J.T.M. Kennis

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.

Proline 68 enhances photoisomerization yield in photoactive yellow protein

In proteins and enzymes, the local environment of an active cofactor plays an important role in controlling the outcome of a functional reaction. In photoactive yellow protein (PYP), it ensures photoisomerization of the chromophore, a prerequisite for formation of a signaling state. PYP is the prototype of a PAS domain, and the preferred model system for the studies of molecular mechanisms of biological light sensing. We investigated the effect of replacing proline-68, positioned near but not in direct contact with the chromophore, with other neutral amino acids (alanine, glycine, and valine), using ultrafast spectroscopy probing the visible and the mid-IR spectral regions, and molecular simulation to understand the interactions tuning the efficiency of light signaling. Transient absorption measurements indicate that the quantum yield of isomerization in the mutants is lower than the yield observed for the wild type. Subpicosecond mid-IR spectra and molecular dynamics simulations of the four proteins reveal that the hydrogen bond interactions around the chromophore and the access of water molecules in the active site of the protein determine the efficiency of photoisomerization. The mutants provide additional hydrogen bonds to the chromophore, directly and by allowing more water molecules access to its binding pocket. We conclude that proline-68 in the wild type protein optimizes the yield of photochemistry by maintaining a weak hydrogen bond with the chromophore, at the same time restraining the entrance of water molecules close to the alkylic part of pCa. This study provides a molecular basis for the structural optimization of biological light sensing.

Femto- to Microsecond Photodynamics of an Unusual Bacteriophytochrome

A bacteriophytochrome from Stigmatella aurantiaca is an unusual member of the bacteriophytochrome family that is devoid of hydrogen bonding to the carbonyl group of ring D of the biliverdin (BV) chromophore. The photodynamics of BV in SaBphP1 wild type and the single mutant T289H reintroducing hydrogen bonding to ring D show that the strength of this particular weak interaction determines excited-state lifetime, Lumi-R quantum yield, and spectral heterogeneity. In particular, excited-state decay is faster in the absence of hydrogen-bonding to ring D, with excited-state half-lives of 30 and 80 ps for wild type and the T289H mutant, respectively. Concomitantly, the Lumi-R quantum yield is two times higher in wild type as compared with the T289H mutant. Furthermore, the spectral heterogeneity in the wild type is significantly higher than that in the T289H mutant. By extending the observable time domain to 25 μs, we observe a new deactivation pathway from the Lumi-R intermediate in the 100 ns time domain that corresponds to a backflip of ring D to the original Pr 15Za isomeric state.

Low-temperature and time-resolved spectroscopic characterization of the LOV2 domain of Avena sativa phototropin 1

The phototropins are plant blue-light receptors that base their light-dependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in light, oxygen or voltage (LOV) domains. The spectroscopic properties of the LOV2 domain of phototropin 1 of Avena sativa (oat) have been investigated by means of low-temperature absorption and fluorescence spectroscopy and by time-resolved fluorescence spectroscopy. The low-temperature absorption spectrum of the LOV2 domain showed a fine structure around 473 nm, indicating heterogeneity in the flavin binding pocket. The fluorescence quantum yield of the flavin cofactor increased from 0.13 to 0.41 upon cooling the sample from room temperature to 77 K. A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K, allowing for an accurate positioning of the flavin triplet state in the LOV2 domain at 16900 cm-1. Fluorescence from the cryotrapped covalent adduct state was extremely weak, with a fluorescence spectrum showing a maximum at 440 nm. Time-resolved fluorescence experiments utilizing a synchroscan streak camera revealed a singlet-excited state lifetime of the LOV2 domain of 2.4 ns. FMN dissolved in aqueous solution showed a pH-dependent lifetime ranging between 2.9 ns at pH 2.0 to 4.7 ns at pH 8.0. No spectral shifting of the flavin emission was observed in the LOV2 domain nor in FMN in aqueous solution.

Short Hydrogen Bonds and Negative Charge in Photoactive Yellow Protein Promote Fast Isomerization but not High Quantum Yield

Biological signal transduction by photoactive yellow protein (PYP) in halophilic purple sulfur bacteria is initiated by trans-to-cis isomerization of the p-coumaric acid chromophore (pCa) of PYP. pCa is engaged in two short hydrogen bonds with protein residues E46 and Y42, and it is negatively charged at the phenolate oxygen. We investigated the role in the isomerization process of the E46 short hydrogen bond and that of the negative charge on the anionic phenolate moiety of the chromophore. We used wild-type PYP and the mutant E46A, in protonated and deprotonated states (referred to as pE46A and dpE46A, respectively), to reduce the number of hydrogen bond interactions between the pCa phenolate oxygen and the protein and to vary the negative charge density in the chromophore-binding pocket. Their effects on the yield and rate of chromophore isomerization were determined by ultrafast spectroscopy. Molecular dynamics simulations were used to relate these results to structural changes in the mutant protein. We found that deprotonated pCa in E46A has a slower isomerization rate as the main part of this reaction was associated with time constants of 1 and 6 ps, significantly slower than the 0.6 ps time constant in wild-type PYP. The quantum yield of isomerization in dpE46A was estimated to be 30 ± 2%, and that of pE46A was 32 ± 3%, very close to the value determined for wtPYP of 32 ± 2%. Relaxation of the isomerized product state I0 to I1 was faster in dpE46A. We conclude that the negative charge on pCa stabilized by the short hydrogen bonds with E46 and Y42 affects the rate of isomerization but not the quantum yield of isomerization. With this information, we propose a scheme for the potential energy surfaces involved in the isomerization and suggest protein motions near the pCa backbone as key events in successful isomerization.

Polarization-controlled optimal scatter suppression in transient absorption spectroscopy

Ultrafast transient absorption spectroscopy is a powerful technique to study fast photo-induced processes, such as electron, proton and energy transfer, isomerization and molecular dynamics, in a diverse range of samples, including solid state materials and proteins. Many such experiments suffer from signal distortion by scattered excitation light, in particular close to the excitation (pump) frequency. Scattered light can be effectively suppressed by a polarizer oriented perpendicular to the excitation polarization and positioned behind the sample in the optical path of the probe beam. However, this introduces anisotropic polarization contributions into the recorded signal. We present an approach based on setting specific polarizations of the pump and probe pulses, combined with a polarizer behind the sample. Together, this controls the signal-to-scatter ratio (SSR), while maintaining isotropic signal. We present SSR for the full range of polarizations and analytically derive the optimal configuration at angles of 40.5° between probe and pump and of 66.9° between polarizer and pump polarizations. This improves SSR by (or compared to polarizer parallel to probe). The calculations are validated by transient absorption experiments on the common fluorescent dye Rhodamine B. This approach provides a simple method to considerably improve the SSR in transient absorption spectroscopy.

The photochemistry of novel flavin-binding photoreceptors

Blue-light sensing using FAD (BLUF) domains are a distinct family of flavin-binding photoreceptors that show no significant relationship to other sensor proteins in sequence or structure. AppA is a two-component protein that can sense blue light at its N-terminal domain, and oxygen tension at its cysteine-rich C-terminal domain, and is shown to control photosynthesis gene expression in response to high-intensity blue-light irradiation and variation of the oxygen tension. This chapter uses femtosecond transient absorption spectroscopy to investigate the photochemical events that lead to the formation of the redshifted signaling state in the AppA and Synechocystis Slr1694 BLUF domains. The time-resolved data are globally analyzed in terms of a kinetic scheme with sequentially interconverting species. The global analysis procedure indicates that six components are required for an adequate description of the time-resolved data.

A simple artificial light harvesting dyad as a mimic of nonphotochemical quenching in green plants

A carotenoid can efficiently quench the Q y energy of phthalocyanine molecule. Target analysis provides evidence for the pivotal role of the carotenoid excited state in the quenching by showing that the spectrum of the quenching species resembles the carotenoid S 1 spectrum. However, energy transfer involving the carotenoid S 1 state alone cannot be solely responsible for the quenching because the process is solvent polarity dependent. This chapter performs a transient absorption measurement on a model carotenoid with 10 double bonds to gain further insights into the process. Solvent polarity-dependent shape changes that cannot be ascribed to the Sl state are detected. Similar changes are reported for several substituted carotenoids and assigned to an intramolecular charge transfer state. Results show that carotenoids can quench tetrapyrrole singlet excited states by means of energy transfer to optically forbidden carotenoid states. Expanding the conjugated system of the carotenoid by one double bond turns the carotenoid from a nonquencher into a strong quencher.