A subgroup of light-driven sodium pumps with an additional Schiff base counterion.

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

Two-photon absorption enhancement for organic acceptor molecules with QD antennas.

The photophysics of an inorganic/organic hybrid system was studied by time-resolved optical spectroscopy, focusing on the goal of increasing the two-photon efficiency of photoresponsive systems. The hybrid system consists of CdS/ZnS core/shell quantum dots (QDs) as energy donor and coumarin derivatives as energy acceptor molecules. The spectral overlap of QD emission and coumarin 343 absorption promotes a Förster resonance energy tranfer (FRET) mechanism leading to a FRET efficiency up to nearly 90%. Additionally, time-correlated single photon counting showed a faster fluorescence decay while acceptor molecules were attached to the QD surface. Femtosecond transient absorption measurements demonstrated an ultrafast FRET reaction. Importantly, FRET was observed also after two-photon excitation of the QDs indicating that the chosen QDs can act as two-photon antennas.

Cyano-tryptophans as dual infrared and fluorescence spectroscopic labels to assess structural dynamics in proteins.

The steady state and time-resolved fluorescence and infrared (IR) properties of 4- and 5-cyanotryptophan (CNTrp) are investigated and compared, and the tryptophan (Trp) analogs are found to be very attractive to study structural and dynamic properties of proteins. The position of the nitrile substitution as well as the solvent environment influences the spectroscopic properties (solvatochromism). Similar to native Trp, electronic (nanosecond) lifetime and emission spectra are modulated by the environment, making CNTrps attractive fluorescent probes to study the structural dynamics of proteins in complex media. The nitrile absorption in the IR region can provide local structural information as it responds sensitively to changes in electrostatics and hydrogen bond (HB) interactions. Importantly, we find that 4CNTrp exhibits a single absorption in the nitrile stretch region, while the model compound 4CN-indole (4CNI) shows two. Even though the spectrum of the model compound is perturbed by a Fermi resonance, we find that 4CNTrp itself is a useful IR label. Moreover, if the nitrile group is substituted at the 5 position, the Trp analog predominantly reports on its HB status. Because the current literature on similar compounds is too limited for a detailed solvatochromic analysis, we extend the available data significantly. Only now are microscopic details such as the mentioned sensitivity to electrostatics coming to light. The vibrational lifetime of the CN moiety (acting on a picosecond time scale in contrast to the nanosecond time scale for fluorescent emission) allows for its application in 2D-IR spectroscopy in the low picosecond range. Taken together, the benefits of CNTrps are that they absorb and emit separately from the naturally occurring Trp and that in these dual fluorescence/vibrational labels, observables of IR and fluorescence spectroscopy are modulated differently by their surroundings. Because IR absorption and fluorescence operate on different time and length scales, they thus provide complementary structural information.

A thin CdSe shell boosts the electron transfer from CdTe quantum dots to methylene blue.

CdTe core and CdTe/CdSe core/shell quantum dots (QD) are investigated with steady state and time-resolved spectroscopic methods. The coating of the CdTe core with a 0.7 nm thick CdSe shell shifts the lowest exciton absorption band to the red by more than 70 nm making the CdTe/CdSe QD an interesting candidate for application in solar energy conversion. Femtosecond transient absorption measurements are applied to study the photoinduced electron transfer (ET) to the molecular acceptor methylene blue (MB). ET times after single excitation of the QD are determined for different MB : QD ratios. The ET reaction is significantly faster in the case of the MB-CdTe/CdSe QD complexes, indicative of an altered charge distribution in the photoexcited heterostructure with a higher electron density in the CdSe shell. As a result of the efficient absorption of incoming light and the faster ET reaction, the amount of reduced MB in the time resolved experiments is higher for CdTe/CdSe QD compared to CdTe QD.

Light-induced antibiotic release from a coumarin-caged compound on the ultrafast timescale.

A synthesis route for puromycin caged with the photo-responsive 7-diethylaminocoumarinyl protecting group carbamate was developed. The inactivation and recovery of the cytotoxic effect of puromycin was tested with a XTT cell viability assay. The uncaging mechanism was studied by ultrafast transient absorption spectroscopy and by time-correlated single photon counting. The combination of these results with quantum-chemical calculations provided detailed insights in dynamics upon excitation. Interestingly, a change of the dipole moment due to structural rearrangements of the amino moiety led to an intermolecular charge transfer on the picosecond time-scale. IR measurements marked the successful uncaging via the release of CO2, resulting from the carbamate linker. This decarboxylation constituted the rate-limiting step of the uncaging reaction and occurred on the subsecond timescale. DEACM-puromycin, thus, represents an efficient photo-activatable antibiotic for in-cell applications.

Connectivity pattern modifies excited state relaxation dynamics of fluorophore-photoswitch molecular dyads.

In order to modulate the emission of BODIPY fluorophores, they were connected to a diarylethene (DAE) photoswitch via phenylene-ethynylene linkers of different lengths and orientations. The latter allowed for modulation of the electronic coupling in the prepared four BODIPY-DAE dyads, which were compared also to appropriate BODIPY and DAE model compounds by steady state as well as time-resolved spectroscopies. In their open isomers, all dyads show comparable luminescence behavior indicative of an unperturbed BODIPY fluorophore. In strong contrast, in the closed isomers the BODIPY emission is efficiently quenched but the deactivation mechanism depends on the nature of the linker. The most promising dyad was rendered water-soluble by means of micellar encapsulation and aqueous suspensions were investigated by fluorescence spectroscopy and microscopy. Our results (i) illustrate that the electronic communication between the BODIPY and DAE units can indeed be fine-tuned by the nature of the linker to achieve fluorescence modulation while maintaining photoswitchability and (ii) highlight potential applications to image and control biological processes with high spatio-temporal resolution.

The ultrafast reactions in the photochromic cycle of water-soluble fulgimide photoswitches.

Photochromic switches are essential for the control and manipulation of nanoscale reactions and processes. The expansion of their application to aqueous environments depends strongly on the development of optimized water-soluble photoswitches. Here we present a femtosecond time-resolved investigation of the photochromic reactions (transition between the open and the closed form) of a water-soluble indolylfulgimide. We observe a pronounced effect of the protic nature of water as a solvent on the ultrafast ring-opening reaction. Typically, the excited state of the closed form has a larger dipole moment than the ground state, which leads to stabilization of the excited state in polar solvents and hence a lifetime (3 ps) longer than in non-polar solvents (2 ps). However, in water, despite the increased solvent polarity and the increased excited state dipole moment, the opposite trend for the excited state lifetime is observed (1.8 ps). This effect is caused by the opening of a new excited state deactivation pathway involving proton transfer reactions.

Ultrafast coherent oscillations reveal a reactive mode in the ring-opening reaction of fulgides.

The ultrafast ring-opening reaction of photochromic fulgides proceeds via conical intersections to the ground state isomers involving activation barriers in the excited state. The coherent oscillations observed in the femtosecond transient absorption signal of a methyl-substituted indolylfulgide were analysed in the framework of vibrational wavepackets to expose a dominant low-frequency mode at ∼80 cm(-1). The quantum chemical calculations in the relaxed excited state geometry of this fulgide revealed that the experimentally observed vibrational normal mode has a dominant contribution to the relevant ring-opening reactive coordinate.

Photo-physical properties of 2-(1-ethynylpyrene)-adenosine: influence of hydrogen bonding on excited state properties.

The photo-physical properties of 2-(1-ethynylpyrene)-adenosine (PyA), a fluorescent probe for RNA dynamics, were examined by solvation studies. The excited-state dynamics display the influence of the vicinity on the spectral features. Combining improved transient absorption and streak camera measurements along with a new analysis method provide a detailed molecular picture of the photophysics. After intramolecular vibrational energy redistribution (IVR), two distinct states are observed. Solvent class (protic/aprotic) and permittivity strongly affect the properties of these states and their population ratio. As a result their emission spectrum is altered, while the fluorescence quantum yield and the overall lifetime remain nearly unchanged. Consequently, the hitherto existing model of the photophysics is herein refined and extended. The findings can serve as basis for improving the information content of measurements with PyA as a label in RNA.

Coherent longitudinal-optical ground-state phonon in CdSe quantum dots triggered by ultrafast charge migration.

We observe the CdSe longitudinal-optical ground-state phonon in the electron transfer system composed of CdSe quantum dots and methylviologen directly by femtosecond absorption spectroscopy. A significant phase shift indicates that the coherent oscillations are triggered by an ultrafast charge migration, which is the consequence of an electron transfer from the photoexcited quantum dot to the molecular acceptor methylviologen. In contrast, the observed coherent phonons in isolated quantum dots stem from the frequency modulation of the quantum dot excited-state spectrum. From the probe wavelength dependence of the longitudinal-optical phonons in the electronic ground state and excited state it is possible to determine a biexciton binding energy of 35 meV.

Observation of pH-dependent back-electron-transfer dynamics in alizarin/TiO2 adsorbates: importance of trap states.

The dependence of the interfacial electron transfer in alizarin-sensitized TiO2 nanoparticles on the sample pH has been examined via transient absorbance spectroscopy in the visible spectral region (443-763 nm). Excitation of the alizarin/TiO2 system with visible pump pulses (lambdaexc = 500 nm) leads to a very fast electron injection (tauinj < 100 fs) over a wide pH range. Back electron transfer shows complicated multiphasic kinetics and strongly depends on the acidity of the solution. The strong dependence of back-electron-transfer dynamics on the ambient pH value is explained by a Nernstian-type change in the semiconductor band energy. Indeed, a variation of pH values over 7 units leads to a approximately 0.42 eV change of the conduction band edge position (i.e., the nominal free energy of the electron in the electrode). Assuming a pH-independent redox potential of the dye, this change was sufficient to push the system to a condition where direct photoinitiated electron injection to intraband gap surface states could be investigated. The existence of an electron-transfer pathway via surface trap states is supported by the similarity of the observed back-electron-transfer kinetics of alizarin/TiO2 at pH 9 and alizarin/ZrO2 reported in earlier work (J. Phys. Chem. B 2000, 104, 8995), where the conduction band edge is approximately 1 eV above the excited state of the dye. The influence of surface trap states on interfacial electron transfer has been studied, and a detailed analysis of their population, depopulation, and relaxation kinetics is performed. Therefore, alizarin adsorbed on the surface of TiO2 nanoparticles is an ideally suited system, where pH-dependent investigations allow a detailed study of the electron dynamics in trap states of TiO2 nanoparticles.

Ultrafast conformational dynamics in cyclic azobenzene peptides of increased flexibility.

Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the cis to trans and the trans to cis photoreactions. For the cis to trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the trans to cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps-1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10 ns.

Primary reactions of sensory rhodopsins.

The first steps in the photocycles of the archaeal photoreceptor proteins sensory rhodopsin (SR) I and II from Halobacterium salinarum and SRII from Natronobacterium pharaonis have been studied by ultrafast pump/probe spectroscopy and steady-state fluorescence spectroscopy. The data for both species of the blue-light receptor SRII suggests that their primary reactions are nearly analogous with a fast decay of the excited electronic state in 300-400 fs and a transition between two red-shifted product states in 4-5 ps. Thus SRII behaves similarly to bacteriorhodopsin. In contrast for SRI at pH 6.0, which absorbs in the orange part of the spectrum, a strongly increased fluorescence quantum yield and a drastically slower and biexponential decay of the excited electronic state occurring on the picosecond time scale (5 ps and 33 ps) is observed. The results suggest that the primary reactions are controlled by the charge distribution in the vicinity of the Schiff base and demonstrate that there is no direct connection between absorption properties and reaction dynamics for the retinal protein family.

Photomodulation of conformational states. I. Mono- and bicyclic peptides with (4-amino)phenylazobenzoic acid as backbone constituent.

The thioredoxin reductase active-site fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141], which is known for its high tendency to assume an almost identical conformation as in the intact enzyme, was backbone cyclized with the photoresponsive (4-amino)phenylazobenzoic acid (APB) to produce a monocyclic and disulfide-bridged bicyclic APB-peptide. Light-induced reversible cis/trans isomerization occurs at identical extents in both the linear and the two cyclic forms. Nuclear magnetic resonance conformational analysis clearly revealed that in the bicyclic APB-peptide both as a trans- and cis-azo-isomer the constraints imparted by the bicyclic structure do not allow the molecule to relax into a defined low energy conformation, thus making the molecule a frustrated system that flip-flops between multiple conformational states. Conversely, the monocyclic APB peptide folds into a well-defined lowest energy structure as a trans-azo-isomer, which upon photoisomerization to the cis-azo configuration relaxes into a less restricted conformational space. First femtosecond spectroscopic analysis of the dynamics of the photoreaction confirm a fast first phase on the femtosecond time scale related to the cis/trans isomerization of the azobenzene moiety followed by a slower phase in the picosecond time scale that involves an adjustment of the peptide backbone. Due to the well- defined photoresponsive two-state transition of this monocyclic peptide molecule, it represents a model system well suited for studying the ultrafast dynamics of conformational transitions by time-resolved spectroscopy.

Identification of a hydrogen bond in the phe M197-->Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X-ray crystallography and FTIR spectroscopy.

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X-ray structure analysis of the Phe M197-->Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875-12886, 1993] between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the pi-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.

Photomodulation of the Conformation of Cyclic Peptides with Azobenzene Moieties in the Peptide Backbone.

The cisright harpoon over left harpoon trans photoisomerization of the azobenzene building block 4-(4-aminophenylazo)benzoic acid incorporated in a cyclic peptide (see scheme) facilitated a two-state transition of the peptide chain from a rigid constrained conformation in the trans isomer into the largely free conformational space of the cis isomer.

Electron transfer dynamics of Rhodopseudomonas viridis reaction centers with a modified binding site for the accessory bacteriochlorophyll.

Femtosecond spectroscopy in combination with site-directed mutagenesis was used to study the influence of histidine L153 in primary electron transfer in the reaction center of Rhodopseudomonas viridis. Histidine was replaced by cysteine, glutamate, or leucine. The exchange to cysteine did not lead to significant changes in the primary reaction dynamics. In the case of the glutamate mutation, the decay of the excited electronic level of the special pair P* is slowed by a factor of 3. The exchange to leucine caused the incorporation of a bacteriopheophytin b instead of a bacteriochlorophyll b molecule at the BA site. As a consequence of this chromophore exchange, the energy level of the electron transfer state P+BA- is lowered to such an extent that repopulation from the next electron transfer intermediate state P+HA- takes place, resulting in a long-lasting P+BA- population. The observed differences in time constants are discussed in the scope of nonadiabatic electron transfer theory considering the influence of the amino acids at position L153 and the chromophore exchange on the energy level of the intermediate state P+BA-. The results show that the high efficiency of primary electron transfer is reduced substantially, if the energy level of P+BA- is lowered or raised by several hundred wave numbers.

Electron transfer from the tetraheme cytochrome to the special pair in the Rhodopseudomonas viridis reaction center: effect of mutations of tyrosine L162.

The structure of the photosynthetic reaction center (RC) from Rhodopseudomonas viridis is known to high resolution. It contains a firmly bound tetraheme cytochrome from which electrons are donated to a special pair (P) of bacteriochlorophylls, which is photooxidized upon absorption of light. Tyrosine at position 162 of the L-subunit of the reaction center (L 162 Y) is a highly conserved residue positioned halfway between P and the proximal heme group (c-559) of the cytochrome. By specific mutagenesis this residue was exchanged against the amino acids phenylalanine (F), glycine (G), methionine (M), leucine (L), tryptophan (W), threonine (T), and histidine (H). All mutants were expressed in Rps. viridis using a recently established transformation system [Laussermair & Oesterhelt (1992) EMBO J. 11, 777-783]. They were shown biochemically to synthesize all four subunits of the RC (cytochrome, subunits L, M, and H) and to assemble them correctly into the membrane. The structures of two mutants (L 162 F and L 162 T) were determined and found not to differ significantly from the wild-type structure. All mutants grew photosynthetically. The absorption spectrum of all the mutants is the same as in WT, but the redox potential of P and of c-559 was changed by the mutations. The kinetics of electron transfer from the heme group to the special pair were measured in chromatophores by flash absorption. As found earlier in the wild type (Y) several exponential components were needed to fit the data.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure, spectroscopic, and redox properties of Rhodobacter sphaeroides reaction centers bearing point mutations near the primary electron donor.

Single mutations of three amino acid residues in the vicinity of the primary electron donor, P, in the reaction center (RC) from Rhodobacter (Rb.) sphaeroides were constructed and characterized in order to study the effects of hydrogen-bonding on the physical properties of P. The mutations, Phe M197-->Tyr, Met L248-->Thr, and Ser L244-->Gly, represent single amino acid changes near P designed to introduce residues found in Rhodopseudomonas (Rps.) viridis and to, thus, probe the effects of nonconserved residues. The mutations were designed to change the nonconserved H-bonding interactions of P in Rb. sphaeroides, at the level of a C2 acetyl, a C9 keto, and a C10 ester carbonyl of P, respectively, to those present in Rps. viridis. The Fourier transform (pre)resonance Raman (FTRR) spectra of P, in its reduced and oxidized states, from reaction centers of these mutants were studied to determine modifications of H-bond interactions of the pi-conjugated C2 acetyl and C9 keto carbonyl groups and the C10 carbomethoxy ester carbonyl groups of P. The vibrational spectra of reduced P in the Met L248-->Thr and Ser L244-->Gly mutants reveal no evidence for changes in the H-bonding pattern of P; this suggests that for Rb. sphaeroides wild type, Ser L244 is not H-bonded to the C10 ester carbonyl of PL. The vibrational spectrum of reduced P from the Phe M197-->Tyr mutant compared to that of wild type can unambiguously be interpreted in terms of the formation of a new H-bond with an acetyl carbonyl of P, specifically PM. Correlating with the new H-bond, the Phe M197-->Tyr mutant exhibits an electronic absorption spectrum where the P absorption band is significantly perturbed. Intact cell and chromatophore photobleaching spectra of the same mutant indicate that the P absorption band has red-shifted by ca. 10 nm; no such behavior is observed for the other mutants. As well, the P-->BPheL electron transfer rate does not seem to strongly depend on the H-bonding of the C2 acetyl carbonyl of PM to a tyrosine residue. The EPR zero-field splitting parameters, E and D, of the primary donor triplet are only slightly modified in the mutant reaction centers, on the order of 1%.(ABSTRACT TRUNCATED AT 400 WORDS)

Tyrosine 162 of the photosynthetic reaction center L-subunit plays a critical role in the cytochrome c2 mediated rereduction of the photooxidized bacteriochlorophyll dimer in Rhodobacter sphaeroides. 1. Site-directed mutagenesis and initial characterization.

Five site-directed mutants were engineered to substitute phenylalanine, serine, leucine, methionine, and glycine for tyrosine residue 162 of the pufL gene in Rhodobacter (R.) sphaeroides. Each of the mutations and the wild-type (WT) genes was expressed in the R. sphaeroides puf deletion strain PUF delta LMX21/3. Initial characterization revealed that all of the mutants were photoheterotrophically competent but that L162G and L162S were impaired. The amounts of mutant reaction centers expressed, the spectral characteristics, and the rates of intraprotein electron transfer and turnover were similar to the values obtained for WT. Kinetic measurements of photooxidized special pair rereduction mediated by the physiological donor cytochrome c2 in intact chemoheterotrophically grown cells revealed that the fast phase was abolished in all mutants and that the overall kinetics of rereduction was drastically slowed. It is concluded that L162Y plays a vital role in facilitating the rapid rereduction of the photooxidized bacteriochlorophyll dimer in R. sphaeroides.