Tilo Mathes

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(2)O) 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 approximately 180 degrees through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch.

The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

BLUF (blue light sensing using FAD) domains belong to a novel group of blue light sensing receptor proteins found in microorganisms. We have assessed the role of specific aromatic and polar residues in the Synechocystis Slr1694 BLUF protein by investigating site-directed mutants with substitutions Y8W, W91F, and S28A. The W91F and S28A mutants formed the red-shifted signaling state upon blue light illumination, whereas in the Y8W mutant, signaling state formation was abolished. The W91F mutant shows photoactivation dynamics that involve the successive formation of FAD anionic and neutral semiquinone radicals on a picosecond time scale, followed by radical pair recombination to result in the long-lived signaling state in less than 100 ps. The photoactivation dynamics and quantum yield of signaling state formation were essentially identical to those of wild type, which indicates that only one significant light-driven electron transfer pathway is available in Slr1694, involving electron transfer from Y8 to FAD without notable contribution of W91. In the S28A mutant, the photoactivation dynamics and quantum yield of signaling state formation as well as dark recovery were essentially the same as in wild type. Thus, S28 does not play an essential role in the initial hydrogen bond switching reaction in Slr1694 beyond an influence on the absorption spectrum. In the Y8W mutant, two deactivation branches upon excitation were identified: the first involves a neutral semiquinone FADH(*) that was formed in approximately 1 ps and recombines in 10 ps and is tentatively assigned to a FADH(*)-W8(*) radical pair. The second deactivation branch forms FADH(*) in 8 ps and evolves to FAD(*-) in 200 ps, which recombines to the ground state in about 4 ns. In the latter branch, W8 is tentatively assigned as the FAD redox partner as well. Overall, the results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from Y8 to FAD, followed by a hydrogen bond rearrangement and radical pair recombination.

In vivo generation of flavoproteins with modified cofactors.

To understand flavoprotein mechanisms and reactivity, biochemical and biophysical methods are usually employed, and differences between wild-type and mutated proteins with altered primary structures are placed under specific consideration. Alternatively, the cofactor can be modified, and modified flavoproteins can be studied accordingly. Here we present an efficient and general method for modifying the cofactor of flavoproteins in vivo. The modified cofactor is incorporated into apoprotein during protein biosynthesis in a riboflavin-auxotrophic Escherichia coli strain, which expresses a bacterial riboflavin transporter to import flavins from the medium. This system was used to introduce roseoflavin into the riboflavin-binding protein dodecin and into microbial blue-light photoreceptors of the BLUF (blue-light sensors using FAD) and LOV (light oxygen voltage) families. The modified photoreceptors showed absorption and fluorescence different from those of proteins carrying their natural cofactor or chromophores in solution, but did not show any photochemical reaction as implied by former physiological studies.

A photochromic histidine kinase rhodopsin (HKR1) that is bimodally switched by ultraviolet and blue light.

Background: Microbial rhodopsins in Chlamydomonas are employed for photoorientation and developmental processes. Results: HKR1 is a UVA-absorbing rhodopsin that is bimodally switched between a UV- and a blue light-absorbing isoform with different light colors. Conclusion: The chromophore of the dark-adapted UV state contains a deprotonated Schiff base stabilized by a 13-cis,15-anti conformation. Significance: This is the initial characterization of the first member of a novel rhodopsin family. Rhodopsins are light-activated chromoproteins that mediate signaling processes via transducer proteins or promote active or passive ion transport as ion pumps or directly light-activated channels. Here, we provide spectroscopic characterization of a rhodopsin from the Chlamydomonas eyespot. It belongs to a recently discovered but so far uncharacterized family of histidine kinase rhodopsins (HKRs). These are modular proteins consisting of rhodopsin, a histidine kinase, a response regulator, and in some cases an effector domain such as an adenylyl or guanylyl cyclase, all encoded in a single protein as a two-component system. The recombinant rhodopsin fragment, Rh, of HKR1 is a UVA receptor (λmax = 380 nm) that is photoconverted by UV light into a stable blue light-absorbing meta state Rh-Bl (λmax = 490 nm). Rh-Bl is converted back to Rh-UV by blue light. Raman spectroscopy revealed that the Rh-UV chromophore is in an unusual 13-cis,15-anti configuration, which explains why the chromophore is deprotonated. The excited state lifetime of Rh-UV is exceptionally stable, probably caused by a relatively unpolar retinal binding pocket, converting into the photoproduct within about 100 ps, whereas the blue form reacts 100 times faster. We propose that the photochromic HKR1 plays a role in the adaptation of behavioral responses in the presence of UVA light.

Blue light induces global and localized conformational changes in the kinase domain of full-length phototropin.

The blue-light photoreceptor phototropin plays a crucial role in optimizing photosynthesis in plants. In the two light-, oxygen-, or voltage-sensitive (LOV) domains of phototropin, the light stimulus is absorbed by the flavin chromophores. The signal is assumed to be transferred via dissociation and unfolding of a conserved J alpha helix element to the serine/threonine kinase domain. We investigated full-length phototropin from the green alga Chlamydomonas reinhardtii by Fourier transform infrared spectroscopy to shed light on the signal transfer within the protein and on the structural response of the kinase. Light-induced structural changes were assigned by comparing signals of the full-length protein with those of the truncated LOV1-LOV2-J alpha and LOV1-LOV2 and with those of deletion mutants. A loss of helicity originating from the J alpha linker helix was observed in LOV1-LOV2-J alpha in agreement with previous studies of LOV2-J alpha. Full-length phototropin showed reversible global conformational changes via several turn elements. These changes were suppressed in a deletion mutant lacking the J alpha linker and are attributed to the kinase domain. The loss of turn structure is interpreted as a light-induced opening of the kinase tertiary structure upon release of the LOV2 domain. Concomitant protonation changes of Asp or Glu residues in the kinase domain were not observed. A light-induced loss in helicity was observed only in the presence of a phototropin-characteristic 54-amino acid extension of the kinase activation loop, which is predicted to be located apart from the catalytic cleft. This response of the extension might play a significant role in the phototropin signaling process.

Redox modulation of flavin and tyrosine determines photoinduced proton-coupled electron transfer and photoactivation of BLUF photoreceptors

Background: Proton-coupled electron transfer is the key step in BLUF photoactivation. Results: Redox modulation of flavin and tyrosine determines electron transfer rates and signaling efficiency and reveals a new photocycle intermediate. Conclusion: Partial charge transfer from tyrosine to flavin takes place prior to full electron transfer. Significance: Mechanistic details of protein-modulated electron transfer processes are crucial to understand biological proton-coupled electron transfer reactions. Photoinduced electron transfer in biological systems, especially in proteins, is a highly intriguing matter. Its mechanistic details cannot be addressed by structural data obtained by crystallography alone because this provides only static information on a given redox system. In combination with transient spectroscopy and site-directed manipulation of the protein, however, a dynamic molecular picture of the ET process may be obtained. In BLUF (blue light sensors using FAD) photoreceptors, proton-coupled electron transfer between a tyrosine and the flavin cofactor is the key reaction to switch from a dark-adapted to a light-adapted state, which corresponds to the biological signaling state. Particularly puzzling is the fact that, although the various naturally occurring BLUF domains show little difference in the amino acid composition of the flavin binding pocket, the reaction rates of the forward reaction differ quite largely from a few ps up to several hundred ps. In this study, we modified the redox potential of the flavin/tyrosine redox pair by site-directed mutagenesis close to the flavin C2 carbonyl and fluorination of the tyrosine, respectively. We provide information on how changes in the redox potential of either reaction partner significantly influence photoinduced proton-coupled electron transfer. The altered redox potentials allowed us furthermore to experimentally describe an excited state charge transfer intermediately prior to electron transfer in the BLUF photocycle. Additionally, we show that the electron transfer rate directly correlates with the quantum yield of signaling state formation.

Flavoproteins Are Potential Targets for the Antibiotic Roseoflavin in Escherichia coli

The riboflavin analog roseoflavin is an antibiotic produced by Streptomyces davawensis. Riboflavin transporters are responsible for roseoflavin uptake by target cells. Roseoflavin is converted to the flavin mononucleotide (FMN) analog roseoflavin mononucleotide (RoFMN) by flavokinase and to the flavin adenine dinucleotide (FAD) analog roseoflavin adenine dinucleotide (RoFAD) by FAD synthetase. In order to study the effect of RoFMN and RoFAD in the cytoplasm of target cells, Escherichia coli was used as a model. E. coli is predicted to contain 38 different FMN- or FAD-dependent proteins (flavoproteins). These proteins were overproduced in recombinant E. coli strains grown in the presence of sublethal amounts of roseoflavin. The flavoproteins were purified and analyzed with regard to their cofactor contents. It was found that 37 out of 38 flavoproteins contained either RoFMN or RoFAD. These cofactors have different physicochemical properties than FMN and FAD and were reported to reduce or completely abolish flavoprotein function.

The Hydrogen-Bond Switch Reaction of the Blrb Bluf Domain of Rhodobacter sphaeroides

The BlrB protein from Rhodobacter sphaeroides is a small 136 amino acid photoreceptor belonging to the BLUF family of blue light receptors. It contains merely the conserved BLUF fold responsible for binding the flavin pigment and a short C-terminal extension of unknown function. We investigated the primary photoreactions of BlrB by picosecond fluorescence and transient absorption spectroscopy. After excitation of the flavin the fluorescence decays in an H/D isotope independent manner with time constants of 21 and 390 ps, indicating a BLUF characteristic heterogeneous excited state quenched by electron transfer. By transient absorption spectroscopy, we observed a rapid relaxation of a vibrationally hot excited state within 6 ps upon excitation at 400 nm. The relaxed excited state evolves biexponentially with 18 ps (27%) and 216 ps (73%) into the signaling state spectrum indicated by a growing absorptive feature at 492 nm. Additionally, a broad triplet feature is observed as residual absorbance at a delay of 5 ns, which we attribute to derive from a significant fraction of free flavin in the sample. The photochemistry of BlrB is similar to other small BLUF proteins in respect to the fast formation of the photoproduct but does not resolve any further intermediates. We compare the photoreaction with other BLUF proteins on the basis of available spectroscopic data and crystal structures. An arginine close to the C2═O carbonyl of the flavin is likely to be a key determinant for the fast electron transfer in BlrB. Additionally, the orientation of the electron-donating tyrosine in respect to the flavin might play a role in the so far unique kinetic separation of the semiquinonic intermediates in Slr1694.

Molecular eyes: proteins that transform light into biological information

Most biological photoreceptors are protein/cofactor complexes that induce a physiological reaction upon absorption of a photon. Therefore, these proteins represent signal converters that translate light into biological information. Researchers use this property to stimulate and study various biochemical processes conveniently and non-invasively by the application of light, an approach known as optogenetics. Here, we summarize the recent experimental progress on the family of blue light receptors using FAD (BLUF) receptors. Several BLUF photoreceptors modulate second messenger levels and thus represent highly interesting tools for optogenetic application. In order to activate a coupled effector protein, the flavin-binding pocket of the BLUF domain undergoes a subtle rearrangement of the hydrogen network upon blue light absorption. The hydrogen bond switch is facilitated by the ultrafast light-induced proton-coupled electron transfer (PCET) between a tyrosine and the flavin in less than a nanosecond and remains stable on a long enough timescale for biochemical reactions to take place. The cyclic nature of the photoinduced reaction makes BLUF domains powerful model systems to study protein/cofactor interaction, protein-modulated PCET and novel mechanisms of biological signalling. The ultrafast nature of the photoconversion as well as the subtle structural rearrangement requires sophisticated spectroscopic and molecular biological methods to study and understand this highly intriguing signalling process.

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.