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The resonance Raman spectra of several synthetic Zn and Mg chlorins were measured in nonpolar solvents in order to characterize the structural elements required for oligomerization of BChl c in chlorosomes of green bacteria. The stretching vibrations of the chlorin and the conjugated keto group were analyzed in terms of the coordination state of the metal ion and the inter- and intramolecular interactions of the C=O group, respectively. In the monomeric Zn chlorin the free carbonyl group gives rise to bands at ca. 1700 cm[sup [minus]1]. In the presence of hydrogen bond donors these bands shift down by about 30 cm[sup [minus]1]. Bands of covalently linked bis(metallochlorins) in the range 1640-1660 cm[sup [minus]1] are assigned to a special hydrogen-bonded ketone, C=O[center dot][center dot][center dot]HO(Me)[center dot][center dot][center dot]M (M = Zn, Mg), formed by a methanol bridge between the ketone of one chlorin and the central metal of the other one. In aggregates constituted by Zn or Mg chlorins which posses a hydroxyl group at position C-3, these modes are observed in the same frequency range. In these aggregates, the hydroxyl substituent mediates the interactions between the metal ion and the keto group. These assignments of the artificial Zn and Mgmore » chlorin aggregates also serve for the elucidation of the supramolecular structures of the native bacteriochlorophyll c aggregates in photosynthetic antenna complexes (chlorosomes) of green bacteria. It is shown that all these aggregates have C=O[center dot][center dot][center dot]HO(R)[center dot][center dot][center dot]M bonds and pentacoordinated central metals with one axial ligand as the most important structural element required for aggregation and self-organization. 33 refs., 10 figs.« less

Chlorosomes are the largest and most efficient natural light-harvesting systems and contain supramolecular assemblies of bacteriochlorophylls that are organized without proteins. Despite a recent structure determination for chlorosomes from Chlorobaculum tepidum (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525), the issue of a possible large structural disorder is still discussed controversially. We have studied individual chlorosomes prepared under very carefully controlled growth condition by a novel 2-dimensional polarization single molecule imaging technique giving polarization information for both fluorescence excitation and emission simultaneously. Contrary to the existing literature data, the polarization degree or modulation depth (M) for both excitation (absorption) and emission (fluorescence) showed extremely narrow distributions. The fluorescence was always highly polarized with M ≈ 0.77, independent of the excitation wavelength. Moreover, the fluorescence spectra of individual chlorosomes were identical within the error limits. These results lead us to conclude that all chlorosomes possess the same type of internal organization in terms of the arrangement of the bacteriochlorophyll c transition dipole moments and their total excitonic transition dipole possess a cylindrical symmetry in agreement with the previously suggested concentric multitubular chlorophyll aggregate organization (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525).

This study aims to prepare (99m)Technetium Pheophorbide-a ((99m)Tc-PH-A) complex and evaluate its efficiency as an infection imaging agent. First, PH-A was obtained from Spirulina maxima algae, and the product compound was confirmed using (1)H NMR and MS (ESI) methods. The PH-A was then labeled with (99m)Tc using the tin chloride method and its biological efficacy as a potential radiotracer for Staphylococcus aureus (S. aureus) infection was evaluated in bacterially infected and sterile inflamed rats. The radiochemical stability of the (99m)Tc-PH-A in human serum was determined by thin-layer radiochromatography (TLRC). The radiochemical purity was 87±3.2% and remained constant at more than 80±0.1% even in serum for 120 min after radiolabeling. These experiments indicated that the ratio of (99m)Tc-PH-A uptake in bacterially infected muscle, as compared to normal muscle, [target/non-target (T/NT)=5.6 at 1h] was over four times higher than that in sterile inflamed muscle (T/NT=1.29 at 1h). Disappearance of activity from the kidney and liver indicated that the urinary and hepatobiliary systems were the normal routes of excretion of the complex. (99m)Technetium Pheophorbide prepared with high yield is able to localize well in the bacterially infected muscle of the rats and (99m)Tc-PH-A may be developed as a radiopharmaceutical agent to distinguish infection from inflammation by nuclear imaging.

Fluorescence decays on a picosecond timescale were recorded by single-photon timing and resolved into exponential components by global data analysis for intact cells of the cyanobacterium Synechococcus 6301 with excitation at 670 nm. Fluorescence decays were measured for cells at F0 (all PS II reaction centres open), at Fm (all PS II reaction centres closed) and at two intermediate levels of PS II reaction centre closure. We found the same set of fluorescence lifetimes to be present at all levels of PS II trap closure; only the relative amplitudes of the various lifetime components changed. We conclude that there is little energy transfer among PS II complexes in this organism and that the variable fluorescence component Fv is principally the consequence of the decreased probability for exciton trapping at individual closed PS II reaction centres.

The influence of double reduction of Q A , the secondary electron acceptor in Photosystem (PS) II, on the primary events of PS II electron transport was studied by picosecond time-resolved fluorescence measurements in isolated PS II membranes. The double reduction was achieved either by chemical treatment or by strong anaerobic illumination. In the presence of doubly reduced Q A fluorescence decay from the PS II reaction centres showed a dominant amount of a fast phase (150–250 ps) similar to open PS II. In addition to two further components of 600 ps and 2–3 ns, a long-lived component of about 10 ns was observed which is characteristic of the doubly reduced state only. The data indicate efficient primaru charge separation as well as the presence of a long-lived radical pair when Q A is doubly reduced. From these results it is concluded that the electrostatic effect of the two electrons on Q A 2− , which is expected to strongly suppress primary charge separation, is neutralized, most likely by protonation at or near the Q A site. Analysis of the decay curves in the framework of the exciton/radical pair equilibrium model indicates the formation of a relaxed radical pair state. Fluorescence quenching due to aerobic photoinhibition was shown to arise from the increase of a fast decaying (300–320 ps) component at the expense of the more slowly decaying components during the photoinhibitory treatment. No long-lived component in the range of about 10 ns was observed in case of aerobic photoinhibition. It is concluded that, in contrast to the anaerobic photoinhibition, no doubly reduced Q A is accumulated during the aerobic photoinhibitory treatment.

The excitation energy transfer and trapping processes in intact cells of Chloroflexus aurantiacus were studied by picosecond time-resolved fluorescence spectroscopy. The fluorescence decay kinetics is investigated over the near infrared emission range between 730 nm and 920 nm using various excitation wavelengths and excitation intensities. The data were analyzed by global decay analysis and are presented as decay-associated spectra (DAS). The specific dependence of the decay kinetics on the excitation wavelength and on the photochemical redox state of the reaction center (RC) allows the identification of the energy transfer and trapping components. The DAS provide evidence for two chlorosomal energy transfer processes. The first one occurs between the chlorosomal bacteriochlorophyll (BChl)- c and the BChl- a 792 complex (B 792 ) in the chlorosomal baseplate with an equilibration time constant of 15–16 ps, while the second one occurs from the B 792 pigments to the BChl- a 806 pigments in the B 806–866 complex with a time constant of 35–40 ps. The overall energy trapping process in whole cells is mainly determined by the kinetics of the primary charge separation process in the RCs. With open RCs (Q A oxidized) the trapping time constant is 70–90 ps, while the trapping process with closed RCs (Q A reduced) takes as long as 180–200 ps. The results on whole cells reported here are interpreted in conjunction with those reported earlier for the various isolated complexes, i.e., two different chlorosome preparations (Holzwarth, A.R., Muller, M.G. and Griebenow, K. (1990) J. Photochem. Photobiol. B 5, 457–465), the B 806–866 complex (Griebenow, K., Muller, M.G. and Holzwarth, A.R. (1991) Biochim. Biophys. Acta 1059, 226–232) and isolated reaction centers (Muller, M.G., Griebenow, K. and Holzwarth, A.R. (1991) Biochim. Biophys. Acta 1098, 1–12). Based on these data, a unified and self-consistent scheme for the primary processes in the whole photosynthetic system of C. aurantiacus is presented. The BChl antenna pigment groups are arranged to form a linear energy transfer cascade with four energy transfer steps from shorter-wavelength- to longer-wavelength-absorbing antenna pools. The overall fluorescence decay kinetics of the photosynthetic system of C. aurantiacus turns out to be ‘trap-limited’ by the reaction center rather than ‘diffusion-limited’ by the energy transfer processes.

The fluorescence kinetics of a D1-D2-cyt-b-559 reaction center complex isolated by short Triton-exposure has been measured with picosecond resolution as a function of temperature below 150 K. The data were analyzed by combined global analysis of data measured with different time resolutions and are presented as decay-associated fluorescence spectra (DAS). Emphasis is given to the resolution and assignment of the fast components below 100 ps. Six lifetime components were generally necessary for a good fit of the data over a long time-range. The shortest lifetime of 13–24 ps (depending on temperature) is attributed to an energy transfer from (accessory) Chlorophyll to P680, presumably via pheophytin. A component in the range of 40–70 ps is attributed to energy transfer from pheophytin to P680. These components can only be properly resolved and assigned from measurements at temperatures below about 40 K. An ultrashort component of 1–6 ps, which had been resolved in our previous measurements (Biochim. Biophys. Acta (1991) 1060, 237–244) was not resolved in the particles studied here. We propose that the absence of the ultrashort component in this study is attributable to the larger chlorophyll content of the present short-term Triton-exposed D1-D2-cyt-b-559 preparation as compared to the previous long-term Triton-exposed RC particles. In the particles studied here the effective charge separation kinetics in the short time-range is rate-limited by relatively slow energy transfer components (lifetimes from 13 to 24 ps) upon preferential excitation of the accessory chlorophylls. In the long time-range a multicomponent charge recombination kinetics giving rise to four fluorescence lifetimes in the range of 1.56–49.7 ns are resolved. All these components show basically identical DAS with maxima near 682 nm.

The energy transfer and charge separation kinetics of a photosystem I (PS I) core particle of an antenna size of 100 chlorophyll/P700 has been studied by combined fluorescence and transient absorption kinetics with picosecond resolution. This is the first combined picosecond study of transient absorption and fluorescence carried out on a PS I particle and the results are consistent with each other. The data were analyzed by both global lifetime and global target analysis procedures. In fluorescence major lifetime components were found to be 12 and 36 ps. The shorter-lived one shows a negative amplitude at long wavelengths and is attributed to an energy transfer process between pigments in the main antenna Chl pool and a small long-wavelength Chl pool emitting around 720 nm whereas the longer-lived component is assigned to the overall charge separation lifetime. The lifetimes resolved in transient absorption are 7–8 ps, 33 ps, and [unk]1 ns. The shortest-lived one is assigned to energy transfer between the same pigment pools as observed also in fluorescence kinetics, the middle component of 33 ps to the overall charge separation, and the long-lived component to the lifetime of the oxidized primary donor P700 + . The transient absorption data indicate an even faster, but kinetically unresolved energy transfer component in the main Chl pool with a lifetime

The fluorescence kinetics of chromatophores from Rhodobacter capsulatus wild type have been measured with picosecond time resolution over the wavelength range from 850 to 940 nm. The data have been analyzed both by global lifetime analysis yielding lifetimes and decay-associated spectra (DAS) and by global target analysis techniques yielding rate constants and species-associated spectra (SAS). In the global lifetime analysis five lifetime components were necessary for a good fit. The lifetimes are: τ1 = 9 ps showing a DAS with positive and negative amplitudes; τ2 = 40 ps with a DAS maximum at ≈ 890 nm; τ3 = 95 ps with a DAS maximum at ≈ 895 nm; τ4 = 260 ps with a DAS similar to that of lifetime component τ3; a fifth lifetime component of 940 ps has nearly negligible amplitude. In the global target analysis several kinetic models were tested. A homogeneous model with sequential energy transfer LHC II ↔ LHC I harr; RC does formally fit the data only if the detailed charge separation and charge recombination processes at the reaction center (RC) are taken into account explicitly. However, despite the good fit, such a model must be excluded as a valid description both on the basis of simple thermodynamic considerations as well as due to the fact that it predicts unreasonable RC rate constants. Instead a heterogeneous model, assuming a mixture of chromatophores with open and closed RCs describes the situation both formally and physically quite well. For such a model the rate constants of the RC electron transfer processes and their free energy values as well as the energy transfer rate constants and SAS of the antenna pools were obtained. The extrapolated RC kinetics for open RCs agrees well with that known from isolated purple bacterial RCs. The rate constants for energy transfer processes among antenna pools and from antenna to RCs indicate that at room temperature the exciton kinetics of the entire antenna system is limited and determined by the RC charge separation, i.e., the exciton decay is trap limited. Our data are compared with the corresponding data for Rb. sphaeroides.

Picosecond time-resolved fluorescence emission spectroscopy at 80 K was used to investigate excitation-energy migration in LHC II isolated from spinach chloroplasts. Time-resolved spectra were obtained for solubilised LHC II trimers and for LHC II in a semi-crystalline aggregated state. In solubilised LHC II, the main decay lifetime is about 4.3 ns. Upon aggregation, the main decay lifetime becomes about 110 ps, indicating the presence of a highly-efficient energy-quenching process. Several red-shifted chlorophyll species can be resolved in aggregated LHC II, but none of these appears to be directly involved in energy quenching. The zeaxanthin / violaxanthin ratio has no influence on the time-resolved fluorescence emission spectrum for LHC II in these states. We suggest that a quenching process similar to that observed in aggregates of isolated LHC II is the basis of the ΔpH-dependent quenching ( q E ) observed in intact thylakoid membranes.

Picosecond time-resolved fluorescence of photosystem I particles isolated from Synechococcus sp. was recorded in the wavelength range from 680 nm to 736 nm for temperatures of 6 o C to 42 o C and -100 o C using the single-photon-timing technique. By global analysis of the data we found four contributing lifetime components at the higher temperatures (τ 1 ≃12 ps, τ 2 ≃35 ps, τ 3 ≃65 ps, τ 4 ≃1000 ps). We attribute τ 1 to an energy transfer between two pigment pools, τ 2 to the charge separation process in the reaction center, component τ 3 is assigned to aggregate and τ 4 to uncoupled chlorophyll emission

The excited state dynamics of trimeric phycoerythrocyanin has been studied by two-color femtosecond transient absorption spectroscopy with a time-resolution better than 200 fs. Upon selective excitation of the short-wavelength phycobiliviolin chromophore at 575 nm absorption bleachings are observed. An isotropic ultrafast decay of the initial bleaching of 585±40 fs has been resolved at short detection wavelength (578 nm). Upon stepwise increase of the detection wavelength up to 617 nm, the bleaching showed a delayed rise above 593 nm with τ rise =380-580 fs

The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and green algae and plays a major role in the capture of sun light for photosynthesis. The LHCII complexes have also been proposed to play a key role in the optimization of photosynthetic efficiency through the process of state 1-state 2 transitions and are involved in down-regulation of photosynthesis under excess light by energy dissipation through non-photochemical quenching (NPQ). We present here the first solid-state magic-angle spinning (MAS) NMR data of the major light-harvesting complex (LHCII) of Chlamydomonas reinhardtii, a eukaryotic green alga. We are able to identify nuclear spin clusters of the protein and of its associated chlorophyll pigments in ¹³C-¹³C dipolar homonuclear correlation spectra on a uniformly ¹³C-labeled sample. In particular, we were able to resolve several chlorophyll 13¹ carbon resonances that are sensitive to hydrogen bonding to the 13¹-keto carbonyl group. The data show that ¹³C NMR signals of the pigments and protein sites are well resolved, thus paving the way to study possible structural reorganization processes involved in light-harvesting regulation through MAS solid-state NMR.

The bacteriochlorophyll-c pigment contained in the chlorosomes of the phototrophic and thermophilic bacterium Chloroflexus aurantiacus is shown to comprise, in the least, 10 constituents, viz. the 2a-epimeric pairs [i.e., the (2aR,7S,8S)- and (2aS,7S,8S)-diastereoisomers in 2:1 ratio] of five bacteriochlorophyllide-c esters. All compounds were fully characterized by 1H and 13C NMR spectroscopy, and by mass spectra. They include the bacteriochlorophyllides-c of geranylgeranio, phytol, and stearol(octadecan-1-ol), which have been reported previously but have not yet been completely characterized, and two novel bacteriochlorophylls-c, viz. the bacteriochlorophyllides-c of the non-isoprenoid alcohols ‘cetol’(hexadecan-1-ol) and ‘oleol’(octadec-9-en-1-ol).

The methyl zinc (3{sup 1}R)- and (3{sup 1}S)-[8Et, 12Me] bacteriopheophorbides d - analogs of the light-harvesting bacteriochlorophylls in photosynthetic bacteria - self-assemble in nonpolar solvents. While in dilute dichloromethane solution both epimers prevail in their monomeric form, complex equilibria of aggregates with gradually red-shifting Q{sub y} absorptions are formed at either higher concentration or lower temperature or upon dilution with hexane. Dynamic {sup 1}H-NMR and FT-IR spectroscopies show that the 3{sup 1}-hydroxy group participates directly in the self-assembly through oxygen ligation to zinc and through hydrogen bridging to the 13{sup 1}-keto group of the ligated hydroxyls in intermediate unsymmetrical dimers. The basic unit, combining three molecules through >C=O...H-O...Zn bonding, and the existence of equilibria between monomers, dimers, and oligomers of varying size depending on the conditions parallel the scheme proposed previously for bacteriochlorophylls c and d. In some respects the CD, {sup 1}H-NMR, and IR spectral behavior of the two epimers indicates diastereoselective conformational and kinetic differences at the levels of dimers and larger oligomers. Inter alia, the self-assembly of a 1:1 mixture of the (3{sup 1}R) and (3{sup 1}S) epimers proceeds more rapidly than that of the separate epimers. 29 refs., 11 figs.

Picosecond to nanosecond fluorescence kinetics as a function of emission wavelength is reported for isolated reaction centers of the purple bacterium Rb. sphaeroides . New kinetic components of 12 ps and about 100 ps lifetime have been found in the fluorescence decay kinetics of the primary donor P*. Various kinetic models of the primary and secondary electron transfer processes are discussed which could possibly explain these new components. The data indicate either the necessity for more complex electron transfer schemes than assumed so far or a substantial heterogeneity in the rates of both the primary and secondary electron transfer steps.

UV—visible spectra of synthetic zinc chlorins in non-polar organic solvents indicate a supramolecular structure similar to the aggregates of bacteriochlorophylls- c in the extramembranous antenna complexes of green photosynthetic bacteria.

Our present understanding of the energy migration and trapping in the photosystems of higher plants is highlighted, summarizing recent results from picosecond fluorescence spectroscopy. The discussion focuses on three main topics: (i) the problem of deciding between trap- vs. diffusion-limited exciton kinetics in photosystem I antenna—reaction centre particles; (ii) the manifestation of heterogeneity for photosystem II in terms of molecular rate constants for the primary processes; (iii) the processes of energy transfer, primary charge separation and charge recombination in isolated D1–D2—cytochrome-b559 reaction centre preparations from photosystem II.

In this study, we have used the method of target analysis to analyze the ps fluorescence kinetics of pea chloroplasts with open ( F 0 ) and closed ( F max ) photosystem II (PS II) centers. Extending the exciton/radical pair equilibrium model (Schatz, G. H., H. Brock, and A. R. Holzwarth. 1988. Biophys. J. 54:397–405) to allow for PS II heterogeneity, we show that two types of PS II (labeled α and β) must be accounted for, each pool being characterized by its own set of molecular rate constants within the model. Simultaneous global target analysis of the data at F 0 and F max results in a detailed description of the molecular kinetics and energetics of the primary processes in both types of PS II units. This characterization revealed that the PS IIα pool accounts for twice as many Chl molecules as PS IIβ, which suggests a PSIIα/PSIIβ reaction center stoichiometry of close to unity. By extrapolation it is shown that the primary charge separation in hypothetical "isolated" β reaction centers is slower than in isolated α reaction centers: in open centers by a factor of 4 (1/ k 1 int = 11 vs 2.9 ps), in closed centers by a factor of 2 (1/ k 1 int = 34 vs 19 ps). Despite this slower charge separation process in PS IIβ, the quantum efficiency of the charge separation process is hardly affected: a charge stabilization yield at F 0 , (i.e., P + IQ A - ) of 86% (as compared to 90% in PS IIα). Reduction of Q A (closing PS II) has distinctly different effects on the primary kinetics of PS IIβ, as compared to PS IIα. In PS IIα the charge separation rate drops by a factor of 6, whereas the charge recombination process is hardly affected. In PS IIβ the charge separation is slowed down by a factor of 3, whereas the charge recombination rate increases by a factor of 5. In terms of changes in standard free energy, the reduction to Q A - lifts the free energy of the radical pair P + I - , relative to the excited state (Chl n / P ) * , by 47 meV in PS IIα and by 67 meV in PS IIβ. The concomitant increase in fluorescence quantum yield is the same for both types of PS II. These results show that PS IIα and PS IIβ exhibit a different molecular functioning with respect to the primary processes, which might have its origin in a different molecular structure of the reaction centers and/or a different local environment of these centers. Location in different parts of the thylakoid membrane might be involved. We also applied different error analysis procedures to determine the error ranges of the values found for the molecular rate constants. It is shown that the commonly used standard error has very little meaning, as it assumes independence of the fit parameters. Instead, an exhaustive search procedure, accounting for all possible correlations between the fit parameters, gives a more realistic view on the accuracy of the fit parameters.

The biochemical and spectroscopic evidence supporting the notion that chlorosomes, the main antenna complexes of the green photosynthetic bacterium Chloroflexus aurantiacus represent a novel type of self-organizing protein-free pigment structure are reviewed. The spectroscopic data from two different chlorosome preparations are compared with those of artificial pigment aggregates and the striking similarities in all spectroscopic aspects are discussed. Thus, chlorosomes represent the first known natural antenna structure where pigment-protein interactions do not determine the pigment arrangement.

The picosecond fluorescence kinetics of closed (quinone acceptor QA reduced) reaction centers isolated from the phototrophic bacterium Chloroflexus aurantiacus shows time constants of ≈ 20 and ≈ 300 ps (amplitude ratio ≈ 1:1), which are nearly independent of temperature (7 to 80 K). Assuming a three-state kinetic model, we tested various assignments of the kinetics to the electron transfer processes. Only two of these assignments seem to be physically reasonable. One of these includes a fast reversible electron transfer between P* and HM, the pheophytin(s) in the M branch of the reaction center which up to now has been considered virtually inactive in electron transfer. The other possible model involves the formation of a monomeric bacteriochlorophyll anion.

The photochemical reactions of the 1-deaza derivative of riboflavin (RF) have been studied by sub-ps time-resolved fluorescence spectroscopy. A potential effect of the solvent polarity and hydrogen bonding capability on the excited state (ES) dynamics was tested by measuring 1-deaza RF in water and, after acetylation of the sugar moiety, also in acetonitrile. Opposite to former reports, which indicated no fluorescence after laser excitation (Spencer et al. [1977] Biochemistry 16: 3586-3594), we find a significant fluorescence in either solvent. For both compounds, the fluorescence decays are biexponential with lifetimes in the ps time domain (1.8 and 12.5 ps for 1-deaza RF, and 3 and 87 ps for Ac 4 -1-deaza RF). In addition, a third, independent fluorescence decay was observed for both compounds. The remarkably slower second decay process for the measurements of the tetra-acetyl derivative in acetonitrile points to the involvement of hydrogen bonding or changes in the protonation/tautomerization state in the ES. In contrast to the parent compound (RF), no triplet state formation can be detected for 1-deaza RF, making the internal conversion and the fluorescence the only decay processes of the ES.

We investigate femtosecond transient absorption dynamics of lutein and beta-carotene. Strong oscillations up to about 400 fs are observed, depending on excitation or detection wavelength and solvent. We propose electronic quantum beats as the origin of these oscillations. They provide direct proof for strong coupling of the 1B(u)(+) with another electronic "dark" state predicted by quantum chemical calculations to be the 1B(u)(-) state resulting in a crossing within a dynamic relaxation model. The overall dynamics can be described well by an optical Bloch equation approach.

For the natural carotenoid 3,3'-dihydroxyisorenieratene (DHIR) and two synthetic derivatives, 3,3'-dihydroxy-16,17,18,16',17',18'-hexanor-Phi,Phi-carotene (DHHC) and Phi,Phi-carotene-3,3'-dione (DHIRQ, isorenieratene-3,3'-dione), steady state absorption experiments and combined density functional and multi-reference configuration interaction calculations were carried out. In addition, femtosecond transient absorption spectra were recorded for DHIR. Due to their marked out-of-plane distortion in DHIR, the phenolic end groups participate only partially in the conjugation system. In the low-energy regime its absorption spectrum with the maximum at 21 700 cm(-1) in acetone solution therefore closely resembles that of beta-carotene, the same as for the T1 energy. Further similarities are also found for the decay kinetics of the optically bright 1(1)Bu+ state of these compounds. After femtosecond excitation, the 1(1)Bu+ population of DHIR decays with a lifetime of 110 fs to the vibrationally hot 2(1)Ag-,v state which in turn relaxes to the 2(1)Ag-,0 state within 500 fs. Decay of the 2(1)Ag-,0 state to the S0 state occurs at a time scale of 12 ps. Demethylation of the phenolic end groups alleviates the steric repulsion by the polyene chain and causes a small red shift (1000 cm(-1)) comparing the absorption spectra of DHHC and DHIR. Oxidation of DHIR leads to drastic changes of the electronic and geometric properties. The quinoid end groups of DHIRQ are fully integrated into the conjugation system, shifting the absorption maximum to 17 800 cm(-1) in acetone solution which thus takes a blue color. The results of the quantum chemical calculations indicate that, in addition to the 2(1)Ag-(S1) state, two dark internal charge-transfer singlet states and the 1(1)Bu- state might be located energetically below the optically bright 1(1)Bu+ (S5) state of DHIRQ.

Fluorescence decays on a picosecond timescale were recorded at various excitation and emission wavelengths for intact cells of the cyanobacterium Synechococcus 6301. Fluorescence decays were measured by single photon timing and resolved into exponential components by global data analysis. Exponential fluorescence decay components were assigned to PS II, PS I and components of the phycobilisome according to their excitation spectra as determined from a time-resolved excitation-emission matrix and according to the effects of PS II reaction centre closure on the lifetimes and relative amplitudes. We have used the experimentally observed decay components from PS II and the phycobilisome core to estimate the effective rate constants for energy transfer within the phycobilisome/PS II complex and for charge separation at PS II. We show that the rate-limiting step for energy migration in the phycobilisome/PS II complex is a relatively slow energy transfer step from the phycobilisome terminal emitters to the PS II core complex. These results also support the idea that adaptation of cells to light-state 2 decreases the extent of energy transfer from phycobilisomes to PS II.

The stationary and time-resolved fluorescence emission spectra of reaction centres (RCs) isolated from the thermophilic phototrophic bacterium Chloroflexus aurantiacus strain Ok-70-fl have been studied. Several lifetime components in the picosecond and nanosecond time range have been resolved at room temperature. A short-lived approx. 3 ps component is related to an energy-transfer process from the excited accessory BChl-αL to the special pair P. This is demonstrated by the existence of positive and negative amplitudes of this component, depending on the emission wavelength. A conformational relaxation of P∗ causing a bathochromic shift is excluded for this time constant due to the absence of this component at an excitation wavelength of 850 nm (P) and also by the temperature dependence of the stationary fluorescence. Two further short-lived components with positive amplitudes and lifetimes of about 7 ps and about 18 ps were also resolved. Their decay-associated spectra (DAS) and emission maxima at about 910–920 nm are similar, showing that their origin is fluorescence from P∗. Their relative amplitudes change strongly, depending on the excitation intensity. They represent the charge separation times of open RCs (7 ps from P∗HQA) and closed RCs (18 ps from P∗ HQA−). The time constant for open RCs is in full agreement with the value reported recently from transient absorption measurements (Becker et al. (1991) Biochim. Biophys. Acta 1057, 299–312). The presence of “heterogeneity” in the primary rates of charge separation in RC preparations proposed recently is discussed. It is suggested that the “heterogeneity” reported by other authors is possibly an open/closed RC heterogeneity.

The fluorescence kinetics of the D 1 -D 2 -cyt- b -559 Photosystem II reaction-center complex have been characterized in the picosecond and nanosecond time ranges. Measurements have been performed in the presence of β-lauryl maltoside under anaerobic conditions, both at 277 K and 77 K. Global analysis of decays recorded at different wavelengths and with different time resolutions yielded the decay-associated emission spectra of six exponential lifetime components, in the range of 1–6 ps up to 35 ns. We report here an ultrafast (τ≈1–6ps) fluorescence lifetime component with dominant amplitude (≥90%), which is thought to reflect the primary charge separation process. Furthermore, we have resolved a component with a 30–40 ps lifetime, its DAS indicating energy transfer. The lifetime of the primary radical pair P 680 + I ∼ , reflected by its recombination fluorescence, was found to be temperature-dependent. At 277 K this lifetime was 30 ns with a relative yield of 0.87, whereas at 77 K the radical-pair lifetime is increased to 35 ns with a relative yield of only 0.24 (both yields are corrected for the emission from uncoupled chlorophylls).

We report simulations of exciton migration and charge separation kinetics in spectrally heterogeneous two-dimensional model antenna/reaction center complexes based on a numerical solution of the Pauli master equation for exciton motion. Experimentally accessible quantities like the charge separation yield, the exciton lifetime(s), the first passage time, the time-resolved and the steady-state fluorescence emission spectra are calculated. The simulated data lend themselves to a direct comparison with experimental data. The diagnostic value of these parameters for studying antenna heterogeneity, antenna structure and trap properties in real photosystems are discussed. Both the ‘slow trap’ and the ‘fast trap’ cases are examined in order to simulate the trap-limited and the diffusion-limited kinetics, respectively. For spectrally heterogeneous antenna sytems we show that conclusions drawn by other authors regarding the effects of antenna optimisation like, e.g., funnelling, on the charge separation yield and exciton kinetics apply only to the hypothetical fast-trap case. In contrast, in the ‘slow-trap case’, which is more relevant for natural photosystems, the influence of spectral and structural optimization on the charge separation yield is quite limited. The most relevant parameters that influence the charge separation yield and the exciton kinetics in the heterogeneous antenna case are the ratio of energy transfer rates for trapping vs. detrapping at the reaction center and the spectral heterogeneity of the antenna. We show that the presence of long-wavelength-absorbing pigments (in relation to reaction center absorption) is still compatible with a high yield of charge separation. However, long-wavelength-absorbing pigments will make a pronounced contribution to the steady-state fluorescence emission spectrum, which has considerable diagnostic value. Funneling is shown to be essential for a short first passage time but less important for achieving a high yield of charge separation.

The present understanding of how interactions between chromophore and protein as well as between chromophore and chromophore in different aggregation states influence the spectral and excited state kinetic properties of phycobiliprotein antenna pigments is discussed. Properties of isolated phycobiliproteins from both cyanobacteria and red algae as well as from cryptophytes and of intact phycobilisomes are covered. The experimental results are discussed in terms of general principles for chromophore coupling and energy transfer. Some controversial topics in this field are outlined.

Magic angle spinning (MAS) solidstate NMR spectroscopy was used to broadly envisage the differences between the [8-Et, 12- Me] BChl c producing bchQR mutant and the [8-Et, 12-Me] BChl d producing bchQRU mutant of the green sulfur bacterium C. tepidum. Twodimensional 13C−13C homonuclear MAS NMR dipolar correlation experiments on uniformly 13C enriched bchQR and bchQRU chlorosomes, revealed a major doubling of the 13C resonances at the 5−C, 131−C, and 14−C position for the bchQRU mutant and a minor doubling at the 5−C, 14−C, and 20−C positions for the bchQRU mutant, indicating the presence of two components in the chlorosomes from both mutants though not in the same ratios.

Summary In this chapter, we discuss the theories applicable for calculating energy transfer kinetics and optical spectra of photosynthetic pigment-protein complexes. The various theoretical approaches for obtaining expressions for rate constants and optical spectra are reviewed. At the extremes we distinguish weak and strong coupling between electronic excitations of the pigments. If the coupling is strong compared to the dynamic and static disorder introduced by the protein environment, then delocalized electronic states are formed after light excitation. The excitation energy relaxes between those delocalized states. In the weak coupling limit localized

The transient absorption kinetics and spectra of isolated reaction centers of photosystem II (PSIIRC) have been modeled using modified Redfield theory. This yielded the site energies of the eight RC pigments, and the intrinsic charge separation rate as well as the charge recombination rate between the excited state and the first radical pair (RP) state. The intrinsic charge separation rate was found to be 0.67 ps−1 for the accessory chlorophyll on the D1 branch as primary electron donor. This is consistent with an average effective primary charge separation time of 5–6 ps. The site energies on the D1 and D2 branches were found asymmetric, with the site energies of all D2 branch pigments substantially higher than for the symmetrically equivalent D1 branch pigments. This is partially at variance with the results of other exciton modeling studies.

Two ²-carotenes are located in the D1–D2 reaction center (RC) of photosystem II (PSII) in the X-ray structure (Loll et al. 2005). Despite the presence of these ²-carotenes and a high yield of reaction center (RC) triplet chlorophyll (Chl) production, no Chl triplet quenching by ²- carotene has been observed so far in the RC of PS II (see Telfer 2005 for a review). We have used nanosecond transient absorption spectroscopy to study the triplet kinetics and photoprotection in intact PS II cores with closed RC from T. elongatus. We report efficient photoprotection by 3Chl-to-Car quenching. We assign the 3Car to the D2 carotene, proposing a photoprotective role to the otherwise inactive branch of the RC

Magic angle spinning (MAS) solidstate NMR spectroscopy was used to investigate the stacking of bacteriochlorophylls (BChl) in the bchQRU mutant of the green sulfur bacterium C. tepidum. This mutant produces [8-Et, 12-Me] BChl d instead of the BChl c in the wild type. Using uniformly 13C enriched bchQRU chlorosomes, a 13C and 1H resonance assignment of the BChl d ring was made using two-dimensional 13C−13C homonuclear and 1H−13C heteronuclear MAS NMR dipolar correlation experiments. The aggregation shifts are largest for the 21−H3, 121−H3, 31−H, and 5-H, which are shifted upfield by −3.3, −2.6, −3.7, and −2.0 ppm, respectively. A comparison of the bchQRU chlorosomes with aggregation shifts for the wild type and chlorin models forming dense aggregates reveals parallel stacking of the [8-Et, 12-Me]BChl d, which is more dense and much more homogeneously ordered than for the BChl c in the wild type. However, the structure is less dense than for the Cd-chlorin models which lack the 31-Me.

The early photosynthetic processes taking place in Photosystem II (PSII), charge separation and electron transfer, can be regulated in many ways, amongst which the intrinsic properties and the internal dynamics of particular cofactors in the electron transfer chain as well as their interactions with the surrounding proteins play a significant role. We compare the time-resolved fluorescence of the dimeric cyanobacterial (Thermosynechococcus elongatus) Photosystem II core particles isolated from WT and from cells expressing only the psbA3 gene product. The D1 protein of the PS II reaction center (RC) is present in three different copies in cyanobacteria: PsbA1, PsbA2 and PsbA3. In the investigated PSII particles the two former genes were knocked-out, resulting in the overexpression of the psbA3 gene, which is not present under physiological light conditions. The psbA3 gene product differs from PsbA1 in over ten amino acid residues, some positioned close to the early electron transfer chain cofactors. The influence of this amino acid alteration, in particular the interaction between the active pheophytin PheoD1 and the D1-130 residue (glutamine/glutamate), on the primary events was investigated in PSII particles with the RCs in either the open or closed state. We are particularly interested in possible changes of the protein dynamics coupled to charge separation.

The energy and electron transfer processes in Photosystem I core and intact particles have been studied. Evidence is provided for location of the ‘red’ chlorophylls outside the core complex. The influence of these forms on the light utilization kinetics has been evaluated. The trapping kinetics in both particles is proven to be trap-limited.

Time-resolved fluorescence measurements were performed on isolated core and intact Photosystem I (PS I) particles and stroma membranes from Arabidopsis thaliana to characterize the type of energy-trapping kinetics in higher plant PS I. Target analysis confirms the previously proposed "charge recombination" model. No bottleneck in the energy flow from the bulk antenna compartments to the reaction center has been found. For both particles a trap-limited kinetics is realized, with an apparent charge separation lifetime of approximately 6 ps. No red chlorophylls (Chls) are found in the PS I-core complex from A. thaliana. Rather, the observed red-shifted fluorescence (700-710 nm range) originates from the reaction center. In contrast, two red Chl compartments, located in the peripheral light-harvesting complexes, are resolved in the intact PS I particles (decay lifetimes 33 and 95 ps, respectively). These two red states have been attributed to the two red states found in Lhca 3 and Lhca 4, respectively. The influence of the red Chls on the slowing of the overall trapping kinetics in the intact PS I complex is estimated to be approximately four times larger than the effect of the bulk antenna enlargement.

It is shown that in all photosystems of oxygenic photosynthesis, with the exception of the largest PS II units, the energy trapping for the excited states is limited by charge separation in the reaction centers, and not by energy transfer from the antenna to the trap. Recent ultrafast spectroscopic studies show that in the reaction centers of oxygenic photosynthesis the primary electron donors are the monomeric accessory chorophylls, and not the “special pairs” P680 or P700. These findings change dramatically our understanding of the ultrafast electron transfer processes in reaction centers. The insight gained from the ultrafast studies on isolated antenna/reaction center units has been applied to unravel the quenching sites in the so-called “non-photochemical quenching” processes that protect in particular photosystem II against photodamage in high light.

Chlorosomcs containing BChl a790 have been isolated from Chloroflexus aurantiacus on sucrose density gradients using the detergents Miranol. Deriphat. N.N-dimethyldodecyl- aminc-N-oxidc, and dodecyl-p-D-rnaltoside. All freshly prepared samples cither lack the poly- peptide of approximately 5 kDa. which appears identical with the 5.6-kDa protein previously assigned the role of BChl c-binding [R. G. Feick and R. C. Fuller. Biochemistry 23, 3693- 3700 (1984)]. or they contain only a minor amount thereof. This polypeptide accumulates in the chlorosomcs in vitro at room temperature within 24 h after isolation. The reaction cannot be prevented simply by addition of the protease inhibitors benzamidinc. F.-caproic ac|d. and phenylmethylsulfonyl fluoride. However, upon denaturation, as required lor gel electrophore- sis, of the freshly isolated chlorosome sample the formation of the 5-kDa polypeptide is inhibit- ed. We conclude that this species, viz. 5.6-kDa protein, is a degradation product of another - as yet unidentified - protein present in the chlorosome preparations. Despite the pronounced proteolytic activity which affords the 5-kDa fragment, the native absorption and fluorescence properties of BChl c and BChl a arc essentially not changed in these chlorosome preparations.

— A fluorescence quantum yield (emission at650–850 nm) of π= (2.3 ± 0.3)10−3 was measured for the red-absorbing form (Pr) of 124-kDa phytochrome from etiolated oat seedlings (Avena sativa) upon excitation in the Soret band at Λexc= 380 nm. The small difference between this value and the previously determined quantum yield with Λexc= 640 nm, π= (3.5 ± 0.4)10−3is attributed to a blue-absorbing emitter responsible for the “anomalous” or “blue” emission of the chromoprotein in the region from ca. 400 to 550 nm. The absorption of Pr at 380 nm is consequently somewhat lower than that measured directly from the spectrum. Processes from upper excited states of the Pr phytochromobilin-derived chromophore other than rapid relaxation to the emitting state are not important. A quantum yield of Φ ' 1.2 times 10−3 is estimated for the blue fluorescence. The proportion of the blue emitters relative to Pr appears to be relatively high.

The concept of a relaxed radical pair state in closed photosystem (PS) II centers (first quinone acceptor reduced) is critically examined on the basis of chlorophyll fluorescence decay data of the green alga Scenedesmus obliquus. Global analysis resulting in the decay-associated fluorescence spectra from closed PS II centers reveals a new PS II lifetime component (τ ≈ 380 ps) in addition to two PS II components (τ ∼ 1.3 and 2.1 ns) resolved earlier. Particular emphasis was given to resolve a potential long-lived (∼ 10 ns) component of small amplitude; however, the longest lifetime found is only 2.1 ns. From comparison of experimental and simulated data we conclude that the maximum relative amplitude of such a potential long-lived component must be 15%) of a long-lived (τ ≥ 3 ns) relaxed radical pair in closed PS II. If at all distinguishable kinetically and energetically from the primary radical pair, a relaxed radical pair would not live longer than 2–3 ns in green algae. The data suggest, however, that the concept of a long-lived relaxed radical pair state is inappropriate for intact PS II.

Decay-associated (time-resolved) fluorescence spectra of isolated bacteriochlorophyll (BChl) a-containing and BChl a-free chlorosomes were measured using a picosecond single-photon timing apparatus in the detection wavelength range 730 – 820 nm. Two different relaxation steps can be resolved in the energy transfer chain. The first relaxation step occurs within the main BChl c pigment pool and the other is due to the energy transfer from one BChl c pool to the BChl a pool in the so-called baseplate. The corresponding equilibration times are approximately 5 ps and approximately 14 ps respectively.

Photoinhibition and photobleaching as a function of time at high fluence rates were studied using picosecond time-resolved emission spectra (TRES) and fluorescence induction in the cyanobacterium Anabaena variabilis. Photoinhibition and photobleaching can be distinguished using these techniques as phenomena which operate on different parts of the photosynthetic apparatus with very different induction periods. Photoinhibition is a short-term irradiation effect (time scale of hours) under our irradiation conditions and operates on photosystem II reaction centres in agreement with earlier studies. On photoinhibition the minimum fluorescence intensity F0 is increased and the maximum fluorescence Fmax is decreased. Both effects lead to a decrease in the variable fluorescence yield. In contrast, photobleaching is a long-term effect (time scale of days) and operates on the phycobilisome antenna. This finding is supported by experiments with sodium azide, a protective agent against photobleaching. Although sodium azide cannot fully prevent the formation of long-lived fluorescence components (detached phycobiliproteins), large negative amplitudes measured in the sodium-azide-protected samples indicate an efficient energy transfer to the reaction centre which is absent in the control.

The interior of chlorosomes, the main antenna system of the photosynthesizing bacterium Chloroßexus aurantiacus, is shown to contain no proteins in a fixed ratio to BChl c and in amounts that could be significant of direct chromophore-protein complexes. This excludes non-covalent chromophore-protein complexing -that has so far been found in all other antennae -as the main organizational principle of the interior architecture for chlorosom es of chlorophyll C. aurantiacus. Rather, these antennae constitute the first case of a chromophore-chromophore aggregate functioning as a photosynthetic light harvesting system.

Picosecond time-resolved fluorescence emission spectra were recorded for cells of the cyanobacterium Synechococcus 6301. Fluorescence decay was measured by single-photon timing and kinetic components were resolved by global data analysis. Time-resolved fluoresence decay components were assigned to PS I, PS II and the phycobilisome terminal emitter by comparing spectra for cells with open PS II centres (fluorescence at Fo) with those for cells with closed PS II centres (fluorescence at Fm) for excitation wavelengths of 620 nm and 670 nm. Time-resolved spectra were recorded under these conditions for cells adapted to state 1 or to state 2. The state 2 transition reduced the amplitude of the PS II fluorescence emission by about 60%, with a complementary increase in the apparent amplitude of the emission from the phycobilisome terminal emitter. Similar changes in amplitude were observed for cells at Fo and at Fm. State transitions had no significant effect on the lifetime of PS II fluorescence decay. These results indicate that state transitions alter the extent of energy transfer from the phycobilisome to PS II.

The excited state kinetics of three different allophycocyanin (AP) complexes has been studied by picosecond fluorescence spectroscopy. Both the fluorescence kinetics and the decay-associated fluorescence spectra of the different complexes can be understood on the basis of a structural model for AP which uses (a) an analogy to the known x-ray determined structure of C-phycocyanin, (b) the biochemical analogies of AP and C-phycocyanin, and (c) the biochemical composition of AP-B (AP-681). A model is developed that describes the excited state kinetics as a mixture of internal conversion processes within a coupled exciton pair and energy transfer processes between exciton pairs. We found excited state relaxation times in the range of 13 ps (AP with linker peptide) up to 66 ps (AP-B). The trimeric aggregates AP 660 and AP 665 show one fast relaxation component each, as was expected on the basis of their symmetry properties. The lower symmetry of AP-B (AP-681) gives rise to two fast lifetime components (τ1 = 23 ps and τ2 = 66 ps) which are attributed to internal conversion and/or energy transfer between excitonic states formed by the coupling of symmetrically and spectrally nonequivalent chromophores. It is proposed that the internal conversion between exciton states of strongly coupled chromophores fulfills the requirements of the small energy gap limit. Thus, internal conversion rates in the order of tens of picoseconds are feasible. The influence of the interaction of the linker peptide on the properties of the AP trimer are manifested in the fluorescence kinetics. Lack of the linker peptide in AP 660 gives rise to a heterogeneity in the chromophore conformations and chromophore-chromophore interactions.

The photophysical properties of 124-kDa phytochrome from etiolated oat shoots and some aspects of its photochemical reactivity are reviewed in terms of the working scheme depicted in Fig. 3. A review discussing these results in greater detail - with the exception of the interaction of the Pr form of phytochrome with ubiquitin - has been recently published in ref. le.

Intact chlorosomes of Chlorobium tepidum were embedded in amorphous ice layers and examined by cryoelectron microscopy to study the long-range organization of bacteriochlorophyll ( BChl) layers. End-on views reveal that chlorosomes are composed of several multi-layer tubules of variable diameter (20-30 nm) with some locally undulating non-tubular lamellae in between. The multi-layered tubular structures are more regular and larger in a C. tepidum mutant that only synthesizes [8-ethyl, 12-methyl]-BChl d. Our data show that wild-type C. tepidum chlorosomes do not have a highly regular, long-range BChl c layer organization and that they contain several multilayered tubules rather than single-layer tubules or exclusively undulating lamellae as previously proposed. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.