Light-oxygen-voltage Research Articles

Modulating Polymerase Activity Through Light-Oxygen-Voltage Domain Insertion.

Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase. In this follow-up study, we studied the activities of previously selected variants in kinetic detail, and we generated additional LOV-polymerase fusion variants based on further insertion criteria. Our results provide mechanistic insights into how LOV domain insertion influences polymerase activity in a light-responsive manner: All active and photoresponsive enzyme variants studied by us to date were partially inhibited (i. e., "turned off") after irradiation with blue light at 470 nm, which can be explained by specific obstructions of the polymerase entry or exit structures (substrate entry channels or product exit channels, or both). Although the effects observed are moderate, we anticipate further engineering strategies that could be used to improve the extent of switchability and possibly to develop a "turn-on mode" insertion.

Nuclear hyperpolarization in electron-transfer proteins: Revealing unexpected light-induced 15N signals with field-cycling magic-angle spinning NMR

The solid-state photo-CIDNP (photo-chemically induced dynamic nuclear polarization) effect allows for nuclear hyperpolarization, i.e., non-Boltzmann nuclear spin population. The effect relies on the light-induced formation of a spin-correlated radical pair (SCRP) and has been observed in various photosynthetic reaction center (RC) proteins and flavin-containing light, oxygen, voltage (LOV) proteins. Both systems exhibit strongly enhanced NMR signals originating from the electron transfer partners. Here, we present experimental data on the magnetic field dependence of the 15N solid-state photo-CIDNP effect in both phototropin LOV1 C57S from Chlamydomonas reinhardtii and the bacterial photosynthetic RC from Rhodobacter sphaeroides. Using a pneumatic field-cycling system, samples containing a frozen solution of the proteins are explored between 0.25 T and 9.4 T. Both systems yield hyperpolarized 15N NMR signals across the entire magnetic field range originating from the electron transfer moieties. Also, in both systems, hyperpolarized signals from unexpected positions are detected between 1.0 T and 2.0 T: position N-1 of the flavin in the LOV1 protein and the τ-N of the axial magnesium-coordinating histidine of the donor. A first attempt to explain the occurrence of these unexpected signals based on quantum chemical calculations is presented.

Singlet oxygen-mediated photochemical cross-linking of an engineered fluorescent flavoprotein iLOV

Genetically-encoded photoactive proteins are integral tools in modern biochemical and molecular biological research. Within this tool box, truncated variants of the phototropin 2 light-oxygen-voltage (LOV) flavoprotein have been developed to photochemically generate singlet oxygen (1O2) in vitro and in vivo, yet the effect of 1O2 on these genetically encoded photosensitizers remains underexplored. In this study, we demonstrate that the "improved" LOV (iLOV) flavoprotein is capable of photochemical 1O2 generation. Once generated, 1O2 induces protein oligomerization via covalent cross-linking. The molecular targets of protein oligomerization by cross-linking are not endogenous tryptophans or tyrosines, but rather primarily histidines. Substitution of surface-exposed histidines for serine or glycine residues effectively eliminates protein cross-linking. When used in biochemical applications, such protein-protein cross-links may interfere with native biological responses to 1O2, which can be ameliorated by substitution of the surface exposed histidines of iLOV or other 1O2-generating flavoproteins.

Optogenetic control of phosphate-responsive genes using single component fusion proteins in Saccharomyces cerevisiae.

Blue light illumination can be detected by Light-Oxygen-Voltage (LOV) photosensing proteins and translated into a range of biochemical responses, facilitating the generation of novel optogenetic tools to control cellular function. Here we develop new variants of our previously described VP-EL222 light-dependent transcription factor and apply them to study the phosphate-responsive signaling (PHO) pathway in the budding yeast Saccharomyces cerevisiae, exemplifying the utilities of these new tools. Focusing first on the VP-EL222 protein itself, we quantified the tunability of gene expression as a function of light intensity and duration, and demonstrated that this system can tolerate the addition of substantially larger effector domains without impacting function. We further demonstrated the utility of several EL222-driven transcriptional controllers in both plasmid and genomic settings, using the PHO5 and PHO84 promoters in their native chromosomal contexts as examples. These studies highlight the utility of light-controlled gene activation using EL222 tethered to either artificial transcription domains or yeast activator proteins (Pho4). Similarly, we demonstrate the ability to optogenetically repress gene expression with EL222 fused to the yeast Ume6 protein. We finally investigated the effects of moving EL222 recruitment sites to different locations within the PHO5 and PHO84 promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (Pi) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionality they can deliver and biological questions that can be probed.

Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography.

Light-oxygen-voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (e.g. phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed in real time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.

A rapid aureochrome opto-switch enables diatom acclimation to dynamic light

Diatoms often outnumber other eukaryotic algae in the oceans, especially in coastal environments characterized by frequent fluctuations in light intensity. The identities and operational mechanisms of regulatory factors governing diatom acclimation to high light stress remain largely elusive. Here, we identified the AUREO1c protein from the coastal diatom Phaeodactylum tricornutum as a crucial regulator of non-photochemical quenching (NPQ), a photoprotective mechanism that dissipates excess energy as heat. AUREO1c detects light stress using a light-oxygen-voltage (LOV) domain and directly activates the expression of target genes, including LI818 genes that encode NPQ effector proteins, via its bZIP DNA-binding domain. In comparison to a kinase-mediated pathway reported in the freshwater green alga Chlamydomonas reinhardtii, the AUREO1c pathway exhibits a faster response and enables accumulation of LI818 transcript and protein levels to comparable degrees between continuous high-light and fluctuating-light treatments. We propose that the AUREO1c-LI818 pathway contributes to the resilience of diatoms under dynamic light conditions.

Red Shift in the Absorption Spectrum of Phototropin LOV1 upon the Formation of a Semiquinone Radical: Reconstructing the Orbital Architecture.

Flavin mononucleotide (FMN) is a ubiquitous blue-light pigment due to its ability to drive one- and two-electron transfer reactions. In both light-oxygen-voltage (LOV) domains of phototropin from the green algae Chlamydomonas reinhardtii, FMN is noncovalently bound. In the LOV1 cysteine-to-serine mutant (C57S), light-induced electron transfer from a nearby tryptophan occurs, and a transient spin-correlated radical pair (SCRP) is formed. Within this photocycle, nuclear hyperpolarization is created by the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect. In a side reaction, a stable protonated semiquinone radical (FMNH·) forms undergoing a significant bathochromic shift of the first electronic transition from 445 to 591 nm. The incorporation of phototropin LOV1-C57S into an amorphous trehalose matrix, stabilizing the radical, allows for application of various magnetic resonance experiments at ambient temperatures, which are combined with quantum-chemical calculations. As a result, the bathochromic shift of the first absorption band is explained by lifting the degeneracy of the molecular orbital energy levels for electrons with alpha and beta spins in FMNH· due to the additional electron.

Light-oxygen-voltage (LOV) domain-derived photosensitizers with the highest quantum yield for superoxide anion or singlet oxygen

Reactive oxygen species (ROS), such as superoxide anion (O2• −) and singlet oxygen (1O2), play important roles in the process of intracellular oxidative stress. Limitations in studies on the intracellular behavior and mechanisms of ROS are generally related to the difficulty in controlling the species, location, and amount of ROS produced within the cell. Therefore, it is necessary to develop genetically encoded photosensitizers that can efficiently and selectively generate O2• − and/or 1O2. We report the genetic encoding photosensitizer LovPSA, a flavoprotein engineered from Oryza sativa japonica phototropin 2 that efficiently generates O2• −. By rationally designing LovPSA to reduce the O2• − produced by charge transfer, we developed the photosensitizer LovPSO, which can efficiently and selectively generate 1O2, and LovPRO, a photosensitizer that can produce both O2• − and 1O2. LovPSO has a unique 1O2 quantum yield (ΦΔ = 0.68). We confirmed that LovPSA, LovPSO, and LovPRO have good thermostability and photostability. In addition, they also exhibit excellent phototoxicity. These results contribute to our current understanding of the interactions between the light-oxygen-voltage (LOV) apo-protein and the chromophore FMN and provide new tools for the study of intracellular ROS mechanisms and photodynamic therapy (PDT).

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 β-scaffold in signal transduction.

Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling.

Phosphatidic acid (PA) is a multifunctional lipid with important metabolic and signaling functions, and efforts to dissect its pleiotropy demand strategies for perturbing its levels with spatiotemporal precision. Previous membrane editing approaches for generating local PA pools used light-mediated induced proximity to recruit a PA-synthesizing enzyme, phospholipase D (PLD), from the cytosol to the target organelle membrane. Whereas these optogenetic PLDs exhibited high activity, their residual activity in the dark led to undesired chronic lipid production. Here, we report ultralow background membrane editors for PA wherein light directly controls PLD catalytic activity, as opposed to localization and access to substrates, exploiting a light-oxygen-voltage (LOV) domain-based conformational photoswitch inserted into the PLD sequence and enabling their stable and nonperturbative targeting to multiple organelle membranes. By coupling organelle-targeted LOVPLD activation to lipidomics analysis, we discovered different rates of metabolism for PA and its downstream products depending on the subcellular location of PA production. We also elucidated signaling roles for PA pools on different membranes in conferring local activation of AMP-activated protein kinase signaling. This work illustrates how membrane editors featuring acute, optogenetic conformational switches can provide new insights into organelle-selective lipid metabolic and signaling pathways.

Reduction midpoint potential of a paradigm light–oxygen–voltage receptor and its modulation by methionine residues

The introduction of methionine residues into the Avena sativa phototropin 1 LOV2 domain raises the reduction midpoint of its flavin-nucleotide chromophore.

The N-Terminal Region of the BcWCL1 Photoreceptor Is Necessary for Self-Dimerization and Transcriptional Activation upon Light Stimulation in Yeast.

The BcWCL1 protein is a blue-light photoreceptor from the fungus Botrytis cinerea. This protein has a central role in B. cinerea circadian regulation and is an ortholog to WC-1 from Neurospora crassa. The BcWCL1 and WC-1 proteins have similar protein domains, including a LOV (Light Oxygen Voltage) domain for light sensing, two PAS (Per Arnt Sim) domains for protein-protein interaction, and a DNA binding domain from the GATA family. Recently, the blue-light response of BcWCL1 was demonstrated in a version without PAS domains (BcWCL1PAS∆). Here, we demonstrated that BcWCL1PAS∆ is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1PAS∆ enables transcriptional activation as a single component in yeast. By using chimeric transcription factors and the luciferase reporter gene, we assessed the transcriptional activity of different fragments of the N-terminal and C-terminal regions of BcWCL1PAS∆, identifying a functional transcriptional activation domain (AD) in the N-terminal region that belongs to the 9aaTAD family. Finally, we determined that the transcriptional activation levels of BcWCL1PAS∆ AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1PAS∆ protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics.

Diversity of function and higher-order structure within HWE sensor histidine kinases

Integral to the protein structure/function paradigm, oligomeric state is typically conserved along with function across evolution. However, notable exceptions such as the hemoglobins show how evolution can alter oligomerization to enable new regulatory mechanisms. Here we examine this linkage in histidine kinases (HKs), a large class of widely distributed prokaryotic environmental sensors. While the majority of HKs are transmembrane homodimers, members of the HWE/HisKA2 family can deviate from this architecture as exemplified by our finding of a monomeric soluble HWE/HisKA2 HK (EL346, a photosensing Light-Oxygen-Voltage (LOV)-HK). To further explore the diversity of oligomerization states and regulation within this family, we biophysically and biochemically characterized multiple EL346 homologs and found a range of HK oligomeric states and functions. Three LOV-HK homologs are primarily dimeric with differing structural and functional responses to light, while two Per-ARNT-Sim (PAS)-HKs interconvert between differentially active monomers and dimers, suggesting dimerization might control enzymatic activity for these proteins. Finally, we examined putative interfaces in a dimeric LOV-HK, finding that multiple regions contribute to dimerization. Our findings suggest the potential for novel regulatory modes and oligomeric states beyond those traditionally defined for this important family of environmental sensors.

Stabilization of a flavoprotein for solid-state photo-CIDNP MAS NMR at room temperature by embedding in a glassy sugar matrix

Hyperpolarization via the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect can be detected in frozen solutions of electron transfer proteins generating a radical-pair upon illumination. The effect has been observed in various natural photosynthetic reaction centers and in light-oxygen-voltage (LOV) sensing domains incorporating a flavin mononucleotide (FMN) as chromophore. In LOV domains, where a highly conserved cysteine is mutated to a flavin to interrupt its natural photochemistry, a radical-pair is generated by electron transfer from a nearby tryptophan to the photoexcited triplet state of FMN. During the photocycle, both the LOV domain and the chromophore are photochemically degraded, e.g., by the formation of singlet oxygen. This limits the time for collection of hyperpolarized nuclear magnetic resonance (NMR) data. We show that embedding of the protein into a trehalose sugar glass matrix stabilizes the protein for 13C solid-state photo-CIDNP NMR experiments which can be conducted at room temperature in a powder sample. Additionally, this preparation allows for incorporation of high amounts of protein further boosting the intensity of the detected signals from FMN and tryptophan at natural abundance. Signal assignment is aided by quantum chemical calculations of absolute shieldings. The underlying mechanism for the surprising absorption-only signal pattern is not yet understood. Comparison to calculated isotropic hyperfine couplings imply that the enhancement is not due to the classical radical-pair mechanism (RPM). Analysis of the anisotropic hyperfine couplings associated with solid-state photo-CIDNP mechanisms also show no simple correlation, suggesting a more complex underlying mechanism.

Network analysis of chromophore binding site in LOV domain

Photoreceptor proteins are versatile toolbox for developing biosensors for optogenetic applications. These molecular tools get activated upon illumination of blue light, which in turn offers a non-invasive method for gaining high spatiotemporal resolution and precise control of cellular signal transduction. The Light-Oxygen-Voltage (LOV) domain family of proteins is a well-recognized system for constructing optogenetic devices. Translation of these proteins into efficient cellular sensors is possible by tuning their photochemistry lifetime. However, the bottleneck is the need for more understanding of the relationship between the protein environment and photocycle kinetics. Significantly, the effect of the local environment also modulates the electronic structure of chromophore, which perturbs the electrostatic and hydrophobic interaction within the binding site. This work highlights the critical factors hidden in the protein networks, linking with their experimental photocycle kinetics. It presents an opportunity to quantitatively examine the alternation in chromophore's equilibrium geometry and identify details which have substantial implications in designing synthetic LOV constructs with desirable photocycle efficiency.

Time-resolved infrared difference spectroscopy in cells: Response of the basic region leucine zipper of aureochrome

Aureochromes are light, oxygen, voltage (LOV) proteins and central blue-light receptors in algae acting as light-gated transcription factors. The C-terminal LOV domain mediates blue-light recognition and the basic region leucine zipper (bZIP) domain binds a specific DNA motif as effector. LOV domains from aureochromes have been successfully applied in optogenetic tools. The light-induced response of aureochromes has been studied by a variety of biophysical techniques, but the mechanism of signal progression from LOV to bZIP remains unclear. We studied the bZIP-LOV module of aureochrome1a from the diatom Phaeodactylum tricornutum using time-resolved rapid-scan FTIR difference spectroscopy. Time-resolved difference spectra of bZIP-LOV in vitro revealed a time constant of 5 s for the formation of a light state dimer of the LOV domains and the concomitant loss of α-helical elements in the bZIP domain. To verify these observations in a near-native environment, in-cell infrared difference spectroscopy (ICIRD) was extended from a steady state to a time-resolved technique using LOV domains in bacterial cells. We established a time-resolved in-cell method with a resolution of 7.6 ms after the laser pulse. Using this technique, the response of bZIP-LOV was followed in living bacterial cells and the light-induced partial unfolding of bZIP was confirmed to take place in cells in a similar time range as in vitro. These results provide structural and kinetic insights into the signaling mechanism of aureochromes. The slow response points to an association of LOV to bZIP in the dark state prior to activation.

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.

Structural basis of signal transduction within PAS regulated Ser/Thr kinase.

To control integral cellular processes, nature has developed molecular switches where the sensory domain senses external cues and controls the function of relevant effector domains. Here, we focus on the superfamily of PAS (Per-ARNT-Sim) sensory domains and their roles in regulating Ser/Thr Kinases (STKs). PAS domains achieve regulatory control by internally binding ligands/cofactors (e.g., O2 binding to heme in FixL; flavins within the Light-Oxygen-Voltage (LOV)-type PAS domains). While aspects of PAS sensing are understood, the signaling mechanism from sensory to effector domain and Protein-Protein Interactions (PPIs) within full-length proteins or complexes remain elusive. To address these, we investigated plant phototropin, Avena sativa Phototropin1 (AsPhot1). AsPhot1 has two LOV domains followed by the STK domain, where the LOV2 regulates the kinase function to maintain essential plant physiology via blue light sensing. We used biochemical assays in combination with HDX-MS to study the signal transduction from LOV2 to the STK domain. Our data shows substantial conformation changes in the dark vs. light state upon photoactivation, highlighting key regions might be necessary for LOV-regulated kinase functions. We also observed the activation of kinase involves destabilizing the linker region between LOV2-STK, which interacts with the n-lobe of the kinase near the ATP binding site. We speculated that upon illumination, rearrangement of these linkers might hold the key to the STK substrate association and downstream functions. Additionally, using a low-resolution cryo-EM map, combined with local HDX-MS data and computational resources (e.g., AlphaFold), we built a model for the LOV2-STK complex. Our model highlights critical PPI sites within AsPhot1 and provides additional insights into understanding the generality of how protein kinases interact with their associated domain via conserved residues and subdomains. These findings may help develop novel optogenetic tools and therapeutics for mammalian PAS regulated kinase.

Engineer RNA-Protein Nanowires as Light-Responsive Biomaterials.

RNA molecules have emerged as increasingly attractive biomaterials with important applications such as RNA interference (RNAi) for cancer treatment and mRNA vaccines against infectious diseases. However, it remains challenging to engineer RNA biomaterials with sophisticated functions such as non-covalent light-switching ability. Herein, light-responsive RNA-protein nanowires are engineered to have such functions. It first demonstrates that the high affinity of RNA aptamer enables the formation of long RNA-protein nanowires through designing a dimeric RNA aptamer and an engineered green fluorescence protein (GFP) that contains two TAT-derived peptides at N- and C- termini. GFP is then replaced with an optogenetic protein pair system, LOV2 (light-oxygen-voltage) protein and its binding partner ZDK (Z subunit of protein A), to confer blue light-controlled photo-switching ability. The light-responsive nanowires are long (>500 nm) in the dark, but small (20-30 nm) when exposed to light. Importantly, the co-assembly of this RNA-protein hybrid biomaterial does not rely on the photochemistry commonly used for light-responsive biomaterials, such as bond formation, cleavage, and isomerization, and is thus reversible. These RNA-protein structures can serve as a new class of light-controlled biocompatible frameworks for incorporating versatile elements such as RNA, DNA, and enzymes.

Sub-Millisecond Photoinduced Dynamics of Free and EL222-Bound FMN by Stimulated Raman and Visible Absorption Spectroscopies.

Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm-1, which covers only part of the spectrum of biologically relevant time scales and Raman shifts. Here we report on a broadband (~200-2200 cm-1) dual transient visible absorption (visTA)/FSRS set-up that can accommodate time delays from a few femtoseconds to several hundreds of microseconds after illumination with an actinic pump. The extended time scale and wavenumber range allowed us to monitor the complete excited-state dynamics of the biological chromophore flavin mononucleotide (FMN), both free in solution and embedded in two variants of the bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The observed lifetimes and intermediate states (singlet, triplet, and adduct) are in agreement with previous time-resolved infrared spectroscopy experiments. Importantly, we found evidence for additional dynamical events, particularly upon analysis of the low-frequency Raman region below 1000 cm-1. We show that fs-to-sub-ms visTA/FSRS with a broad wavenumber range is a useful tool to characterize short-lived conformationally excited states in flavoproteins and potentially other light-responsive proteins.