Light, Oxygen, Voltage Protein Research Articles

Photoinduced bleaching in an efficient singlet oxygen photosensitizing protein: Identifying a culprit in the flavin-binding LOV-based protein SOPP3

• Bleaching of FMN in SOPP3 depends on electron transfer with surrounding molecules. • Amino acids in SOPP3 mediate the bleaching reaction of bound FMN. • Despite FMN bleaching, SOPP3 is still be a viable intracellular optogenetic actuator. Light-oxygen-voltage (LOV) proteins that bind flavin mononucleotide (FMN) have received attention as optogenetic systems that can photosensitize the production of singlet molecular oxygen, O 2 (a 1 Δ g ). Although the seminal compound in this family, mini singlet oxygen generator (miniSOG), does not make O 2 (a 1 Δ g ) in appreciable yield, it is a convenient standard against which other singlet oxygen photosensitizing proteins (SOPPs) can be judged. SOPP3 is a re-engineered version of miniSOG that selectively produces O 2 (a 1 Δ g ) in high yield but, when localized in a cell, the protein-encased FMN readily bleaches upon irradiation. In contrast, the bleaching of FMN in miniSOG is slow under the same conditions. The available evidence indicates that O 2 (a 1 Δ g ) is not directly involved in the bleaching reaction. Rather, electron-transfer reactions between FMN and molecules in the surrounding medium, likely mediated by residues in the LOV protein, accelerate FMN bleaching. In this regard, and identifying a culprit, replacing a glutamine that H-bonds to FMN in miniSOG with either leucine (Q103L) or valine (Q103V) increases the 3 FMN lifetime and, thereby, increases both the O 2 (a 1 Δ g ) yield and the bleaching rate. Although the same electron transfer reactions likely influence these respective observations, the O 2 (a 1 Δ g ) yield principally reflects the kinetic competition between two processes that remove/deactivate 3 FMN: (1) quenching by ground state oxygen, O 2 (X 3 Σ g - ), to produce O 2 (a 1 Δ g ), and (2) electron transfer to produce the FMN radical anion. Photobleaching principally reflects the accumulated electron-transfer-mediated depletion of ground state FMN, which, over time in the dark, is partly regenerated by compensating redox reactions. Despite the bleaching, irradiation of SOPP3 induces morphological change in a cell. This result, which contradicts conclusions from previous experiments with a lower light dose, indicates that SOPP3 may indeed be a viable optogenetic actuator.

Real-Time Tracking of Proton Transfer from the Reactive Cysteine to the Flavin Chromophore of a Photosensing Light Oxygen Voltage Protein

LOV (light oxygen voltage) proteins are photosensors ubiquitous to all domains of life. A variant of the short LOV protein from Dinoroseobacter shibae (DsLOV) exhibits an exceptionally fast photocycle. We performed time-resolved molecular spectroscopy on DsLOV-M49S and characterized the formation of the thio-adduct state with a covalent bond between the reactive cysteine (C72) and C4a of the FMN. By use of a tunable quantum cascade laser, the weak absorption change of the vibrational band of S-H stretching vibration of C57 was resolved with a time resolution of 10 ns. Deprotonation of C72 proceeded with a time constant of 12 μs which tallies the rise of the thio-adduct state. These results provide valuable information for the mechanistic interpretation of light-induced structural changes in LOV domains, which involves the choreographed sequence of proton transfers, changes in electron density distributions, spin alterations of the latter, and transient bond formation and breakage. Such molecular insight will help develop new optogenetic tools based on flavin photoreceptors.

Dimeric allostery mechanism of the plant circadian clock photoreceptor ZEITLUPE.

In Arabidopsis thaliana, the Light-Oxygen-Voltage (LOV) domain containing protein ZEITLUPE (ZTL) integrates light quality, intensity, and duration into regulation of the circadian clock. Recent structural and biochemical studies of ZTL indicate that the protein diverges from other members of the LOV superfamily in its allosteric mechanism, and that the divergent allosteric mechanism hinges upon conservation of two signaling residues G46 and V48 that alter dynamic motions of a Gln residue implicated in signal transduction in all LOV proteins. Here, we delineate the allosteric mechanism of ZTL via an integrated computational approach that employs atomistic simulations of wild type and allosteric variants of ZTL in the functional dark and light states, together with Markov state and supervised machine learning classification models. This approach has unveiled key factors of the ZTL allosteric mechanisms, and identified specific interactions and residues implicated in functional allosteric changes. The final results reveal atomic level insights into allosteric mechanisms of ZTL function that operate via a non-trivial combination of population-shift and dynamics-driven allosteric pathways.

Structural determinants underlying the adduct lifetime in the LOV proteins of Pseudomonas putida.

The primary photochemistry is similar among the flavin-bound sensory domains of light-oxygen-voltage (LOV) photoreceptors, where upon blue-light illumination a covalent adduct is formed on the microseconds time scale between the flavin chromophore and a strictly conserved cysteine residue. In contrast, the adduct-state decay kinetics vary from seconds to days or longer. The molecular basis for this variation among structurally conserved LOV domains is not fully understood. Here, we selected PpSB2-LOV, a fast-cycling (τrec 3.5min, 20°C) short LOV protein from Pseudomonas putida that shares 67% sequence identity with a slow-cycling (τrec 2467min, 20°C) homologous protein PpSB1-LOV. Based on the crystal structure of the PpSB2-LOV in the dark state reported here, we used a comparative approach, in which we combined structure and sequence information with molecular dynamic (MD) simulations to address the mechanistic basis for the vastly different adduct-state lifetimes in the two homologous proteins. MD simulations pointed toward dynamically distinct structural region, which were subsequently targeted by site-directed mutagenesis of PpSB2-LOV, where we introduced single- and multisite substitutions exchanging them with the corresponding residues from PpSB1-LOV. Collectively, the data presented identify key amino acids on the Aβ-Bβ, Eα-Fα loops, and the Fα helix, such as E27 and I66, that play a decisive role in determining the adduct lifetime. Our results additionally suggest a correlation between the solvent accessibility of the chromophore pocket and adduct-state lifetime. The presented results add to our understanding of LOV signaling and will have important implications in tuning the signaling behavior (on/off kinetics) of LOV-based optogenetic tools.

Site-Specific Photoinduced Dynamics of the LOV Protein EL222 Monitored by Multiple Genetically Encoded Infrared Probes

Proteins containing the light-oxygen-voltage (LOV) domain are photosensory receptors that mediate biological actions in response to blue light. For instance, the bacterial transcription factor EL222 regulates gene expression in a light-regulated manner and has found use in optogenetic applications. Upon excitation of the embedded flavin mononucleotide (FMN) cofactor, EL222 undergoes structural changes that ultimately drive its association with DNA. However, our knowledge of the light-adapted state(s) and the molecular mechanism underlying the transition between dark and lit states is incomplete.

Photobiologically Directed Assembly of Gold Nanoparticles.

In nature, photoreceptor proteins undergo molecular responses to light, that exhibit supreme fidelity in time and space and generally occur under mild reaction conditions. To unlock these traits for material science, the light-induced homodimerization of light-oxygen-voltage (LOV) photoreceptors is leveraged to control the assembly of gold nanoparticles. Conjugated to genetically encodable LOV proteins, the nanoparticles are monodispersed in darkness but rapidly assemble into large aggregates upon blue-light exposure. The study establishes a new modality for reaction control in macromolecular chemistry and thus augurs enhanced precision in space and time in diverse applications of gold nanoparticles.

A Metastable Photoinduced Protein-Flavin Adduct in Choline Oxidase, an Enzyme Not Involved in Light-Dependent Processes.

Photoinduced formation of protein-flavin adducts is crucial in photoresponsive proteins containing light-oxygen-voltage (LOV) domains. LOV proteins typically share an N-terminal sensor domain with FMN and a C-terminal effector domain such as a kinase, a phosphodiesterase, or a DNA-binding protein. Light absorption by FMN results in a covalent flavin-cysteine adduct, which allosterically translates to the linked effector domain. Photoinduced protein-flavin adducts have not been reported in enzymes not involved in light-dependent processes. Here, we have used fluorescence, pH effects, and mutagenesis to follow up a serendipitous observation of an unusual fluorescence excitation spectrum in choline oxidase at alkaline pH (M. Ghanem and G. Gadda, unpublished observations). Physiologically, choline oxidase oxidizes choline to betaine through two FAD-associated reactions and is not a photoenzyme. The enzyme-bound flavin showed a progressive shift of the fluorescence excitation maximum (λex) from 468 to 399 nm with increasing pH values between pH 6.0 and 10.0, consistent with a metastable photoinduced protein-flavin adduct. In contrast, the maximal λem was independent of pH, with values of ∼526 nm. For comparison, fluorescence spectra of FAD in bulk solution had maximal values differing by ≤2 nm at different pH values, with λex at 453 nm and λem at 527 nm. The unusual behavior of the enzyme persisted in the mutated S101A enzyme variant but was eliminated in the H466Q variant, suggesting that the photoinduced species is likely a C4a-N-histidyl-FAD. The results provide evidence that metastable photoinduced formation of a flavin-protein adduct can occur in an enzyme that is not a photoreceptor or a photoenzyme.

Dimerization of LOV domains of Rhodobacter sphaeroides (RsLOV) studied with FRET and stopped-flow experiments.

The bacterium Rhodobacter sphaeroides has a short LOV (light-oxygen-voltage) domain, which is not connected to an effector domain but has an α-helix extension at the N-terminus as well as a helix-turn-helix (HTH) motiv at the C-terminus. These extensions offer possibilities for interactions with effector enzymes or DNA. Whereas many LOV domains show a tendency to form dimers in the light state, RsLOV is unique in that it is a dimer in the dark state but dissociates into monomers after blue-light excitation. We studied the kinetics of this dimerization process by a combination of FRET spectroscopy and stopped-flow experiments with a time resolution of ≈10 ms. Although excitation of the flavin chromophore in dye-labeled LOV domains leads to considerable FRET from flavin to the dye, the typical adduct formation between flavin and a nearby cysteine still occurs with considerable yield. We obtain a rate constant for LOV-LOV dimerization in the range (0.8-1.8) × 105 M-1 s-1, and an equilibrium constant of the dark-state dimer in the range (3.0-7.0) × 10-6 M. Dissociation of the dimers in the light state and reforming of dimers after return to the dark state was monitored using an anti-FRET effect caused by excitonic interaction between dye labels on different monomers. Reforming of the dark state dimers is slower than recovery of the flavin-cysteinyl adduct, indicating that light-induced conformational changes in the LOV domain persist for much longer time than the adduct lifetime.

Single mutation in a novel bacterial LOV protein yields a singlet oxygen generator.

Mr4511 from Methylobacterium radiotolerans is a 164 amino acid protein built of a flavin mononucleotide (FMN) binding, blue-light responsive LOV (Light, Oxygen, Voltage) core domain plus flanking regions. In contrast to the majority of LOV domains, Mr4511 lacks a tryptophan residue that was previously identified as a major quencher for the FMN triplet state in photosensitizers for singlet oxygen (SO) engineered from these photoreceptors. Here we show that for Mr4511 it is sufficient to only mutate the reactive cysteine responsible for the photocycle (Cys71) in the native protein to generate an efficient SO photosensitizer: both C71S and C71G variants exhibit SO quantum yields of formation, ΦΔ, around 0.2 in air-saturated solutions. Under oxygen saturated conditions, ΦΔ reaches ∼0.5 in deuterated buffer. The introduction of Trp112 in the canonical position for LOV domains dramatically lowers ΦΔ to values comparable to miniSOG, one of the early FMN binding proteins touted as a SO sensitizer. Besides its SO properties, Mr4511 is also exceedingly robust against denaturation with urea and is more photostable than free FMN.

Small-angle X-ray scattering study of the kinetics of light-dark transition in a LOV protein.

Light, oxygen, voltage (LOV) photoreceptors consist of conserved photo-responsive domains in bacteria, archaea, plants and fungi, and detect blue-light via a flavin cofactor. We investigated the blue-light induced conformational transition of the dimeric photoreceptor PpSB1-LOV-R66I from Pseudomonas putida in solution by using small-angle X-ray scattering (SAXS). SAXS experiments of the fully populated light- and dark-states under steady-state conditions revealed significant structural differences between the two states that are in agreement with the known structures determined by crystallography. We followed the transition from the light- to the dark-state by using SAXS measurements in real-time. A two-state model based on the light- and dark-state conformations could describe the measured time-course SAXS data with a relaxation time τREC of ~ 34 to 35 min being larger than the recovery time found with UV/vis spectroscopy. Unlike the flavin chromophore-based UV/vis method that is sensitive to the local chromophore environment in flavoproteins, SAXS-based assay depends on protein conformational changes and provides with an alternative to measure the recovery kinetics.

Mechanistic Basis of the Fast Dark Recovery of the Short LOV Protein DsLOV from Dinoroseobacter shibae.

Light, oxygen, voltage (LOV) proteins, a ubiquitously distributed class of photoreceptors, regulate a wide variety of light-dependent physiological responses. Because of their modular architecture, LOV domains, i.e., the sensory domains of LOV photoreceptors, have been widely used for the construction of optogenetic tools. We recently described the structure and function of a short LOV protein (DsLOV) from the marine phototropic bacterium Dinoroseobacter shibae, for which, in contrast to other LOV photoreceptors, the dark state represents the physiologically relevant signaling state. Among bacterial LOV photoreceptors, DsLOV possesses an exceptionally fast dark recovery, corroborating its function as a "dark" sensor. To address the mechanistic basis of this unusual characteristic, we performed a comprehensive mutational, kinetic, thermodynamic, and structural characterization of DsLOV. The mechanistic basis of the fast dark recovery of the protein was revealed by mutation of the previously noted uncommon residue substitution at position 49 found in DsLOV. The substitution of M49 with different residues that are naturally conserved in LOV domains tuned the dark-recovery time of DsLOV over 3 orders of magnitude, without grossly affecting its overall structure or the light-dependent structural change observed for the wild-type protein. Our study thus provides a striking example of how nature can achieve LOV photocycle tuning by subtle structural alterations in the LOV domain active site, highlighting the easy evolutionary adaptability of the light sensory function. At the same time, our data provide guidance for the mutational photocycle tuning of LOV domains, with relevance for the growing field of optogenetics.

Seeing the Light: The Roles of Red- and Blue-Light Sensing in Plant Microbes.

Plants collect, concentrate, and conduct light throughout their tissues, thus enhancing light availability to their resident microbes. This review explores the role of photosensing in the biology of plant-associated bacteria and fungi, including the molecular mechanisms of red-light sensing by phytochromes and blue-light sensing by LOV (light-oxygen-voltage) domain proteins in these microbes. Bacteriophytochromes function as major drivers of the bacterial transcriptome and mediate light-regulated suppression of virulence, motility, and conjugation in some phytopathogens and light-regulated induction of the photosynthetic apparatus in a stem-nodulating symbiont. Bacterial LOV proteins also influence light-mediated changes in both symbiotic and pathogenic phenotypes. Although red-light sensing by fungal phytopathogens is poorly understood, fungal LOV proteins contribute to blue-light regulation of traits, including asexual development and virulence. Collectively, these studies highlight that plant microbes have evolved to exploit light cues and that light sensing is often coupled with sensing other environmental signals.

Converting Nature's Switches Into Scientists' Tools: How Biophysical Insights Lay The Foundation For Artificial Control Of Protein Activity

Environmental cues regulate many biological processes, coordinating cellular pathways to respond to changing conditions. Such regulation is often initiated by sensory protein domains which expand their chemical repertoire by using small molecule ligands to convert environmentally‐triggered changes into altered protein/protein interactions. Several families of these domains have evolved with remarkable diversity in their inputs and outputs. Using a combination of biophysics, biochemistry and synthetic chemistry, we seek to gain insight into the mechanistic controls of such environmental sensing domains for both fundamental understanding and subsequent artificial control.Here I will discuss examples of our work exploring how the Light‐Oxygen‐Voltage (LOV) protein domains convert changes in a common blue light stimulus into control of a wide range of output functions. Such photosensory domains utilize flavin chromophores and a photochemically‐generated protein/flavin adduct as a trigger for the allosteric control of effectors as diverse as kinases to DNA‐binding domains. While high‐resolution structures of inhibited LOV proteins have provided insights into certain regulatory principles, it has been difficult to apply similar techniques to spontaneously‐activated “dark noise” and photoactivated conformations that are essential for a full understanding of control. We have tackled this challenge by combining data from several biophysical approaches, including HDX‐MS and EPR spectroscopy, letting us observe and characterize these states. This information has shown several commonalities of LOV signaling in diverse classes of sensory proteins, and further, develop structure‐based mutations to shift these proteins among these states. Doing so, we have taken advantage of this mechanistic understanding to develop the artificial regulation of such systems, both in vitro and in living cells. Taken together, our work provides an integrated view of a fascinating class of natural switches and suggests routes by which these can be manipulated to achieve desired therapeutic and/or technological outcomes.Support or Funding InformationSupport from NIH R01 GM106239 is gratefully acknowledged.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electron transfer pathways in a light, oxygen, voltage (LOV) protein devoid of the photoactive cysteine

Blue-light absorption by the flavin chromophore in light, oxygen, voltage (LOV) photoreceptors triggers photochemical reactions that lead to the formation of a flavin-cysteine adduct. While it has long been assumed that adduct formation is essential for signaling, it was recently shown that LOV photoreceptor variants devoid of the photoactive cysteine can elicit a functional response and that flavin photoreduction to the neutral semiquinone radical is sufficient for signal transduction. Currently, the mechanistic basis of the underlying electron- (eT) and proton-transfer (pT) reactions is not well understood. We here reengineered pT into the naturally not photoreducible iLOV protein, a fluorescent reporter protein derived from the Arabidopsis thaliana phototropin-2 LOV2 domain. A single amino-acid substitution (Q489D) enabled efficient photoreduction, suggesting that an eT pathway is naturally present in the protein. By using a combination of site-directed mutagenesis, steady-state UV/Vis, transient absorption and electron paramagnetic resonance spectroscopy, we investigate the underlying eT and pT reactions. Our study provides strong evidence that several Tyr and Trp residues, highly conserved in all LOV proteins, constitute the eT pathway for flavin photoreduction, suggesting that the propensity for photoreduction is evolutionary imprinted in all LOV domains, while efficient pT is needed to stabilize the neutral semiquinone radical.

Structure of a LOV protein in apo-state and implications for construction of LOV-based optical tools

Unique features of Light-Oxygen-Voltage (LOV) proteins like relatively small size (~12–19 kDa), inherent modularity, highly-tunable photocycle and oxygen-independent fluorescence have lately been exploited for the generation of optical tools. Structures of LOV domains reported so far contain a flavin chromophore per protein molecule. Here we report two new findings on the short LOV protein W619_1-LOV from Pseudomonas putida. First, the apo-state crystal structure of W619_1-LOV at 2.5 Å resolution reveals conformational rearrangements in the secondary structure elements lining the chromophore pocket including elongation of the Fα helix, shortening of the Eα-Fα loop and partial unfolding of the Eα helix. Second, the apo W619_1-LOV protein binds both natural and structurally modified flavin chromophores. Remarkably different photophysical and photochemical properties of W619_1-LOV bound to 7-methyl-8-chloro-riboflavin (8-Cl-RF) and lumichrome imply application of these variants as novel optical tools as they offer advantages such as no adduct state formation, and a broader choice of wavelengths for in vitro studies.

Glutamine Amide Flip Elicits Long Distance Allosteric Responses in the LOV Protein Vivid.

Light-oxygen-voltage (LOV) domains sense blue light through the photochemical formation of a cysteinyl-flavin covalent adduct. Concurrent protonation at the flavin N5 position alters the hydrogen bonding interactions of an invariant Gln residue that has been proposed to flip its amide side chain as a critical step in the propagation of conformational change. Traditional molecular dynamics (MD) and replica-exchange MD (REMD) simulations of the well-characterized LOV protein Vivid (VVD) demonstrate that the Gln182 amide indeed reorients by ∼180° in response to either adduct formation or reduction of the isoalloxazine ring to the neutral semiquinone, both of which involve N5 protonation. Free energy simulations reveal that the relative free energies of the flipped Gln conformation and the flipping barrier are significantly lower in the light-adapted state. The Gln182 flip stabilizes an important hinge-bβ region between the PAS β-sheet and the N-terminal cap helix that in turn destabilizes an N-terminal latch region against the PAS core. Release of the latch, observed both experimentally and in the simulations, is known to mediate light-induced VVD dimerization. This computational study of a LOV protein, unprecedented in its agreement with experiment, provides an atomistic view of long-range allosteric coupling in a photoreceptor.

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY

Light-oxygen-voltage (LOV) domain-containing proteins function as small light-activated modules capable of imparting blue light control of biological processes. Their small modular nature has made them model proteins for allosteric signal transduction and optogenetic devices. Despite intense research, key aspects of their signal transduction mechanisms and photochemistry remain poorly understood. In particular, ordered water has been identified as a possible key mediator of photocycle kinetics, despite the lack of ordered water in the LOV active site. Herein, we use recent crystal structures of a fungal LOV protein ENVOY to interrogate the role of Thr(101) in recruiting water to the flavin active site where it can function as an intrinsic base to accelerate photocycle kinetics. Kinetic and molecular dynamic simulations confirm a role in solvent recruitment to the active site and identify structural changes that correlate with solvent recruitment. In vivo analysis of T101I indicates a direct role of the Thr(101) position in mediating adaptation to osmotic stress, thereby verifying biological relevance of ordered water in LOV signaling. The combined studies identify position 101 as a mediator of both allostery and photocycle catalysis that can impact organism physiology.

Signaling States of a Short Blue-Light Photoreceptor Protein PpSB1-LOV Revealed from Crystal Structures and Solution NMR Spectroscopy

Light-Oxygen-Voltage (LOV) domains represent the photo-responsive domains of various blue-light photoreceptor proteins and are widely distributed in plants, algae, fungi, and bacteria. Here, we report the dark-state crystal structure of PpSB1-LOV, a slow-reverting short LOV protein from Pseudomonas putida that is remarkably different from our previously published "fully light-adapted" structure [1]. A direct comparison of the two structures provides insight into the light-activated signaling mechanism. Major structural differences involve a~11Å movement of the C terminus in helix Jα, ~4Å movement of Hβ-Iβ loop, disruption of hydrogen bonds in the dimer interface, and a~29° rotation of chain-B relative to chain-A as compared to the light-state dimer. Both crystal structures and solution NMR data are suggestive of the key roles of a conserved glutamine Q116 and the N-cap region consisting of A'α-Aβ loop and the A'α helix in controlling the light-activated conformational changes. The activation mechanism proposed here for the PpSB1-LOV supports a rotary switch mechanism and provides insights into the signal propagation mechanism in naturally existing and artificial LOV-based, two-component systems and regulators.

LOV and BLUF flavoproteins’ regulatory photoreceptors of microorganisms and photosensory actuators in optogenetic systems

In recent years, it has been shown that LOV (light, oxygen, voltage) and BLUF (Blue Light sensing Using FAD) photosensory proteins are functioning as photoreceptors of light-regulated processes not only in eukaryotes but also in numerous prokaryotes. In bacterial photoreceptors, LOV and BLUF domains with attached flavin chromophores are often associated with different effector domains, which possess enzymatic and other functions, constituting modular light-switchable systems. At present, some progress in uncovering the photoactivation mechanisms of such systems has been achieved. They are based on the chromophore photoreaction-induced changes in the photosensory domain structures and subsequent signal transduction to the effector domains. Understanding of the signal transduction principles in LOV and BLUF photosensors is important for designing, on their basis, photo-switchable enzymes and transcriptional systems applied in optogenetics—a new field in cell biology and biotechnology. The structural aspects of signal transduction by light-activated LOV and BLUF photoreceptors and their regulatory functions in bacteria, as well as some recent advances in using LOV and BLUF photosensors as activators in optogenetic systems for regulation of cellular processes are discussed.

Signal transduction in light-oxygen-voltage receptors lacking the adduct-forming cysteine residue.

Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.