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Summary Photosystem I (PSI) converts photons into electrons with a nearly 100% quantum efficiency. Its minimal energy requirement for photochemistry corresponds to a 700-nm photon, representing the well-known “red limit” of oxygenic photosynthesis. Recently, some cyanobacteria containing the red-shifted pigment chlorophyll f have been shown to harvest photons up to 800 nm. To investigate the mechanism responsible for converting such low-energy photons, we applied steady-state and time-resolved spectroscopies to the chlorophyll-f-containing PSI and chlorophyll-a-only PSI of various cyanobacterial strains. Chlorophyll-f-containing PSI displays a less optimal energetic connectivity between its pigments. Nonetheless, it consistently traps long-wavelength excitations with a surprisingly high efficiency, which can only be achieved by lowering the energy required for photochemistry, i.e., by “breaking the red limit”. We propose that charge separation occurs via a low-energy charge-transfer state to reconcile this finding with the available structural data excluding the involvement of chlorophyll f in photochemistry.

The heterologous expression of the far-red absorbing chlorophyll (Chl) f in organisms that do not synthesize this pigment has been suggested as a viable solution to expand the solar spectrum that drives oxygenic photosynthesis. In this study, we investigate the functional binding of Chl f to the Photosystem I (PSI) of the cyanobacterium Synechococcus 7002, which has been engineered to express the Chl f synthase gene. By optimizing growth light conditions, one-to-four Chl f pigments were found in the complexes. By using a range of spectroscopic techniques, isolated PSI trimeric complexes were investigated to determine how the insertion of Chl f affects excitation energy transfer and trapping efficiency. The results show that the Chls f are functionally connected to the reaction center of the PSI complex and their presence does not change the overall pigment organization of the complex. Chl f substitutes Chl a (but not the Chl a red forms) while maintaining efficient energy transfer within the PSI complex. At the same time, the introduction of Chl f extends the photosynthetically active radiation of the new hybrid PSI complexes up to 750 nm, which is advantageous in far-red light enriched environments. These conclusions provide insights to engineer the photosynthetic machinery of crops to include Chl f and therefore increase the light-harvesting capability of photosynthesis.

The Lhca4 antenna complex of plant Photosystem I (PSI) is characterized by extremely red-shifted and broadened absorption and emission bands from its low-energy chlorophylls (Chls). The mixing of a charge-transfer (CT) state with the excited state manifold causing these so-called red forms results in highly complicated multi-component excited energy transfer (EET) kinetics within the complex. The two-dimensional electronic spectroscopy experiments presented here reveal that EET towards the CT state occurs on three timescales: fast from the red Chls (within 1 ps), slower (5–7 ps) from the stromal side Chls, and very slow (100–200 ps) from a newly discovered 690 nm luminal trap. The excellent agreement between the experimental data with the previously presented Redfield–Fo¨rster exciton model of Lhca4 strongly supports the equilibration scheme of the bulk excitations with the dynamically localized CT on the stromal side. Thus, a complete picture of the energy transfer pathways leading to the population of the CT final trap within the whole Lhca4 complex is presented. In view of the environmental sensitivity of the CT contribution to the Lhca4 energy landscape, we speculate that one role of the CT states is to regulate the EET from the peripheral antenna to the PSI core.

Cells are extremely crowded, and a central question in biology is how this affects the intracellular water. Here, we use ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy to observe the random orientational motion of water molecules inside living cells of three prototypical organisms: Escherichia coli, Saccharomyces cerevisiae (yeast), and spores of Bacillus subtilis. In all three organisms, most of the intracellular water exhibits the same random orientational motion as neat water (characteristic time constants ~9 and ~2 ps for the first-order and second-order orientational correlation functions), whereas a smaller fraction exhibits slower orientational dynamics. The fraction of slow intracellular water varies between organisms, ranging from ~20% in E. coli to ~45% in B. subtilis spores. Comparison with the water dynamics observed in solutions mimicking the chemical composition of (parts of) the cytosol shows that the slow water is bound mostly to proteins, and to a lesser extent to other biomolecules and ions., The cytoplasm’s crowdedness leads one to expect that cell water is different from bulk water. By measuring the rotational motion of water molecules in living cells, Tros et al. find that apart from a small fraction of water solvating biomolecules, cell water has the same dynamics as bulk water.

Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu– and Arg+ are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.

An infrared pump-probe setup using rapid polarization modulation has been developed to perform time-resolved vibrational anisotropy measurements. A photo-elastic modulator is used as a rapidly switchable half-wave plate, enabling the measurement of transient absorptions for parallel and perpendicular polarizations of the pump and probe pulses on a shot-to-shot basis. In this way, infrared intensity fluctuations are nearly completely canceled, significantly enhancing the accuracy of the transient-anisotropy measurement. The method is tested on the OD-stretch vibration of HDO in H2O, for which the signal-to-noise ratio is found to be 4 times better than with conventional methods.

The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis-trans and trans-cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates. Additionally, static FTIR difference measurements of the Dronpa-M159T mutant correspond very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone chromophore in the cis anion and trans neutral forms in the bright "on" and dark "off" states, respectively. Green excitation of the on state is followed by dominant radiative decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1 ns, and additionally reveals spectral changes belonging to the species decaying with a 1.1 ns time constant, associated with both protein and chromophore modes. A 1 ms measurement of the on state identifies bleach features that correspond to those seen in the static off-minus-on Fourier transform infrared (FTIR) difference spectrum, indicating that thermal protonation of the hydroxyphenyl oxygen proceeds within this time window. Blue excitation of the off state directly resolves the formation of the primary photoproduct with 0.6 and 14 ps time constants, which is stable on the nanosecond time scale. Assignment of the primary photoproduct to the cis neutral chromophore in the electronic ground state is supported by the frequency positions expected relative to those for the nonplanar distorted geometry for the off state. A 1 ms measurement of the off state corresponds closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the Arg66 side chain to be complete.