Quenching of fluorescence by triplet excited states in chloroplasts

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5–4.0 μs duration dye laser flash of varying intensities as an excitation source. The yield (Ф) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectroscopy at 515 nm, while the yield Ф following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10–12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from Ф utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (10¹⁶ photon · cm⁻²) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1–2 μs after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules.

At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse.

A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a singlet-triplet bimolecular rate constant of γ_st = 10⁻⁸ cm³ · s⁻¹, the following observations can be accounted for: (1) the rapid initial drop in Ф and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.     

Transfer and trapping of excitation energy in photosystem II as studied by chlorophyll alpha 2 fluorescence quenching by dinitrobenzene and carotenoid triplet. The matrix model

1. The curves representing the reciprocal fluorescence yield of chlorophyll alpha of Photosystem II (PS II) in Chlorella vulgaris as a function of the concentration of m-dinitrobenzene in the states P Q and P Q-, are found to be straight parallel lines; P is the primary donor and Q the primary acceptor of PS II. In the weakly trapping state P Q- the half-quenching of dinitrobenzene is about 0.2 mM, in vitro it is of the order of 10 mM. The fluorescence yield as a function of the concentration of a quencher is described for three models for the energy transfer between the units, and the matrix model. If it is assumed that the rate constant of quenching by dinitrobenzene is high and thus the number of dinitrobenzene molecules per reaction center low, it can be concluded that the pigment system of PS II in C. vulgaris is a matrix of chlorophyll molecules in which the reaction centers are embedded. Theoretical and experimental evidence is consistent with such an assumption. For Cyanidium caldarium the zero fluorescence yield phi 0 and its quenching by dinitrobenzene were found to be much smaller than the corresponding quantities for C. vulgaris. Nevertheless, our measurements on C. caldarium could be interpreted by the assumption that the essential properties (rate constants, dinitrobenzene quenching) of PS II are the same for these two species belonging to such widely different groups. 2. The measured dinitrobenzene concentrations required for half-quenching in vivo and other observations are explained by (non-rate-limiting) energy transfer between the chlorophyll alpha molecules of PS II and by the assumptions that dinitrobenzene is approximately distributed at random in the membrane and does not diffuse during excitation. 3. The fluorescence kinetics of C. vulgaris during a 350 ns laser flash of variable intensity could be simulated on a computer using the matrix model. From the observed fluorescence quenching by the carotenoid triplet (CT) and the measurement of the the number of CT per reaction center via difference absorption spectroscopy, the rate constant for quenching of CT is calculated to be kT = 3.3 . 10(11)s-1 which is almost equal to the rate constant of trapping by an open reaction center (Duysens, L.N.M. (1979) CIBA Foundation Symposium 61 (New Series), pp. 323--340). 4. The fluorescence quenching by CT in non-treated spinach chloroplasts after a 500 ns laser flash (Breton, J., Geacintov, N.E. and Swenberg, C.E. (1979) Biochim, Biophys. Acta 548, 616--635) could be explained within the framework of the matrix model when the value for kT is used as given in point 3. 5. The observations mentioned under point 1 indicate that the fluorescence yield phi 0 for centers in trapping state P Q is probably for a fraction exceeding 0.8 emitted by PS II.     

The triplet exciton of chlorophyll a and carotenoid in solution and in photosynthetic antenna proteins

We have observed the development and decay of triplet excitons formed in the ‘antenna’ chlorophyll $\text{a}\text{b}$ protein complex by high-intensity laser excitation. The carotenoid triplet (³Car) appeared 5 ns after excitation in the protein isolation, commonly termed CP-II; the risetime in a larger antenna particle, called LHC (light-harvesting complex) was 12 ns. The quantum yield of ³Car in CP-II decreased 11-fold as intensity was increased from 10¹⁶ to 2 · 10¹⁷ photons/cm² per pulse. The effect is attributed to exciton annihilation during the initial period of triplet formation. Above 5 · 10¹⁶ photons/cm² per s, the ³Car lifetime decreases substantially from its low intensity value of 8.7 μs. A comparison of the transient absorption spectrum of CP-II with those of chlorophyll and carotenoid in vitro indicates that ‘trapped’ chlorophyll triplets formed at high intensities. We present a simple model of destructive interaction between ³Car and chlorophyll triplets which is compatible with the observed increased rate of ³Car decay. Indirect evidence suggests similar effects occur in LHC.     

Reactions between primary and secondary acceptors of photosystem II in Chlorella pyrenoidosa under anaerobic conditions as studied by chlorophyll fluorescence.

The kinetics of the fluorescence yield phi of chlorophyll a in Chlorella pyrenoidosa were studied under anaerobic conditions in the time range from 50 mus to several minutes after short (t 1/2 = 30 ns or 5 mus) saturating flashes. The fluorescence yield "in the dark" increased from phi = 1 at the beginning to phi approximately 5 in about 3 h when single flashes separated by dark intervals of about 3 min were given. After one saturating flash, phi increased to a maximum value (4-5) at 50 mus, then phi decreased to about 3 with a half time of about 10 ms and to the initial value with a half time of about 2 s. When two flashes separated by 0.2 s were given, the first phase of the decrease after the second flash occurred within 2 ms. After one flash given at high initial fluorescence yield, the 10-ms decay was followed by a 10 s increase to the initial value. After the two flashes 0.2 s apart, the rapid decay was not followed by a slow increase. These and other experiments provided additional evidence for and extend an earlier hypothesis concerning the acceptor complex of Photosystem II (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250-256; Velthuys, B. R. and Amesz. J. (1974) Biochim. Biophys. Acta 333, 85-94): reaction center 2 contains an acceptor complex QR consisting of an electron-transferring primary acceptor molecule Q, and a secondary electron acceptor R, which can accept two electrons in succession, but transfers two electrons simultaneously to a molecule of the tertiary acceptor pool, containing plastoquinone (A). Furthermore, the kinetics indicate that 2 reactions centers of System I, excited by a short flash, cooperate directly or indirectly in oxidizing a plastohydroquinone molecule (A2-). If initially all components between photoreaction 1 and 2 are in the reduced state the following sequence of reactions occurs after a flash has oxidised A2- via System I: Q-R2- + A leads to Q-R + A2- leads to QR- + A2-. During anaerobiosis two slow reactions manifest themselves: the reduction of R (and A) within 1 s, presumably by an endogenous electron donor D1, and the reduction of Q in about 10 s when R is in the state R- and A in the state A2-. An endogenous electron donor, D2, and Q- complete in reducing the photooxidized donor complex of System II in reactions with half times of the order of 1 s.     

Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures

The formation of the triplet state of carotenoids (detected by an absorption peak at 515 nm) and the photo-oxidation of the primary donor of Photosystem II, P-680 (detected by an absorption increase at 820 nm) have been measured by flash absorption spectroscopy in chloroplasts in which the oxygen evolution was inhibited by treatment with Tris. The amount of each transient form has been followed versus excitation flash intensity (at 590 or 694 nm). At low excitation energy the quantum yield of triplet formation (with the Photosystem II reaction center in the state Q⁻) is about 30% that of P-680 photo-oxidation. The yield of carotenoid triplet formation is higher in the state Q⁻ than in the state Q, in nearly the same proportion as chlorophyll a fluorescence. It is concluded that, for excited chlorophyll a, the relative rates of intersystem crossing to the triplet state and of fluorescence emission are the same in vivo as in organic solvent. At high flash intensity the signal of P-680⁺ completely saturates, whereas that of carotenoid triplet continues to increase.

The rate of triplet-triplet energy transfer from chlorophyll a to carotenoids has been derived from the rise time of the absorption change at 515 nm, in chloroplasts and in several light-harvesting pigment-protein complexes. In all cases the rate is very high, around 8 · 10⁷ s⁻¹ at 294 K. It is about 2–3 times slower at 5 K. The transitory formation of chlorophyll triplet has been verified in two pigment-protein complexes, at 5 K.     

Rate of carotenoid triplet formation in solubilized light-harvesting complex II (LHCII) from spinach.

In the present study the rate of triplet transfer from chlorophyll to carotenoids in solubilized LHCII was investigated by flash spectroscopy using laser pulses of approximately 2 ns for both pump and probe. Special attention has been paid to calibration of the experimental setup and to avoid saturation effects. Carotenoid triplets were identified by the pronounced positive peak at approximately 507 nm in the triplet-singlet difference spectra. DeltaOD (507 nm) exhibits a monoexponential relaxation kinetics with characteristic lifetimes of 2-9 micros (depending on the oxygen content) that was found to be independent of the pump pulse intensity. The rise of DeltaOD (507 nm) was resolved via a pump probe technique where an optical delay of up to 20 ns was used. A thorough analysis of these experimental data leads to the conclusion that the kinetics of carotenoid triplet formation in solubilized LHCII is almost entirely limited by the lifetime of the excited singlet state of chlorophyll but neither by the pulse width nor by the rate constant of triplet-triplet transfer. Within the experimental error the rate constant of triplet-triplet transfer from chlorophyll to carotenoids was estimated to be kTT > (0.5 ns)-1. This value exceeds all data reported so far by at least one order of magnitude. The implications of this finding are briefly discussed.     

Fluorometric equipment for monitoring P680+• reduction in PS II preparations and green leaves

Newly developed equipment is described that permits the monitoring of laser flash induced transients of the normalised chlorophyll-a fluorescence quantum yield in isolated PS II preparations and whole leaves with a high time resolution. The essential operational unit of the set-up is a rapidly gated photomultiplier. In this way, the fluorescence artefact, due to the high intensity excitation laser flash, is sufficiently suppressed and the dead time of the signal response is reduced to about 500 ns. It is shown that the fluorescence rise kinetics in the s time-domain, after flash excitation is strongly dependent on the redox state of the primary electron donor of PS II (P680). At high excitation energies, the decay of carotenoid triplets, which are very efficient quenchers of chlorophyll singlet states, dominates the rise kinetics of the flash induced fluorescence yield in the s time domain.     

Triplet state dynamics in peridinin-chlorophyll-a-protein: a new pathway of photoprotection in LHCs?

This work investigates the interaction of carotenoid and chlorophyll triplet states in the peridinin-chlorophyll-a-protein (PCP) of Amphidinium carterae using step-scan Fourier transform infrared spectroscopy. We identify two carotenoid triplet state lifetimes of approximately 13 and approximately 42 mus in the spectral region between 1800 and 1100 cm(-1) after excitation of the 'blue' and 'red' peridinin (Per) conformers and the Q(y) of chlorophyll-a (Chl-a). The fast and slow decaying triplets exhibit different spectral signatures in the carbonyl region. The fast component generated at all excitation wavelengths is from a major conformer with a lactone stretching mode bleach at 1745 cm(-1). One (1720 cm(-1)) and two (1720 cm(-1) and 1741 cm(-1)) different Per conformers are observed for the slow component upon 670- and 530-480-nm excitation, respectively. The above result implies that (3)Per triplets are formed via two different pathways, corroborating and complementing visible triplet-singlet (T-S) spectra (Kleima et al., Biochemistry (2000), 39, 5184). Surprisingly, all difference spectra show that Per and Chl-a modes are simultaneously present during the (3)Per decay, implying significant involvement of (3)Chl-a in the (3)Per state. We suggest that this Per-Chl-a interaction via a delocalized triplet state lowers the (3)Per energy and thus provides a general, photoprotection mechanism for light-harvesting antenna complexes.     

Transfer and trapping of excitation energy in Photosystem II as studied by chlorophyll a2 fluorescence quenching by dinitrobenzene and carotenoid triplet. The matrix model

1. The curves representing the reciprocal fluorescence yield of chlorophyll a of Photosystem II (PS II) in Chlorella vulgaris as a function of the concentration of m-dinitrobenzene in the states P Q and P Q^-, are found to be straight parallel lines; P is the primary donor and Q the primary acceptor of PS II. In the weakly trapping state P Q^- the half-quenching of dinitrobenzene is about 0.2 mM, in vitro it is of the order of 10 mM. The fluorescence yield as a function of the concentration of a quencher is described for three models for the structure of pigment systems: the model of separate units, the model of limited energy transfer between the units, and the matrix model. If it is assumed that the rate constant of quenching by dinitrobenzene is high and thus the number of dinitrobenzene molecules per reaction center low, it can be concluded that the pigment system of PS II in C. vulgaris is a matrix of chlorophyll molecules in which the reaction centers are embedded. Theoretical and experimental evidence is consistent with such an assumption.

For Cyanidium caldarium the zero fluorescence yield Ф₀ and its quenching by dinitrobenzene were found to be much smaller than the corresponding quantities for C. vulgaris. Nevertheless, our measurements on C. caldarium could be interpreted by the assumption that the essential properties (rate constants, dinitrobenzene quenching) of PS II are the same for these two species belonging to such widely different groups.

2. The measured dinitrobenzene concentrations required for half-quenching in vivo and other observations are explained by (non-rate-limiting) energy transfer between the chlorophyll a molecules of PS II and by the assumptions that dinitrobenzene is approximately distributed at random in the membrane and does not diffuse during excitation.

3. The fluorescence kinetics of C. vulgaris during a 350 ns laser flash of variable intensity could be simulated on a computer using the matrix model. From the observed fluorescence quenching by the carotenoid triplet (C^T) and the measurement of the number of C^T per reaction center via difference absorption spectroscopy, the rate constant for quenching of C^T is calculated to be k_T = 3.3 · 10¹¹ s⁻¹ which is almost equal to the rate constant of trapping by an open reaction center (Duysens, L.N.M. (1979) CIBA Foundation Symposium 61 (New Series), pp. 323–340).

4. The fluorescence quenching by C^T in non-treated spinach chloroplasts after a 500 ns laser flash (Breton, J., Geacintov, N.E. and Swenberg, C.E. (1979) Biochim. Biophys. Acta 548, 616–635) could be explained within the framework of the matrix model when the value for k_T is used as given in point 3.

5. The observations mentioned under point 1 indicate that the fluorescence yield Ф₀ for centers in trapping state P Q is probably for a fraction exceeding 0.8 emitted by PS II.     

Singlet-triplet fusion in Rhodopseudomonas sphaeroides chromatophores. A probe of the organization of the photosynthetic apparatus

Chromatophores from various strains of Rhodopseudomonas sphaeroides were excited with laser flashes lasting about 20 ns. Fluorescence from the antenna bacteriochlorophyll of the photosynthetic apparatus was measured both during the laser flash, and during a weak Xe flash following the laser flash. Strong laser flashes caused severe quenching of the fluorescence, which could be correlated with the formation of triplet states of the antenna pigments. Triplet states of both BChl and carotenoids acted as quenchers, but bacteriochlorophyll triplets were the more effective of the two. In the double-flash experiments, the reciprocal of the fluorescence yield was proportional to the concentration of triplet quenchers remaining at the time of the second flash. This relationship indicates that singlet excitations can migrate over large domains in the antenna, rather than being restricted by boundaries separating individual reaction centers. Comparisons of chromatophores from different strains and from cells grown under different conditions showed that excitations are concentrated rapidly in the antenna complexes with the longest wavelength absorption bands (B870), and that the migration of excitations to trapping sites is relatively insensitive to the amount of antenna bacteriochlorophyll absorbing at shorter wavelengths (B800–B850). This suggests that the B870 complexes are organized in the membrane so as to interconnect many reaction centers, and that the B800–B850 complexes are arranged peripherally.     

Quenching of chlorophyll fluorescence by triplets in solubilized light-harvesting complex II (LHCII)

The quenching of chlorophyll fluorescence by triplets in solubilized trimeric light harvesting complexes was analyzed by comparative pump-probe experiments that monitor with weak 2-ns probe pulses the fluorescence yield and changes of optical density, DeltaOD, induced by 2-ns pump pulses. By using a special array for the measurement of the probe fluorescence (Sch?del R., F. Hillman, T. Schr?tter, K.-D. Irrgang, J. Voight, and G. Biophys. J. 71:3370-3380) the emission caused by the pump pulses could be drastically reduced so that even at highest pump pulse intensities, IP, no significant interference with the signal due to the probe pulse was observed. The data obtained reveal: a) at a fixed time delay of 50 ns between pump and probe pulse the fluorescence yield of the latter drastically decreased with increasing IP, b) the recovery of the fluorescence yield in the microseconds time domain exhibits kinetics which are dependent on IP, c) DeltaOD at 507 nm induced by the pump pulse and monitored by the probe pulse with a delay of 50 ns (reflecting carotenoid triplets) increases with IP without reaching a saturation level at highest IP values, d) an analogous feature is observed for the bleaching at 675 nm but it becomes significant only at very high IP values, e) the relaxation of DeltaOD at 507 nm occurs via a monophasic kinetics at all IP values whereas DeltaOD at 675 nm measured under the same conditions is characterized by a biphasic kinetics with tau values of about 1 microseconds and 7-9 microseconds. The latter corresponds with the monoexponential decay kinetics of DeltaOD at 507 nm. Based on a Stern-Volmer plot, the time-dependent fluorescence quenching is compared with the relaxation kinetics of triplets. It is shown that the fluorescence data can be consistently described by a quenching due to triplets.     

Generation of fluorescence quenchers from the triplet states of chlorophylls in the major light-harvesting complex II from green plants

Laser flash-induced changes of the fluorescence yield were studied in aggregates of light-harvesting complex II (LHCII) on a time scale ranging from microseconds to seconds. Carotenoid (Car) and chlorophyll (Chl) triplet states, decaying with lifetimes of several microseconds and hundreds of microseconds, respectively, are responsible for initial light-induced fluorescence quenching via singlet-triplet annihilation. In addition, at times ranging from milliseconds to seconds, a slow decay of the light-induced fluorescence quenching can be observed, indicating the presence of additional quenchers generated by the laser. The generation of the quenchers is found to be sensitive to the presence of oxygen. It is proposed that long-lived fluorescence quenchers can be generated from Chl triplets that are not transferred to Car molecules. The quenchers could be Chl cations or other radicals that are produced directly from Chl triplets or via Chl triplet-sensitized singlet oxygen. Decay of the quenchers takes place on a millisecond to second time scale. The decay is slowed by a few orders of magnitude at 77 K indicating that structural changes or migration-limited processes are involved in the recovery. Fluorescence quenching is not observed for trimers, which is explained by a reduction of the quenching domain size compared to that of aggregates. This type of fluorescence quenching can operate under very high light intensities when Chl triplets start to accumulate in the light-harvesting antenna.     

Triplet excited state spectra and dynamics of carotenoids from the thermophilic purple photosynthetic bacterium <Emphasis Type="Italic">Thermochromatium tepidum</Emphasis>

Light-harvesting complex 2 from the anoxygenic phototrophic purple bacterium Thermochromatium tepidum was purified and studied by steady-state absorption, fluorescence and flash photolysis spectroscopy. Steady-state absorption and fluorescence measurements show that carotenoids play a negligible role as supportive energy donors and transfer excitation to bacteriochlorophyll-a with low energy transfer efficiency of ~30%. HPLC analysis determined that the dominant carotenoids in the complex are rhodopin and spirilloxanthin. Carotenoid excited triplet state formation upon direct (carotenoid) or indirect (bacteriochlorophyll-a Q_x band) excitation shows that carotenoid triplets are mostly localized on spirilloxanthin. In addition, no triplet excitation transfer between carotenoids was observed. Such specific carotenoid composition and spectroscopic results strongly suggest that this organism optimized carotenoid composition in the light-harvesting complex 2 in order to maximize photoprotective capabilities of carotenoids but subsequently drastically suppressed their supporting role in light-harvesting process.     

A master equation theory of fluorescence induction, photochemical yield, and singlet-triplet exciton quenching in photosynthetic systems.

A master equation theory is formulated to describe the dependence of the fluorescence yield (phi) in photosynthetic systems on the number of photons (Y) absorbed per photosynthetic unit (or domain). This theory is applied to the calculation of the dependence of the fluorescence yield on Y in (a) fluorescence induction, and (b) singlet exciton-triplet excited-state quenching experiments. In both cases, the fluorescence yield depends on the number of previously absorbed photons per domain, and thus evolves in a nonlinear manner with increasing Y. In case a, excitons transform the photosynthetic reaction centers from a quenching state to a nonquenching state, or a lower efficiency of quenching state; subsequently, absorbed photons have a higher probability of decaying by radiative pathways and phi increases as Y increases. In case b, ground-state carotenoid molecules are converted to long-lived triplet excited-state quenchers, and phi decreases as Y increases. It is shown that both types of processes are formally described by the same theoretical equations that relate phi to Y. The calculated phi (Y) curves depend on two parameters m and R, where m is the number of reaction centers (or ground-state carotenoid molecules that can be converted to triplets), and R is the ratio phi (Y leads to infinity)/(Y leads to 0). The finiteness of the photosynthetic units is thus taken into account. The m = 1 case corresponds to the "puddle" model, and m leads to infinity to the "lake," or matrix, model. It is shown that the experimental phi (Y) curves for both fluorescence induction and singlet-triplet exciton quenching experiments are better described by the m leads to infinity cases than the m = 1 case.     

Effects of heavy central metal on the ground and excited states of chlorophyll

Chlorophylls, owing to their adjustable π-electron system and intense, well-separated electronic transitions, can serve as convenient intrinsic spectroscopic probes of ligand–metal center interactions. They are also interesting for their photosensitizing properties. In order to examine the heavy-atom effects on the chlorophyll triplet state, a key intermediate in chlorophyll–photosensitized reactions, the synthesis of a novel Pt(II)-substituted chlorophyll a was carried out, and the effects of the substitution on steady-state and transient photophysical properties of chlorophyll were studied by absorption and fluorescence spectroscopies, and by laser flash photolysis. The presence of highly electronegative platinum as the central ion increases the energies of the chlorophyll main absorption transitions. As laser flash photolysis experiments show, in air-equilibrated solutions, chlorophyll triplets are efficiently quenched by molecular oxygen. Interestingly, this quenching by oxygen is more effective with metal-containing pigments, in spite of the increased spin–orbit coupling, introduced with the central metals. This points to occurrence of nonspecific interactions of molecular oxygen with metallochlorophylls. The differences in the effects exerted on the pigment triplet by the central metal become distinct after the removal of oxygen. The lifetime of a Pt-chlorophyll triplet remains very short, in the range of only a few microseconds, unlike in the free-base and Mg- and Zn-substituted chlorophylls. Such drastic shortening of the triplet lifetime can be attributed to a large heavy-atom effect, implying that strong interactions must occur between the central Pt(II) ion and the chlorophyll macrocycle, which lead to a more efficient spin–orbit coupling in Pt-chlorophyll than in Pt-porphyrins.Electronic Supplementary Material Supplementary material is available for this article at .     

Rapidly reversible chlorophyll fluorescence quenching induced by pulses of supersaturating light in vivo

The saturation pulse method provides a means to distinguish between photochemical and non-photochemical quenching, based on the assumption that the former is suppressed by a saturating pulse of light (SP) and that the latter is not affected by the SP. Various types of non-photochemical quenching have been distinguished by their rates of dark relaxation in the time ranges of seconds, minutes, and hours. Here we report on a special type of non-photochemical quenching, which is rapidly induced by a pulse of high-intensity light, when PS II reaction centers are closed, and rapidly relaxes again after the pulse. This high-intensity quenching, HIQ, can be quantified by pulse-amplitude-modulation (PAM) fluorimetry (MULTI-COLOR-PAM, high sensitivity combined with high time resolution) via the quasi-instantaneous post-pulse fluorescence increase that precedes recovery of photochemical quenching in the 100–400-µs range. The HIQ amplitude increases linearly with the effective rate of quantum absorption by photosystem II, reaching about 8% of maximal fluorescence yield. It is not affected by DCMU, is stimulated by anoxic conditions, and is suppressed by energy-dependent non-photochemical quenching (NPQ). The HIQ amplitude is close to proportional to the square of maximal fluorescence yield, Fm′, induced by an SP and varied by NPQ. These properties are in line with the working hypothesis of HIQ being caused by the annihilation of singlet excited chlorophyll a by triplet excited carotenoid. Significant underestimation of maximal fluorescence yield and photosystem II quantum yield in dark-acclimated samples can be avoided by use of moderate SP intensities. In physiologically healthy illuminated samples, NPQ prevents significant lowering of effective photosystem II quantum yield by HIQ, if excessive SP intensities are avoided.

     

Photophysical properties of a photocytotoxic fluorinated chlorin conjugated to four beta-cyclodextrins

A meso-tetrakis(pentafluorophenyl)-chlorin with the reduced pyrrole ring linked to an isoxazolidine ring (FC) has been conjugated to four beta-cyclodextrins (CDFC). The CDFC exhibits excellent water solubility and is a potent photosensitizer towards proliferating NCTC 2544 human keratinocytes. The study by conventional steady state absorption and fluorescence spectroscopies and by time-resolved femto- and nanosecond laser flash spectroscopies suggests that in ethanol and pH 7 buffer the beta-cyclodextrins embed the highly hydrophobic tetrakis(pentafluorophenyl)-chlorin macrocycle and strongly interact with the chlorin rings in the singlet and triplet manifolds. In these solvents, femtosecond spectroscopy suggests that the conjugate undergoes a rapid relaxation in the upper excited singlet states induced by photochemical and/or conformation change(s) at a rate of about 5 ps(-1) to fluorescent states whose lifetime is approximately 8 ns. This interaction is destroyed upon addition of Triton X100 to buffer. Both FC and CDFC strongly fluoresce (Phi(F) approximately 0.5) in micelles. Similar behavior is observed at the triplet level. In ethanol and water, the initial transient triplet state absorbance decays within 1-3 mus yielding a longer lived triplet with spectral properties indistinguishable from that of original difference absorbance spectra. The determination of the molar absorbance in the 440-460 nm region ( approximately 35 000 M(-1) cm(-1)) leads to an estimate of approximately 0.2 for the triplet formation quantum yield of FC in toluene and of FC and CDFC in Triton X100 micelles. Quenching of the CDFC triplets by dioxygen in buffer produces (1)O(2) in a good yield consistent with the effective photocytotoxicity of the chlorin-cyclodextrins conjugate towards cultured NCTC 2544 human keratinocytes. By contrast, FC which aggregates in buffer produces little if any (1)O(2).     

Further evidence for dissipative energy migration via triplet states in photosynthesis. The protective mechanism of carotenoids in Rhodopseudomonas spheroides chromatophores.

The protection action of carotenoids against irreversible photodestruction was discovered in photosynthetic bacteria by Stanieda and coworkers. In green plant material it was found by Wolff and Witt (1969) Z. Naturforsch, 24b, 1031-1037 and (1972) Proc. 2nd. Int. Congr. Photosynthesis Res. Stresa (Forti, G., Avron, M. and Melandri, A., eds.), Vol. 2, pp. 931-936, Dr. W. Junk, N. V. Publ. The Hague) that the formation of special carotenoid triplet states (via very rapid energy transfer from excited chlorophylls) and their fast radiationless decay in tau1/2 approximately 3 microns is at least one mechanism for the protective action of carotenoids to irreversible photooxidation of the chlorophylls. Hence, it is anticipated that the same mechanism might be realized also in bacteria. The present study gives evidence for such a "triplet valve" to be established also in bacteria. This conclusion was derived from the following observations: 1. The light-induced difference spectrum shows a bleaching of a carotenoid at three characteristic wavelength between 400 and 500 nm. A positive peak around 533 nm indicates the formation of a carotenoid triplet state. 2. The absorption changes can be induced by red light which excites only bacteriochlorophyll. This indicates an energy transfer from bacteriochlorophyll to carotenoids. 3. The light-induced carotenoid triplets decay radiationless in 3 microns in air-saturated aqueous suspensions of the chromatophores. 4. The carotenoid triplet formation occurs only at actinic flash intensities where the photosynthesis becomes saturated. 5. Addition of dithionite, which blocks photosynthesis, markedly increases the extent of carotenoid triplet formation. The different types of exciton migration within the photosynthetic unit are discussed, especially the routes leading to the dissipation of excess excitation energy.     

Correlation between flash-induced oxygen evolution and fluorescence yield kinetics in the 0 to 16 mus range in Chlorella pyyrenoidosa during incubation with hydroxylamine.

Following flash excitation, oxygen pulses and fluorescence kinetics in the time range 0-16 mus were studied in the alga Chlorella pyrenoidosa during incubation with various concentrations of hydroxylamine. The obtained results could be explained considering four effects of hydroxylamine. 1. Hydroxylamine removes (reduces) oxidizing equivalents, generated in the water-splitting system by flash excitation. This process does not markedly affect the fluorescence yield kinetics between 0 and 16 mus following the ignition of a flash and reaches a constant rate within a few minutes, but possibly within a few seconds, after addition of hydroxylamine. In a sequence of flashes separated by dark time td, the steady-state oxygen yield in the flashes is exp(-ktd), the yield at td=0 being taken equal to 1, where k=(0.1 + beta[NH2OH])s-1, with [NH2OH] in mM and beta=0.6 mM-1, provided [NH2OH]greater than or equal to 0.5 mM. 2. An inhibition between Z, the physiological donor and the oxidized reaction center pigment P+ occurs, proceeding as exp (-kiti)where ti is the incubation time with hydroxylamine and ki=(alpha[NH2OH]) min-1, with [NH2OH] in mM and alpha=0.14 mM-1. This process not only inhibits oxygen evolution capability, but also decreases the amplitude of the fluorescence yield difference deltaphi=phi(16 mus)-phi(2 mus) induced by a flesh in the steady state. In a fraction of the reaction centers this inhibition occurs "immediately" after the addition of hydroxylamine. These observations, combined with the conslusion of Cheniae and Martin (1971, Plant Physiol. 47, 568-575) that the inhibition of the Hill reaction is related to the extraction of bound manganese indicate that the reaction between Z and P+ requires bound manganese. 3. In the inhibited centers a second donor for P+, D, connected to an entry site for the artificial electron donor hydroxylamine becomes apparent. 4. A flash-induced oxygen uptake signal was observed in the presence of hydroxylamine, which was shown to be caused by a system II reaction. The effects under (1) and (4) were reversed in the dark if hydroxylamine was removed by washing. The effects under (2) and (3) were reversed during illumination of a washed sample.