Dark Reversion Research Articles

Sin señas particulares (2020) – espacios, tiempos y tramas de la distopía y lo in/posthumano en la frontera México-EEUU

The film Sin señas particulares (2020) is set in a dystopian landscape in northern Mexico, devastated by the drug trafficking and gang violence that has taken possession of vast territories of the country, in which a mother searches for her son. The protagonist will encounter within this scenario not only the liminal space of the border, that separates and at the same time unites the here and there sides of the colonial order, but also with the limits of that same order which, with its “conditions of extreme mortality” (Rivera Garza), governed by necropolitics (Mbembe), presents itself as the dark reverse of the humanist conception (Braidotti) and points towards an end of humanism in order to make room for other ways of thinking and living the world, though in the modality of dystopia.

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 seconds to 50 minutes 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.

Phytochromes transmit photoperiod information via the evening complex in Brachypodium

BackgroundDaylength is a key seasonal cue for animals and plants. In cereals, photoperiodic responses are a major adaptive trait, and alleles of clock genes such as PHOTOPERIOD1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in adapting barley and wheat to northern latitudes. How monocot plants sense photoperiod and integrate this information into growth and development is not well understood.ResultsWe find that phytochrome C (PHYC) is essential for flowering in Brachypodium distachyon. Conversely, ELF3 acts as a floral repressor and elf3 mutants display a constitutive long day phenotype and transcriptome. We find that ELF3 and PHYC occur in a common complex. ELF3 associates with the promoters of a number of conserved regulators of flowering, including PPD1 and VRN1. Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in short days and is necessary for rapid flowering in response to long days. PHYC is in the active Pfr state at the end of the day, but we observe it undergoes dark reversion over the course of the night.ConclusionsWe propose that PHYC acts as a molecular timer and communicates information on night-length to the circadian clock via ELF3.

Modulation of warm temperature-sensitive growth using a phytochrome B dark reversion variant, phyB[G515E], in Arabidopsis and rice

IntroductionAmbient temperature-induced hypocotyl elongation in Arabidopsis seedlings is sensed by the epidermis-localized phytochrome B (phyB) and transduced into auxin biosynthesis via a basic helix-loop-helix transcription factor, phytochrome-interacting factor 4 (PIF4). Once synthesized, auxin travels down from the cotyledons to the hypocotyl, triggering hypocotyl cell elongation. Thus, the phyB–PIF4 module involved in thermosensing and signal transduction is a potential genetic target for engineering warm temperature-insensitive plants. ObjectivesThis study aims to manipulate warm temperature-induced elongation of plants at the post-translational level using phyB variants with dark reversion, the expression of which is subjected to heat stress. MethodsThe thermosensitive growth response of Arabidopsis was manipulated by expressing the single amino acid substitution variant of phyB (phyB[G515E]), which exhibited a lower dark reversion rate than wild-type phyB. Other variants with slow (phyB[G564E]) or rapid (phyB[S584F]) dark reversion or light insensitivity (phyB[G767R]) were also included in this study for comparison. Warming-induced transient expression of phyB variants was achieved using heat shock-inducible promoters. Arabidopsis PHYB[G515E] and PHYB[G564E] were also constitutively expressed in rice in an attempt to manipulate the heat sensitivity of a monocotyledonous plant species. ResultsAt an elevated temperature, Arabidopsis seedlings transiently expressing PHYB[G515E] under the control of a heat shock-inducible promoter exhibited shorter hypocotyls than those expressing PHYB and other PHYB variant genes. This warm temperature-insensitive growth was related to the lowered PIF4 and auxin responses. In addition, transgenic rice seedlings expressing Arabidopsis PHYB[G515E] and PHYB[G564E] showed warm temperature-insensitive shoot growth. ConclusionTransient expression of phyB variants with altered dark reversion rates could serve as an effective optogenetic technique for manipulating PIF4–auxin-mediated thermomorphogenic responses in plants.

Light-Induced Forward and Reverse Intersystem Crossing in Green Fluorescent Proteins at Cryogenic Temperatures.

Combining fluorescence and phosphorescence kinetics, we characterize forward and reverse intersystem crossing (FISC and RISC, respectively) between the singlet and triplet manifolds S ↔ T in photoswitchable (rsEGFP2) and non-photoswitchable (EGFP) green fluorescent proteins upon continuous 488 nm laser excitation at cryogenic temperatures (CTs). Both proteins behave very similarly, with T1 absorption spectra showing a visible peak at 490 nm (10 mM-1 cm-1) and a vibrational progression in the near-infrared (720 to 905 nm). The dark lifetime of T1 is 21-24 ms at 100 K and very weakly temperature-dependent up to 180 K. Above 180 K, T1 lifetimes reduce rapidly to few milliseconds as found at room temperature (RT). FISC and RISC quantum yields are 0.3 and 0.1%, respectively, for both proteins. The light-induced RISC channel becomes faster than the dark reversal at power densities as low as 20 W cm-2. We discuss implications for fluorescence (super resolution-) microscopy at CT and RT.

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone, and side-chains in EL222.

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A'α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A'α extension of the LOV domain in controlling EL222 photocycle length.

Temperature Sensing in Plants.

Temperature is a key environmental cue that influences the distribution and behavior of plants globally. Understanding how plants sense temperature and integrate this information into their development is important to determine how plants adapt to climate change and to apply this knowledge to the breeding of climate-resilient crops. The mechanisms of temperature perception in eukaryotes are only just beginning to be understood, with multiple molecular phenomena with inherent temperature dependencies, such as RNA melting, phytochrome dark reversion, and protein phase change, being exploited by nature to create thermosensory signaling networks. Here, we review recent progress in understanding how temperature sensing in four major pathways in Arabidopsis thaliana occurs: vernalization, cold stress, thermomorphogenesis, and heat stress. We discuss outstanding questions in the field and the importance of these mechanisms in the context of breeding climate-resilient crops.

Biliverdin incorporation into the cyanobacteriochrome SPI1085g3 from Spirulina.

Cyanobacteriochromes (CBCRs) bind linear tetrapyrrole chromophores, mostly phycocyanobilin (PCB), and exhibit considerable spectral diversity with a high potential for biotechnological applications. Particular attention has been given to the conversion into intrinsic biliverdin (BV) incorporation due to the absence of PCB in mammalian cells. Our recent study discovered that a red/green CBCR of Spirulina subsalsa, SPI1085g3, was covalently attached to PCB and exhibited strong red fluorescence with a unique red/dark switch. In this study, we found that SPI1085g3 could be modestly chromophorylated with BV and absorb somewhat shifted (10 nm) red light, while the single C448S mutant could efficiently bind BV and exhibit unidirectional photoconversion and moderate dark reversion. The fluorescence in its dark-adapted state was switched off by red light, followed by a moderate recovery in the dark, and these were properties similar to those of PCB-binding SPI1085g3. Furthermore, by introducing the CY motif into the conserved CH motif for chromophore attachment, we developed another variant, C448S_CY, which showed increased BV-binding efficiency. As expected, C448S_CY had a significant enhancement in fluorescence quantum yield, reaching that of PCB-binding SPI1085g3 (0.14). These BV-binding CBCRs offer an improved platform for the development of unique photoswitchable fluorescent proteins compared with PCB-binding CBCRs.

Enhancing the Inhomogeneous Photodynamics of Canonical Bacteriophytochrome.

The ability of phytochromes to act as photoswitches in plants and microorganisms depends on interactions between a bilin-like chromophore and a host protein. The interconversion occurs between the spectrally distinct red (Pr) and far-red (Pfr) conformers. This conformational change is triggered by the photoisomerization of the chromophore D-ring pyrrole. In this study, as a representative example of a phytochrome-bilin system, we consider biliverdin IXα (BV) bound to bacteriophytochrome (BphP) from Deinococcus radiodurans. In the absence of light, we use an enhanced sampling molecular dynamics (MD) method to overcome the photoisomerization energy barrier. We find that the calculated free energy (FE) barriers between essential metastable states agree with spectroscopic results. We show that the enhanced dynamics of the BV chromophore in BphP contributes to triggering nanometer-scale conformational movements that propagate by two experimentally determined signal transduction pathways. Most importantly, we describe how the metastable states enable a thermal transition known as the dark reversion between Pfr and Pr, through a previously unknown intermediate state of Pfr. We present the heterogeneity of temperature-dependent Pfr states at the atomistic level. This work paves a way toward understanding the complete mechanism of the photoisomerization of a bilin-like chromophore in phytochromes.

Not All Photoactive Yellow Proteins Are Built Alike: Surprises and Insights into Chromophore Photoisomerization, Protonation, and Thermal Reisomerization of the Photoactive Yellow Protein Isolated from Salinibacter ruber.

We demonstrate that the Halorhodospira halophila (Hhal) photoactive yellow protein (PYP) is not representative of the greater PYP family. The photodynamics of the PYP isolated from Salinibacter ruber (Srub) is characterized with a comprehensive range of spectroscopic techniques including ultrafast transient absorption, photostationary light titrations, Fourier transform infrared, and cryokinetics spectroscopies. We demonstrate that the dark-adapted pG state consists of two subpopulations differing in the protonation state of the chromophore and that both are photoactive, with the protonated species undergoing excited-state proton transfer. However, the primary I0 photoproduct observed in the Hhal PYP photocycle is absent in the Srub PYP photodynamics, which indicates that this intermediate, while important in Hhal photodynamics, is not a critical intermediate in initiating all PYP photocycles. The excited-state lifetime of Srub PYP is the longest of any PYP resolved to date (∼30 ps), which we ascribe to the more constrained chromophore binding pocket of Srub PYP and the absence of the critical Arg52 residue found in Hhal PYP. The final stage of the Srub PYP photocycle involves the slowest known thermal dark reversion of a PYP (∼40 min vs 350 ms in Hhal PYP). This property allowed the characterization of a pH-dependent equilibrium between the light-adapted pB state with a protonated cis chromophore and a newly resolved pG' intermediate with a deprotonated cis chromophore and pG-like protein conformation. This result demonstates that protein conformational changes and chromophore deprotonation precede chromophore reisomerization during the thermal recovery of the PYP photocycle.

The Keto-Enol Tautomerism of Biliverdin in Bacteriophytochrome: Could it Explain the Bathochromic Shift in the Pfr Form?†.

Phytochromes are ubiquitous photoreceptors found in plants, eukaryotic algae, bacteria and fungi. Particularly, when bacteriophytochrome is irradiated with light, a Z-to-E (photo)isomerization takes place in the biliverdin chromophore as part of the Pr-to-Pfr conversion. This photoisomerization is concomitant with a bathochromic shift in the Q-band. Based on experimental evidence, we studied a possible keto-enol tautomerization of BV, as an alternative reaction channel after its photoisomerization. In this contribution, the noncatalyzed and water-assisted reaction pathways for the lactam-lactim interconversion through consecutive keto-enol tautomerization of a model BV species were studied deeply. It was found that in the absence of water molecules, the proton transfer reaction is unable to take place at normal conditions, due to large activation energies, and the endothermic formation of lactim derivatives prevents its occurrence. However, when a water molecule assists the process by catalyzing the proton transfer reaction, the activation free energy lowers considerably. The drastic lowering in the activation energy for the keto-enol tautomerism is due to the stabilization of the water moiety through hydrogen bonds along the reaction coordinate. The absorption spectra were computed for all tautomers. It was found that the UV-visible absorption bands are in reasonable agreement with the experimental data. Our results suggest that although the keto-enol equilibrium is likely favoring the lactam tautomer, the equilibrium could eventually be shifted in favor of the lactim, as it has been reported to occur in the dark reversion mechanism of bathy phytochromes.

A photoproduct of DXCF cyanobacteriochromes without reversible Cys ligation is destabilized by rotating ring twist of the chromophore.

Cyanobacteriochrome photoreceptors (CBCRs) ligate linear tetrapyrrole chromophores via their first (canonical) Cys residue and show reversible photoconversion triggered by light-dependent Z/E isomerization of the chromophore. Among the huge repertoire of CBCRs, DXCF CBCRs contain a second Cys residue within the highly conserved Asp-Xaa-Cys-Phe (DXCF) motif. In the typical receptors, the second Cys covalently attaches to the 15Z-chromophore in the dark state and detaches from the 15E-chromophore in the photoproduct state, whereas atypical ones that lack reversible ligation activity show red-shifted absorption in the dark state due to a more extended π-conjugated system. Moreover, some DXCF CBCRs show blue-shifted absorption in the photoproduct state due to the twisted geometry of the rotating ring. During the process of rational color tuning of a certain DXCF CBCR, we unexpectedly found that twisted photoproducts of some variant molecules showed dark reversion to the dark state, which prompted us to hypothesize that the photoproduct is destabilized by the twisted geometry of the rotating ring. In this study, we comprehensively examined the photoproduct stability of the twisted and relaxed molecules derived from the same CBCR scaffolds under dark conditions. In the DXCF CBCRs lacking reversible ligation activity, the twisted photoproducts showed faster dark reversion than the relaxed ones, supporting our hypothesis. By contrast, in the DXCF CBCRs exhibiting reversible ligation activity, the twisted photoproducts showed no detectable photoconversion. Reversible Cys adduct formation thus results in drastic rearrangement of the protein-chromophore interaction in the photoproduct state, which would contribute to the previously unknown photoproduct stability.

Domain Organization in Plant Blue-Light Receptor Phototropin2 of Arabidopsis thaliana Studied by Small-Angle X-ray Scattering.

Phototropin2 (phot2) is a blue-light (BL) receptor protein that regulates the BL-dependent activities of plants for efficient photosynthesis. Phot2 is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) to absorb BL, and a kinase domain. Photo-activated LOV domains, especially LOV2, play a major role in photo-dependent increase in the phosphorylation activity of the kinase domain. The atomic details of the overall structure of phot2 and the intramolecular mechanism to convert BL energy to a phosphorylation signal remain unknown. We performed structural studies on the LOV fragments LOV1, LOV2, LOV2-linker, and LOV2-kinase, and full-length phot2, using small-angle X-ray scattering (SAXS). The aim of the study was to understand structural changes under BL irradiation and discuss the molecular mechanism that enhance the phosphorylation activity under BL. SAXS is a suitable technique for visualizing molecular structures of proteins in solution at low resolution and is advantageous for monitoring their structural changes in the presence of external physical and/or chemical stimuli. Structural parameters and molecular models of the recombinant specimens were obtained from SAXS profiles in the dark, under BL irradiation, and after dark reversion. LOV1, LOV2, and LOV2-linker fragments displayed minimal structural changes. However, BL-induced rearrangements of functional domains were noted for LOV2-kinase and full-length phot2. Based on the molecular model together with the absorption measurements and biochemical assays, we discuss the intramolecular interactions and domain motions necessary for BL-enhanced phosphorylation activity of phot2.

Two hybrid histidine kinases, TcsB and the phytochrome FphA, are involved in temperature sensing in Aspergillus nidulans.

The adaptation of microorganisms to different temperatures is an advantage in habitats with steadily changing conditions and raises the question about temperature sensing. Here we show that in the filamentous fungus Aspergillus nidulans, the hybrid histidine kinase TcsB and phytochrome are involved in temperature-induced gene transcription. Temperature-activated phytochrome fed the signal into the HOG MAP kinase pathway. There is evidence that the photoreceptor phytochrome fulfills a temperature sensory role in plants and bacteria. The effects in plants are based on dark reversion from the active form of phytochrome, Pfr, to the inactive form, Pr. Elevated temperature leads to higher dark reversion rates, and hence, temperature sensing depends on light. In A. nidulans and in Alternaria alternata, the temperature response was light-independent. In order to understand the primary temperature response of phytochrome, we performed spectral analyses of recombinant FphA from both fungi. Spectral properties after heat stress resembled the spectrum of free biliverdin, suggesting conformational changes and a softening of the binding pocket of phytochrome, possibly mimicking photoactivation. We propose a novel function for fungal phytochrome as temperature sensor.

Physicochemical modeling of the phytochrome-mediated photothermal sensing

Light and temperature cues share many common signaling events towards plant photothermal morphogenesis. Particularly, the red (R)/far-red (FR)-absorbing phytochrome photoreceptors also function as temperature sensors, suggesting that light and temperature responses are intimately associated with each other. Here, we present data from physicochemical modeling of temperature sensing and thermomorphogenic patterning of hypocotyl growth, which illustrate that the two seemingly distinct stimulating cues are tightly coupled through physicochemical principles and temperature effects can be described as a function of infra-red (IR) thermal radiation. It is possible that the dark reversion from the FR-absorbing Pfr to the R-absorbing Pr phytochromes is essentially an IR-mediated thermal conversion. We propose that the phytochromes modulate photothermal responses by monitoring R:IR ratios, as they sense R:FR ratios during photomorphogenesis.

Evidence for weak interaction between phytochromes Agp1 and Agp2 from Agrobacteriumfabrum.

During bacterial conjugation, plasmid DNA is transferred from cell to cell. In Agrobacteriumfabrum, conjugation is regulated by the phytochrome photoreceptors Agp1 and Agp2. Both contribute equally to this regulation. Agp1 and Agp2 are histidine kinases, but, for Agp2, we found no autophosphorylation activity. A clear autophosphorylation signal, however, was obtained with mutants in which the phosphoaccepting Asp of the C-terminal response regulator domain is replaced. Thus, the Agp2 histidine kinase differs from the classical transphosphorylation pattern. We performed size exclusion, photoconversion, dark reversion, autophosphorylation, chromophore assembly kinetics and fluorescence resonance energy transfer measurements on mixed Agp1/Agp2 samples. These assays pointed to an interaction between both proteins. This could partially explain the coaction of both phytochromes in the cell.

Fluorescence Properties of a Novel Cyanobacteriochrome GAF Domain from Spirulina that Exhibits Moderate Dark Reversion.

Cyanobacteriochromes (CBCRs) are biliproteins for photoreception that are present in cyanobacteria. These proteins possess one or more unique cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that can covalently bind the linear tetrapyrrole (bilin). Light absorption triggers the photoisomerization of bilin between the 15Z and 15E photostates. The 15E photoproduct of some CBCR GAF domains can revert to the stable 15Z state in the absence of light. In some cases, this property makes these domains function as sensors of light intensity or as red/dark optogenetic switches. However, there have been few reports regarding the applicability of these fluorescent properties. Here, we report a red/green cyanobacteriochrome GAF domain from Spirulina subsalsa, designated SPI1085g3, which exhibited photoconversion from the red-absorbing dark state (Pr, λmax = 642 nm) to the orange-absorbing photoproduct state (Po, λmax = 590 nm), and exhibited moderate dark reversion (t1/2 = 3.3 min) from the Po state to the Pr state. The SPI1085g3 Pr state exhibited intense red fluorescence (λmax = 662 nm), with a quantum yield of 0.14. The fluorescence was switched off by red light irradiation and increased in the dark. Replacement of Cys448 of SPI1085g3 with Ser resulted in a slightly improved fluorescence quantum yield and nearly 13-fold faster dark reversion (t1/2 = 15.2 s) than that of the wild type. This novel red/dark-switchable fluorescent biliprotein expands the present repertoire and diversity of photoswitchable fluorescent protein candidates.

Molecular characterization of DXCF cyanobacteriochromes from the cyanobacterium Acaryochloris marina identifies a blue-light power sensor

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors that sense a wide range of wavelengths from ultraviolet to far-red. The primary photoreaction in these reactions is a Z/E isomerization of the double bond between rings C and D. After this isomerization, various color-tuning events establish distinct spectral properties of the CBCRs. Among the various CBCRs, the DXCF CBCR lineage is widely distributed among cyanobacteria. Because the DXCF CBCRs from the cyanobacterium Acaryochloris marina vary widely in sequence, we focused on these CBCRs in this study. We identified seven DXCF CBCRs in A. marina and analyzed them after isolation from Escherichia coli that produces phycocyanobilin, a main chromophore for the CBCRs. We found that six of these CBCRs covalently bound a chromophore and exhibited variable properties, including blue/green, blue/teal, green/teal, and blue/orange reversible photoconversions. Notably, one CBCR, AM1_1870g4, displayed unidirectional photoconversion in response to blue-light illumination, with a rapid dark reversion that was temperature-dependent. Furthermore, the photoconversion took place without Z/E isomerization. This observation indicated that AM1_1870g4 likely functions as a blue-light power sensor, whereas typical CBCRs reversibly sense two light qualities. We also found that AM1_1870g4 possesses a GDCF motif in which the Asp residue is swapped with the next Gly residue within the DXCF motif. Site-directed mutagenesis revealed that this swap is essential for the light power-sensing function of AM1_1870g4. This is the first report of a blue-light power sensor from the CBCR superfamily and of photoperception without Z/E isomerization among the bilin-based photoreceptors.

PCH1 and PCHL promote photomorphogenesis in plants by controlling phytochrome B dark reversion

Phytochrome B (phyB) is the primary red light photoreceptor in plants, and regulates both growth and development. The relative levels of phyB in the active state are determined by the light conditions, such as direct sunlight or shade, but are also affected by light-independent dark reversion. Dark reversion is a temperature-dependent thermal relaxation process, by which phyB reverts from the active to the inactive state. Here, we show that the homologous phyB-binding proteins PCH1 and PCHL suppress phyB dark reversion, resulting in plants with dramatically enhanced light sensitivity. Moreover, far-red and blue light upregulate the expression of PCH1 and PCHL in a phyB independent manner, thereby increasing the response to red light perceived by phyB. PCH1 and PCHL therefore provide a node for the molecular integration of different light qualities by regulation of phyB dark reversion, allowing plants to adapt growth and development to the ambient environment.

Hyperactivity of the Arabidopsis cryptochrome (cry1) L407F mutant is caused by a structural alteration close to the cry1 ATP-binding site

Plant cryptochromes (cry) act as UV-A/blue light receptors. The prototype, Arabidopsis thaliana cry1, regulates several light responses during the life cycle, including de-etiolation, and is also involved in regulating flowering time. The cry1 photocycle is initiated by light absorption by its FAD chromophore, which is most likely fully oxidized (FADox) in the dark state and photoreduced to the neutral flavin semiquinone (FADH°) in its lit state. Cryptochromes lack the DNA-repair activity of the closely related DNA photolyases, but they retain the ability to bind nucleotides such as ATP. The previously characterized L407F mutant allele of Arabidopsis cry1 is biologically hyperactive and seems to mimic the ATP-bound state of cry1, but the reason for this phenotypic change is unclear. Here, we show that cry1L407F can still bind ATP, has less pronounced photoreduction and formation of FADH° than wild-type cry1, and has a dark reversion rate 1.7 times lower than that of the wild type. The hyperactivity of cry1L407F is not related to a higher FADH° occupancy of the photoreceptor but is caused by a structural alteration close to the ATP-binding site. Moreover, we show that ATP binds to cry1 in both the dark and the lit states. This binding was not affected by cry1's C-terminal extension, which is important for signal transduction. Finally, we show that a recently discovered chemical inhibitor of cry1, 3-bromo-7-nitroindazole, competes for ATP binding and thereby diminishes FADH° formation, which demonstrates that both processes are important for cry1 function.