Femtosecond visible transient absorption spectroscopy of chlorophyll-f-containing photosystem II.

The recently discovered, chlorophyll-f-containing, far-red photosystem II (FR-PSII) supports far-red light photosynthesis. Participation and kinetics of spectrally shifted far-red pigments are directly observable and separated from that of bulk chlorophyll-a We present an ultrafast transient absorption study of FR-PSII, investigating energy transfer and charge separation processes. Results show a rapid subpicosecond energy transfer from chlorophyll-a to the long-wavelength chlorophylls-f/d The data demonstrate the decay of an ∼720-nm negative feature on the picosecond-to-nanosecond timescales, coinciding with charge separation, secondary electron transfer, and stimulated emission decay. An ∼675-nm bleach attributed to the loss of chl-a absorption due to the formation of a cation radical, PD1+•, is only fully developed in the nanosecond spectra, indicating an unusually delayed formation. A major spectral feature on the nanosecond timescale at 725 nm is attributed to an electrochromic blue shift of a FR-chlorophyll among the reaction center pigments. These time-resolved observations provide direct experimental support for the model of Nürnberg et al. [D. J. Nürnberg et al., Science 360, 1210-1213 (2018)], in which the primary electron donor is a FR-chlorophyll and the secondary donor is chlorophyll-a (PD1 of the central chlorophyll pair). Efficient charge separation also occurs using selective excitation of long-wavelength chlorophylls-f/d, and the localization of the excited state on P720* points to a smaller (entropic) energy loss compared to conventional PSII, where the excited state is shared over all of the chlorin pigments. This has important repercussions on understanding the overall energetics of excitation energy transfer and charge separation reactions in FR-PSII.

Femtosecond infrared spectroscopy of chlorophyll f-containing photosystem I.

The recent discovery of extremely red-shifted chlorophyll f pigments in both photosystem I (PSI) and photosystem II has led to the conclusion that chlorophyll f plays a role not only in the energy transfer, but also in the charge separation processes [Nürnberg et al., Science, 2018, 360, 1210-1213]. We have employed ultrafast transient infrared absorption spectroscopy to study the contribution of far-red light absorbing chlorophyll f to energy transfer and charge separation processes in far-red light-grown PSI (FRL-PSI) from the cyanobacterium Chroococcidiopsis thermalis PCC 7203. We compare the kinetics and spectra of FRL-grown PSI excited at 670 nm and 740 nm wavelengths to those of white light-grown PSI (WL-PSI) obtained at 675 nm excitation. We report a fast decay of excited state features of chlorophyll a and complete energy transfer from chlorophyll a to chlorophyll f in FRL-PSI upon 670 nm excitation, as indicated by a frequency shift in a carbonyl absorption band occurring within a 1 ps timescale. While the WL-PSI measurements support the assignment of initial charge separation to A-1+˙A0-˙ [Di Donato et al., Biochemistry, 2011, 50, 480-490] from the kinetics of a distinct cation feature at 1710 cm-1, in the case of FRL-PSI, small features at 1715 cm-1 from the chlorophyll cation are present from sub-ps delays instead, supporting the replacement of the A-1 pigment with chlorophyll f. Comparisons of nanosecond spectra show that charge separation proceeds with 740 nm excitation, which selectively excites chlorophyll f, and modifications in specific carbonyl absorption bands assigned to P700+˙ minus P700 and A1-˙ minus A1 indicate dielectric differences of FRL-PSI compared to WL-PSI in one or both of the two electron transfer branches of FRL-PSI.

Femtosecond Visible Transient Absorption Spectroscopy of Chlorophyll f-Containing Photosystem I.

Photosystem I (PSI) from Chroococcidiopsis thermalis PCC 7203 grown under far-red light (FRL; >725 nm) contains both chlorophyll a and a small proportion of chlorophyll f. Here, we investigated excitation energy transfer and charge separation using this FRL-grown form of PSI (FRL-PSI). We compared femtosecond transient visible absorption changes of normal, white-light (WL)-grown PSI (WL-PSI) with those of FRL-PSI using excitation at 670 nm, 700 nm, and (in the case of FRL-PSI) 740 nm. The possibility that chlorophyll f participates in energy transfer or charge separation is discussed on the basis of spectral assignments. With selective pumping of chlorophyll f at 740 nm, we observe a final ∼150 ps decay assigned to trapping by charge separation, and the amplitude of the resulting P700+•A1-• charge-separated state indicates that the yield is directly comparable to that of WL-PSI. The kinetics shows a rapid 2 ps time constant for almost complete transfer to chlorophyll f if chlorophyll a is pumped with a wavelength of 670 nm or 700 nm. Although the physical role of chlorophyll f is best supported as a low-energy radiative trap, the physical location should be close to or potentially within the charge-separating pigments to allow efficient transfer for charge separation on the 150 ps timescale. Target models can be developed that include a branching in the formation of the charge separation for either WL-PSI or FRL-PSI.

Ultrafast infrared observation of exciton equilibration from oriented single crystals of photosystem II.

In oxygenic photosynthesis, two photosystems work in series. Each of them contains a reaction centre that is surrounded by light-harvesting antennae, which absorb the light and transfer the excitation energy to the reaction centre where electron transfer reactions are driven. Here we report a critical test for two contrasting models of light harvesting by photosystem II cores, known as the trap-limited and the transfer-to-the trap-limited model. Oriented single crystals of photosystem II core complexes of Synechococcus elongatus are excited by polarized visible light and the transient absorption is probed with polarized light in the infrared. The dichroic amplitudes resulting from photoselection are maintained on the 60 ps timescale that corresponds to the dominant energy transfer process providing compelling evidence for the transfer-to-the-trap limitation of the overall light-harvesting process. This finding has functional implications for the quenching of excited states allowing plants to survive under high light intensities.

Photoisomerization and proton transfer in the forward and reverse photoswitching of the fast-switching M159T mutant of the Dronpa fluorescent protein.

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.

Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa.

The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright "on" and dark "off" states via photoisomerization and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0-Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand ß8 containing Thr159 as well as an altered A-C dimer interface involving strands ß7, ß8, ß10, and ß11. The Met159Thr mutation increases the cavity volume for the p-hydroxybenzylidene-imidazolinone chromophore as a result of both the side chain difference and the backbone positional differences.

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa.

The reversible photoswitching between the 'on' and 'off' states of the fluorescent protein Dronpa involves photoisomerization as well as protein side-chain rearrangements, but the process of interconversion remains poorly characterized. Here we use time-resolved infrared measurements to monitor the sequence of these structural changes, but also of proton transfer events, which are crucial to the development of spectroscopic contrast. Light-induced deprotonation of the chromophore phenolic oxygen in the off state is a thermal ground-state process, which follows ultrafast (9 ps) trans-cis photoisomerization, and so does not involve excited-state proton transfer. Steady-state infrared difference measurements exclude protonation of the imidazolinone nitrogen in both the on and off states. Pump-probe infrared measurements of the on state reveal a weakening of the hydrogen bonding between Arg66 and the chromophore C=O, which could be central to initiating structural rearrangement of Arg66 and His193 coinciding with the low quantum yield cis-trans photoisomerization.