BLUF Photoreceptor Research Articles

Unveiling the Photoactivation Mechanism of BLUF Photoreceptor Protein through Hybrid Quantum Mechanics/Molecular Mechanics Free-Energy Calculation

Unveiling the Photoactivation Mechanism of BLUF Photoreceptor Protein through Hybrid Quantum Mechanics/Molecular Mechanics Free-Energy Calculation

Redox Properties of Flavin in BLUF and LOV Photoreceptor Proteins from Hybrid QM/MM Molecular Dynamics Simulation.

Flavins play an important role in many oxidation and reduction processes in biological systems. For example, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are common cofactors found in enzymatic proteins that use the special redox properties of these flavin molecules for their catalytic or photoactive functions. The redox potential of the flavin is strongly affected by its (protein) environment; however, the underlying molecular interactions of this effect are still unknown. Using hybrid quantum mechanics/molecular mechanics (QM/MM) simulation techniques, we have studied the redox properties of flavin in the gas phase, aqueous solution, and two different protein environments, in particular, a BLUF and a LOV photoreceptor domain. By mapping the changes in electrostatic potential and solvent structure, we gain insight into how specific polarization of the flavin by its environment tunes the reduction potential. We find also that accurate calculation of the reduction potentials of these systems by using the hybrid QM/MM approach is hampered by a too limited sampling of the counterion configurations and by artifacts at the QM/MM boundary. We make suggestions for how these issues can be overcome.

Structural characterization of the BLUF photoreceptor OaPAC using time-resolved crystallography

Structural characterization of the BLUF photoreceptor OaPAC using time-resolved crystallography

A novel BLUF photoreceptor modulates the Xanthomonas citri subsp. citri-host plant interaction.

Plant-pathogen interaction is influenced by multiple environmental factors, including temperature and light. Recent works have shown that light modulates not only the defense response of plants but also the pathogens virulence. Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker, an important plant disease worldwide. The Xcc genome presents four genes encoding putative photoreceptors: one bacteriophytochrome and three blue light photoreceptors, one LOV and two BLUFs (bluf1: XAC2120 and bluf2: XAC3278). The presence of two BLUFs proteins is an outstanding feature of Xcc. In this work we show that the bluf2 gene is functional. The mutant strain, XccΔbluf2, was constructed demonstrating that BLUF2 regulates swimming-type motility, adhesion to leaves, exopolysaccharide production and biofilm formation, features involved in the Xcc virulence processes. An important aspect during the plant-pathogen interaction is the oxidative response of the host and the consequent reaction of the pathogen. We observed that ROS detoxification is regulated by Xcc bluf2 gene. The phenotypes of disease in orange plants produced by WT and XccΔbluf2 strains were evaluated, observing different phenotypes. Altogether, these results show that BLUF2 negatively regulates virulence during citrus canker. This work constitutes the first report on BLUF-like receptors in plant pathogenic bacteria.

Structural and functional analysis of the Acinetobacter baumannii BlsA photoreceptor and regulatory protein.

The Acinetobacter baumannii BlsA photoreceptor has an N-terminal (NT) BLUF domain and a C-terminal (CT) amino acid sequence with no significant homology to characterized bacterial proteins. In this study, we tested the biological role of specific residues located in these BlsA regions. Site-directed mutagenesis, surface motility assays at 24°C and protein overexpression showed that residues Y7, Q51 and W92 are essential for not only light-regulated motility, but also BlsA’s solubility when overexpressed in a heterologous host. In contrast, residues A29 and F32, the latter representing a difference when compared with other BLUF-containing photoreceptors, do not play a major role in BlsA’s biological functions. Analysis of the CT region showed that the deletion of the last five BlsA residues has no significant effect on the protein’s light-sensing and motility regulatory functions, but the deletion of the last 14 residues as well as K144E and K145E substitutions significantly alter light-regulated motility responses. In contrast to the NT mutants, these CT derivatives were overexpressed and purified to homogeneity to demonstrate that although these mutations do not significantly affect flavin binding and photocycling, they do affect BlsA’s photodynamic properties. Notably, these mutations map within a potential fifth α-helical component that could play a role in predicted interactions between regulatory partners and BlsA, which could function as a monomer according to gel filtration data. All these observations indicate that although BlsA shares common structural and functional properties with unrelated photoreceptors, it also exhibits unique features that make it a distinct BLUF photoreceptor.

Electron-Coupled Double Proton Transfer in the Slr1694 BLUF Photoreceptor: A Multireference Electronic Structure Study.

Photoreceptor proteins control vital cellular responses to light. The photocycle of the Slr1694 blue light using flavin photoreceptor is initiated by photoexcitation to a locally excited state within the flavin, followed by electron transfer from Tyr8 to the flavin and a proton relay from Tyr8 to the flavin via an intervening glutamine. Herein, the two-dimensional excited state potential energy surfaces associated with this double proton-transfer reaction are computed using the complete active space self-consistent-field method and multiconfigurational perturbation theory, including the protein and solvent environment with electrostatic embedding. The double proton-transfer reaction was found to be energetically unfavorable in the ground state and locally excited state but energetically favorable in the charge-transfer state corresponding to electron transfer from Tyr8 to the flavin. These results indicate that the proton-coupled electron transfer process is sequential, with electron transfer preceding double proton transfer, and that the double proton-transfer reaction is also sequential, with proton transfer from Tyr8 to Gln50 followed by proton transfer from Gln50 to the flavin. The barrier is lower for the first proton-transfer reaction, and both barriers are significantly influenced by geometrical changes within the active site, particularly the proton donor-acceptor distance as well as the protein environment. These calculations provide insight into the impact of protein reorganization and electrostatics on the excited electronic states prior to and during the double proton-transfer reaction. This interplay between excited states and the environment has implications for other photoreceptor proteins.

Integration of Temperature and Blue-Light Sensing in Acinetobacter baumannii Through the BlsA Sensor.

BlsA is a BLUF photoreceptor present in Acinetobacter baumannii, responsible for modulation of motility, biofilm formation and virulence by light. In this work, we have combined physiological and biophysical evidences to begin to understand the basis of the differential photoregulation observed as a function of temperature. Indeed, we show that blsA expression is reduced at 37°C, which correlates with negligible photoreceptor levels in the cells, likely accounting for absence of photoregulation at this temperature. Another point of control occurs on the functionality of the BlsA photocycle itself at different temperatures, which occurs with an average quantum yield of photoactivation of the signaling state of 0.20±0.03 at 15°C<T<25°C, but is practically inoperative at T>30°C, as a result of conformational changes produced in the nanocavity of FAD. This effect would be important when the photoreceptor is already present in the cell to avoid almost instantaneously further signaling process when it is no longer necessary, for example under circumstances of temperature changes possibly faced by the bacteria. This complex interplay between light and temperature would provide the bacteria clues of environmental location and dictate/modulate light photosensing in A.baumannii.

Photoactivation Mechanism of a Bacterial Light-Regulated Adenylyl Cyclase

Light-regulated enzymes enable organisms to quickly respond to changing light conditions. We characterize a photoactivatable adenylyl cyclase (AC) from Beggiatoa sp. (bPAC) that translates a blue light signal into the production of the second messenger cyclic AMP. bPAC contains a BLUF photoreceptor domain that senses blue light using a flavin chromophore, linked to an AC domain. We present a dark state crystal structure of bPAC that closely resembles the recently published structure of the homologous OaPAC from Oscillatoria acuminata. To elucidate the structural mechanism of light-dependent AC activation by the BLUF domain, we determined the crystal structures of illuminated bPAC and of a pseudo-lit state variant. We use hydrogen–deuterium exchange measurements of secondary structure dynamics and hypothesis-driven point mutations to trace the activation pathway from the chromophore in the BLUF domain to the active site of the cyclase. The structural changes are relayed from the residues interacting with the excited chromophore through a conserved kink of the BLUF β-sheet to a tongue-like extrusion of the AC domain that regulates active site opening and repositions catalytic residues. Our findings not only show the specific molecular pathway of photoactivation in BLUF-regulated ACs but also have implications for the general understanding of signaling in BLUF domains and of the activation of ACs.

Molecular mechanism of the dark-state recovery in BLUF photoreceptors

Photoactivation of the bacterial photoreceptor BLUF is achieved by a unique tautomerization and rotation of the conserved Gln residue. To identify interactions controlling the lifetime of the high-energy ZZ-imide tautomer, we consider thermal dark-state recovery of the BLUF photoreceptor BlrB using MD and QM/MM calculations. We found that protonation/deprotonation of Gln51 by protonated His73 via Tyr9 was energetically favorable. The rate-limiting steps correspond to rearrangements of hydrogen bonds and rotation of the protonated Gln51 side chain. Notably, strengthening the hydrogen bond between protonated Gln51 and flavin O4 plays a central role in extending the lifetime of the photoactivated state.

Role of active site conformational changes in photocycle activation of the AppA BLUF photoreceptor.

Blue light using flavin adenine dinucleotide (BLUF) proteins are essential for the light regulation of a variety of physiologically important processes and serve as a prototype for photoinduced proton-coupled electron transfer (PCET). Free-energy simulations elucidate the active site conformations in the AppA (activation of photopigment and puc expression) BLUF domain before and following photoexcitation. The free-energy profile for interconversion between conformations with either Trp104 or Met106 closer to the flavin, denoted Trpin/Metout and Trpout/Metin, reveals that both conformations are sampled on the ground state, with the former thermodynamically favorable by ∼3 kcal/mol. These results are consistent with the experimental observation of both conformations. To analyze the proton relay from Tyr21 to the flavin via Gln63, the free-energy profiles for Gln63 rotation were calculated on the ground state, the locally excited state of the flavin, and the charge-transfer state associated with electron transfer from Tyr21 to the flavin. For the Trpin/Metout conformation, the hydrogen-bonding pattern conducive to the proton relay is not thermodynamically favorable on the ground state but becomes more favorable, corresponding to approximately half of the configurations sampled, on the locally excited state. The calculated energy gaps between the locally excited and charge-transfer states suggest that electron transfer from Tyr21 to the flavin is more facile for configurations conducive to proton transfer. When the active site conformation is not conducive to PCET from Tyr21, Trp104 can directly compete with Tyr21 for electron transfer to the flavin through a nonproductive pathway, impeding the signaling efficiency.

Editorial: Optogenetic Tools in the Molecular Spotlight.

Editorial: Optogenetic Tools in the Molecular Spotlight.

Revealing the functional states in the active site of BLUF photoreceptors from electrochromic shift calculations.

Photoexcitation with blue light of the flavin chromophore in BLUF photoreceptors induces a switch into a metastable signaling state that is characterized by a red-shifted absorption maximum. The red shift is due to a rearrangement in the hydrogen bond pattern around Gln63 located in the immediate proximity of the isoalloxazine ring system of the chromophore. There is a long-lasting controversy between two structural models, named Q63A and Q63J in the literature, on the local conformation of the residues Gln63 and Tyr21 in the dark state of the photoreceptor. As regards the mechanistic details of the light-activation mechanism, rotation of Gln63 is opposed by tautomerism in the Q63A and Q63J models, respectively. We provide a structure-based simulation of electrochromic shifts of the flavin chromophore in the wild type and in various site-directed mutants. The excellent overall agreement between experimental and computed data allows us to evaluate the two structural models. Compelling evidence is obtained that the Q63A model is incorrect, whereas the Q63J is fully consistent with the present computations. Finally, we confirm independently that a keto–enol tautomerization of the glutamine at position 63, which was proposed as molecular mechanism for the transition between the dark and the light-adapted state, explains the measured 10 to 15 nm red shift in flavin absorption between these two states of the protein. We believe that the accurateness of our results provides evidence that the BLUF photoreceptors absorption is fine-tuned through electrostatic interactions between the chromophore and the protein matrix, and finally that the simplicity of our theoretical model is advantageous as regards easy reproducibility and further extensions.

Photoinduced Electron Transfer Facilitates Tautomerization of the Conserved Signaling Glutamine Side Chain in BLUF Protein Light Sensors

The BLUF domain (sensor of blue light using flavin adenine dinucleotide) from a bacterial photoreceptor protein AppA undergoes a cascade of chemical transformations, including hydrogen bond rearrangements around the flavin adenine dinucleotide (FAD) chromophore, in response to light illumination. These transformations are initiated by photoinduced electron and proton transfer from a tyrosine residue to the photoexcited flavin which is assisted by a glutamine residue. According to the recent studies, the proton-coupled electron transfer leads to formation of a radical-pair intermediate Tyr•···FADH• and a tautomeric EE form of glutamine in the ground electronic state. This intermediate is a precursor of the light-induced state of the BLUF photoreceptor implicated in biological signaling. In order to describe evolution of the radical pair, we computed reaction pathways on the ground state potential energy surface employing quantum-chemical calculations in the DFT PBE0/cc-pVDZ approximation for a molecular cluster mimicking the chromophore containing pocket of the AppA BLUF protein. We found a minimum-energy pathway comprised of the following consecutive reaction steps: (1) rotation of the imidic group of the EE glutamine side chain around the Cγ-Cδ bond; (2) flip of the OεH group and formation of the ZE form of the glutamine side chain; and (3) biradical recombination via coupled proton and electron transfer, leading to the ZZ form of the glutamine side chain. The potential-energy barriers for stages 1-3 do not exceed 9 kcal/mol. Energy barrier 3 describing the ZE to ZZ glutamine tautomerization is significantly smaller in the BLUF model than in isolated glutamine, since tautomerization in BLUF is facilitated by electron transfer and radical recombination. Thus, our study shows that tautomerization of the conserved glutamine is coupled to the light-induced electron transfer process in BLUF and, thus, is a viable candidate for the photoactivation mechanism which at present is very much debated.

Ultrafast mid-infrared spectroscopy by chirped pulse upconversion in 1800-1000cm^−1 region

Broadband femtosecond mid-infrared pulses can be converted into the visible spectral region by chirped pulse upconversion. We report here the upconversion of pump probe transient signals in the frequency region below 1800cm(-1), using the nonlinear optical crystal AgGaGeS4, realizing an important expansion of the application range of this method. Experiments were demonstrated with a slab of GaAs, in which the upconverted signals cover a window of 120cm(-1), with 1.5cm(-1) resolution. In experiments on the BLUF photoreceptor Slr1694, signals below 1 milliOD were well resolved after baseline correction. Possibilities for further optimization of the method are discussed. We conclude that this method is an attractive alternative for the traditional MCT arrays used in most mid-infrared pump probe experiments.

Hydrogen Bond Switching among Flavin and Amino Acids Determines the Nature of Proton-Coupled Electron Transfer in BLUF Photoreceptors

BLUF domains are flavin-binding photoreceptors that can be reversibly switched from a dark-adapted state to a light-adapted state. Proton-coupled electron transfer (PCET) from a conserved tyrosine to the flavin that results in a neutral flavin semiquinone/tyrosyl radical pair constitutes the photoactivation mechanism of BLUF domains. Whereas in the dark-adapted state PCET occurs in a sequential fashion where electron transfer precedes proton transfer, in the light-adapted state the same radical pair is formed by a concerted mechanism. We propose that the altered nature of the PCET process results from a hydrogen bond switch between the flavin and its surrounding amino acids that preconfigures the system for proton transfer. Hence, BLUF domains represent an attractive biological model system to investigate and understand PCET in great detail.

A way to sense light intensity: Multiple-excitation of the BLUF photoreceptor TePixD suppresses conformational change

TePixD, a cyanobacterial sensor of blue light using flavin adenine dinucleotide (FAD) (BLUF) which exists in a decamer form, was found to exhibit photoreaction sensitive to light intensity. While the number of excited molecules increased monotonically as the laser power increased, the number of decamers exhibiting a global conformational change initially increased, and then decreased with the increase of excitation intensity. This unusual power dependence was analyzed based on a Poisson distribution equation, demonstrating that decamers containing more than one excited monomer subunit do not undergo conformational change. Our results suggest that TePixD functions not only as a photosensor, but also by sensing light intensity.

Natural and Engineered Photoactivated Nucleotidyl Cyclases for Optogenetic Applications

Cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers that regulate metabolic and behavioral responses in diverse organisms. We describe purification, engineering, and characterization of photoactivated nucleotidyl cyclases that can be used to manipulate cAMP and cGMP levels in vivo. We identified the blaC gene encoding a putative photoactivated adenylyl cyclase in the Beggiatoa sp. PS genome. BlaC contains a BLUF domain involved in blue-light sensing using FAD and a nucleotidyl cyclase domain. The blaC gene was overexpressed in Escherichia coli, and its product was purified. Irradiation of BlaC in vitro resulted in a small red shift in flavin absorbance, typical of BLUF photoreceptors. BlaC had adenylyl cyclase activity that was negligible in the dark and up-regulated by light by 2 orders of magnitude. To convert BlaC into a guanylyl cyclase, we constructed a model of the nucleotidyl cyclase domain and mutagenized several residues predicted to be involved in substrate binding. One triple mutant, designated BlgC, was found to have photoactivated guanylyl cyclase in vitro. Irradiation with blue light of the E. coli cya mutant expressing BlaC or BlgC resulted in the significant increases in cAMP or cGMP synthesis, respectively. BlaC, but not BlgC, restored cAMP-dependent growth of the mutant in the presence of light. Small protein sizes, negligible activities in the dark, high light-to-dark activation ratios, functionality at broad temperature range and physiological pH, as well as utilization of the naturally occurring flavins as chromophores make BlaC and BlgC attractive for optogenetic applications in various animal and microbial models.

A Q63E Rhodobacter sphaeroides AppA BLUF Domain Mutant Is Locked in a Pseudo-Light-Excited Signaling State

The AppA BLUF photoreceptor from Rhodobacter sphaeroides contains a conserved key residue, Gln63, that is thought to undergo a shift in hydrogen-bonding interactions when a bound flavin is light excited. In this study we have characterized two substitution mutants of Gln63 (Q63E, Q63L) in the context of two constructs of the BLUF domain that have differing lengths, AppA1-126 and AppA17-133. Q63L mutations in both constructs exhibit a blue-shifted flavin absorption spectrum as well as a loss of the photocycle. Altered fluorescence emission and fluorescence quenching of the Q63L mutant indicate significant perturbations of hydrogen bonding to the flavin and surrounding amino acids which is confirmed by (1)H-(15)N HSQC NMR spectroscopy. The Q63E substitution mutant is constitutively locked in a lit signaling state as evidenced by a permanent 3 nm red shift of the flavin absorption, quenching of flavin fluorescence emission, analysis of (1)H-(15)N HSQC spectra, and the inability of full-length AppA Q63E to bind to the PpsR repressor. The significance of these findings on the mechanism of light-induced output signaling is discussed.

Structural Refinement of a Key Tryptophan Residue in the BLUF Photoreceptor AppA by Ultraviolet Resonance Raman Spectroscopy

The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. The BLUF domain is of special interest because it uses a rigid flavin rather than an isomerizable chromophore, such as a rhodopsin or phytochrome, for its light-activation process. Crystal and solution structures of several BLUF domains were recently obtained, and their overall structures are consistent. However, there is a key ambiguity regarding the position of a conserved tryptophan (Trp-104 in AppA), in that this residue was found either close to flavin (Trpin conformation) or exposed to the solvent (Trpout conformation). The location of Trp-104 is a crucial factor in understanding the photocycle mechanism of BLUF domains, because this residue has been shown to play an essential role in the activation of AppA. In this study, we demonstrated a Trpin conformation for the BLUF domain of AppA through direct observation of the vibrational spectrum of Trp-104 by ultraviolet resonance Raman spectroscopy, and also observed light-induced conformational and environmental changes in Trp-104. This study provides a structural basis for future investigations of the photocycle mechanism of BLUF proteins.

Hydrogen Bond Switching among Flavin and Amino Acid Side Chains in the BLUF Photoreceptor Observed by Ultrafast Infrared Spectroscopy

BLUF domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino acid side chains, including a highly conserved tyrosine and glutamine. The participation of particular amino acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD •− and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, showing that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay on H/D exchange. Subsequent protonation of FAD •− to result in FADH • on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH • C=N stretch marker mode, with tyrosine as the likely proton donor. FADH • is reoxidized in 67 ps (180 ps in D 2O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch shows infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by ∼180° through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch.