Photocobilins integrate B12 and bilin photochemistry for enzyme control.

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.

Elementary Reactions in the Functional Triads of the Blue-Light Photoreceptor BLUF Domain.

The blue light using the flavin (BLUF) domain is one of the smallest photoreceptors in nature, which consists of a unique bidirectional electron-coupled proton relay process in its photoactivation reaction cycle. This perspective summarizes our recent efforts in dissecting the photocycle into three elementary processes, including proton-coupled electron transfer (PCET), proton rocking, and proton relay. Using ultrafast spectroscopy, we have determined the temporal sequence, rates, kinetic isotope effects (KIEs), and concertedness of these elementary steps. Our findings provide important implications for illuminating the photoactivation mechanism of the BLUF domain and suggest an engineering platform to characterize intricate reactions involving proton motions that are ubiquitous in nonphotosensitive protein machines.

The Carbonyl Group in ß2 of the Carotenoid Tunes the Photocycle Kinetics in Orange Carotenoid Protein.

Adaptation to rapid environmental changes is crucial for maintaining optimal photosynthetic efficiency and is ultimately key to the survival of all photosynthetic organisms. Like most of them, cyanobacteria protect their photosynthetic apparatus against rapidly increasing light intensities by nonphotochemical quenching (NPQ). In cyanobacteria, NPQ is controlled by Orange Carotenoid Protein (OCP) photocycle. OCP is the only known photoreceptor that uses carotenoid for its light activation. How carotenoid drives and controls this unique photoactivation process is still unknown. However, understanding and potentially controlling the OCP photocycle may open up new possibilities for improving photosynthetic biomass. Here we investigate the effect of the carbonyl group in the ß2 ring of the carotenoid on the OCP photocycle. We report microsecond to minute OCP light activation kinetics and Arrhenius plots of the two OCP forms: Canthaxanthin-bound OCP (OCPCAN) and echinenone-bound OCP (OCPECH). The difference between the two carotenoids is the presence of a carbonyl group in the ß2-ring located in the N-terminal domain of the protein. A combination of temperature-dependent spectroscopy, flash photolysis, and pump-probe transient absorption allows us to report the previously unresolved OCP intermediate associated primarily with the absorption bleach (OCPB). OCPB dominates the photokinetics in the µs to subms time range for OCPCAN and in the µs to ms range for OCPECH. We show that in OCPCAN the OCP photocycle steps are always faster than in OCPECH: from 2 to almost 20 times depending on the step. These results suggest that the presence of the carbonyl group in the ß2-ring of the carotenoid accelerates the OCP photocycle.

Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation.

The blue-light photoreceptor YtvA from Bacillus subtilis has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2═O group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of ∼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV ß-scaffold in signal transduction.

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.

Crucial Residue for Tuning Thermal Relaxation Kinetics in the Biliverdin-binding Cyanobacteriochrome Photoreceptor Revealed by Site-saturation Mutagenesis.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to the phytochromes sensing red and far-red light reversibly. Only the cGMP phosphodiesterase/Adenylate cyclase/FhlA (GAF) domain is needed for chromophore incorporation and proper photoconversion. The CBCR GAF domains covalently ligate linear tetrapyrrole chromophores and show reversible photoconversion between two light-absorbing states. In most cases, the two light-absorbing states are stable under dark conditions, but in some cases, the photoproduct state undergoes thermal relaxation back to the dark-adapted state during thermal relaxation. In this study, we examined the engineered CBCR GAF domain, AnPixJg2_BV4. AnPixJg2_BV4 covalently binds biliverdin IX-alpha (BV) and shows reversible photoconversion between a far-red-absorbing Pfr dark-adapted state and an orange-absorbing Po photoproduct state. Because the BV is an intrinsic chromophore of mammalian cells and absorbs far-red light penetrating into deep tissues, BV-binding CBCR molecules are useful for the development of optogenetic and bioimaging tools used in mammals. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis on the Phe319 position. We succeeded in obtaining variant molecules with higher chromophore-binding efficiency and higher molar extinction coefficient. Furthermore, we observed a wide variation in thermal relaxation kinetics, with an 81-fold difference between the slowest and fastest rates. Both molecules with relatively slow and fast thermal relaxation would be advantageous for optogenetic control.

Conserved Signal Transduction Mechanisms and Dark Recovery Kinetic Tuning in the Pseudomonadaceae Short Light, Oxygen, Voltage (LOV) Protein Family.

Light-Oxygen-Voltage (LOV) flavoproteins transduce a light signal into variable signaling outputs via a structural rearrangement in the sensory core domain, which is then relayed to fused effector domains via α-helical linker elements. Short LOV proteins from Pseudomonadaceae consist of a LOV sensory core and N- and C-terminal α-helices of variable length, providing a simple model system to study the molecular mechanism of allosteric activation. Here we report the crystal structures of two LOV proteins from Pseudomonas fluorescens - SBW25-LOV in the fully light-adapted state and Pf5-LOV in the dark-state. In a comparative analysis of the Pseudomonadaceae short LOVs, the structures demonstrate light-induced rotation of the core domains and splaying of the proximal A'α and Jα helices in the N and C-termini, highlighting evidence for a conserved signal transduction mechanism. Another distinguishing feature of the Pseudomonadaceae short LOV protein family is their highly variable dark recovery, ranging from seconds to days. Understanding this variability is crucial for tuning the signaling behavior of LOV-based optogenetic tools. At 37 °C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. To investigate this remarkable difference in dark recovery rates, we targeted three residues lining the solvent channel entrance to the chromophore pocket where we introduced mutations by exchanging the non-conserved amino acids from SBW25-LOV into Pf5-LOV and vice versa. Dark recovery kinetics of the resulting mutants, as well as MD simulations and solvent cavity calculations on the crystal structures suggest a correlation between solvent accessibility and adduct lifetime.

Multifaceted photoreceptor compositions in dual phototrophic systems - A genomic analysis.

For microbes and their hosts, sensing of external cues is essential for their survival. For example, in the case of plant associated microbes, the light absorbing pigment composition of the plant as well as the ambient light conditions determine the well-being of the microbe. In addition to light sensing, some microbes can utilize xanthorhodopsin based proton pumps and bacterial photosynthetic complexes that work in parallel for energy production. They are called dual phototrophic systems. Light sensing requirements in these type of systems are obviously demanding. In nature, the photosensing machinery follows mainly the same composition in all organisms. However, the specific role of each photosensor in specific light conditions is elusive. In this study, we provide an overall picture of photosensors present in dual phototrophic systems. We compare the genomes of the photosensor proteins from dual phototrophs to those from similar microbes with "single" phototrophicity or microbes without phototrophicity. We find that the dual phototrophic bacteria obtain a larger variety of photosensors than their light inactive counterparts. Their rich domain composition and functional repertoire remains similar across all microbial photosensors. Our study calls further investigations of this particular group of bacteria. This includes protein specific biophysical characterization in vitro, microbiological studies, as well as clarification of the ecological meaning of their host microbial interactions.

Difference in the Charge-Separation Energetics between Distinct Conformers in the PixD Photoreceptor.

Blue light using flavin (BLUF) domain proteins are photoreceptors in various organisms. The PixD BLUF domain can adopt two conformations, W91out and W91in, with Trp91 either proximal or distal to flavin (FMN). Using a quantum mechanical/molecular mechanical/polarizable continuum model approach, the energetics of charge-separated and biradical states in the two conformations were investigated. In the W91out conformation, the charge-separated state (FMN•-) is more stable than the photoexcited state (FMN*), whereas it is less stable due to an electrostatic repulsive interaction with the Ser28 side chain in the W91in conformation. This leads to a lower activation energy for the charge separation in the W91out conformation, resulting in a faster charge separation compared to that in the W91in conformation. In the W91out conformation, the radical state (FMNH•) is more stable than FMN•- and forms from FMN•-, leading to reorientation of the Gln50 side chain adjacent to FMN and formation of a hydrogen bond between Gln50 and FMN. Subsequently, a signaling state forms through charge recombination. In contrast, in the W91in conformation, FMN•- cannot proceed further, returning to the dark-adapted state, as FMNH• is less stable. Thus, formation of the signaling state exclusively occurs in the W91out conformation.

Length and Charge of the N-terminus Regulate the Lifetime of the Signaling State of Photoactive Yellow Protein.

Photoactive yellow protein (PYP) is one of the most extensively studied photoreceptors. Nevertheless, the role of the N-terminus in the photocycle and structural transitions is still elusive. Here, we attached additional amino acids to the N-terminus of PYP and investigated the effect of the length and charge of additional N-terminal residues using circular dichroism, two-dimensional nuclear magnetic resonance (2D-NMR), transient absorption (TA), and transient grating (TG) spectroscopic techniques. TA experiments showed that, except for negatively charged residues (5D-PYP), additional N-terminal residues of PYP generally enable faster dark recovery from the putative signaling state (pB2) to the ground state (pG). TG data showed that although the degree of structural changes can be controlled by adjusting specific amino acid residues in the extended N-terminus of N-terminal extended PYPs (NE-PYPs), the dark recovery times of wt-PYP and NE-PYPs, except for 5D-PYP, are independent of the structural differences between pG and pB2 states. These results demonstrate that the recovery time and the degree of structural change can be regulated by controlling the length and sequence of N-terminal residues of PYP. The findings in this study emphasize the need for careful attention to the remaining amino acid residues when designing recombinant proteins for genetic engineering purposes.

Spectroscopic Validation of Crystallographic Structures of a Protein Active Site by Chiroptical Spectroscopy.

Out-of-plane distortions of a cofactor molecule in a protein active site are functionally important, and in photoreceptors, it has been proposed that they are crucial for spectral tuning and energy storage in photocycle intermediates. However, these subtle structural features are often beyond the grasp of structural biology. This issue is strikingly exemplified by photoactive yellow protein: its 14 independently determined crystal structures exhibit considerable differences in the dihedral angles defining the chromophore geometry, even though most of these are at excellent resolution. Here we developed a strategy to verify cofactor distortions in crystal structures by using quantum chemical calculations and chiroptical spectroscopy, particularly Raman optical activity and electronic circular dichroism spectroscopies. Based on this approach, we identify seven crystal structures with the chromophore geometries inconsistent with the experimentally observed data. The strategy implemented here promises to be widely applicable to uncovering cofactor distortions at active sites and to studies of reaction intermediates.

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process.

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.

Introduction of reversible cysteine ligation ability to the biliverdin-binding cyanobacteriochrome photoreceptor.

Cyanobacteriochrome (CBCR) photoreceptors are distantly related to the canonical red/far-red reversible phytochrome photoreceptors. In the case of the CBCRs, only the GAF domain is required for chromophore incorporation and photoconversion. The GAF domains of CBCR are highly diversified into many lineages to sense various colors of light. These CBCR GAF domains are divided into two types: those possessing only the canonical Cys residue and those with both canonical and second Cys residues. The canonical Cys residue stably ligates to the chromophore in both cases. The second Cys residue mostly shows reversible adduct formation with the chromophore during photoconversion for spectral tuning. In this study, we focused on the CBCR GAF domain AnPixJg2_BV4, which possesses only the canonical Cys residue. AnPixJg2_BV4 covalently ligates to the biliverdin (BV) chromophore and shows far-red/orange reversible photoconversion. Because BV is a mammalian intrinsic chromophore, BV-binding molecules are advantageous for in vivo optogenetic and bioimaging tool development. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis and serendipitously obtained a unique variant molecule that showed far-red/blue reversible photoconversion, in which the Cys residue was introduced near the chromophore. This introduced Cys residue functioned as the second Cys residue that reversibly ligated with the chromophore. Because the position of the introduced Cys residue is distinct from the known second Cys residues, the variant molecule obtained in this study would expand our knowledge about the spectral tuning mechanism of CBCRs and contribute to tool development.

Effects of N- and C-terminal regions on oligomeric formation of blue light sensor protein SyPixD.

A sensor of blue-light using flavin adenine dinucleotide (BLUF) is a typical blue light photoreceptor domain that is found in many photosensor proteins in bacteria and some eukaryotic algae. SyPixD in Synechocystis is one of the well-studied BLUF proteins. In the dark state, it forms a decamer and, upon photoexcitation, a dissociation reaction takes place to yield dimers. Such change in the intermolecular interactions of the protomers is important for the biological function. The effect of the N- and C-terminal sequences on the stability of SyPixD oligomeric states and photoreactions of SyPixD were studied to understand how the oligomeric form is maintained with weak interaction. It was found that a few residues that frequently persist at the N-terminus after removing a tag for purification are sensitive to the stability of the decamer structure. Even two or three residues at the N-terminus considerably reduces decamer stability, whereas four or more residues completely prevent decamer formation. Unexpectedly, truncating C-terminal sequences, which locate far from any protomer interface and of which structure is undetermined in crystal structure, also destabilizes the decamer structure. This destabilization is also apparent from the dissociation reaction dynamics detected by the transient grating and transient absorption measurements. The dissociation reaction is faster and the yield increases when the C-terminus does not contain seven amino acid residues. Photoexcitation induces a conformational change in the C-terminus of the decamer but not the dimer.

The molecular mechanism of light-induced bond formation and breakage in the cyanobacteriochrome TePixJ.

Cyanobacteriochromes (CBCRs) are small and versatile photoreceptor proteins with high potential for biotechnological applications. Among them, the so-called DXCF-CBCRs exhibit an intricate secondary photochemistry: miliseconds after activation with light, a covalent linkage between a conserved cysteine residue and the light-absorbing tetrapyrrole chromophore is reversibly formed or broken. We employed time-resolved IR spectroscopy over ten orders of magnitude in time in conjunction with 2D-IR spectroscopy to investigate the molecular mechanism of this intriguing reaction in the DXCF-CBCR model system TePixJ from T. elongatus. The crosspeak pattern in the 2D-IR spectrum facilitated the assignment of the dominant signals to vibrational modes of the chromophore, which in turn enabled us to construct a mechanistic model for the photocycle reactions from the time-resolved IR spectra. Here, we assigned the time-resolved signals to several proton transfer steps and distinct geometric changes of the chromophore. We propose a model that describes how these events lead to the rearrangement of charges in the chromophore binding pocket, which serves as the trigger for the light-induced bond formation and breakage with the nearby cysteine.

Evidence for an early green/red photocycle that precedes the diversification of GAF domain photoreceptor cyanobacteriochromes.

Phytochromes are linear tetrapyrrole-binding photoreceptors in eukaryotes and bacteria, primarily responding to red and far-red light signals reversibly. Among the GAF domain-based phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show various optical properties covering the entire visible region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here, we utilize ancestral sequence reconstruction and biochemical verification to show that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore is crucial to perceive green light. The ancestral cyanobacteriochromes show only modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense the incident green/red light ratio. Many cyanobacteria can utilize green light for photosynthesis using phycobilisome light-harvesting complexes. The green/red sensing cyanobacteriochromes may have allowed better acclimation to changing light environments by rearranging the absorption capacity of the phycobilisome through chromatic acclimation.

Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction.

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

Novel cyanobacteriochrome photoreceptor with the second Cys residue showing atypical orange/blue reversible photoconversion.

Cyanobacteriochromes (CBCRs) are cyanobacterial linear tetrapyrrole-binding photoreceptors distantly related to phytochromes. Only the GAF domain is needed for chromophore incorporation and proper photoconversion of the CBCRs. Most CBCR GAF domains possess the canonical Cys residue stably ligating to the chromophore. DXCF-type CBCR GAF domains also possess a second Cys residue within the DXCF motif. This second Cys residue reversibly ligates to the C10 of the chromophore. The Cys adduct formation is mostly observed for the dark-adapted state but not for the photoproduct state. In this study, we discovered novel CBCR GAF domains with a DXCI motif instead of the DXCF motif. Since these CBCR GAF domains are categorized into two subfamilies (DXCI-1 and DXCI-2), the GAF domains from each subfamily were analyzed. Although the CBCR GAF domain belonging to the DXCI-2 subfamily showed orange/green reversible photoconversion without transient Cys ligation, the CBCR GAF domain belonging to the DXCI-1 subfamily showed reversible photoconversion between an orange-absorbing dark-adapted state and a blue-absorbing photoproduct state. This indicates that the second Cys residue is covalently bound to the C10 of the chromophore in the photoproduct state but not in the dark-adapted state. Since the covalent bond formation in the photoproduct state is atypical, site-directed mutagenesis was conducted to understand the molecular mechanism of this GAF domain. The Ile residue within the DXCI motif may be key for covalent bond formation in the photoproduct state.

Residue alterations within a conserved hydrophobic pocket influence light, oxygen, voltage photoreceptor dark recovery.

Light, oxygen, voltage (LOV) photoreceptors are widely distributed throughout all kingdoms of life, and have in recent years, due to their modular nature, been broadly used as sensor domains for the construction of optogenetic tools. For understanding photoreceptor function as well as for optogenetic tool design and fine-tuning, a detailed knowledge of the photophysics, photochemistry, and structural changes underlying the LOV signaling paradigm is instrumental. Mutations that alter the lifetime of the photo-adduct signaling state represent a convenient handle to tune LOV sensor on/off kinetics and, thus, steady-state on/off equilibria of the photoreceptor (or optogenetic switch). Such mutations, however, should ideally only influence sensor kinetics, while being benign with regard to the nature of the structural changes that are induced by illumination, i.e., they should not result in a disruption of signal transduction. In the present study, we identify a conserved hydrophobic pocket for which mutations have a strong impact on the adduct-state lifetime across different LOV photoreceptor families. Using the slow cycling bacterial short LOV photoreceptor PpSB1-LOV, we show that the I48T mutation within this pocket, which accelerates adduct rupture, is otherwise structurally and mechanistically benign, i.e., light-induced structural changes, as probed by NMR spectroscopy and X-ray crystallography, are not altered in the variant. Additional mutations within the pocket of PpSB1-LOV and the introduction of homologous mutations in the LOV photoreceptor YtvA of Bacillus subtilis and the Avena sativa LOV2 domain result in similarly altered kinetics. Given the conserved nature of the corresponding structural region, the here identified mutations should find application in dark-recovery tuning of optogenetic tools and LOV photoreceptors, alike.