Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10(13) photons.cm-2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10(13)--10(16) photons-cm-2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part I. In the intact alga

The wavelength-resolved fluorescence emission kinetics of the accessory pigments and chlorophyll a in Porphyridium cruentum have been studied by picosecond laser spectroscopy. Direct excitation of the pigment B-phycoerythrin with a 530 nm, 6 ps pulse produced fluorescence emission from all of the pigments as a result of energy transfer between the pigments to the reaction centre of Photosystem II. The emission from B-phycoerythrin at 576 nm follows a nonexponential decay law with a mean fluorescence lifetime of 70 ps, whereas the fluorescence from R-phycocyanin (640 nm), allophycocyanin (660 nm) and chlorophyll a (685 nm) all appeared to follow an exponential decay law with lifetimes of 90 ps, 118 ps and 175 ps respectively. Upon closure of the Photosystem II reaction centres with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination the chlorophyll a decay became non-exponential, having a long component with an apparent lifetime of 840 ps. The fluorescence from the latter three pigments all showed finite risetimes to the maximum emission intensity of 12 ps for R-phycocyanin, 24 ps for allophycocyanin and 50 ps for chlorophyll a.A kinetic analysis of these results indicates that energy transfer between the pigments is at least 99% efficient and is governed by an exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ transfer function. The apparent exponential behaviour of the fluorescence decay functions of the latter three pigments is shown to be a direct result of the energy transfer kinetics, as are the observed risetimes in the fluorescence emissions.     

Energy transfer from photosystem II to photosystem I in Porphyridium cruentum

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of Porphyridium cruentum which had been frozen to ?196 °C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at ?196 °C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part I. In the intact alga

The wavelength-resolved fluorescence emission kinetics of the accessory pigments and chlorophyll a in Porphyridium cruentum have been studied by pico-second laser spectroscopy. Direct excitation of the pigment B-phycoerythrin with a 530 nm, 6 ps pulse produced fluorescence emission from all of the pigments as a result of energy transfer between the pigments to the reaction centre of Photosystem II. The emission from B-phycoerythrin at 576 nm follows a nonexponential decay law with a mean fluorescence lifetime of 70 ps, whereas the fluorescence from R-phycocyanin (640 nm), allophycocyanin (660 nm) and chlorophyll a (685 nm) all appeared to follow an exponential decay law with lifetimes of 90 ps, 118 ps and 175 ps respectively. Upon closure of the Photosystem II reaction centres with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination the chlorophyll a decay became non-exponential, having a long component with an apparent lifetime of 840 ps. The fluorescence from the latter three pigments all showed finite risetimes to the maximum emission intensity of 12 ps for R-phycocyanin, 24 ps for allophycocyanin and 50 ps for chlorophyll a. A kinetic analysis of these results indicates that energy transfer between the pigments is at least 99% efficient and is governed by an exp --At1/2 transfer function. The apparent exponential behaviour of the fluorescence decay functions of the latter three pigments is shown to be a direct result of the energy transfer kinetics, as are the observed risetimes in the fluorescence emissions.     

Energy distribution in the photochemical apparatus of Porphyridium cruentum in state I and state II

Fluorescence of Porphyridium cruentum in state I (cells equilibrated in light absorbed predominantly by Photosystem I) and in state II (cells equilibrated in light absorbed appreciably by Photosystem II) was examined to determine how the distribution of excitation energy was altered in the transitions between state I and state II. Low temperature emission spectra of cells frozen in state I and state II confirmed that a larger fraction of the excitation energy is delivered to Photosystem II in state I. Low temperature measurements showed that the yield of energy transfer from Photosystem II to Photosystem I was greater in state II and calculations indicated that the photochemical rate constant for such energy transfer was approximately twice as large in state II. Measurements at low temperature also showed that the cross sections and the spectral properties of the photosystems did not change in the transitions between state I and state II. In agreement with predictions made from the parameters measured at low temperature, the action spectra for oxygen evolution measured at room temperature were found to be the same in state I and state II.     

Picosecond kinetics of light harvesting and photoprotective quenching in wild-type and mutant phycobilisomes isolated from the cyanobacterium Synechocystis PCC 6803

In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCP(r)), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APC(Q)(660)) with a molecular quenching rate that is faster than (1 ps)(-1). Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APC(Q)(660) and OCP or by excitation energy transfer from APC(Q)(660) to the S(1) state of the carotenoid--a distinction that is very hard, if not impossible, to make in vivo.     

Highly efficient energy transfer from a carbonyl carotenoid to chlorophyll a in the main light harvesting complex of Chromera velia

We report on energy transfer pathways in the main light-harvesting complex of photosynthetic relative of apicomplexan parasites, Chromera velia. This complex, denoted CLH, belongs to the family of FCP proteins and contains chlorophyll (Chl) a, violaxanthin, and the so far unidentified carbonyl carotenoid related to isofucoxanthin. The overall carotenoid-to-Chl-a energy transfer exhibits efficiency over 90% which is the largest among the FCP-like proteins studied so far. Three spectroscopically different isofucoxanthin-like molecules were identified in CLH, each having slightly different energy transfer efficiency that increases from isofucoxanthin-like molecules absorbing in the blue part of the spectrum to those absorbing in the reddest part of spectrum. Part of the energy transfer from carotenoids proceeds via the ultrafast S₂ channel of both the violaxanthin and isofucoxanthin-like carotenoid, but major energy transfer pathway proceeds via the S₁/ICT state of the isofucoxanthin-like carotenoid. Two S₁/ICT-mediated channels characterized by time constants of ~ 0.5 and ~ 4 ps were found. For the isofucoxanthin-like carotenoid excited at 480 nm the slower channel dominates, while those excited at 540 nm employs predominantly the fast 0.5 ps channel. Comparing these data with the excited-state properties of the isofucoxanthin-like carotenoid in solution we conclude that, contrary to other members of the FCP family employing carbonyl carotenoids, CLH complex suppresses the charge transfer character of the S₁/ICT state of the isofucoxanthin-like carotenoid to achieve the high carotenoid-to-Chl-a energy transfer efficiency.     

Exploring the mechanism(s) of energy dissipation in the light harvesting complex of the photosynthetic algae Cyclotella meneghiniana

Photosynthetic organisms have developed vital strategies which allow them to switch from a light-harvesting to an energy dissipative state at the level of the antenna system in order to survive the detrimental effects of excess light illumination. These mechanisms are particularly relevant in diatoms, which grow in highly fluctuating light environments and thus require fast and strong response to changing light conditions. We performed transient absorption spectroscopy on FCPa, the main light-harvesting antenna from the diatom Cyclotella meneghiniana, in the unquenched and quenched state. Our results show that in quenched FCPa two quenching channels are active and are characterized by differing rate constants and distinct spectroscopic signatures. One channel is associated with a faster quenching rate (16 ns^− 1) and virtually no difference in spectral shape compared to the bulk unquenched chlorophylls, while a second channel is associated with a slower quenching rate (2.7 ns^− 1) and exhibits an increased population of red-emitting states. We discuss the origin of the two processes in the context of the models proposed for the regulation of photosynthetic light-harvesting. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Factors affecting energy transfer from phycobilisomes to thylakoids in Anacystis nidulans

Short illumination with white light of dark-maintained Anacystis nidulans prior to immersion in liquid nitrogen resulted in a marked change of fluorescence emission characteristics at 77 K. The fluorescence of Photosystem II-associated membrane bound pigments increases, while the emission due to phycobilins decreases. This effect seems to be due to a light-dependent alteration in the extent of contact between phycobilisomes and thylakoids, since the effect is reversible in the dark and is abolished by short glutaraldehyde fixation. The preillumination effect is not inhibited by DCMU. Emission spectra obtained with actively growing and CO₂-starved cells indicate that the light-dependent increase in energy transfer from phycobilins to chlorophyll depends upon the physiological state of the cells.     

Mechanism of the light state transition in photosynthesis. I. Analysis of the kinetics of cytochrome f oxidation in State 1 and State 2 in the red alga, Porphyridium cruentum

The kinetics of photooxidation and reduction of cytochrome f were examined spectrophotometrically in the red alga Porphyridium cruentum in light State 1 and light State 2. Experiments were performed on intact cells that had been chemically fixed and stabilized in the light states. The cytochrome f turnover was measured during conditions of linear electron transport driven by both photosystems and during several cyclic reactions mediated by the long-wavelength Photosystem (PS) I. The data show that the rate of photooxidation of cytochrome f increased in State 2 when the cells were activated by subsaturating intensities of green light absorbed primarily by the phycobilisome. No differences in kinetics were found between algae in State 1 or State 2 when they were activated by light absorbed primarily by the chlorophyll of PS I. The results confirm that changes in energy distribution between the two photosystems occur as a result of the light state transition and verify that the redistribution of excitation results in the predicted changes in electron transport.     

Studies on chromophore coupling in isolated phycobiliproteins. I. Picosecond fluorescence kinetics of energy transfer in phycocyanin 645 from Chroomonas sp.

By applying the single-photon timing method the fluorescence kinetics of phycocyanin 645 from Chroomonas sp. has been measured as a function of both the excitation and emission wavelength using low-intensity excitation. The fluorescence kinetics were found to be dominated by a fast (15 ps) and a slow (1.44 ns) decay component. The relative yields and amplitudes of these components depended strongly on both the excitation and emission wavelengths. A component with a small relative amplitude and a lifetime (τ) in the range of 360–680 ps has been found as well. The fast decay component is attributed to intramolecular energy transfer from sensitizing to fluorescing chromophores. Our results are discussed in relation to a chromophore coupling model suggested previously (Jung, J., Song, P.-S., Paxton, R.J., Edelstein, M.S., Swanson, R. and Hazen, E.E. (1980) Biochemistry 19, 24–32).     

Polarized absorption picosecond kinetics as a probe of energy transfer in phycobilisomes of Synechococcus 6301

The energy transfer between C-phycocyanin chromophores in intact phycobilisomes of Synechococcus 6301 is shown to lead to an anisotropy relaxation with a lifetime of 10 ± 2 ps. However, due to the molecular order within the hexameric units of C-phycocyanin the anisotropy does not decay to zero. The Förster dipole-dipole mechanism of energy transfer can qualitatively explain these data provided that there is no back transfer of excitation energy and that the chromophore distribution is non-random. The rate of energy transfer in phycobilisomes between C-phycocyanin and allophycocyanin can best be described by a double exponential with lifetimes of 12 ± 3 and 84 ± 8 ps.     

Native and in vitro-associated phycobilisomes of Nostoc sp. Composition,energy transfer,and effect of antibodies

The relationship of the structure and function of the light-harvesting antennae in the blue-green alga Nostoc sp. was further elucidated by reconstitution experiments. Separated phycoerythrin-phycocyanin complexes and allophycocyanin fractions were reassociated as described earlier (Canaani, O., Lipschultz, C.A. and Gantt, E. (1980) FEBS Lett. 115, 225–229) into functional phycobilisomes with a 70% yield. Native and reassociated physobilisomes had molar ratios of about 1.4:1.1:1.0 of phycoerythrin:phycocyanin:allophycocyanim. Energy transfer was demonstrated by their fluorescence emission maximum at approx. 675 nm (20°C), and their excitation spectra (emission wavelength 680 nm) which reflected the contribution of the three constitutive phycobiliproteins. Scans of Coomassie blue-stained SDS-polyacrylamide gels showed that the polypeptide composition of native and reassociated phycobilisomes was virtually indistinguishable. Reassociation of phycobilisomes was dependent on the interaction of allophycocyanin and phycocyanin, because it could be blocked with antisera to phycocyanin and allophycocyanin, but not to phycoerythrin. In addition, reassociation did not occur when a 31 000 Da polypeptide, which is part of the phycoerythrin-phycocyanin complex, was reduced in size (by 4000 Da). These results suggest that at least two domains are required for functional reassociation of phycobilisomes involving phycocyanin and allophycocyanin.     

Effect of adaptation to high light intensity on the kinetics of energy transfer from phycobilisomes to photosystem II in Anabaena cylindrica

Transfer efficiencies between phycobilisomes and photosystem II antenna chlorophylls were determined on membrane fragments isolated from low and high light adapted Anabaena cells. The observed increase in energy transfer in high light adapted cells is a consequence of shorter interchromophore distances and a decrease in the number of jumps of the exciting photons. Calculation of the rates of energy transfer and the coupling energies indicate that the weak interaction inferred for energy transfer between phycobilisome and photosystem II in low light adapted cells is replaced by an intermediate interaction in high light adapted cells.Abbreviations LLA low light adapted - HLA high light adapted - PBS phycobilisome - PS photosystem     

Picosecond time-resolved fluorescence studies on excitation energy transfer in a histidine 117 mutant of the D2 protein of photosystem II in Synechocystis 6803

The role of the peripheral reaction center chlorophyll a molecule associated with His117 of the D2 polypeptide in photosystem II was investigated in Synechocystis sp. PCC 6803 using a combination of steady state, pump-probe, and picosecond time-resolved fluorescence spectroscopy. Data were obtained from intact cells and isolated thylakoid membranes of a control mutant and a D2-H117T mutant, both of which lacked photosystem I. Excitation energy transfer and trapping were investigated by analyzing the data with a kinetic model that used an exact numerical solution of the Pauli master equation, taking into account available photosystem II spectral and structural information. The results of our kinetic analysis revealed the observed difference in excited-state dynamics between the H117T mutant and the control to be consistent with a retardation of the rate of excitation energy transfer from the peripheral chlorophyll of D2 (Chl at His117) to the electron-transfer pigments and an increase of the rate constant for charge recombination in the H117T mutant. The kinetic model was able to account for the experimentally observed changes in absorption cross section and fluorescence decay kinetics between the control and mutant by invoking changes in only these two rate constants. The results rule out quenching of excitation by a chlorophyll cation radical as a mechanism responsible for the lower efficiency of excitation energy utilization in the H117T mutant. Our work also demonstrates the importance of the chlorophyll associated with His117 of the D2 protein for excitation energy transfer to the PSII electron-transfer pigments and for the effective stabilization of the primary radical pair.     

An alternative model for photosystem II/light harvesting complex II in grana membranes based on cryo-electron microscopy studies.

The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes. These membranes have an ultrastructure that consists of tightly stacked 'grana' regions interconnected by unstacked membrane regions. The structure of isolated grana membranes has been studied here by cryo-electron microscopy. The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes. Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions. These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself. This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems.     

Picosecond energy transfer in Porphyridium cruentum and Anacystis nidulans.

Picosecond energy transfer is measured in Anacystis nidulans and Porphyridium cruentum. Fluorescence is sensitized by a 6-ps laser flash, at 530 nm. The time dependence of fluorescence is measured with reference to the laser pulse. Fluorescence is recorded from phycoerythrin (576 nm), R-phycocyanin (640 nm), allophycocyanin (666 nm), Photosystem II chlorophyll (690 nm) and long wave length chlorophyll (715 nm). Energy transfer measurements are made at 37 degrees C, 23 degrees C, and 0 degrees C, and 77 degrees K. It is shown that the rate of energy transfer can be varied with temperature. In both A. nidulans and P. cruentum there is a sequential transfer of excitation energy from phycoerythrin to phycocyanin to allophycocyan to Photosystem II chlorophyll fluorescence. The long wavelength chlorophyll fluorescence at 715 nm, however, does not always follow a sequential transfer of excitation energy. Depending on the temperature, fluorescence at 715 nm can precede fluorescence from phycocyanin.     

Interaction between light harvesting chlorophyll-a/b protein (LHCII) kinase and cytochrome b6/f complex. In vitro control of kinase activity

We have previously reported that the cytochrome b6/f complex may be involved in the redox activation of light harvesting chlorophyll-a/b protein complex of photosystem II (LHCII) kinase in higher plants (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210). The aim of this work was to establish whether a relation between the cytochrome b6/f and LHCII kinase activation can be demonstrated in vitro. Preparations enriched in cytochrome b6/f obtained from spinach thylakoids by detergent extraction and precipitation with ammonium sulfate followed by different procedures of purification, contained various amounts of LHCII kinase activity. Analysis of the cytochrome b6/f content and kinase activity of fractions obtained by histone-Sepharose and immunoaffinity columns, immunoprecipitation and sucrose density centrifugation, indicate functional association of kinase and cytochrome b6/f. Phosphorylation of LHCII by fractions containing both cytochrome b6/f and kinase was enhanced by addition of plastoquinol-1. LHCII phosphorylation and kinase activation could be obtained in fractions prepared by use of beta-D-octyl glucoside but not when 3-[(cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate was used as the solubilizing detergent. Kinase activity could be inhibited by halogenated quinone analogues (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2,3-diiodo-5-t-butyl-p-benzoquinone) known to inhibit cytochrome b6/f activity. However, kinase activity was inhibited by these analogues in all preparations including those which could not phosphorylate LHCII. We thus propose that the redox activation of LHCII phosphorylation is mediated by kinase interaction with cytochrome b6/f while the deactivation may be related to a distinct quinone binding site of the enzyme molecule.