Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates. Oxidized flavin needs to be reduced for regeneration of the enzyme, which can be performed in vitro by a photoreduction with blue light. Here, we employed this photoreduction to study characteristic structural changes associated with the transition from oxidized to fully reduced flavin in PyrH from Streptomyces rugosporus as a model for tryptophan-5-halogenases. The effect of the presence of bromide and chloride or the absence of any halides on the UV-vis spectrum of the enzyme demonstrated a halide-dependent structure of the flavin-binding pocket. Light-induced FTIR difference spectroscopy was applied and the signals assigned by selective isotope labeling of the protein moiety. The identified structural changes in α-helix and ß-sheet elements were strongly dependent on the presence of bromide, chloride, the substrate tryptophan, and the product 5-chloro-tryptophan, respectively. We identified a clear allosteric coupling in solution at ambient conditions between cofactor-binding site and substrate-binding site that is active in both directions, despite their separation by a tunnel. We suggest that this coupling constitutes a fine-tuned mechanism for the promotion of the enzymatic reaction of flavin-dependent halogenases in dependence of halide and substrate availability.

Light-induced Trpin/Metout Switching During BLUF Domain Activation in ATP-bound Photoactivatable Adenylate Cyclase OaPAC.

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in an energetically unfavorable conformation for the conversion to cAMP. However, FTIR spectroscopic experiments confirm that this conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the early events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Early changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed.

Core-shell microgels synthesized in continuous flow: deep insight into shell growth using temperature-dependent FTIR.

While core-shell microgels have been intensively studied in their fully synthesized state, the formation mechanism of the shell has not been completely understood. Such insight is decisive for a customization of microgel properties for applications. In this work, microgels based on a N-isopropylmethacrylamide (NiPMAM) core and a N-n-propylacrylamide (NnPAM) shell are synthesized in a continuous flow reactor. The shell growth is studied depending on the solution's time of residence inside the reactor. PCS experiments reveal a significant decrease of the volume phase transition temperatures of the core and the shell, with increasing residence time. At early stages, a decreased swelling capacity is found before a discrete NnPAM shell is formed. Temperature-dependent FTIR spectroscopy shows that the decreased swelling capacity originates from a pronounced interpenetrated network (IPN) between NnPAM and NiPMAM. AFM images resolve heterogeneously distributed shell material after 3 min, pointing to an aggregation of NnPAM domains before the distinct shell forms. The combination of diffusional properties, AFM images and vibrational information confirms a deeply interpenetrated network already at early stages of the precipitation polymerization, in which the shell material heavily influences the swelling properties.

Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates.

pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK' = 6.6) and EGF-CoGFP-mCRISPRed (pK' = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein-drug conjugates in cancer therapy.

Plant Cryptochromes Illuminated: A Spectroscopic Perspective on the Mechanism.

Plant cryptochromes are central blue light receptors for the control of land plant and algal development including the circadian clock and the cell cycle. Cryptochromes share a photolyase homology region with about 500 amino acids and bind the chromophore flavin adenine dinucleotide. Characteristic for plant cryptochromes is a conserved aspartic acid close to flavin and an exceptionally long C-terminal extension. The mechanism of activation by excitation and reduction of the chromophore flavin adenine dinucleotide has been controversially discussed for many years. Various spectroscopic techniques have contributed to our understanding of plant cryptochromes by providing high time resolution, ambient conditions and even in-cell approaches. As a result, unifying and differing aspects of photoreaction and signal propagation have been revealed in comparison to members from other cryptochrome subfamilies. Here, we review the insight from spectroscopy on the flavin photoreaction in plant cryptochromes and present the current models on the signal propagation from flavin reduction to dissociation of the C-terminal extension.

The World of Algae Reveals a Broad Variety of Cryptochrome Properties and Functions.

Algae are photosynthetic eukaryotic (micro-)organisms, lacking roots, leaves, and other organs that are typical for land plants. They live in freshwater, marine, or terrestrial habitats. Together with the cyanobacteria they contribute to about half of global carbon fixation. As primary producers, they are at the basis of many food webs and they are involved in biogeochemical processes. Algae are evolutionarily distinct and are derived either by primary (e.g., green and red algae) or secondary endosymbiosis (e.g., diatoms, dinoflagellates, and brown algae). Light is a key abiotic factor needed to maintain the fitness of algae as it delivers energy for photosynthesis, regulates algal cell- and life cycles, and entrains their biological clocks. However, excess light can also be harmful, especially in the ultraviolet range. Among the variety of receptors perceiving light information, the cryptochromes originally evolved as UV-A and blue-light receptors and have been found in all studied algal genomes so far. Yet, the classification, biophysical properties, wavelength range of absorbance, and biological functions of cryptochromes are remarkably diverse among algal species, especially when compared to cryptochromes from land plants or animals.

DASH cryptochrome 1, a UV-A receptor, balances the photosynthetic machinery of Chlamydomonas reinhardtii.

Drosophila, Arabidopsis, Synechocystis, Homo (DASH) cryptochromes belong to the cryptochrome/photolyase family and can act as DNA repair enzymes. In bacteria and fungi, they also can play regulatory roles, but in plants their biological functions remain elusive. Here, we characterize CRY-DASH1 from the green alga Chlamydomonas reinhardtii. We perform biochemical and in vitro photochemical analysis. For functional characterization, a knock-out mutant of cry-dash1 is used. CRY-DASH1 protein is localized in the chloroplast and accumulates at midday. Although the photoautotrophic growth of the mutant is significantly reduced compared to the wild-type (WT), the mutant has increased levels of photosynthetic pigments and a higher maximum photochemical efficiency of photosystem II (PS II). Hyper-stacking of thylakoid membranes occurs together with an increase in proteins of the PS II reaction center, D1 and its antenna CP43, but not of their transcripts. CRY-DASH1 binds fully reduced flavin adenine dinucleotide and the antenna 5,10-methenyltetrahydrofolate, leading to an absorption peak in the UV-A range. Supplementation of white light with UV-A increases photoautotrophic growth of the WT but not of the cry-dash1 mutant. These results suggest a balancing function of CRY-DASH1 in the photosynthetic machinery and point to its role as a photoreceptor for the UV-A range separated from the absorption of photosynthetic pigments.

C-Terminal Extension of a Plant Cryptochrome Dissociates from the ß-Sheet of the Flavin-Binding Domain.

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting ß-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the ß-sheet signal, implying an association of the CCT close to the only ß-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that ß-sheet reorganization causes the CCT release and restructuring.

Resolving Structural Changes of Photoreceptors in Living Escherichia coli via In-cell Infrared Difference Spectroscopy.

Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions in vivo differ dramatically from those selected for in vitro experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules. Fourier transform infrared (FTIR) difference spectroscopy is a well-suited method to study the light-induced structural responses of photoreceptors including changes in cofactor, side chains and secondary structure. Here, we describe a protocol to study the response of cofactor and protein in living E. coli cells via in-cell infrared difference (ICIRD) spectroscopy using the attenuated total reflection (ATR) configuration. Proteins are overexpressed in E. coli, the cells are transferred into saline solution and the copy number per cell is determined using fluorescence spectroscopy. The suspension is centrifuged and the concentrated cells transferred onto the ATR cell inside the FTIR spectrometer. The thermostatted cell is sealed and illuminated from the top with an LED. Intensity spectra are recorded before and after illumination to generate the difference spectrum of the receptor inside the living cell. With ICIRD spectroscopy, structural changes of soluble photoreceptors are resolved in a near-native environment. The approach works in H2O at ambient conditions, is label free, without any limitations in protein size and does not require any purification step. Graphic abstract: In-cell infrared difference spectroscopy on photoreceptors in living E. coli using attenuated total reflection.

Peripheral Methionine Residues Impact Flavin Photoreduction and Protonation in an Engineered LOV Domain Light Sensor.

Proton-coupled electron transfer reactions play critical roles in many aspects of sensory phototransduction. In the case of flavoprotein light sensors, reductive quenching of flavin excited states initiates chemical and conformational changes that ultimately transmit light signals to downstream targets. These reactions generally require neighboring aromatic residues and proton-donating side chains for rapid and coordinated electron and proton transfer to flavin. Although photoreduction of flavoproteins can produce either the anionic (ASQ) or neutral semiquinone (NSQ), the factors that favor one over the other are not well understood. Here we employ a biologically active variant of the light-oxygen-voltage (LOV) domain protein VVD devoid of the adduct-forming Cys residue (VVD-III) to probe the mechanism of flavin photoreduction and protonation. A series of isosteric and conservative residue replacements studied by rate measurements, fluorescence quantum yields, FTIR difference spectroscopy, and molecular dynamics simulations indicate that tyrosine residues facilitate charge recombination reactions that limit sustained flavin reduction, whereas methionine residues facilitate radical propagation and quenching and also gate solvent access for flavin protonation. Replacement of a single surface Met residue with Leu favors formation of the ASQ over the NSQ and desensitizes photoreduction to oxidants. In contrast, increasing site hydrophilicity by Gln substitution promotes rapid NSQ formation and weakens the influence of the redox environment. Overall, the photoreactivity of VVD-III can be understood in terms of redundant electron donors, internal hole quenching, and coupled proton transfer reactions that all depend upon protein conformation, dynamics, and solvent penetration.

Site-by-site tracking of signal transduction in an azidophenylalanine-labeled bacteriophytochrome with step-scan FTIR spectroscopy.

Signal propagation in photosensory proteins is a complex and multidimensional event. Unraveling such mechanisms site-specifically in real time is an eligible but a challenging goal. Here, we elucidate the site-specific events in a red-light sensing phytochrome using the unnatural amino acid azidophenylalanine, vibrationally distinguishable from all other protein signals. In canonical phytochromes, signal transduction starts with isomerization of an excited bilin chromophore, initiating a multitude of processes in the photosensory unit of the protein, which eventually control the biochemical activity of the output domain, nanometers away from the chromophore. By implementing the label in prime protein locations and running two-color step-scan FTIR spectroscopy on the Deinococcus radiodurans bacteriophytochrome, we track the signal propagation at three specific sites in the photosensory unit. We show that a structurally switchable hairpin extension, a so-called tongue region, responds to the photoconversion already in microseconds and finalizes its structural changes concomitant with the chromophore, in milliseconds. In contrast, kinetics from the other two label positions indicate that the site-specific changes deviate from the chromophore actions, even though the labels locate in the chromophore vicinity. Several other sites for labeling resulted in impaired photoswitching, low structural stability, or no changes in the difference spectrum, which provides additional information on the inner dynamics of the photosensory unit. Our work enlightens the multidimensionality of the structural changes of proteins under action. The study also shows that the signaling mechanism of phytochromes is accessible in a time-resolved and site-specific approach by azido probes and demonstrates challenges in using these labels.

Smart membranes by electron beam cross-linking of copolymer microgels.

Poly(N-isopropylacrylamide) (pNIPAM) based copolymer microgels were used to create free-standing, transferable, thermoresponsive membranes. The microgels were synthesized by copolymerization of NIPAM with N-benzylhydrylacrylamide (NBHAM). Monolayers of these colloidal gels were subsequently cross-linked using an electron gun leading to the formation of a connected monolayer. Furthermore, the cross-linked microgel layer is detached from the supporting material by dissolving the substrate. These unique systems can be used as transferable, thermoresponsive coatings and as thermoresponsive membranes. As a proof of principle for the use of such membranes we studied the ion transport through them at different temperatures revealing drastic changes when the lower critical solution temperature of the copolymer microgels is reached.

A quantum cascade laser setup for studying irreversible photoreactions in H2O with nanosecond resolution and microlitre consumption.

Time-resolved infrared spectroscopy on irreversible reactions requires in general an exchange of sample for thousands of acquisitions leading to high sample consumption. Here, we present a setup employing a modern quantum cascade laser (QCL) as a probe light source to record time-resolved difference spectra of irreversible photoreactions in H2O. The combination of the focused QCL with a pressure-tolerant flow cell and a micrometre stage orthogonal to the flow allowed us to drastically reduce the sample consumption. We investigated the irreversible photoreduction of the cofactor flavin mononucleotide (FMN) in H2O, which is a common reaction taking place in biological photoreceptors. A broad time range from 20 nanoseconds to 1 second was accessible, because the approach minimized any signal drift by the flow. Kinetics were recorded at 46 selected wavenumbers consuming 12 microlitres for a complete dataset. The tuning range of 1490-1740 cm-1 included relevant carbonyl vibrations and the region of strong water absorption at around 1650 cm-1. A continuous dataset in the spectral dimension was generated by applying a fit with a sum of Lorentzians. Subsequent global analysis allowed us to resolve reference spectra and kinetics of the photoreaction proceeding from the triplet excited state via the intermediate flavin anion radical to the product, the fully reduced state of FMN. Accordingly, the neutral radical state is not populated in the disproportionation. The approach strongly facilitates the spectroscopic access to irreversible reactions of flavin-containing photoreceptors and photoenzymes with high time resolution and small sample consumption.

Tailored flavoproteins acting as light-driven spin machines pump nuclear hyperpolarization.

The solid-state photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect generates non-Boltzmann nuclear spin magnetization, referred to as hyperpolarization, allowing for high gain of sensitivity in nuclear magnetic resonance (NMR). Well known to occur in photosynthetic reaction centers, the effect was also observed in a light-oxygen-voltage (LOV) domain of the blue-light receptor phototropin, in which the functional cysteine was removed to prevent photo-chemical reactions with the cofactor, a flavin mononucleotide (FMN). Upon illumination, the FMN abstracts an electron from a tryptophan to form a transient spin-correlated radical pair (SCRP) generating the photo-CIDNP effect. Here, we report on designed molecular spin-machines producing nuclear hyperpolarization upon illumination: a LOV domain of aureochrome1a from Phaeodactylum tricornutum, and a LOV domain named 4511 from Methylobacterium radiotolerans (Mr4511) which lacks an otherwise conserved tryptophan in its wild-type form. Insertion of the tryptophan at canonical and novel positions in Mr4511 yields photo-CIDNP effects observed by 15N and 1H liquid-state high-resolution NMR with a characteristic magnetic-field dependence indicating an involvement of anisotropic magnetic interactions and a slow-motion regime in the transient paramagnetic state. The heuristic biomimetic design opens new categories of experiments to analyze and apply the photo-CIDNP effect.

In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells.

Proteins are usually studied in well-defined buffer conditions, which differ substantially from those within a host cell. In some cases, the intracellular environment has an impact on the mechanism, which might be missed by in vitro experiments. IR difference spectroscopy previously has been applied to study the light-induced response of photoreceptors and photoenzymes in vitro Here, we established the in-cell IR difference (ICIRD) spectroscopy in the transmission and attenuated total reflection configuration to investigate the light-induced response of soluble proteins in living bacterial cells. ICIRD spectroscopy on the light, oxygen, or voltage (LOV) domains of the blue light receptors aureochrome and phototropin revealed a suppression of the response of specific secondary structure elements, indicating that the intracellular environment affects LOV photoreceptor mechanisms in general. Moreover, in-cell fluorescence spectroscopy disclosed that the intracellular environment slows down the recovery of the light-induced flavin adduct. Segment-resolved ICIRD spectroscopy on basic-region leucine zipper (bZIP)-LOV of aureochrome 1a from the diatom Phaeodactylum tricornutum indicated a signal progression from the LOV sensor to the bZIP effector independent of unfolding of the connecting A'α-helix, an observation that stood in contrast to in vitro results. This deviation was recapitulated in vitro by emulating the intracellular environment through the addition of the crowding agent BSA, but not by sucrose polymers. We conclude that ICIRD spectroscopy is a noninvasive, label-free approach for assessing conformational changes in receptors in living cells at ambient conditions. As demonstrated, these near-native responses may deviate from the mechanisms established under in vitro conditions.

Core-shell microgels as thermoresponsive carriers for catalytic palladium nanoparticles.

Responsive core-shell microgels are promising systems for a stabilization of Pd nanoparticles and control of their catalytic activity. Here, poly-N-n-propylacrylamide (PNNPAM) was copolymerized with methacrylic acid to yield microgel core particles, which were subsequently coated with an additional, acid-free poly-N-isopropylmethacrylamide (PNIPMAM) shell. Both core and core-shell systems were used as pH- and temperature-responsive carrier systems for the incorporation of palladium nanoparticles. The embedded nanoparticles were found to have a uniform size distribution with diameters at around 20 nm. Their catalytic activity was investigated by following the kinetics of the reduction of p-nitrophenol to p-aminophenol using UV-vis spectroscopy. For the PNNPAM microgel core, the temperature dependence of the rate constant followed the Arrhenius equation, which is an unusual behaviour for thermoresponsive carrier systems but common for passive systems such as polyelectrolyte brushes. In contrast, the catalytic activity of nanoparticles embedded in microgel core-shell systems decreased drastically at the volume phase transition temperature (44 °C) of the PNIPMAM shell. Accordingly, a promising architecture of passive nanoparticle-carrying core and thermoresponsive shell was realized successfully.

Tongue Refolding in the Knotless Cyanobacterial Phytochrome All2699.

Phytochromes regulate central responses of plants and microorganisms such as shade avoidance and photosystem synthesis. Canonical phytochromes comprise a photosensory module of three domains. The C-terminal phytochrome-specific (PHY) domain interacts via a tongue element with the bilin chromophore in the central GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain. The bilin isomerizes upon illumination with red light, transforming the receptor from the Pr state to the Pfr state. The "knotless" phytochrome All2699 from the cyanobacterium Nostoc sp. PCC7120 comprises three GAF domains as a sensory module and a histidine kinase as an effector. GAF1 and GAF3 both bind a bilin, and GAF2 contains a tongue-like element. We studied the response of All2699, GAF1-GAF2, and GAF1 to red light by Fourier transform infrared difference spectroscopy, including a 13C-labeled protein moiety for assignment. In GAF1-GAF2, a refolding of the tongue from ß-sheet to α-helix and an upshift of the ring D carbonyl stretch from 1700 to 1712 cm-1 were observed. Therefore, GAF1-GAF2 is regarded as the smallest model system available to study the tongue response and interaction with the chromophore. Replacement of an arginine in the tongue with proline (R387P) did not affect the unfolding of the ß-sheet to Pfr but strongly impaired α-helix formation. In contrast, the Y55H mutation close to bilin ring D did not interfere with conversion to Pfr. Strikingly, the presence of GAF3 in the full-length All2699 diminished the response of the tongue and generated the signal pattern found for GAF1 alone. These results point to a regulatory or integrative role of GAF3 in All2699 that is absent in canonical phytochromes.

Calculation of the Geometries and Infrared Spectra of the Stacked Cofactor Flavin Adenine Dinucleotide (FAD) as the Prerequisite for Studies of Light-Triggered Proton and Electron Transfer.

Flavin cofactors, like flavin adenine dinucleotide (FAD), are important electron shuttles in living systems. They catalyze a wide range of one- or two-electron redox reactions. Experimental investigations include UV-vis as well as infrared spectroscopy. FAD in aqueous solution exhibits a significantly shorter excited state lifetime than its analog, the flavin mononucleotide. This finding is explained by the presence of a "stacked" FAD conformation, in which isoalloxazine and adenine moieties form a π-complex. Stacking of the isoalloxazine and adenine rings should have an influence on the frequency of the vibrational modes. Density functional theory (DFT) studies of the closed form of FAD in microsolvation (explicit water) were used to reproduce the experimental infrared spectra, substantiating the prevalence of the stacked geometry of FAD in aqueous surroundings. It could be shown that the existence of the closed structure in FAD can be narrowed down to the presence of only a single water molecule between the third hydroxyl group (of the ribityl chain) and the N7 in the adenine ring of FAD.

Nuclear spin-hyperpolarization generated in a flavoprotein under illumination: experimental field-dependence and theoretical level crossing analysis.

The solid-state photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect generates non-equilibrium nuclear spin polarization in frozen electron-transfer proteins upon illumination and radical-pair formation. The effect can be observed in various natural photosynthetic reaction center proteins using magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, and in a flavin-binding light-oxygen-voltage (LOV) domain of the blue-light receptor phototropin. In the latter system, a functionally instrumental cysteine has been mutated to interrupt the natural cysteine-involving photochemistry allowing for an electron transfer from a more distant tryptophan to the excited flavin mononucleotide chromophore. We explored the solid-state photo-CIDNP effect and its mechanisms in phototropin-LOV1-C57S from the green alga Chlamydomonas reinhardtii by using field-cycling solution NMR. We observed the 13C and, to our knowledge, for the first time, 15N photo-CIDNP signals from phototropin-LOV1-C57S. Additionally, the 1H photo-CIDNP signals of residual water in the deuterated buffer of the protein were detected. The relative strengths of the photo-CIDNP effect from the three types of nuclei, 1H, 13C and 15N were measured in dependence of the magnetic field, showing their maximum polarizations at different magnetic fields. Theoretical level crossing analysis demonstrates that anisotropic mechanisms play the dominant role at high magnetic fields.

Time-Resolved Infrared and Visible Spectroscopy on Cryptochrome aCRY: Basis for Red Light Reception.

Cryptochromes function as flavin-binding photoreceptors in bacteria, fungi, algae, land plants, and insects. The discovery of an animal-like cryptochrome in the green alga Chlamydomonas reinhardtii has expanded the spectral range of sensitivity of these receptors from ultraviolet A/blue light to almost the complete visible spectrum. The broadened light response has been explained by the presence of the flavin neutral radical as a chromophore in the dark. Concomitant with photoconversion of the flavin, an unusually long-lived tyrosyl radical with a red-shifted ultraviolet-visible spectrum is formed, which is essential for the function of the receptor. In this study, the microenvironment of this key residue, tyrosine 373, was scrutinized using time-resolved Fourier transform infrared spectroscopy on several variants of animal-like cryptochrome and density functional theory for band assignment. The reduced tyrosine takes on distinct hydrogen bond scenarios depending on the presence of the C-terminal extension and of a neighboring cysteine. Upon radical formation, all variants showed a signal at 1400 cm-1, which we assigned to the ν7'a marker band of the CO stretching mode. The exceptionally strong downshift of this band cannot be attributed to a loss of hydrogen bonding only. Time-resolved ultraviolet-visible spectroscopy on W322F, a mutant of the neighboring tryptophan residue, revealed a decrease of the tyrosyl radical lifetime by almost two orders of magnitude, along with a shift of the absorbance maximum from 416 to 398 nm. These findings strongly support the concept of a π-π stacking as an apolar interaction between Y373 and W322 to be responsible for the characteristics of the tyrosyl radical. This concept of radical stabilization has been unknown to cryptochromes so far but might be highly relevant for other homologs with a tetrad of tryptophans and tyrosines as electron donors.