OXIDATIVE PHOTODYNAMIC PROCESSES IN THE PHOTOSYSTEM I OF THERMOPHILIC CYANOBACTERIA SYNECHOCOCCUS ELONGATUS AT HIGH TEMPERATURE

 

 

Department of Biophysics, Faculty of Biology, Moscow State University, Moscow, 119899 Russia

¹Department of Cell Physiology and Immunology, Faculty of Biology, Moscow State University, Moscow, Russia.

 

Abstract

 

Relationships were investigated between the thermoactivated enhancement of the millisecond delayed fluorescence in PSI and the bleaching of photosynthetic pigments in Synechococcus elongatus membranes at 60-80^oC under light- and reagent-dependent stimulation of the fluorescence yield. It was shown that the light and temperature dependencies of the chlorophyll bleaching rate in the membranes at 60-80^oC were similar to light and temperature curves of the PS I delayed fluorescence. Bromide quenchers of triplet-excited chlorophyll suppressed almost completely the chlorophyll oxidative destruction and decreased the fluorescence yield by 70%. The rate of chlorophyll bleaching was also reduced greatly under anaerobic conditions and in the presence of sodium ascorbate. It was observed that the long-wavelength fraction of chlorophyll bleached more rapidly than the bulk chlorophyll. The results show that thermoactivation of delayed fluorescence and oxidative reactions are due mainly to accumulation of triplet chlorophyll as a result of carotenoid inactivation and enhancement of backward electron transfer in PSI upon heating above 60^oC.

 

 

Abbreviations: DF, delayed fluorescence; PSA, photosynthetic apparatus; PSI, photosystem I; RC, reaction center; Chl, chlorophyll; I, light flux density.

 

1. Introduction

 

The delayed fluorescence of chlorophyll (DF) is an informative characteristic of the backward electron transfer in the reaction centers (RC) as well as of the functional activity of the photosynthetic apparatus (PSA) in vivo and in vitro under various physical and chemical factors (Hauvax, Lannoye, 1985; Rubin et al., 1987). Temperature dependencies of the steady-state DF, i.e., the positions of the fluorescence maximum on the thermogram provide information about the cold and heat resistance of the PSA of higher plants (Fork et al., 1985). However, the value of the method decreases greatly for intact cells and tissues due to problems of interpreting the results obtained on these objects. The use of subcellular samples is limited because of their thermolability.

The PSA of thermophilic cyanobacteria is comparable to that of higher plants and, moreover, it is resistant at the subcellular level to high temperatures. A previous study showed that thermograms of the steady-state DF in the millisecond time domain for isolated membranes from thermophilic cyanobacteria Synechococcus elongatus have two separate peaks (Kaurov et al., 1988). The low-temperature band of delayed fluorescence with a maximum at 54^oC, resulting from recombination reactions in photosystem II (PS II), represents the main component on the thermograms of intact chloroplasts and algal cells (Veselovsky, Veselova, 1983). The high-temperature band with a peak at 78^oC originates from the backward electron transfer from the iron-sulfur centers Fa and Fb to P₇₀₀ in PS I (Vos, Van Gorkom, 1988). The possibility of simple separation of the PSI and PSII fluorescence bands makes Synechococcus elongatus suitable for investigating thermoinduced changes in PS I of thermophiles by the fluorescence method.

Heating cyanobacterial membranes from 60 to 78^oC enhanced both the fluorescence yield and the oxidative photodynamic destruction of photosynthetic pigments (Kaurov et al., 1993). It was assumed that the thermally dependent increase in concentration of excited chlorophyll (Chl) triplets is the main factor inducing these processes. The increases in DF yield and oxidative reaction rate in this case are due, respectively, to the triplet-singlet conversion and generation of singlet oxygen (Krasnovsky, 1986), both of which are important in photodynamic reactions (Spikes, Boomer, 1991; Merzlyak, Pogosyan, 1986). Kaurov et al. (1993) showed that quenchers of the Chl triplets, such as halogen and nitrate anions, efficiently reduced the PSI-generated delayed fluorescence. The rise of the DF and the suppression of oxidative processes in PSI were observed under anaerobic conditions.

The present study concerns the delayed fluorescence in PSI in relation to the oxidative destruction of pigments in the membranes of thermophilic cyanobacteria under light-, temperature- and reagent-dependent stimulation of the fluorescence yield.

 

2. Materials and methods

 

S. elongatus cells were cultured on Kratz-Myers aseptic inorganic medium (Kratz, Myers, 1955) at 55 ^oC. Air supplemented with 0,2% CO₂ was bubbled through the incubation mixture. The cell culture was illuminated with white light from fluorescent lamps. The light intensity was 1500 lux during early phase of logarithmic growth and gradually increased, reaching 6000 lux at the end of this phase. To obtain membranes fragments, the cells were treated with lysozyme. The resultant mass of spheroplasts was destroyed with glass beads on a 302 Homogenizer (Poland) (see details in Kaurov et al., 1988). Membrane fragments were suspended in a buffer containing 10 mM Hepes-NaOH, with pH 7.5. Lysozyme and Hepes-NaOH were from Serva (Germany). The chlorophyll concentration in the suspension was 30 mg ml^-1. The molar ratio P₆₈₀:P₇₀₀ in the membranes did not exceed 1:6.

The millisecond DF of chlorophyll was registered with the help of an electronic phosphoroscope 'Photos' designed at the biological faculty of Moscow State University. Excitation was induced by a red light LED matrix (AL307BM light-emitting diodes), placed along the internal perimeter of the measuring chamber. The spectral sensitivity range of the fluorescence sensor was above 680 nm. The intensity of excitation pulses (I) was varied in the range 0,1-25,0 W m^-2. The timing protocol parameters were: time between excitation and recording of fluorescence, 3 ms; recording time, 5 ms; time of fluorescence excitation, 25 ms. The fluorescence was registered at 60, 70, 75, 78 and 80^oC. The temperatures of the samples, which were placed in a cuvette, were measured by a chromel-copel thermocouple with a precision of +1^oC. The samples were heated at a rate of 5^oC per minute using a spiral heater placed into a hermetic capsule inside the cuvette.

Oxygen was removed from samples by gassing the membrane suspension with argon for 10 min.

The oxidative photodynamic bleaching of photosynthetic pigments in membranes was estimated after the 10-min light exposure of the samples at 60, 65, 70, 78 and 80^oC. After the exposure samples were cooled to room temperature, and the absorption spectra were measured with a Hitachi 150-200 spectrophotometer (Japan). The rate of pigment bleaching was estimated from the decrease in absorbance at 490 nm (carotenoids) and 650-720 nm (chlorophyll) as a result of illumination as compared to the control sample kept in darkness at the same temperature.

 

3. Results

 

The experimental curves of the light dependencies of the rate of Chl bleaching in membranes of S. elongatus at temperatures from 60 to 80^oC were similar to the light curves of the PS I-generated delayed fluorescence (Fig. 1A). As shown in Fig. 1B, a linear correlation was observed between the chlorophyll bleaching rate and DF intensity at irradiances up to 8 W m^-2.

The maximum DF intensity as well as the maximum Chl bleaching were observed at I = 8 W m^-2; DF and Chl bleaching did not change with further increase in illumination to 20 W m^-2. This might result from the light saturation of backward electron transfer (charge recombination) in the reaction centers of PSI. However, the rate of chlorophyll bleaching began to rise again at irradiances above 20 W m^-2. This rise was possibly due to direct photo-oxidation of chlorophyll under the supraoptimal light and high temperature conditions (60-80^oC).

Fig. 2A shows the dependencies of fluorescence yield and chlorophyll bleaching rate versus temperature at I = 8 W m^-2. As seen in the figure, the increase in temperature from 60 to 80^oC under light saturation of delayed fluorescence stimulated DF intensity and the rate of Chl bleaching in membranes. These characteristics achieved their maximal values at 75-80^oC. Figure 2B shows a similar thermally activated increase both in Chl bleaching rate and in delayed fluorescence intensity; probably this result indicates the existence of a common thermodependent mechanism underlying these processes.

It was mentioned above, that halogen and nitrate anions efficiently decrease the yield of PSI delayed fluorescence (Kaurov et al., 1993), probably due to their ability to quench the triplet excitation of chlorophyll (Terenin, 1967). Fig. 3A shows the DF intensity and the Chl bleaching rate as a function of MgBr₂ concentration at 75^oC and I = 8 W m^-2. These results are typical of halogen ion action in the temperature range from 60 to 80^oC. As seen in the figure, the reagent dramatically reduced the delayed fluorescence intensity and the rate of chlorophyll oxidation. Fig. 3B shows the linear correlation between changes of these characteristics, induced by MgBr₂ addition. As seen in the figure, the Chl bleaching in membrane preparation was almost completely blocked at 1 mM MgBr₂ (point F). At this concentration, the fluorescence yield constituted 30% of the initial level (point A). On the contrary, under weak light (I= 0,1 W m^-2) the value of delayed fluorescence was equal to about 5% and the rate of Chl bleaching exceeded 25% (point 1) of the respective values measured at 8 W m^-2 (point 6) (Fig. 1B). A nearly 100% inhibition of Chl bleaching in the membranes by Br^- ions indicates a strong dependence of photooxidative reactions on the concentration of triplet chlorophyll. Since Br^- quenched the delayed fluorescence by no more than 70% , the contribution of triplet excitation to delayed fluorescence did not exceed 70% at 78^oC.

It was shown earlier that both the intensity of PSI-generated DF in S. elongatus membranes and the thermoresistance of PSI-dependent electron transport increase dramatically under anaerobic conditions (Kaurov et al., 1993). We have shown that Chl bleaching under anaerobiosis is also inhibited almost completely at irradiances below 17 W m^-2 in the temperature range from 60 to 80^oC (Fig. 2A, curve 5). Similar results were obtained by adding 2 mM sodium ascorbate (Fig. 2A, curve 4) which is an efficient antioxidant and is able to deactivate singlet oxygen (Krasnovsky, 1994; Chou, Khan, 1983) when the functional activity of carotenoids, the main quenchers of ¹O₂ in photosynthetic systems (Foot, 1976), is low.

Bleaching of Chl at high temperature was accompanied by a shift of an absorption long-wavelength maximum of this pigment to shorter wavelengths. Fig. 4 shows the kinetics of Chl bleaching and the kinetics of the shift of chlorophyll absorption maximum, obtained at 78^oC in the dark, and in the light at I=1 and 17 W m^-2 after 10 minutes incubation. In the dark control sample chlorophyll bleached by only 5% from the initial level, and its absorption maximum at 684 nm remained unshifted. However, after light exposure at irradiance of 1 W m^-2, the chlorophyll absorption decreased by 24% and the absorption maximum shifted to 678 nm. At a light intensity of 17 W m^-2, the chlorophyll absorption decreased by 45% and the absorption maximum shifted to 676 nm. The shift of the chlorophyll absorption maximum to shorter wavelengths can result from the preferential degradation of the long-wave fractions of this pigment in the antenna. As seen in the figure, the long-wave forms of chlorophyll bleached more rapidly at weak light intensity, than the bulk chlorophyll. For example, the degradation of long-wave forms of the pigment at an irradiance of 1 W m^-2 reached 75% of the degradation at 17 W m^-2, whereas the bleaching of total Chl in low light (1 W m^-2) made up only 50 % of chlorophyll degradation at 17 W m^-2.

 

4. Discussion

 

Our data show a correlation between the light curves of the PSI-generated delayed fluorescence and the rate of light-induced Chl bleaching in S. elongatus membranes at irradiances below 20 W m^-2 at 60-80^oC (Fig. 1B). This evidences for a linear relationship between the rate of oxidative processes and the rate of the backward electron transfer from iron-sulfur centers Fa and Fb to chlorophyll P₇₀₀ in membranes of thermophilic cyanobacteria.

It is known that the equilibrium ratio of singlet to triplet states in the primary radical pair with separated charges is 1:3 (Hoff, 1986). In previous experiments, the yield of ³P₇₀₀ resulting from the charge recombination varies from 30% (Shuvalov et al., 1986) to 85% (Setif et al., 1985; Polm, Brettel, 1998). Evidently, the increase in the chlorophyll triplet concentration should stimulate the DF yield, due to triplet-singlet conversion, and promote oxidative reactions by the generation of singlet oxygen. Indeed, we have shown that the Chl bleaching in membranes of thermophiles is almost completely suppressed by bromide ions (exogenous quenchers of chlorophyll triplets), whereas the DF is quenched maximally by 70%, thus reflecting the role of triplet states in both processes. However, under normal conditions, the chlorophyll triplet forms are efficiently quenched by carotenoids (Van Gorkom et al., 1985; Jursinic, 1986). This process is probably inhibited in thermophilic cyanobacteria at high temperatures. In fact we observed that the rate of carotenoid bleaching under illumination increased with temperature up to 60^oC and did not change in the interval from 60 to 80^oC (Fig. 5). Thus, bleaching of carotenoids at high temperature, unlike bleaching of chlorophyll (Fig. 2A, curve 2), does not depend on oxidative reactions induced in PSI. This may be accounted for by the properties of the carotenoids located close to the PSI reaction center (Nugent, 1999; Joliot, Joliot, 1999). Probably, these carotenoids, capable of the efficient quenching of ³P₇₀₀, adjacent molecules of ³Chl, and ¹O₂, are rapidly inactivated under illumination at temperatures of about 60^oC. Indeed, the long-wavelength forms of chlorophyll located in the vicinity of PSI centers are predominantly destroyed by the light (Fig. 4). Thus, the rises of DF intensity and of the rate of chlorophyll destruction upon heating the membranes from 60 to 80^oC are mainly due to the acceleration of charge recombination in PSI rather than by the decreased efficiency of the chlorophyll triplet quenching by carotenoids.

The differential scanning calorimetric trace of the isolated membranes showed an endothermic transition with a peak at 71^oC. This peak is absent in PSII preparations and is related to polypeptide denaturation (results not shown). Therefore, the increases in DF intensity and the rate of chlorophyll degradation upon heating of membranes from 60 to 80^oC are accompanied by structural degradation of the PSI complex and soluble proteins. Heating the membranes from thermophilic cyanobacteria at 80^oC in darkness led to almost complete degradation of ferredoxin, a surface-located protein, whereas [4Fe-4S] clusters Fx, Fa and Fb were destroyed by 50% (Kaurov et al., 1999). These events should stimulate the rate of backward electron transfer from iron-sulfur centers to P₇₀₀.

The main suggested ways of energy deactivation in the PSI of thermophilic cyanobacteria are presented in Fig. 6. Upon heating to 60^oC, the carotenoids located in the vicinity of PSI and involved in reaction 1 are inactivated. Further heating from 60 to 80^oC gradually inactivates forward electron transport to iron-sulfur clusters of [4Fe-4S] type, Fx, Fa, Fb (reaction 2) as well as electron transfer to ferredoxin and oxygen (reaction 3). The rate of backward electron transfer from the iron-sulfur centers rises accordingly (reaction 4 and 5). Stimulation of reactions 4 and 5 and suppression of reaction 1 facilitate the formation of chlorophyll triplets and thereby induce oxidative reactions by generation of singlet oxygen (reaction 6). Heating also enhances the rate constant of triplet-singlet conversion in excited chlorophyll molecules (reaction 7). The elevations in rates of both charge recombination and triplet-singlet conversion increase the fluorescence yield (reaction 8).

Thus, the photodynamic oxidative destruction of PSI components at temperatures above 60^oC is induced by inhibition of triplet chlorophyll deactivation by carotenoids in the vicinity of P₇₀₀ and by singlet oxygen generation. Predominant bleaching of the long-wavelength fraction of chlorophyll provides evidence that oxidative reactions are located close to the reaction center of PSI. Activation of oxidative processes upon heating from 60 to 80^oC is due mainly to inhibition of forward electron transfer and the respective stimulation of charge recombination in PSI. Heating above 80^oC results in a complete loss of PSI functional activity, due to denaturation of core proteins.

According to views based on earlier studies, photo-oxidative reactions in photosynthetic apparatus and generation of activated oxygen forms are mainly related to the functioning of PSII. Our results suggest that PSI is also able to induce oxidative reactions, which contribute to the decrease in photosynthetic activity of thermophilic cyanobacteria under light and supraoptimal temperature conditions. A considerable role of singlet forms of oxygen in oxidative photodynamic reactions in photoinhibited centers of PSI was demonstrated on spinach thylakoids (Baba et al., 1995). Degradation of PSI components, which is accompanied by the increase in the content of chlorophyll triplets, was also observed on cucumber leaves under illumination at low temperature (Sonoike et al., 1995). This provides evidence of similar pathways of oxidative degradation of the PSI components at low and high temperatures.

 

This study was supported by the International Scientific Fund and the Russian Foundation of Basic Research.

 

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