dynamics of the Meta-F intermediate of both the PCM and the full-length (PCM and output module) variant of the bathy phytochrome Agp2 from Agrobacterium fabrum. In both cases, this intermediate represents a branching point of the phototransformation, since it opens an unproductive reaction channel back to the initial state and a productive pathway to the final active state, including the functional protein structural changes. It is shown that the functional quantum yield, i.e. the events of tongue refolding per absorbed photons, is lower by a factor of ca. two than the quantum yield of the primary photochemical process. However, the kinetic data derived from the spectroscopic experiments imply an increased formation of the final active state upon increasing photon flux or elevated temperature under photostationary conditions. Accordingly, the branching mechanism does not only account for the phytochrome's function as a light intensity sensor but may also modulate its temperature sensitivity.

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/β-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.

Bacteriophytochromes harboring a biliverdin IXα (BV) chromophore undergo photoinduced reaction cascades to switch between physiologically inactive and active states. Employing vibrational spectroscopic and computational methods, we analyzed the role of propionic substituents of BV in the transformations between parent states Pr and Pfr in prototypical (Agp1) and bathy (Agp2) phytochromes from Agrobacterium fabrum. Both proteins form adducts with BV monoesters (BVM), esterified at propionic side chain B (PsB) or C (PsC), but in each case, only one monoester adduct is reactive. In the reactive Agp2-BVM-B complex (esterified at ring B), the Pfr dark state displays the structural properties characteristic of bathy phytochromes, including a protonated PsC. As in native Agp2, PsC is deprotonated in the final step of the Pfr phototransformation. However, the concomitant α-helix/β-sheet secondary structure change of the tongue is blocked at the stage of unfolding of the coiled loop region. This finding and the shift of the tautomeric equilibrium of BVM toward the enol form are attributed to the drastic changes in the electrostatic potential. The calculations further suggest that deprotonation of PsC and the protonation state of His278 control the reactivity of the enol tautomer, thereby accounting for the extraordinarily slow thermal reversion. Although strong perturbations of the electrostatic potential are also found for Agp1-BVM, the consequences for the Pr-to-Pfr phototransformation are less severe. Specifically, the structural transition of the tongue is not impaired and thermal reversion is even accelerated. The different response of Agp1 and Agp2 to monoesterification of BV points to different photoconversion mechanisms.

The tiny picoalga, Ostreococcus tauri, originating from the Thau Lagoon is a member of the marine phytoplankton. Because of its highly reduced genome and small cell size, while retaining the fundamental requirements of a eukaryotic photosynthetic cell, it became a popular model organism for studying photosynthesis or circadian clock-related processes. We analyzed the spectroscopic properties of the photoreceptor domain of the histidine kinase rhodopsin Ot-HKR that is suggested to be involved in the light-induced entrainment of the Ostreococcus circadian clock. We found that the rhodopsin, Ot-Rh, dark state absorbs maximally at 505 nm. Exposure to green-orange light led to the accumulation of a blue-shifted M-state-like absorbance form with a deprotonated Schiff base. This Ot-Rh P400 state had an unusually long lifetime of several minutes. A second long-living photoproduct with a red-shifted absorbance, P560, accumulated upon illumination with blue/UVA light. The resulting photochromicity of the rhodopsin is expected to be advantageous to its function as a molecular control element of the signal transducing HKR domains. The light intensity and the ratio of blue vs green light are reflected by the ratio of rhodopsin molecules in the long-living absorbance forms. Furthermore, dark-state absorbance and the photocycle kinetics vary with the salt content of the environment substantially. This observation is attributed to anion binding in the dark state and a transient anion release during the photocycle, indicating that the salinity affects the photoinduced processes.

Early research on the four microbial rhodopsins discovered in the archaeal Halobacterium salinarum revealed a structural template that served as a scaffold for two different functions: light-driven ion transport and phototaxis. Bacteriorhodopsin and halorhodopsin are proton and chloride pumps, respectively, while sensory rhodopsin I and II are responsible for phototactic behavior of the archaea. Halorhodopsins have been identified in various other species. Besides this group of archaeal halorhodopsins distinct chloride transporting rhodopsins groups have recently been identified in other organism like Flavobacteria or Cyanobacteria. Halorhodopsin from Natronomonas pharaonis is the best-studied homologue because of its facile expression and purification and its advantageous properties, which was the reason to introduce this protein as neural silencer into the new field of optogenetics. Two other major families of genetically encoded silencing proteins, proton pumps and anion channels, extended the repertoire of optogenetic tools. Here, we describe the functional and structural characteristics of halorhodopsins. We will discuss the data in light of common principles underlying the mechanism of ion pumps and sensors and will review biophysical and biochemical aspects of neuronal silencers.

Phytochromes are bimodal photoreceptors which, upon light absorption by the tetrapyrrole chromophore, can be converted between a red-absorbing state (Pr) and far-red-absorbing state (Pfr). In bacterial phytochromes, either Pr or Pfr are the thermally stable states, thereby constituting the classes of prototypical and bathy phytochromes, respectively. In this work, we have employed vibrational spectroscopies to elucidate the origin of the thermal stability of the Pfr states in bathy phytochromes. Here, we present the first detailed spectroscopic analysis of RpBphP6 (Rhodopseudomas palustris), which together with results obtained for Agp2 (Agrobacterium tumefaciens) and PaBphP (Pseudomonas aeruginosa) allows identifying common structural properties of the Pfr state of bathy phytochromes, which are (1) a homogenous chromophore structure, (2) the protonated ring C propionic side chain of the chromophore and (3) a retarded H/D exchange at the ring D nitrogen. These properties are related to the unique strength of the hydrogen bonding interactions between the ring D N-H group with the side chain of the conserved Asp194 (PaBphP numbering). As revealed by a comparative analysis of homology models and available crystal structures of Pfr states, these interactions are strengthened by an Arg residue (Arg453) only in bathy but not in prototypical phytochromes.

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation. Here, we report a vibrational spectroscopic study on bathy phytochromes that demonstrates that the formation of the photoactivated state and thus (de) activation of the output module is based on proton translocations in the chromophore pocket coupling chromophore and protein structural changes. These proton transfer steps, involving the tetrapyrrole and a nearby histidine, also enable thermal back-isomerization of the chromophore via keto-enol tautomerization to afford the initial dark state. Thus, the same proton re-arrangements inducing the (de) activation of the output module simultaneously initiate the reversal of this process, corresponding to a negative feedback mechanism.

The second GAF domain of AnPixJ, AnPixJg2, a bilin-binding protein from the cyanobacterium Anabaena PCC 7120, undergoes a photoinduced interconversion between a red-absorbing state, Pr, and a green-absorbing state, Pg. Combining ultraviolet-vis (UV-vis), infrared, resonance Raman (RR), and magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, we have studied this cyanobacteriochrome (CBCR) assembled with phycocyanobilin (PCB) either in vivo or in vitro. In both assembly routes, the spectroscopic data of the Pr state reveal nearly identical chromophore structures with a protonated (cationic) bilin. However, unlike the native (in vivo assembly) Pg photoproduct, in which the bilin retains protonation, the Pg generated from the in vitro-assembled AnPixJg2 harbors a deprotonated (neutral) bilin chromophore at pH 7.8. IR difference spectroscopy further reveals the transfer of a proton from the bilin to a side-chain carboxylate on an amino acid, probably Asp291. Besides the change in protonation state, the bilin structure is very similar in the in vitro- and in vivo-assembled Pg photoproducts. The chromophore of the in vitro Pg becomes protonated when the pH is increased to 10, presumably because of a partial reversal of protein misfolding. Most remarkably, the electronic transitions remain unchanged and are very similar to those of the native Pg. Thus, bilin protonation is not a key parameter for controlling the energies of the electronic transitions in AnPixJg2. Possible alternative molecular mechanisms for color tuning are discussed.

Mithilfe eines optimierten Metallzentrums ermöglichen Hydrogenasen die Umsetzung von Wasserstoff. Resonanz-Raman-Spektroskopie wird als neue Methode zur Charakterisierung des aktiven Zentrums dieser Biokatalysatoren vorgestellt. Ein kombinierter spektroskopischer und theoretischer Ansatz gibt Einblicke in die Struktur und die photochemischen Eigenschaften des [NiFe]-Zentrums einer sauerstofftoleranten Hydrogenase.

Angewandte Chemie International Edition is one of the prime chemistry journals in the world, publishing research articles, highlights, communications and reviews across all areas of chemistry.

[NiFe]-Hydrogenasen katalysieren die biologische Umwandlung von Wasserstoff mithilfe eines maßgeschneiderten bimetallischen Zentrums. In ihrer Zuschrift auf S. 5267 ff. nutzen P. Hildebrandt, I. Zebger, M. Horch et al. die Resonanz-Raman-Spektroskopie als neue Charakterisierungstechnik für dieses aktive Zentrum, die eine Möglichkeit zur direkten Beobachtung der Fe-CO/CN-Schwingungsmoden bietet. Ihr kombinierter spektroskopischer und theoretischer Ansatz gibt Einblicke in die Struktur und die photochemischen Eigenschaften des [NiFe]-Zentrums.

Rhodopsin, a seven transmembrane helix (TM) receptor, binds its ligand 11-cis-retinal via a protonated Schiff base. Coupling to the G-protein transducin (G(t)) occurs after light-induced cis/trans-retinal isomerization, which leads through photoproducts into a sequence of metarhodopsin (Meta) states: Meta I ⇌ Meta IIa ⇌ Meta IIb ⇌ Meta IIbH(+). The structural changes behind this three-step activation scheme are mediated by microswitch domains consisting of conserved amino acids. Here we focus on Tyr223(5.58) as part of the Y(5.58)X(7)K(R)(5.66) motif. Mutation to Ala, Phe, or Glu results in specific impairments of G(t)-activation measured by intrinsic G(t) fluorescence. UV-vis/FTIR spectroscopy of rhodopsin and its complex with a C-terminal G(t)α peptide allows the assignment of these deficiencies to specific steps in the activation path. Effects of mutation occur already in Meta I but do not directly influence deprotonation of the Schiff base during formation of Meta IIa. Absence of the whole phenol ring (Y223A) allows the activating motion of TM6 in Meta IIb but impairs the coupling to G(t). When only the hydroxyl group is lacking (Y223F), Meta IIb does not accumulate, but the activity toward G(t) remains substantial. From the FTIR features of Meta IIbH(+) we conclude that proton uptake to Glu134(3.49) is mandatory for Tyr223(5.58) to engage in the interaction with the key player Arg135(3.50) predicted by X-ray analysis. This polar interaction is partially recovered in Y223E, explaining its relatively high activity. Only the phenol side chain of tyrosine provides all characteristics for accumulation of the active state and G-protein activation.

The visual pigment rhodopsin belongs to the group of GPCR's and is so far the best characterized member. Although the structure is known since 2000, the activation mechanism is still not completely understood. Activation is triggered by the light-induced conversion of the covalently bound inverse agonist, 11-cis retinal, to the all-trans geometry, constituting an agonist. We have studied the activation mechanism by Fourier transform infrared difference spectroscopy, a technique capable of monitoring molecular changes of proteins and co-factors. Therefore, this technique could also be of importance for the study of general GPCR's. Some of our recent results will be reviewed. They include the study of the retinal-free opsin state, which can be activated by low pH. This observation has been relevant for the recently published structures of opsin. Next, we show how the technique can be used to study conformational changes of the active state induced by the interacting G-protein (here a peptide derived from the Gα C-terminus known to be responsible for the interaction). In addition, the technique reveals conformational changes of the peptide. Further, the different ionic locks keeping the receptor in its inactive state are addressed. They encompass the Schiff base-counterion, the ERY motif at the end helix C, and the interaction of this motif with two further glutamic acids on helix F. This interaction is thought to keep also the β-adrenergic receptor in its inactive conformation. Finally, a method will be proposed which will allow the study of the ligand-induced activation of general GPCR´s. The essential feature is a receptor monolayer, allowing free access of the ligand. The required increase in sensitivity is realized by surface-enhanced infrared spectroscopy.

The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV-Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV-Vis and FTIR experiments. Three intermediates have been trapped at -25, -45 and -120 degrees C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at -70 and -140 degrees C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore-protein interactions.

Membrane proteins are molecular machines that transport ions, solutes, or information across the cell membrane. Electrophysiological techniques have unraveled many functional aspects of ion channels but suffer from the lack of structural sensitivity. Here, we present spectroelectrochemical data on vibrational changes of membrane proteins derived from a single monolayer. For the seven-helical transmembrane protein sensory rhodopsin II, structural changes of the protein backbone and the retinal cofactor as well as single ion transfer events are resolved by surface-enhanced IR difference absorption spectroscopy (SEIDAS). Angular changes of bonds versus the membrane normal have been determined because SEIDAS monitors only those vibrations whose dipole moment are oriented perpendicular to the solid surface. The application of negative membrane potentials (DeltaV = -0.3 V) leads to the selective halt of the light-induced proton transfer at the stage of D75, the counter ion of the retinal Schiff base. It is inferred that the voltage raises the energy barrier of this particular proton-transfer reaction, rendering the energy deposited in the retinal by light excitation insufficient for charge transfer to occur. The other structural rearrangements that accompany light-induced activity of the membrane protein, are essentially unaffected by the transmembrane electric field. Our results demonstrate that SEIDAS is a generic approach to study processes that depend on the membrane potential, like those in voltage-gated ion channels and transporters, to elucidate the mechanism of ion transfer with unprecedented spatial sensitivity and temporal resolution.

Molecular vibrations can be excited via two physical mechanisms: the absorption of light quanta and the inelastic scattering of photons (Fig. 2.1) (Herzberg 1945). Direct absorption of photons is achieved by irradiation of molecules with poly-chromatic light that includes photons of energy matching the energy difference hn k between two vibrational energy levels, the initial (i, e.g., ground state) and the final (f , e.g., first excited state) vibrational state. hn k ¼ hn f À hn i ð2:1Þ As these energy differences are in the order of 0.5 and 0.005 eV, light with wavelengths longer than 2.5 mm, that is infrared (IR) light, is sufficient to induce the vibrational transitions. Thus, vibrational spectroscopy that is based on the direct absorption of light quanta is denoted as IR absorption or IR spectroscopy. The physical basis of IR light absorption is very similar to light absorption in the ultraviolet (UV)–visible (vis) range, which causes electronic transitions or combined electronic–vibrational (vibronic) transitions. Thus, UV–vis absorption spectroscopy can, in principle, also provide information about molecular vibrations. However, for molecules in the condensed phase at ambient temperature, the vibrational fine structure of the absorption spectra is only poorly resolved , if at all, such that vibrational spectroscopy of biomolecules by light absorption is restricted to the IR range. Fig. 2.1 Illustration of the excitation of molecular vibrations in IR (top) and Raman (bottom) spectroscopy. In IR spectroscopy, the vibrational transitions are induced by absorption of light quanta from a continuous light source in the IR spectral region. Vibrational Raman transitions correspond to inelastic scattering (n R ; thin arrow) of the incident monochromatic light ðn 0 Þ whereas the elastic scattering ðn 0 Þ is represented by the thick arrow.

Activation of family A G-protein-coupled receptors involves a rearrangement of a conserved interhelical cytoplasmic hydrogen bond network between the E(D)RY motif on transmembrane helix 3 (H3) and residues on H6, which is commonly termed the cytoplasmic “ionic lock.” Glu1343.49 of the E(D)RY motif also forms an intrahelical salt bridge with neighboring Arg1353.50 in the dark-state crystal structure of rhodopsin. We examined the roles of Glu1343.49 and Arg1353.50 on H3 and Glu2476.30 and Glu2496.32 on H6 on the activation of rhodopsin using Fourier transform infrared spectroscopy of wild-type and mutant pigments reconstituted into lipid membranes. Activation of rhodopsin is pH-dependent with proton uptake during the transition from the inactive Meta I to the active Meta II state. Glu1343.49 of the ERY motif is identified as the proton-accepting group, using the Fourier transform infrared protonation signature and the absence of a pH dependence of activation in the E134Q mutant. Neutralization of Arg1353.50 similarly leads to pH-independent receptor activation, but with structural alterations in the Meta II state. Neutralization of Glu2476.30 and Glu2496.32 on H6, which are involved in interhelical interactions with H3 and H7, respectively, led to a shift toward Meta II in the E247Q and E249Q mutants while retaining the pH sensitivity of the equilibrium. Disruption of the interhelical interaction of Glu2476.30 and Glu2496.32 on H6 with H3 and H7 plays its role during receptor activation, but neutralization of the intrahelical salt bridge between Glu1343.49 and Arg1353.50 is considerably more critical for shifting the photoproduct equilibrium to the active conformation. These conclusions are discussed in the context of recent structural data of the β2-adrenergic receptor.

Fourier transform infrared spectroscopy was used to analyze the chromophore structure in the parent states Pr and Pfr of plant phytochrome phyA and the respective photoproducts lumi-R and lumi-F. The spectra were obtained from phyA adducts assembled with either uniformly or selectively isotope-labeled phytochromobilin and phycocyanobilin. The interpretation of the experimental spectra is based on the spectra of chromophore models calculated by density functional theory. Global 13C-labeling of the tetrapyrrole allows for the discrimination between chromophore and protein bands in the Fourier transform infrared difference spectra. All infrared difference spectra display a prominent difference band attributable to a stretching mode with large contributions from the methine bridge between the inner pyrrole rings (B-C stretching). Due to mode coupling, frequencies and isotopic shifts of this mode suggest that the Pr chromophore may adopt a distorted ZZZssa or ZZZasa geometry with a twisted A-B methine bridge. The transition to lumi-R is associated with only minor changes of the amide I bands indicating limited protein structural changes during the isomerization site of the C-D methine bridge. Major protein structural changes occur upon the transition to Pfr in which the chromophore adopts a ZZEssa or ZZEasa-like state. In addition, specific interactions with the protein alter the structure of the B-C methine bridge as concluded from the substantial downshift of the respective stretching mode. These interactions are removed during the photoreaction to lumi-F (ZZE→ZZZ), which involves only small protein structural changes.