Ultrafast energy transfer and excited state coupling in an artificial photosynthetic antenna.

We have studied the energy transfer dynamics in an artificial light-harvesting dyad composed of a phthalocyanine (Pc) covalently linked to a carotenoid (Car). The combination of high temporal resolution transient absorption spectroscopy with global and target analysis allowed us to quantify the efficiency of the energy transfer from the S2 excited state of the Car to the Pc at 37%, close to values observed in some natural light-harvesting complexes. In addition, following selective excitation of the Pc, we have identified the spectral signatures of the S1 excited state of the Car which appear within the ≈30 fs time resolution of our measurement. This strongly indicates excited state coupling between the S1 state of Car and the Qx state of Pc, with important implications for the regulation of photosynthetic activity.

Disentangling picosecond events that complicate the quantitative use of the calcium sensor YC3.60.

Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein (EYFP). Depending on the calcium concentration, the efficiency of FRET from donor ECFP to acceptor EYFP is changing. In this study, we have recorded time-resolved fluorescence spectra of ECFP, EYFP, and YC3.60 in aqueous solution with picosecond time resolution, using different excitation wavelengths. Detailed insight in the FRET kinetics was obtained by using global and target analyses of time- and wavelength-resolved fluorescence of purified YC3.60 in calcium-free and calcium-bound conformations. The results clearly demonstrate that for both conformations, there are two distinct donor populations: a major one giving rise to FRET and a minor one not able to perform FRET. The transfer time for the calcium-bound conformation is 21 ps, whereas it is in the order of 1 ns for the calcium-free conformation. Ratio imaging of acceptor and donor fluorescence intensities of YC3.60 is usually applied to measure Ca(2+) concentrations in living cells. From the obtained results, it is clear that the intensity ratio is strongly influenced by the presence of donor molecules that do not take part in FRET, thereby significantly affecting the quantitative interpretation of the results.

Charge separation is virtually irreversible in photosystem II core complexes with oxidized primary quinone acceptor.

X-ray structures of the Photosystem II (PSII) core revealed relatively large interpigment distances between the CP43 and CP47 antenna complexes and the reaction center (RC) with respect to the interpigment distances in a single unit. This finding questions the possibility of fast energy equilibration among the antenna and the RC, which has been the basic explanation for the measured PSII fluorescence kinetics for more than two decades. In this study, we present time-resolved fluorescence measurements obtained with a streak-camera setup on PSII core complexes from Thermosynechococcus elongatus at room temperature (RT) and at 77 K. Kinetic modeling of the RT data obtained with oxidized quinone acceptor Q(A), reveals that the kinetics are best described by fast primary charge separation at a time scale of 1.5 ps and slow energy transfer from the antenna into the RC, which results in an energy equilibration time between the antenna and the RC of about 44 ps. This model is consistent with structure-based computations. Primary radical pair formation was found to be a virtually irreversible process. Energy equilibration within the CP43 and CP47 complexes is shown to occur at a time scale of 8 ps. Kinetic modeling of the 77 K data reveals similar energy transfer time scales in the antenna units and among the antenna and the RC as at RT, respectively, 7 and 37 ps. We conclude that the energy transfer from the CP43/CP47 antenna to the RC is the dominant factor in the total charge separation kinetics in intact PSII cores.

Charge separation and energy transfer in the photosystem II core complex studied by femtosecond midinfrared spectroscopy.

The core of photosystem II (PSII) of green plants contains the reaction center (RC) proteins D1D2-cytb559 and two core antennas CP43 and CP47. We have used time-resolved visible pump/midinfrared probe spectroscopy in the region between 1600 and 1800 cm(-1) to study the energy transfer and charge separation events within PSII cores. The absorption difference spectra in the region of the keto and ester chlorophyll modes show spectral evolution with time constants of 3 ps, 27 ps, 200 ps, and 2 ns. Comparison of infrared (IR) difference spectra obtained for the isolated antennas CP43 and CP47 and the D1D2-RC with those measured for the PSII core allowed us to identify the features specific for each of the PSII core components. From the presence of the CP43 and CP47 specific features in the spectra up to time delays of 20-30 ps, we conclude that the main part of the energy transfer from the antennas to the RC occurs on this timescale. Direct excitation of the pigments in the RC evolution associated difference spectra to radical pair formation of PD1+PheoD1- on the same timescale as multi-excitation annihilation and excited state equilibration within the antennas CP43 and CP47, which occur within approximately 1-3 ps. The formation of the earlier radical pair ChlD1+PheoD1-, as identified in isolated D1D2 complexes with time-resolved mid-IR spectroscopy is not observed in the current data, probably because of its relatively low concentration. Relaxation of the state PD1+PheoD1-, caused by a drop in free energy, occurs in 200 ps in closed cores. We conclude that the kinetic model proposed earlier for the energy and electron transfer dynamics within the D1D2-RC, plus two slowly energy-transferring antennas C43 and CP47 explain the complex excited state and charge separation dynamics in the PSII core very well. We further show that the time-resolved IR-difference spectrum of PD1+PheoD1- as observed in PSII cores is virtually identical to that observed in the isolated D1D2-RC complex of PSII, demonstrating that the local structure of the primary reactants has remained intact in the isolated D1D2 complex.

Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein.

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is approximately 0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.

(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system.

The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3- carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes.

Excitation energy transfer pathways in Lhca4.

EET in reconstituted Lhca4, a peripheral light-harvesting complex from Photosystem I of Arabidopsis thaliana, containing 10 chlorophylls and 2 carotenoids, was studied at room temperature by femtosecond transient absorption spectroscopy. Two spectral forms of Lut were observed in the sites L1 and L2, characterized by significantly different interactions with nearby chlorophyll a molecules. A favorable interpretation of these differences is that the efficiency of EET to Chls is about two times lower from the "blue" Lut in the site L1 than from the "red" Lut in the site L2 due to fast IC in the former case. A major part of the energy absorbed by the "red" Lut, approximately 60%-70%, is transferred to Chls on a sub-100-fs timescale from the state S(2) but, in addition, minor EET from the hot S(1) state within 400-500 fs is also observed. EET from the S(1) state to chlorophylls occurs also within 2-3 ps and is ascribed to Vio and/or "blue" Lut. EET from Chl b to Chl a is biphasic and characterized by time constants of approximately 300 fs and 3.0 ps. These rates are ascribed to EET from Chl b spectral forms absorbing at approximately 644 nm and approximately 650 nm, respectively. About 25% of the excited Chls a decays very fast-within approximately 15 ps. This decay is proposed to be related to the presence of the interacting Chls A5 and B5 located next to the carotenoid in the site L2 and may imply some photoprotective role for Lhca4 in the photosystem I super-complex.

Energy and electron transfer in photosystem II reaction centers with modified pheophytin composition.

Energy and electron transfer in Photosystem II reaction centers in which the photochemically inactive pheophytin had been replaced by 13(1)-deoxo-13(1)-hydroxy pheophytin were studied by femtosecond transient absorption-difference spectroscopy at 77 K and compared to the dynamics in untreated reaction center preparations. Spectral changes induced by 683-nm excitation were recorded both in the Q(Y) and in the Q(X) absorption regions. The data could be described by a biphasic charge separation. In untreated reaction centers the major component had a time constant of 3.1 ps and the minor component 33 ps. After exchange, time constants of 0.8 and 22 ps were observed. The acceleration of the fast phase is attributed in part to the redistribution of electronic transitions of the six central chlorin pigments induced by replacement of the inactive pheophytin. In the modified reaction centers, excitation of the lowest energy Q(Y) transition produces an excited state that appears to be localized mainly on the accessory chlorophyll in the active branch (B(A) in bacterial terms) and partially on the active pheophytin H(A). This state equilibrates in 0.8 ps with the radical pair. B(A) is proposed to act as the primary electron donor also in untreated reaction centers. The 22-ps (pheophytin-exchanged) or 33-ps (untreated) component may be due to equilibration with the secondary radical pair. Its acceleration by H(B) exchange is attributed to a faster reverse electron transfer from B(A) to. After exchange both and are nearly isoenergetic with the excited state.

Stark spectroscopy of the light-harvesting complex II in different oligomerisation states.

The electric field-induced absorption changes (Stark effect) of light-harvesting complex II (LHCII) in different oligomerisation states-monomeric, trimeric and aggregated-have been probed at 77 K. All the chlorophyll (Chl) a molecules exhibit electro-optic properties in the Q(y) absorption region characterized by a change in dipole moment /Deltamu-->/ =0.6+/-0.06D/f and polarizability, Tr(Deltaalpha;) approximately 55+/-5 A(3)/f(2) upon electronic excitation, which are similar to those of unbound monomeric Chl a, indicating the absence of strong delocalization of the excitations which would be expected in the presence of strong excitonic interactions. The Stark effect in the Chl b absorption region is significantly bigger with /Deltamu-->/ values of the order of 2.0+/-0.2 D/f and it is attributed to strong interactions with neoxanthin molecules. Clear oligomerisation-dependent differences are observed in the carotenoid region, mainly due to the appearance of a new xanthophyll absorption band at 509 in the spectra of trimers and oligomers. It is ascribed to some lutein molecules, in agreement with previous experimental observations. The electro-optic properties of these lutein molecules are significantly different from those of the other xanthophylls in LHCII, which do not exhibit such a big change in dipole moment upon electronic excitation (/Deltamu-->/ =14.6+/-2.0 D/f). Upon aggregation of LHCII some extra absorption appears on the red side of the main Chl a Q(y) absorption band. In contrast to an earlier suggestion [J. Phys. Chem., A 103 (1999) 2422], no indications are found for the charge-transfer character of the corresponding band. The assignments of the S(2) electronic transitions of neoxanthin and lutein in LHCII and possible origins of the Stark effect are discussed.