Miroslav Kloz

Carotenoid Photoprotection in Artificial Photosynthetic Antennas

A series of phthalocyanine-carotenoid dyads in which a phenylamino group links a phthalocyanine to carotenoids having 8-11 backbone double bonds were examined by visible and near-infrared femtosecond pump-probe spectroscopy combined with global fitting analysis. The series of molecules has permitted investigation of the role of carotenoids in the quenching of excited states of cyclic tetrapyrroles. The transient behavior varied dramatically with the length of the carotenoid and the solvent environment. Clear spectroscopic signatures of radical species revealed photoinduced electron transfer as the main quenching mechanism for all dyads dissolved in a polar solvent (THF), and the quenching rate was almost independent of carotenoid length. However, in a nonpolar solvent (toluene), quenching rates displayed a strong dependence on the conjugation length of the carotenoid and the mechanism did not include charge separation. The lack of any rise time components of a carotenoid S(1) signature in all experiments in toluene suggests that an excitonic coupling between the carotenoid S(1) state and phthalocyanine Q state, rather than a conventional energy transfer process, is the major mechanism of quenching. A pronounced inhomogeneity of the system was observed and attributed to the presence of a phenyl-amino linker between phthalocyanine and carotenoids. On the basis of accumulated work on various caroteno-phthalocyanine dyads and triads, we have now identified three mechanisms of tetrapyrrole singlet excited state quenching by carotenoids in artificial systems: (i) Car-Pc electron transfer and recombination; (ii)(1) Pc to Car S(1) energy transfer and fast internal conversion to the Car ground state; (iii) excitonic coupling between (1)Pc and Car S(1) and ensuing internal conversion to the ground state of the carotenoid. The dominant mechanism depends upon the exact molecular architecture and solvent environment. These synthetic systems are providing a deeper understanding of structural and environmental effects on the interactions between carotenoids and tetrapyrroles and thereby better defining their role in controlling natural photosynthetic systems.

Wavelength-modulated femtosecond stimulated raman spectroscopy—approach towards automatic data processing

A new wavelength modulator based on a custom-made chopper blade and a slit placed in the Fourier plane of a pulse shaper was used to detect explicitly the first derivative of the time-resolved femtosecond stimulated Raman spectroscopy (FSRS) signals. This approach resulted in an unprecedented reduction of the non-coherent background that results from population transfer by the Raman pump inherent to FSRS experiments. The method of Fourier peak filtering was implemented as a powerful tool for reducing both the remaining non-coherent and coherent background associated with FSRS experiments. The method was demonstrated on β-carotene and a similar synthetic aryl carotenoid. The experiments confirm earlier FSRS results on β-carotene but suggest some reinterpretation. Strong bleaching signals of ground state vibrations were observed and interpreted as an inseparable part of the time-resolved FSRS experiment. New long-lived Raman features were observed in β-carotene and the synthetic aryl carotenoid and assigned to a combination of conformational changes and solvent rearrangement. More complex wavelength modulation methods are proposed in the development of more robust FSRS experiments.

Proton-Coupled Electron Transfer Constitutes the Photoactivation Mechanism of the Plant Photoreceptor UVR8.

UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used as a versatile optogenetic tool. Instead of an exogenous chromophore, UVR8 uniquely employs tryptophan side chains to accomplish UV-B photoreception. UV-B absorption by homodimeric UVR8 induces monomerization and hence signaling, but the underlying photodynamic mechanisms are not known. Here, by using a combination of time-resolved fluorescence and absorption spectroscopy from femto- to microseconds, we provide the first experimental evidence for the UVR8 molecular signaling mechanism. The results indicate that tryptophan residues at the dimer interface engage in photoinduced proton coupled electron transfer reactions that induce monomerization.

Triplet–triplet annihilation upconversion followed by FRET for the red light activation of a photodissociative ruthenium complex in liposomes

Three molecules in a single lipid bilayer to trigger high-energy photochemistry with low-energy photons.

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.

Molecular Origin of Photoprotection in Cyanobacteria Probed by Watermarked Femtosecond Stimulated Raman Spectroscopy

Photoprotection is fundamental in photosynthesis to avoid oxidative photodamage upon excess light exposure. Excited chlorophylls (Chl) are quenched by carotenoids, but the precise molecular origin remains controversial. The cyanobacterial HliC protein belongs to the Hlip family ancestral to plant light-harvesting complexes, and binds Chl a and β-carotene in 2:1 ratio. We analyzed HliC by watermarked femtosecond stimulated Raman spectroscopy to follow the time evolution of its vibrational modes. We observed a 2 ps rise of the C=C stretch band of the 2Ag– (S1) state of β-carotene upon Chl a excitation, demonstrating energy transfer quenching and fast excess-energy dissipation. We detected two distinct β-carotene conformers by the C=C stretch frequency of the 2Ag– (S1) state, but only the β-carotene whose 2Ag– energy level is significantly lowered and has a lower C=C stretch frequency is involved in quenching. It implies that the low carotenoid S1 energy that results from specific pigment–protein or pigment–pigment interactions is the key property for creating a dissipative energy channel. We conclude that watermarked femtosecond stimulated Raman spectroscopy constitutes a promising experimental method to assess energy transfer and quenching mechanisms in oxygenic photosynthesis.

Spectral watermarking approach to femtosecond Raman spectroscopy - ultrafast Raman spectroscopy with broadband shaped pulses

A new method for recording Raman spectra was developed that dramatically improves the recovery of signal pulses and automates the reduction of baseline problems. Instead of using a narrowband Raman source, an experiment is performed using shaping of a broadband source or a manifold of narrowband sources. This allows locking of the signal in specific structures using carefully crafted watermarks that can be recovered from the measured data with high fidelity. This approach uses unique properties of Raman scattering, thus allowing a direct recording of Raman signals free of fluorescence and fixed-pattern-noise. Low cost technology for generating the required pulse-shapes was developed and demonstrated. The methodology is applicable for any Raman experiment but primarily targets ultrafast Raman experiments (Femtosecond stimulated Raman spectroscopy) where a lack of robust methods for parasitic signal rejection has been the major obstacle in the practical development of the field in the last decade.

Femtosecond stimulated Raman spectroscopy in 1D and 2D — Direct observation of intramolecular motions and intermolecular interactions

Summary form only given. It is exactly half century now since the discovery of stimulated Raman scattering (SRS) (1). Despite numerous proof-of-the-principle experiments it is only about a decade since the mechanism got a general analytical use through the special phenomena called femtosecond stimulated Raman scattering (FSRS). When two strongly different light pulses are time spatially overlapped in a sample, one spectrally ultra narrow and one ultra short in time, the entire Raman spectra of the sample are imprinted on the broad spectral envelope of the ultra short pulse with a high signal gain. While the spectral resolution is determined by the narrow pulse the time gating precision is set by the ultrafast pulse so the time-energy resolution is no longer bound by the time-bandwidth uncertainty principle. This mechanism was recently successfully harnessed in mapping the fastest know bio reactions (2, 3) yet robust FSRS experiment is still under development (4). The supreme time resolution of FSRS opened the door for a fully coherent time domain 2D-Raman experiments (5) where the vibrational motion is mapped beyond the period of single oscillation. After initial optimism 2D Raman signals were discovered to be strongly overwhelmed by parasitic cascading signals (6) and at the moment it is unclear if the problem is fully solvable. Frequency domain 2D Raman experiments were proposed as well and their applicability is currently being evaluated. Never the less 1D Raman techniques are already obtaining strong recognition as an irreplaceable toll for studding of vibrations with low IR cross section.