Rolf Diller

Ultrafast dynamics of phytochrome from the cyanobacterium synechocystis, reconstituted with phycocyanobilin and phycoerythrobilin.

Femtosecond time-resolved transient absorption spectroscopy was employed to characterize for the first time the primary photoisomerization dynamics of a bacterial phytochrome system in the two thermally stable states of the photocycle. The 85-kDa phytochrome Cph1 from the cyanobacterium Synechocystis PCC 6803 expressed in Escherichia coli was reconstituted with phycocyanobilin (Cph1-PCB) and phycoerythrobilin (Cph1-PEB). The red-light-absorbing form Pr of Cph1-PCB shows an approximately 150 fs relaxation in the S(1) state after photoexcitation at 650 nm. The subsequent Z-E isomerization between rings C and D of the linear tetrapyrrole-chromophore is best described by a distribution of rate constants with the first moment at (16 ps)(-1). Excitation at 615 nm leads to a slightly broadened distribution. The reverse E-Z isomerization, starting from the far-red-absorbing form Pfr, is characterized by two shorter time constants of 0.54 and 3.2 ps. In the case of Cph1-PEB, double-bond isomerization does not take place, and the excited-state lifetime extends into the nanosecond regime. Besides a stimulated emission rise time between 40 and 150 fs, no fast relaxation processes are observed. This suggests that the chromophore-protein interaction along rings A, B, and C does not contribute much to the picosecond dynamics observed in Cph1-PCB but rather the region around ring D near the isomerizing C(15) [double bond] C(16) double bond. The primary reaction dynamics of Cph1-PCB at ambient temperature is found to exhibit very similar features as those described for plant type A phytochrome, i.e., a relatively slow Pr, and a fast Pfr, photoreaction. This suggests that the initial reactions were established already before evolution of plant phytochromes began.

Ultrafast Infrared Spectroscopy of Riboflavin: Dynamics, Electronic Structure, and Vibrational Mode Analysis

Femtosecond time-resolved infrared spectroscopy was used to study the vibrational response of riboflavin in DMSO to photoexcitation at 387 nm. Vibrational cooling in the excited electronic state is observed and characterized by a time constant of 4.0 +/- 0.1 ps. Its characteristic pattern of negative and positive IR difference signals allows the identification and determination of excited-state vibrational frequencies of riboflavin in the spectral region between 1100 and 1740 cm (-1). Density functional theory (B3LYP), Hartree-Fock (HF) and configuration interaction singles (CIS) methods were employed to calculate the vibrational spectra of the electronic ground state and the first singlet excited pipi* state as well as respective electronic energies, structural parameters, electronic dipole moments and intrinsic force constants. The harmonic frequencies of the S 1 excited state calculated by the CIS method are in satisfactory agreement with the observed band positions. There is a clear correspondence between computed ground- and excited-state vibrations. Major changes upon photoexcitation include the loss of the double bond between the C4a and N5 atoms, reflected in a downshift of related vibrations in the spectral region from 1450 to 1720 cm (-1). Furthermore, the vibrational analysis reveals intra- and intermolecular hydrogen bonding of the riboflavin chromophore.

Photochemical quantum yield of bacteriorhodopsin from resonance Raman scattering as a probe for photolysis.

Abstract Bacteriorhodopsin (BR) is a retinal-binding protein in the purple membrane of Halobacteria where it acts as a light-driven proton pump. The quantum yield, γ B , of the primary photoreaction of BR which initiates a cyclic reaction of the chromophore was measured. A liquid suspension of purple membranes was flown across a cw laser beam where the BR chromophore was partially photolyzed. After the sample had left the photolysis beam it was probed by recording resonance Raman spectra in the region of the strong and characteristic bands of the retinal chromophores (1500–1600 cm −1 ). The ethylenic band of the parent species at 1529 cm −1 could be separated with good accuracy from those of its photoproducts by fitting procedures. In this way the relative intensity decrease of the 1529 cm −1 band was monitored as a function of the power of the photolysis beam. From the quantitative relation between these two quantities which besides γ B still contains the intensity profile of the photolysis laser, the velocity of the flowing sample and the extinction coefficient of the chromophore, the quantum yield could be deduced. From photolysis experiments at 502, 514, 568 and 647 nm, a mean value of 0.67 was obtained for γ B . Combining this value with literature data for the quantum yield of proton release from purple membranes it was concluded that on the average not more than one proton is translocated across the membrane per each photochemical reaction cycle of BR. Our value of 0.67 for γ B is of the same magnitude as the quantum yield for the primary photochemical reaction of the retinal chromophore in rhodopsin which initiates the visual process in photoreceptor cells of vertebrates.

Subpicosecond Midinfrared Spectroscopy of the Pfr Reaction of Phytochrome Agp1 from Agrobacterium tumefaciens

Phytochromes are light-sensing pigments found in plants and bacteria. For the first time, the P(fr) photoreaction of a phytochrome has been subject to ultrafast infrared vibrational spectroscopy. Three time constants of 0.3 ps, 1.3 ps, and 4.0 ps were derived from the kinetics of structurally specific marker bands of the biliverdin chromophore of Agp1-BV from Agrobacterium tumefaciens after excitation at 765 nm. VIS-pump-VIS-probe experiments yield time constants of 0.44 ps and 3.3 ps for the underlying electronic-state dynamics. A reaction scheme is proposed including two kinetic steps on the S(1) excited-state surface and the cooling of a vibrationally hot P(fr) ground state. It is concluded that the upper limit of the E-Z isomerization of the C(15) = C(16) methine bridge is given by the intermediate time constant of 1.3 ps. The reaction scheme is reminiscent of that of the corresponding P(r) reaction of Agp1-BV as published earlier.

FEMTOSECOND TIME-RESOLVED INFRARED LASER STUDY OF THE J-K TRANSITION OF BACTERIORHODOPSIN

Abstract The J − K transition of the bacteriorhodopsin photocycle was monitored by sub-picosecond time-resolved infrared spectroscopy. IR difference spectra in the region between 1670 and 1600 cm −1 were taken at 1.5 and 9 ps, respectively, after photoexcitation of BR 570 at 540 nm. Spectral shifts of the bands at 1607 and 1661 cm −1 reflect the chromophoric conformational changes during the J to K transition. Kinetics, taken at 1640 and 1607 cm −1 show rise times determined by the dephasing times of the vibrational modes. The partial decrease of the bleach signal at 1640 cm −1 is interpreted as a recovery of the vibrationally cooled BR 570 electronic ground state and provides a new method to measure the photocycle quantum yield. The development of the bleach at 1661 cm −1 occurred faster than 500 fs, suggesting an almost instantaneous protein response to the electronic excitation.

Resonance Raman study of intermediates of the halorhodopsin photocycle

The resonance Raman (RR) study of the retinal protein halorhodopsin (HR578) was extended to two of its photoproducts: HR and HRL 410 RR spectra of both species were recorded in H2O and D2O and compared with the RR spectra of the intermediates L550 and M412 from the bacteriorhodopsin photocycle. HR520 was found to be a protonated Schiff base in the 13‐cis configuration and HRL 410 a deprotonated Schiff base in the 13‐cis configuration.

Ultrafast infrared spectroscopy of bacteriorhodopsin.

Picosecond infrared spectroscopy is developed and used for the first time to study the dynamics of photoexcited bacteriorhodopsin (BR). Both spectral and time-resolved data are obtained. The results open an entirely new approach to investigations of the BR photocycle. The infrared difference spectrum (K minus BR570) recorded at ambient temperature between 1,560 and 1,700 cm-1 is not identical with the spectrum reported for a frozen sample. Three bands of the K state at 1,622, 1,610, and 1,580 cm-1 and the bleaching at 1,637 cm-1 (C = NH stretch) are seen. These new spectral lines appear in less than 10 ps.

Unusual spectral properties of bacteriophytochrome Agp2 result from a deprotonation of the chromophore in the red-absorbing form Pr.

Background: Typical phytochromes include a protonated chromophore in the parent states (Pr and Pfr) that transiently deprotonates during photoconversion. Results: In Agp2, the pKa of the chromophore is lowered from >11 to 7.6 during the conversion from Pfr to Pr. Conclusion: Chromophore protonation affects light-induced and thermal Pr to Pfr conversion. Significance: Agp2 can act as integrated light and pH sensor. Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pKa is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pKa shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.

ESIPT and Photodissociation of 3-Hydroxychromone in Solution: Photoinduced Processes Studied by Static and Time-Resolved UV/Vis, Fluorescence, and IR Spectroscopy

The spectral properties of fluorescence sensors such as 3-hydroxychromone (3-HC) and its derivatives are sensitive to interaction with the surrounding medium as well as to substitution. 3-HC is a prototype system for other derivatives because it is the basic unit of all flavonoides undergoing ESIPT and is not perturbed by a substituent. In this study, the elementary processes and intermediate states in the photocycle of 3-HC as well as its anion were identified and characterized by the use of static and femtosecond time-resolved spectroscopy in different solvents (methylcyclohexane, acetonitrile, ethanol, and water at different pH). Electronic absorption and fluorescence spectra and lifetimes of the intermediate states were obtained for the normal, tautomer and anionic excited state, while mid-IR vibrational spectra yielded structural information on ground and excited states of 3-HC. A high sensitivity on hydrogen-bonding perturbations was observed, leading to photoinduced anion formation in water, while in organic solvents, different processes are suggested, including slow picosecond ESIPT and contribution of the trans-structure excited state or a different stable solvation state with different direction of OH. The formation of the latter could be favored by the lack of a substituent increasing contact points for specific solute-solvent interactions at the hydroxyl group compared to substituted derivatives. The effect of substituents has to be considered for the design of future fluorescence sensors based on 3-HC.

The yrast bands in 77Kr and 76Kr

Abstract High-spin states of both parities in 77 Kr have been studied in the reaction 63 Cu( 16 O, pn). On the basis of γγ- and nγ-coincidence experiments, we extended the yrast bands up to spin I π = 25 2 + and 21 2 t- . Doppler-shift attenuation and recoil-distance measurements have been performed for some fifteen states. In addition, lifetimes of some yrast states in 76 Kr have been determined in the reaction 63 Cu( 16 O, p2n). Transition energies and E2 strengths in 76 Kr have been interpreted with the IBA-2 and different collective and microscopic triaxial rotor models, those in 77 Kr with the triaxial rotor plus quasiparticle model.