Cascade Transformation of the Ansamycin Benzoquinone Core into Benzoxazole Influencing Anticancer Activity and Selectivity.

The metal-free cascade transformation of geldanamycin benzoquinone core is proposed at relatively mild conditions. This approach yields new benzoxazole ansamycin antibiotics and enables their functionalization in an atom-economic manner, irrespective of the type of amine used. The analysis of the heterocyclization course reveals the dependence of its rate on the nature of the para-substituent within the benzylamine moiety (EDG/EWG) and the strength of the base. The reduction of the ansamycin core enables an increase in anticancer potency and selectivity.

Regioselective approach to colchiceine tropolone ring functionalization at C(9) and C(10) yielding new anticancer hybrid derivatives containing heterocyclic structural motifs.

The influence of base type, temperature, and solvent on regioselective C(9)/C(10) "click" modifications within the tropolone ring of colchiceine (2) is investigated. New ether derivatives of 2, bearing alkyne, azide, vinyl, or halide aryl groups enable assembly of the alkaloid part with heterocycles or important biomolecules such as saccharides, geldanamycin or AZT into hybrid scaffolds by dipolar cycloaddition (CuAAC) or Heck reaction. Compared to colchicine (1) or colchiceine (2), ether congeners, as e.g. 3e [IC50s(3e) ∼ 0.9 nM], show improved or similar anticancer effects, whereby the bulkiness of the substituents and the substitution pattern of the tropolone proved to be essential. Biological studies reveal that expanding the ether arms by terminal basic heterocycles as quinoline or pyridine, decreases the toxicity in HDF cells at high anticancer potency (IC50s ∼ 1-2 nM). Docking of ether and hybrid derivatives into the colchicine pocket of αGTP/ß tubulin dimers reveals a relationship between the favourable binding mode and the attractive anticancer potency.

Light- and temperature-dependent dynamics of chromophore and protein structural changes in bathy phytochrome Agp2.

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.

Anticancer activity and toxicity of new quaternary ammonium geldanamycin derivative salts and their mixtures with potentiators.

Geldanamycin (GDM) has been modified by different type neutral/acidic/basic substituents (1-7) and by quinuclidine motif (8), transformed into ammonium salts (9-13) at C(17). These compounds have been characterised by spectroscopic and x-ray methods. Derivative 8 shows better potency than GDM in MCF-7, MDA-MB-231, A549 and HeLa (IC50s = 0.09-1.06 µM). Transformation of 8 into salts 9-13 reduces toxicity (by 11-fold) at attractive potency, e.g. MCF-7 cell line (IC50∼2 µM). Our studies show that higher water solubility contributes to lower toxicity of salts than GDM in healthy CCD39Lu and HDF cells. The use of 13 mixtures with potentiators PEI and DOX enhanced anticancer effects from IC50∼2 µM to IC50∼0.5 µM in SKBR-3, SKOV-3, and PC-3 cancer cells, relative to 13. Docking studies showed that complexes between quinuclidine-bearing 8-13 and Hsp90 are stabilised by extra hydrophobic interactions between the C(17)-arms and K58 or Y61 of Hsp90.

Modulation of Light Energy Transfer from Chromophore to Protein in the Channelrhodopsin ReaChR.

The function of photoreceptors relies on efficient transfer of absorbed light energy from the chromophore to the protein to drive conformational changes that ultimately generate an output signal. In retinal-binding proteins, mainly two mechanisms exist to store the photon energy after photoisomerization: 1) conformational distortion of the prosthetic group retinal, and 2) charge separation between the protonated retinal Schiff base (RSBH+) and its counterion complex. Accordingly, energy transfer to the protein is achieved by chromophore relaxation and/or reduction of the charge separation in the RSBH+-counterion complex. Combining FTIR and UV-Vis spectroscopy along with molecular dynamics simulations, we show here for the widely used, red-activatable Volvox carteri channelrhodopsin-1 derivate ReaChR that energy storage and transfer into the protein depends on the protonation state of glutamic acid E163 (Ci1), one of the counterions of the RSBH+. Ci1 retains a pKa of 7.6 so that both its protonated and deprotonated forms equilibrate at physiological conditions. Protonation of Ci1 leads to a rigid hydrogen-bonding network in the active-site region. This stabilizes the distorted conformation of the retinal after photoactivation and decelerates energy transfer into the protein by impairing the release of the strain energy. In contrast, with deprotonated Ci1 or removal of the Ci1 glutamate side chain, the hydrogen-bonded system is less rigid, and energy transfer by chromophore relaxation is accelerated. Based on the hydrogen out-of-plane (HOOP) band decay kinetics, we determined the activation energy for these processes in dependence of the Ci1 protonation state.

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.

Tracking Pore Hydration in Channelrhodopsin by Site-Directed Infrared-Active Azido Probes.

In recent years, gating and transient ion-pathway formation in the light-gated channelrhodopsins (ChRs) have been intensively studied. Despite these efforts, a profound understanding of the mechanistic details is still lacking. To track structural changes concomitant with the formation and subsequent collapse of the ion-conducting pore, we site-specifically introduced the artificial polarity-sensing probe p-azido-l-phenylalanine (azF) into several ChRs by amber stop codon suppression. The frequently used optogenetic actuator ReaChR (red-activatable ChR) exhibited the best expression properties of the wild type and the azF mutants. By exploiting the unique infrared spectral absorption of azF [νas(N3) ∼ 2100 cm-1] and its sensitivity to polarity changes, we monitored hydration changes at various sites of the pore region and the inner gate by stationary and time-resolved infrared spectroscopy. Our data imply that channel closure coincides with a dehydration event occurring between the interface of the central and the inner gate. In contrast, the extracellular ion pathway seems to be hydrated in the open and closed states to similar extents. Mutagenesis of sites in the inner gate suggests that it acts as an intracellular entry funnel, whose architecture and composition modulate water influx and efflux within the channel pore. Our results highlight the potential of genetic code expansion technology combined with biophysical methods to investigate channel gating, particularly hydration dynamics at specific sites, with a so far unprecedented spatial resolution.

Role of the Propionic Side Chains for the Photoconversion of Bacterial Phytochromes.

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/ß-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 arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin.

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (Gt) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the Gt α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, Gt α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling.

Proton transfer reactions in the red light-activatable channelrhodopsin variant ReaChR and their relevance for its function.

Channelrhodopsins (ChRs) are light-gated ion channels widely used for activating selected cells in large cellular networks. ChR variants with a red-shifted absorption maximum, such as the modified Volvox carteri ChR1 red-activatable channelrhodopsin ("ReaChR," λmax = 527 nm), are of particular interest because longer wavelengths allow optical excitation of cells in deeper layers of organic tissue. In all ChRs investigated so far, proton transfer reactions and hydrogen bond changes are crucial for the formation of the ion-conducting pore and the selectivity for protons versus cations, such as Na+, K+, and Ca2+ (1). By using a combination of electrophysiological measurements and UV-visible and FTIR spectroscopy, we characterized the proton transfer events in the photocycle of ReaChR and describe their relevance for its function. 1) The central gate residue Glu130 (Glu90 in Chlamydomonas reinhardtii (Cr) ChR2) (i) undergoes a hydrogen bond change in D → K transition and (ii) deprotonates in K → M transition. Its negative charge in the open state is decisive for proton selectivity. 2) The counter-ion Asp293 (Asp253 in CrChR2) receives the retinal Schiff base proton during M-state formation. Starting from M, a photocycle branching occurs involving (i) a direct M → D transition and (ii) formation of late photointermediates N and O. 3) The DC pair residue Asp196 (Asp156 in CrChR2) deprotonates in N → O transition. Interestingly, the D196N mutation increases 15-syn-retinal at the expense of 15-anti, which is the predominant isomer in the wild type, and abolishes the peak current in electrophysiological measurements. This suggests that the peak current is formed by 15-anti species, whereas 15-syn species contribute only to the stationary current.

Synthesis, antiproliferative activity and molecular docking of thiocolchicine urethanes.

A number of naturally occurring compounds such as paclitaxel, vinblastine, combretastatin, and colchicine exert their therapeutic effect by changing the dynamics of tubulin and its polymer form, microtubules. The identification of tubulin as a potential target for anticancer drugs has led to extensive research followed by clinical development of numerous compounds from several families. In this paper we report on the design, synthesis and in vitro evaluation of a group of thiocolchicine derivatives, modified at ring-B, labelled here compounds 4-14. These compounds have been obtained in a simple reaction of 7-deacetyl-10-thiocolchicine 3 with eleven different alcohols in the presence of triphosgene. These novel agents have been checked for anti-proliferative activity against four human cancer cell lines and their mode of action has been confirmed as colchicine binding site inhibition (CBSI) using molecular docking. Molecular simulations provided rational tubulin binding models for the tested compounds. On the basis of in vitro tests, derivatives 4-8 and 14 demonstrated the highest potency against MCF-7, LoVo and A549 tumor cell lines (IC50 values = 0.009-0.014 µM). They were more potent and characterized by a higher selectivity index than several standard chemotherapeutics including cisplatin and doxorubicin as well as unmodified colchicine. Further, studies revealed that colchicine and its several derivatives arrested MCF-7 cells in mitosis, while its selected derivatives caused microtubule depolymerization.

Complex Photochemistry within the Green-Absorbing Channelrhodopsin ReaChR.

Channelrhodopsins (ChRs) are light-activated ion channels widely employed for photostimulation of excitable cells. This study focuses on ReaChR, a chimeric ChR variant with optimal properties for optogenetic applications. We combined electrophysiological recordings with infrared and UV-visible spectroscopic measurements to investigate photocurrents and photochemical properties of ReaChR. Our data imply that ReaChR is green-light activated (λmax = 532 nm) with a non-rhodopsin-like action spectrum peaking at 610 nm for stationary photocurrents. This unusual spectral feature is associated with photoconversion of a previously unknown light-sensitive, blue-shifted photocycle intermediate L (λmax = 495 nm), which is accumulated under continuous illumination. To explain the complex photochemical reactions, we propose a symmetrical two-cycle-model based on the two C15=N isomers of the retinal cofactor with either syn- or anti-configuration, each comprising six consecutive states D, K, L, M, N, and O. Ion conduction involves two states per cycle, the late M- (M2) with a deprotonated retinal Schiff base and the consecutive green-absorbing N-state that both equilibrate via reversible reprotonation. In our model, a fraction of the deprotonated M-intermediate of the anti-cycle may be photoconverted-as the L-state-back to its inherent dark state, or to its M-state pendant (M') of the syn-cycle. The latter reaction pathway requires a C13=C14, C15=N double-isomerization of the retinal chromophore, whereas the intracircular photoconversion of M back to D involves only one C13=C14 double-bond isomerization.

Formation and decay of the arrestin·rhodopsin complex in native disc membranes.

In the G protein-coupled receptor rhodopsin, light-induced cis/trans isomerization of the retinal ligand triggers a series of distinct receptor states culminating in the active Metarhodopsin II (Meta II) state, which binds and activates the G protein transducin (Gt). Long before Meta II decays into the aporeceptor opsin and free all-trans-retinal, its signaling is quenched by receptor phosphorylation and binding of the protein arrestin-1, which blocks further access of Gt to Meta II. Although recent crystal structures of arrestin indicate how it might look in a precomplex with the phosphorylated receptor, the transition into the high affinity complex is not understood. Here we applied Fourier transform infrared spectroscopy to monitor the interaction of arrestin-1 and phosphorylated rhodopsin in native disc membranes. By isolating the unique infrared signature of arrestin binding, we directly observed the structural alterations in both reaction partners. In the high affinity complex, rhodopsin adopts a structure similar to Gt-bound Meta II. In arrestin, a modest loss of ß-sheet structure indicates an increase in flexibility but is inconsistent with a large scale structural change. During Meta II decay, the arrestin-rhodopsin stoichiometry shifts from 1:1 to 1:2. Arrestin stabilizes half of the receptor population in a specific Meta II protein conformation, whereas the other half decays to inactive opsin. Altogether these results illustrate the distinct binding modes used by arrestin to interact with different functional forms of the receptor.

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin.

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.

Synthesis, antiproliferative activity and molecular docking of Colchicine derivatives.

In order to create more potent anticancer agents, a series of five structurally different derivatives of Colchicine have been synthesised. These compounds were characterised spectroscopically and structurally and their antiproliferative activity against four human tumour cell lines (HL-60, HL-60/vinc, LoVo, LoVo/DX) was evaluated. Additionally the activity of the studied compounds was calculated using computational methods involving molecular docking of the Colchicine derivatives to ß-tubulin. The experimental and computational results are in very good agreement indicating that the antimitotic activity of Colchicine derivatives can be readily predicted using computational modeling methods.

Regioselective long-range proton transfer in new rifamycin antibiotics: a process in which crown ethers act as stronger Brønsted bases than amines.

Water-mediated proton transfer in six new derivatives of 3-formylrifamycin SV that contain crown, aza-crown, and benzo-crown ether rings were investigated by FTIR and NMR spectroscopy. (1)H-(1)H COSY couplings provide evidence for the formation of zwitterionic structures of the aza-crown and crown ether derivatives of rifamycin, in which a proton from one of the phenolic groups is transferred to tertiary and secondary nitrogen atoms. The increased intensity of the continuous absorption in the mid-infrared region together with the NMR data indicate proton transfer from the phenol group of the rifamycin core to the cavity of the benzo-crown ether ring. This proton transfer is achieved by formation of hydronium (H3O(+)) or Zundel ions (H5O2(+)), which form intermolecular hydrogen bonds with the oxygen atoms of the crown ether. DFT calculations are in agreement with the spectroscopic data and allow visualization of the structures of all new rifamycin derivatives, characterized by different intramolecular protonation sites.

Early formation of the ion-conducting pore in channelrhodopsin-2.

Channelrhodopsins (ChRs) are light-gated ion channels that are widely used in optogenetics. They allow precise control of neuronal activity with light, but a detailed understanding of how the channel is gated and the ions are conducted is still lacking. The recent determination of the X-ray structural model in the closed state marks an important milestone. Herein the open state structure is presented and the early formation of the ion conducting pore is elucidated in atomic detail using time-resolved FTIR spectroscopy. Photo-isomerization of the retinal-chromophore causes a downward movement of the highly conserved E90, which opens the pore. Molecular dynamic (MD) simulations show that water molecules invade through this opened pore, Helix 2 tilts and the channel fully opens within ms. Since E90 is a highly conserved residue, the proposed E90-Helix2-tilt (EHT) model might describe a general activation mechanism and provides a new avenue for further mechanistic studies and engineering.

Light-dark adaptation of channelrhodopsin C128T mutant.

Channelrhodopsins are microbial type rhodopsins that operate as light-gated ion channels. Largely prolonged lifetimes of the conducting state of channelrhodopsin-2 may be achieved by mutations of crucial single amino acids, i.e. cysteine 128. Such mutants are of great scientific interest in the field of neurophysiology because they allow neurons to be switched on and off on demand (step function rhodopsins). Due to their slow photocycle, structural alterations of these proteins can be studied by vibrational spectroscopy in more detail than possible with wild type. Here, we present spectroscopic evidence that the photocycle of the C128T mutant involves three different dark-adapted states that are populated according to the wavelength and duration of the preceding illumination. Our results suggest an important role of multiphoton reactions and the previously described side reaction for dark state regeneration. Structural changes that cause formation and depletion of the assumed ion conducting state P520 are only small and follow larger changes that occur early and late in the photocycle, respectively. They require only minor structural rearrangements of amino acids near the retinal binding pocket and are triggered by all-trans/13-cis retinal isomerization, although additional isomerizations are also involved in the photocycle. We will discuss an extended photocycle model of this mutant on the basis of spectroscopic and electrophysiological data.

Structure of the biliverdin cofactor in the Pfr state of bathy and prototypical phytochromes.

Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZE → ZZZ) Pfr to Pr back-conversion.

The Functionality of the DC Pair in a Rhodopsin Guanylyl Cyclase from Catenaria anguillulae.

Rhodopsin guanylyl cyclases (RGCs) belong to the class of enzymerhodopsins catalyzing the transition from GTP into the second messenger cGMP, whereas light-regulation of enzyme activity is mediated by a membrane-bound microbial rhodopsin domain, that holds the catalytic center inactive in the dark. Structural determinants for activation of the rhodopsin moiety eventually leading to catalytic activity are largely unknown. Here, we investigate the mechanistic role of the D283-C259 (DC) pair that is hydrogen bonded via a water molecule as a crucial functional motif in the homodimeric C. anguillulae RGC. Based on a structural model of the DC pair in the retinal binding pocket obtained by MD simulation, we analyzed formation and kinetics of early and late photocycle intermediates of the rhodopsin domain wild type and specific DC pair mutants by combined UV-Vis and FTIR spectroscopy at ambient and cryo-temperatures. By assigning specific infrared bands to S-H vibrations of C259 we are able to show that the DC pair residues are tightly coupled. We show that deprotonation of D283 occurs already in the inactive L state as a prerequisite for M state formation, whereas structural changes of C259 occur in the active M state and early cryo-trapped intermediates. We propose a comprehensive molecular model for formation of the M state that activates the catalytic moiety. It involves light induced changes in bond strength and hydrogen bonding of the DC pair residues from the early J state to the active M state and explains the retarding effect of C259 mutants.