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 λ_em = 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 λ_exc = 630 or λ_exc = 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 τ_m 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 F_max 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.     

Fluorescence lifetime spectra of in vivo bacteriochlorophyll at room temperature

Fluorescence lifetime spectra of Rhodopseudomonas sphaeroides chromatophores have been measured at room temperature by phase fluorimetry at 82 MHz in order to investigate the heterogeneity of the emission. The total fluorescence was decomposed into two main components. A constant component, F_c, centered at 865 nm, represents about 50% of the total emission from dark-adapted chromatophores (F_o) and has a lifetime of 0.55 ns. A variable component is centered at 890 nm. Upon closing the reaction centers, 5-fold increases take place in both emission yield and lifetime of this component. In the dark-adapted state, its lifetime is about 50 ps and its contribution to the total fluorescence is 70% at 890 nm. In the presence of sodium dithionite, a long-lifetime component ($\text{τ}{}_{\mathrm{<mtext>D</mtext>}}\text{? 4}\text{ns}$) is observed. This probably arises from radical pair recombination between P⁺ and I^? (P, the primary electron donor, is a dimer of bacteriochlorophyll; I, the primary electron acceptor, is a molecule of bacteriopheophytin). Its spectrum is nearly identical to that of the variable component. This emission seems to be present also under nonreducing conditions, although with a much weaker intensity than when the electron acceptor quinone is prereduced.     

Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl₂-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where A was found to be 0.052 $\text{ps}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl₂ produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.     

Probing the kinetics of Photosystem I and Photosystem II fluorescence in pea chloroplasts on a picosecond pulse fluorometer

Picosecond fluorescence kinetics of pea chloroplasts have been investigated at room temperature using a pulse fluorometer with a resolution time of 10^?11 s. Fluorescence has been excited by both a ruby and neodymium-glass mode-locked laser and has been recorded within the 650 to 800 nm spectral region.We have found three-component kinetics of fluorescence from pea chloroplasts with lifetimes of 80, 300 and 4500 ps, respectively. The observed time dependency of the fluorescence of different components on the functional state of the photosynthetic mechanism as well as their spectra enabled us to conclude that Photosystem I fluoresces with a lifetime of 80 ps (τ_I) and Photosystem II fluoresces with a lifetime of 300 ps (τ_II). Fluorescence with a lifetime of 4500 ps (τ_III) may be interpreted as originating from chlorophyll monomeric forms which are not involved in photosynthesis.It was determined that the rise time of Photosystem I and Photosystem II fluorescence after 530 nm photoexcitation is 200 ps, which corresponds to the time of energy migration to them from carotenoids.     

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.     

Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K II. ATP-dependent quenching

In intact, uncoupled type B chloroplasts from spinach, added ATP causes a slow light-induced decline ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{min}$) of chlorophyll a fluorescence at room temperature. Fluorescence spectra were recorded after fast cooling to 77 K and normalized with fluorescein as an internal standard. Related to the fluorescence quenching at room temperature, an increase in Photosystem (PS) I fluorescence (F₇₃₅) and a decrease in PS II fluorescence (F₆₉₅) were observed in the low-temperature spectra. The change in the $\text{F}{}_{735}\text{F}{}_{695}$ ratio was abolished by the presence of methyl viologen. Fluorescence induction at 77 K of chloroplasts frozen in the quenched state showed lowered variable (F_v) and initial (F₀) fluorescence at 690 nm and an increase in F₀ at 735 nm. The results are interpreted as indicating an ATP-dependent change of the initial distribution of excitation energy in favor of PS I, which is controlled by the redox state of the electron-transport chain and, according to current theories, is caused by phosphorylation of the light-harvesting complex.     

Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K I. ΔpH-dependent quenching

The nature of the light-induced ΔpH-dependent decline of chlorophyll a fluorescence in intact and broken spinach chloroplasts was investigated. Fluorescence spectra at 77 K of chloroplasts frozen in the low-fluorescent (high ΔpH) state showed increased ratios of the band peak at 735 nm (Photosystem (PS) I fluorescence) to the peak at 695 nm (PS II fluorescence). The increase in the $\text{F}{}_{735}\text{F}{}_{695}$ ratio at 77 K was related to the extent of fluorescence quenching at room temperature. Normalization of low-temperature spectra with fluorescein as an internal standard revealed a lowering of F₆₉₅ that was not accompanied by an increase in F₇₃₅: preillumination before freezing decreased both F₆₉₅ and, to a lesser extent, F₇₃₅ in the spectra recorded at 77 K. Fluorescence induction of chloroplasts frozen in the low-fluorescent state showed a markedly decreased variable fluorescence (F_v) of PS II, but no concomitant increase in initial fluorescence (F₀) of PS I. Thus, the buildup of a proton gradient at the thylakoid membrane, as reflected by fluorescence quenching at room temperature, affects low-temperature fluorecence emission in a manner entirely different from the effect of removal of Mg²⁺, which is thought to alter the distribution of excitation energy in favor of PS I. The ΔpH-dependent quenching therefore cannot be caused by such change in energy distribution and is suggested to reflect increased thermal deactivation.     

Changes in the redox state of the secondary acceptor of Photosystem II associated with light-induced thylakoid protein phosphorylation

Thylakoid membrane protein phosphorylation affects photochemical reactions of Photosystem II. Incubation of thylakoids in the light with ATP leads to: (1) an increase in the amplitude of three components (4–6, 25–45 and 280–300 μs) of delayed light emission after a single flash without any change in their kinetics; (2) a reduction of the flash-dependent binary oscillations of chlorophyll a fluorescence yield associated with electron transfer from the primary quinone acceptor, Q, to the secondary quinone acceptor, B; (3) an increase in the $\text{B}{}^{\mathrm{?}}\text{B}$ ratio resulting from an increase in stability of the semiquinone anion during dark adaptation; and (4) no change in the redox state of the plastoquinone pool as determined by flash-induced photooxidation of the Photosystem I reaction center, P-700. All the above observations are reversible upon dephosphorylation of the thylakoid membranes. These data are explained by a protein phosphorylation-induced stabilization of the bound semiquinone anion, B^?. It is proposed that this increased stability may be due to an alteration in the accessibility of an endogenous reductant to B, or to an increase in dissipative cycling of charge around Photosystem II.     

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm.Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.     

Picosecond fluorescence kinetics and energy transfer in chloroplasts and algae

Single-photon timing with picosecond resolution is used to investigate the kinetics of the fluorescence emission of chlorophyll a in chloroplasts from spinach and pea and in the algae Chlorella pyrenoidosa and Chlamydomonas reinhardii. The fluorescence decay is best described by three exponential components in all species. At low light intensity and with open reaction centers of Photosystem II (F₀), we find lifetimes of approx. 100, 400 and 1100 ps for the three components. Closing the reaction centers by addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea plus hydroxylamine and by increasing light intensity produces only minor changes in the almost constant fast- and medium-lifetime components; however, there is a dramatic increase in the yield of the slow component, by a factor of about 20, accompanied by only a modest increase in the lifetime to 2200 ps (F_max). In good agreement with previous fluorescence lifetime measurements, we find an increase in the averaged lifetime of the three components from 0.5 to 2.0 ns, which is proportional to the 4-fold increase in the total fluorescence yield. Our time-resolved results are inconsistent with models which are based on the proportionality between lifetime and yield and which involve a homogeneous origin of fluorescence that is sensitive to the state of the reaction centers. We conclude that the variable part of the fluorescence, which is dominated by the slow phase, reflects the kinetics of charge recombination in the reaction center, as proposed previously (Klimov, V.V., Allakhverdiev, S.I. and Paschenko, V.Z. (1978) Dokl. Akad. Nauk S.S.S.R. 242, 1204–1207). The modest increase in lifetime of the slow phase indicates the presence of some energy transfer between photosynthetic units.     

Chemiluminescence in the reaction of cytochrome c with hydrogen peroxide

(1) Aqueous solutions of 1–10 μM ferricytochrome c treated with 100 μM–100 mM H₂O₂ at pH 8.0 emit chemiluminescence with quantum yield $\text{Ф ? 10}{}^{\mathrm{?9}}$ and absolute maximum intensity $\text{I}{}_{\mathrm{<mtext>max</mtext>}}\text{? 10}{}^5\text{hv/}\text{s}$ per cm³ (λ = 440), and exhibit exponential decay with a rate constant of 0.15 s^?1. (2) The emission spectrum of the chemiluminescence covers the range 380–620 nm with the maximum at 460 ± 10 nm. (3) Neither cytochrome c nor haemin fluoresce in the spectral region of the chemiluminescence. In the reaction course with H₂O₂, a weak fluorescence in the region 400–620 nm with λ_max = 465–510 nm (λ_exc 315–430 nm) gradually arises. This originates from tryptophan oxidation products of the formylkynurenine type or from imidazole derivatives, respectively. (4) Frozen solutions (77 K) of cytochrome c exhibit phosphorescence typical of tryptophan (λ_exc = 280 nm, λ_em = 450 nm). During the peroxidation, an additional phosphorescence gradually appears in the range 480–620 nm with λ_max = 530 nm (λ_exc = 340 nm). This originates from oxidative degradation products of tryptophan. (5) There are no red bands in the chemiluminescence spectra of cytochrome c or haemin. This result suggests that singlet molecular oxygen $\text{O}{}_2\text{(}{}^1\text{Δ}{}_{\mathrm{<mtext>g</mtext>}}\text{)}$ is not involved in either peroxidation or chemiluminescence. (6) The haem Fe³⁺ group and H₂O₂ appear to be crucial for the chemiluminescence. It is suggested that the generation of electronically excited, light-emitting states is coupled to the production of conformational out-of-equilibrium states of peroxy-Fe-protoporphyrin IX compounds.     

Effect of photosystem II reaction center closure on nanosecond fluorescence relaxation kinetics

The fluorescence decay of chlorophyll in spinach thylakoids was measured as a function of the degree of closure of Photosystem II reaction centers, which was set for the flowed sample by varying either the preillumination by actinic light or the exposure of the sample to the exciting pulsed laser light. Three exponential kinetic components originating in Photosystem II were fitted to the decays; a fourth component arising from Photosystem I was determined to be negligible at the emission wavelength of 685 nm at which the fluorescence decays were measured. Both the lifetimes and the amplitudes of the components vary with reaction center closure. A fast (170–330 ps) component reflects the trapping kinetics of open Photosystem II reaction centers capable of reducing the plastoquinone pool; its amplitude decreases gradually with trap closure, which is incompatible with the concept of photosynthetic unit connectivity where excitation energy which encounters a closed trap can find a different, possibly open one. For a connected system, the amplitude of the fast fluorescence component is expected to remain constant. The slow component (1.7–3.0 ns) is virtually absent when the reaction centers are open, and its growth is attributable to the appearance of closed centers. The middle component (0.4–1.7 ns) with approximately constant amplitude may originate from centers that are not functionally linked to the plastoquinone pool. To explain the continuous increase in the lifetimes of all three components upon reaction center closure, we propose that the transmembrane electric field generated by photosynthetic turnover modulates the trapping kinetics in Photosystem II and thereby affects the excited state lifetime in the antenna in the trap-limited case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PQ plastoquinone - PSI and PSII Photosystem I and II - Q_A and Q_B primary and secondary quinone acceptor of PSII     

The reconstitution of a Photosystem II protein complex, P-700-chlorophyll a-protein complex and light-harvesting chlorophyll ab-protein

A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophylla protein complex and light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The $\text{F}{}_{685}\text{F}{}_{695}$ ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The $\text{F}{}_{681}\text{F}{}_{697}$ ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex (F₆₈₅ and F₆₉₅) and the P-700-chlorophyll a-protein complex (F₇₃₅) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.     

Photosystem II reactions in oxidant-treated chloroplast fragments.

Treatment of Photosystem II fragments with the oxidant K₂IrCl₆ destroys approximately 50% of the bulk chlorophyll and results in fragments that are twofold enriched in P680 (the Photosystem II reaction-center chlorophyll) and cytochrome b₅₅₉. The fragments retain a fully competent reaction center, as evidenced by P680 photooxidation and subsequent reduction in a back reaction with the primary electron acceptor ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5}\text{ms}$ at 25 dgK). The K₂IrCl₆-treated fragments contain no photoactive or chemically detectable C-550 and do not exhibit any variable fluorescence. These results imply that the Photosystem II primary electron acceptor is unaffected by oxidant treatment. It therefore may be concluded that neither C-550 nor the fluorescence quencher, Q, functions as the primary electron acceptor of Photosystem II.     

Fluorescence emission spectra of Photosystem I,Photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at ?196 °C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at ?196 °C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at ?196 °C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at ?196 °C.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

An analysis of repressor overproduction by the lambda cIIIs mutant.

Efficient lysogenization of Escherichia coli K12 by bacteriophage λ requires the high level of synthesis of the phage repressor shortly after infection. This high level of synthesis of repressor requires the action of the λ eII and cIII proteins. Certain mutants of λ (λcIIIs) appear to have excess $\text{c}\text{II}\text{c}\text{III}$ activity and can lysogenize more efficiently than λ⁺. The basis for the enhanced lysogenization is that, while two or more infecting phage are necessary for λ⁺ to lysogenize, a single infecting λcIIIs particle is sufficient for lysogenization. Also, repressor levels in cells infected with λcIIIs are higher than in those infected with λ⁺. I report here that repressor overproduction by λcIIIs (1) is due to a much higher rate of repressor synthesis than that of λ⁺; (2) is most marked at low multiplicities of infection, possibly because λcIIIs produces repressor much more efficiently than λ⁺ as a singly infecting phage.