Joachim Wendler

Studies on Chromophore Coupling in Isolated Phycobiliproteins: II. Picosecond Energy Transfer Kinetics and Time-Resolved Fluorescence Spectra of C-Phycocyanin from Synechococcus 6301 as a Function of the Aggregation State.

The fluorescence kinetics of C-Phycocyanin in the monomeric, trimeric, and hexameric aggregation states has been measured as a function of the emission wavelength with picosecond resolution using the single-photon timing technique. All the decay curves measured at the various emission wavelengths were analyzed simultaneously by a global data analysis procedure. A sum of four exponentials was required to fit the data for the monomers and trimers. Only in the case of the hexamers, a three-exponential model function proved to be nearly sufficient to describe the experimental decays. The lifetime of those fluorescence components reflecting energy transfer decreased with increasing aggregation. This is due to the increased number of efficient acceptor molecules next to a donor in the higher aggregates. In all aggregates the shortest-lived component, ranging from 50 ps for monomer to 10 ps for hexamers, is observed as a decay term (positive amplitude) at short emission wavelength. At long emission wavelength it turns into a rise term (negative amplitude). The lifetime of a second ps-component ranges from 200 ps for monomers to 50 ps for hexamers. The long-lived (ns) fluorescence is inhomogeneous in monomers and trimers, showing two lifetimes of approximately 0.6 and 1.3 ns. The latter one carries the larger amplitude. The amplitudes of the kinetic components in the fluorescence decays are presented as time-resolved component spectra. A theoretical model has been derived to rationalize the observed fluorescence kinetics. Using symmetry arguments, it is shown that the fluorescence kinetics of C-Phycocyanin is expected to be characterized by three exponential kinetic components, independent of the aggregation state. An analytical expression is derived, which allows us to gain a detailed understanding of the origin of the different kinetic components and their associated time-resolved spectra. Numerical calculations of time-resolved spectra are compared with the experimental data.

Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of Photosystem I fluorescence☆

The time-resolved fluorescence emission and excitation spectra of Chlorella vulgaris cells have been measured by single-photon timing with picosecond resolution. In a three-exponential analysis the time-resolved excitation spectra recorded at 685 and 706 nm emission wavelength with closed PS II reaction centers show large variations of the preexponential factors of the different decay components as a function of wavelength. At λem = 685 nm the major contribution to the fluorescence decay originates from two components with life-times of 2.1–2.4 and 1.2–1.3 ns. A short-lived component with life-times of 0.1–0.16 ns of relatively small amplitude is also found. When the emission is detected at 706 nm, the short-lived component with a life-time of less than 0.1 ns predominates. Time-resolved emission spectra using λexc = 630 or λexc = 652 nm show a spectral peak of the two longer-lived components at about 680–685 nm, whereas the fast component is red-shifted as compared to the others and shows a maximum at about 690 nm. The emission spectrum observed upon excitation at 696 nm with closed PS II reaction centers shows a large increase in the amplitude of the fast component with a lifetime of 80–100 ps as compared to that at 630 nm excitation. At almost open Photosystem II (PS II) reaction centers (F0), the life-time of the fast component decreased from 150–160 ps at 682 nm to less than 100 ps at 720 nm emission wavelength. We conclude that at least two pigment pools contribute to the fast component. One is attributed to PS II and the other to Photosystem I (PS I). They have life-times of approx. 180 ps and 80 ps, respectively. The 80 ps (PS I) contribution has a spectral maximum slightly below 700 nm, whereas the 180 ps (PS II) spectrum peaks at 680–685 nm. The spectra of the middle decay component τm and its sensitivity to inhibitors of PS II suggest that this component is not preferentially related to LHC II but arises mainly from Chl a pigments probably associated with a second type of PS II centers. The amplitudes of the fast (180 ps, PS II) component and the long-lived decay show an opposite dependence on the state of the PS II centers and confirm our earlier conclusion that the contribution of PS II to the fast component probably disappears at the Fmax state (Haehnel W., Holzwarth, A.R. and Wendler, J. (1983) Photochem. Photobiol. 34, 435–443). Our data are discussed in terms of α,β-heterogeneity in PS II centers.

State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F(0) level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (tau approximately 85 ps, lambda(max) (em) approximately 695-700 nm), open PS II alpha-centers (tau approximately 300 ps, lambda(max) (em) = 685 nm), and open PS II beta-centers (tau approximately 600 ps, lambda(max) (em) = 685 nm). A fourth component of very low amplitude (tau approximately 2.2-2.3 ns, lambda(max) (em) = 685 nm) derives from dead chlorophyll. (b) At the F(max) level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F(0) level, from closed PS II alpha-centers (tau approximately 2.2 ns, lambda(max) (em) = 685 nm) and from closed PS beta-centers (tau approximately 1.2 ns, lambda(max) (em) = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of alpha- and beta-units brought about by the reversible migration of light-harvesting complexes between alpha-centers and beta-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II alpha,beta-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202).

Picosecond study of energy-transfer kinetics in phycobilisomes of Synechococcus 6301 and the mutant AN 112

Excitation-energy-transfer kinetics in isolated phycobilisomes from the cyanobacterium Synechococcus 6301 (Anacystis nidulans) and the mutant AN 112 (rods containing one hexameric C-phycocyanin unit only) was investigated by picosecond absorption and fluorescence techniques. The different chromophores in the phycobilisomes were selectively excited. A lifetime component of about 10 ps was found for both C-phycocyanin and allophycocyanin in both types of phycobilisomes. We assign these signals to a transfer of excitation energy from sensitizing (‘s’) to fluorescing (‘f’) chromophores within C-phycocyanin and allophycocyanin units. A 10 ps component was also observed in the anisotropy relaxation measurements. The anisotropy decay is attributed mainly to differently oriented transition dipole moments of ‘s’- and ‘f’-chromophores and partially to ‘f’ → ‘f’ transfer. An absorption recovery signal of τ ≈ 90 ps at λ ≤ 630 nm in phycobilisomes of Synechococcus 6301 is reduced to 40–50 ps in AN 112 phycobilisomes. This is rationalized in terms of a decreased rod → core transfer time in the shorter rods of AN 112. The 40–50 ps lifetime of fluorescence and absorption recovery in AN 112 phycobilisomes is assigned mainly to a rate-limiting transfer step between C-phycocyanin and the allophycocyanin core. A decay component of allophycocyanin τ ≈ 50 ps was observed both in absorption recovery measurements and in fluorescence decay. It is assigned to energy transfer to the terminal chromophores. The final emitter(s) of the phycobilisomes from AN 112 have fluorescence lifetimes of 1.9 and 1.3 ns. We find a good correlation in the fluorescence kinetics between the decay times of phycocyanin and allophycocyanin and the fluorescence risetimes of the terminal emitters.

Fluorescence decay kinetics in phycobilisomes isolated from the bluegreen alga Synechococcus 6301

Abstract The picosecond fluorescence and energy-transfer kinetics of isolated phycobilisomes from Synechococcus 6301 were studied under low intensity excitation. Different combinations of excitation and emission wavelengths were used in order to monitor selectively the fluorescence of the pigments phycocyanin and allophycocyanin. The relatively long overall energy-transfer time of 120 ps from the phycocyanin rods to the allophycocyanin-core is rationalized in terms of the special structure of the rods being built up of several phycocyanin hexamers in this alga species. The fluorescence lifetime of the terminal chromophores in the core was determined to be 1.8–1.9 ns depending on the excitation wavelength. A fast decay component of 20 ± 10 ps which is most prominent at short emission wavelengths is assigned to arise mainly from energy transfer within the C-phycocyanin-units from ‘sensitizing’ to ‘fluorescing’ chromophores.

Picosecond time-resolved energy transfer in phycobilisomes isolated from the red alga Porphyridium cruentum☆

Abstract Energy-transfer kinetics in isolated phycobilisomes of the red alga Porphyridium cruentum has been probed by picosecond absorption and fluorescence techniques upon selective excitation of the individual phycobiliproteins by a tunable picosecond laser. The fluorescence decays of B-phycoerythrin and R-phycocyanin were found to be non-exponential. It does not, however, follow an exp (− 2At 1 2 ) decay law. The main components in the fluorescence decays of B-phycoerythrin and R-phycocyanin have lifetimes of approx. 60 and approx. 40 ps, respectively, as a result of energy transfer. In addition a second decay component with small relative amplitude is required for a good description of the energy-transfer kinetics. This component has a lifetime in the range of approx. 200 ps (B-phycoerythrin) and 550 ps (R-phycocyanin). The fluorescence decay in the main emission band is non-uniform with two components of 1.0 and 1.8 ns. The energy transfer processes were found to occur sequentially from B-phycoerythrin to R-phycocyanin and allophycocyanin, in agreement with an earlier study (Searle, G.F.W., Barber, J., Porter, G. and Tredwell, C.J. (1978) Biochim. Biophys. Acta 501, 246–256). Measurements of transient absorption anisotropy revealed the presence of two processes leading to fast depolarization in B-phycoerythrin. The anisotropy decay times have values of 12 and 150 ps. The shorter one is attributed to intramolecular energy transfer within B-phycoerythrin monomers. the longer one either arises from transfer within the B-phycoerythrin pigment bed or is related to the transfer from B-phycoerythrin to R-phycocyanin. Rising-terms are observed in the fluorescence kinetics of the indirectly excited pigments. Their rate constants agree well with those determined from the decay of the directly excited pigments. The characteristic energy-transfer time from B-phycoerythrin at the periphery to the terminal emitter is around 70 ps. This fast transfer ensures an efficiency of better than 98%. The overall energy-transfer kinetics in these hemiellipsoidal phycobilisomes is found to be very similar to that found in hemidiscoidal phycobilisomes.

PICOSECOND TIME RESOLVED ENERGY TRANSFER IN ISOLATED PHYCOBILISOMES FROM RHODELLA VIOLACEA (RHODOPHYCEAE)

Energy‐transfer kinetics in isolated phycobilisomes (PBS) of the red alga Rhodella violacea have been measured by detecting both ground state recovery and fluorescene rise or decay using a synchronously pumped cavity‐dumped dye laser as excitation source. For the first time the constituent phycobiliproteins of PBS have been excited selectively, thus allowing the kinetics of both directly and indirectly excited pigments to be followed. Energy‐transfer between the phyeobiliproteins, which was found to proceed extremely fast, is governed by nonexponential kinetics at low excitation intensities. When analyzed in a biexponential model, the main components of the fluorescence of B‐phycoerythrin (B‐PE) and C‐phycocyanin (C‐PC) decay with τ= 34 ps and τ= 25 ps, respectively. Evidence is presented that transfer between the biliprotein pigments is close to a single‐step process with some contribution of homotransfer. Fluorescence quantum yields of PBS have been determined as a function of the excitation wavelength and were found to reflect a dissociation equilibrium involving ca. 10% dissociated PBS at the concentrations studied.

Picosecond time-resolved and stationary fluorescence of oat phytochrome highly enriched in the native 124 kDa protein

Abstract Photophysical data of the first excited singlet state have been determined by picosecond fluorescence decay and fluorescence quantum yield measurements of a phytochrome preparation highly enriched in the native (124 kDa) form. A lifetime of 48 ± 3 ps for the P r form has been measured, with a flourescence yield of 2.9 · 10 −3 . These photophysical data are identical within the error limits with those reported for the large (114 and 118 kDa) and small (60 kDa) P r phytochromes (Wendler, J.; Holzwarth, A.R.; Braslavsky, S.E. and Schaffner K. (1984) Biochim. Biophys. Acta 786, 213–221). Using the radiative lifetime of 14 ns for P r , a fluorescence yield of 3.4 · 10 −3 is calculated. Our lifetime and fluorescence data for native phytochrome are thus also consistent with the known radiative lifetime. No change in the fluorescence lifetime with temperature could be found in the range 275–298 K. The implications of our data with respect to the reported photochemical differences between the large and native phytochromes, in particular the higher phototransformation yield of the latter, are discussed. The percentage of P fr at photoequilibrium, as obtained under our conditions ( A 665 = 0.10/cm, in phosphate buffer at 277 K), is P ftr 660 = 0.75 ± 0.03.

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

Abstract 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).

Wavelength-resolved fluorescence decay and fluorescence quantum yield of large phytochrome from oat shoots

Abstract Measurements of both wavelength-resolved fluorescence decay and fluorescence quantum yield of large (119 kDa) Pr phytochrome and small (60 kDa) Pr phytochrome for the first time provide a consistent set of the photophysical parameters of the excited state of Pr. At 275 K, the lifetimes of both large and small Pr have been determined to be 45 ± 10 ps, while the fluorescence yields are 2.0 10−3 and 1.5 10−3, respectively.