Kintake Sonoike 1, Hideki Hatanaka 1, Sakae Katoh 1 and Shigeru Itoh 2. Tokyo, 113 Japan 2 National Institute for Basic Biology, Okazaki, 444 Japan

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1 Plant Cell Physiol. 31(6): (1990) JSPP 1990 Heat-Stability of Iron-Sulfur Centers and P-700 in Photosystem I Reaction Center Complexes Isolated from the Thermophilic Cyanobacterium Synechococcus elongatus Kintake Sonoike 1, Hideki Hatanaka 1, Sakae Katoh 1 and Shigeru Itoh 2 1 Department of Biology, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan 2 National Institute for Basic Biology, Okazaki, 444 Japan Stabilities of iron-sulfur centers and reaction center chlorophyll P-700 in Photosystem I reaction center complex (CPl-a), isolated by sodium dodecyl sulfate treatment from the thermophilic cyanobacterium Synechococcus elongatus, were studied by EPR and optical spectroscopy. P- 700 was destroyed by treatment at temperatures above 80 C for 5 minutes with a half inactivation temperature of 93 C. The three iron-sulfur centers F A, F B and F x showed similar thermal stabilities and were half inactivated at about 70 C. Thus, the isolated Photosystem I reaction center complexes of S. elongatus are still highly resistant to heat. Key words: EPR spectroscopy Heat-stability Iron-sulfur centers Photosystem I Reaction center complex Synechococcus elongatus. The thermophilic cyanobacterium Synechococcus elongatus, which has been harvested from a hot spring in Beppu, Kyushu (Yamaoka et al. 1978), shows a rapid growth at temperatures between 50 and 60 C. A series of experiments on the thermal properties of photosynthesis in this cyanobacterium have shown that its thermophily is supported by the heat resistance of individual proteins. Soluble proteins such as cytochrome e-553 and ferredoxin were stable up to 60 C (Koike and Katoh 1979, Koike et al. 1982) and ferredoxin-nadp oxidoreductase (FNR) showed the maximum activity at 60 C (Koike and Katoh 1980). Electron transport activities in cells and isolated thylakoid membranes are also heat-stable: temperatures required for the half inactivation were 81 C, 81 C, 99 C and 99 C for turnover of P-430 (F A /F B ), A 2 (Fx), A, (phylloquinone) and P-700, respectively (Koike et al. 1982). However, because inactivation of the iron-sulfur centers have been determined only photochemically, it is not clear whether the F A /F B and F x denature at the same temperature, or the Abbreviations: Ao, Ai and A 2> primary (chlorophyll 690), secondary (phylloquinone) and tertiary (iron-sulfur center, Fx) electron acceptors in Photosystem I reaction center complex; P-700, Photosystem I reaction center chlorophyll; P-430, the optically detected electron acceptor iron-sulfur centers (normally iron-sulfur centers F A and F B ). decrease of P-430 signal is due to the denaturation of F x, which mediates electron transfer between phylloquinone and F A /F B in electron transfer pathway in PS I reaction center as shown below. hv P-700 > Ao(chlorophyll 690) A,(phylloquinone) P-430(F A /FB) The high stability of the PS I electron transfer components as well as the development of the purification method (Takahashi et al. 1982, Boekema et al. 1987) seems to be an important basis for the successful crystallization of PS I reaction center (Witt et al. 1987), whose tertiary structure is now being analyzed by X-ray scattering (Witt et al. 1988). The CPl-a reaction center complex studied in this work contains all these functional components (Takahashi and Katoh 1982) and composed of two kda core polypeptides (products of psaa and psab genes) and four 8-14 kda polypeptides including the 10 kda F A /F B polypeptide (psac gene product) as shown elsewhere (Enami et al. 1990). We here report the heat stabilities of the ironsulfur centers F A, F B and F x, and reaction center chlorophyll P-700 in CPl-a complex of S. elongatus, using EPR and photochemical techniques. 86S

2 866 K. Sonoike, H. Hatanaka, S. Katoh and S. Itoh Materials and Methods I A Flash 5. elongatus was grown at 55 C for two days and thylakoid membranes and PS I reaction center complex (CPl-a) were prepared as described previously (Takahashi et al. 1982). For heat-treatment, samples suspended in 50mM Tris/HCl (ph 7.5) and 10 mm NaCl (2 mg Chl/ml) were incubated at indicated temperatures for 5 min and then rapidly cooled by dipping into ice cold water. Chemical determination of P-700 was carried but by measuring differences in absorbance at nm between ferricyanide(0.3 mm)- oxidized and ascorbate(10mm)/tmpd(l mm)-reduced samples containing 20 ng Chl/ml with a Hitachi 320 spectrophotometer (Sonoike and Katoh 1988). Because the differential absorption coefficients of P-700 varies considerably under influence of detergents, the value of 85 mm"'cm"' determined with CPl-a (Sonoike and Katoh 1989) was used. P-430 was determined by measuring the rapid phase (t, /2, ~1 ms) of the biphasic decay kinetics of flash-induced bleaching at 430 nm in the presence of 10//M methyl viologen (Hiyama and Ke 1971). EPR signals were measured with a Bruker EPR-200 X-band spectrometer using an Oxford Instruments ESR-900 continuous liquid helium flow cryostat (Matsuura and Itoh 1985). Samples (1.2 mg Chl/ml) were incubated with dithionite and benzyl viologen for 5 minutes in the dark under argon atmosphere, or illuminated in the presence of dithionite and benzyl viologen with a white light from a slide projector for about 2 min at room temperature and frozen in liquid nitrogen under illumination. Results Thermal stabilities of P-700 and P-430 in the reaction center complex (CPl-a) isolated from S. elongatus were studied spectrophotometrically. Fig. 1A shows the flashinduced absorption changes at 430 nm in CPl-a treated at different temperatures for 5 minutes. Magnitudes of P , which was re-reduced by phenazine methosulfate (slow phase with a half decay time of 50 ms), and P-430", which was rapidly reoxidized by methyl viologen (fast phase with a half decay time of 1 ms), decreased significantly after treatment at 65 C and more strongly at 75 C (traces b and c, respectively). Fig. IB indicates that the magnitudes of the two signals steeply decreased as temperature was raised above 55 C, showing almost identical half inactivation temperatures of about 72 C. Effect of heat-treatment on P-700 itself was examined by' measuring chemically reduced-minus-oxidized absorption changes at 698 nm (Fig. IB). P-700 was little affected by the treatment below 80 C but decreased at higher temperatures showing a half inactivation temperature of about 10 ms Incubation Temperature CO Fig. 1 (A) Time courses of flash-induced absorption change at 430 nm in CPl-a PS I reaction center complexes (a), untreated complexes; (b) and (c), the complexes were treated at 65 C and 75 C for 5 minutes, respectively. (B) Heat-stabilities of flash induced P- 700 oxidation and P-430 reduction, and chemically determined P-700 in CPl-a complex. The reaction mixture contained 50 mm Tris/HCl (ph 7.5), lomm NaCl, 10/iM methyl viologen, 4/a* phenazine methosulfate, 10mM sodium ascorbate and about 2//g chlorophyll/ml. Signal obtained with 160 flashes fired at 1 Hz were averaged. Closed circles, flash-oxidized P-700; open triangles, flash-reduced P-430; open circles, chemically determined P-700.

3 Heat-stability of Photosystem I reaction center C. Thus, P-700 per se is more stable than P-430 (or other components). The half inactivation temperature of P-700 in the CPl-a preparation is, however, lower than that observed in thylakoid membranes from the same organism (about 99 C, Koike et al. 1982). The difference spectrum of P-700 in the red region was not affected by treatment at 90 C except a slight enhancement of bleaching around 680 nm (data not shown). This suggests that antenna chlorophylls became unstable in heat-treated sample. Koike et al. (1982) reported a shift of the P-700 band from 703 nm to 700 nm on heat-treatment of the thylakoid membranes. No blue-shift of the red band was detected in CPl-a which already shows the shifted maximum at 697 nm (Takahashi et al. 1982). Fig. 2A shows EPR signals of F A and F B, which were reduced by incubating CPl-a complexes in the presence of dithionite/benzyl viologen at ph 10 for 5 minutes in the dark under argon atmosphere. When the complexes were heated at or above 65 C for 5 min, the size of both F A and F B signals decreased (traces b and c). Fig. 2B, in which signal amplitudes are plotted against the treatment temperature, shows that the two EPR components have similar heat-stabilities, both showing half inactivation temperatures of about 70 C. Reduction of F A and F B appears to X T3 be incomplete in the dark because EPR spectra show two small features at g = 1.86 (FA) and at g=2.06 (F B ) (indicated by arrows). Nugent et al. (1981) showed that these features disappear due to spin-spin interaction when the two iron-sulfur centers are both reduced in the same PS I reaction center. Illumination of samples under the reducing conditions at room temperature and also during freezing gave rise to EPR spectra without the g= 1.86 and g=2.06 features, indicating full reduction of F A and F B (Fig. 3A). Magnitudes of photoreduced F A and F B signals determined with the complexes treated at various temperatures are shown in Fig. 3B. Again, half inactivation temperatures of the two iron-sulfur centers were about 70 C. These temperature dependencies of F A and F B are well correlated to that of the optically measured P-430 (see Fig. IB). Thus, the decrease in the magnitude of photoinduced P-430 by heating is directly ascribed to the destruction of F A /F B. EPR features of F x also appeared at g=1.83 and g= 1.76 on reduction in the light (Fig. 3A). Fig. 3B shows that the signal amplitude of F x increased as temperature was raised above 55 C to reach a maximum at 65 C C. This suggests that reduction of F x is incomplete even in the light in the complexes treated at temperatures below 55 C. The in- 2.2 Zl g-value SO Incubation Temperature CO Fig. 2 (A) Effect of heat-treatment on the EPR signals of chemically reduced iron-sulfur centers in CPl-a complexes. Untreated complexes (a) or complexes incubated for 5 minutes at 65 C (b) and 75 C (c) were suspended in a medium containing 200 mm glycine/naoh (ph 10), saturating amounts of sodium dithionite and 0.5 mm benzyl viologen. Signals were measured twice at 20 K and averaged. EPR parameters: Microwave frequency, 9.6 GHz; microwave power, 10raW;modulation amplitude, 10 G; receiver gain, 1 x 10 5 ; scan time, 100 s. Arrows indicate the g= 1.86 and g=2.06 features. (B) Heat-stabilities of iron-sulfur centers. Open circles, g=1.93 signal (FA); open triangles, g=1.92 signal (F B ).

4 868 K. Sonoike, H. Hatanaka, S. Katoh and S. Itoh g - value Incubation Temperature CO Fig. 3 (A) Effect of heat-treatment on the EPR signals of photochemically reduced iron-sulfur centers in CPl-a complexes. Untreated complexes (a) and complexes incubated for 5 minutes at 65 C (b) and 75 C (c) were suspended in the medium described in Fig. 2, left at room temperature for 2 minutes and then frozen in liquid nitrogen both under illumination with a strong white light. Signals were measured twice at 8 K and averaged. Microwave frequency, 9.6 GHz; microwave power, 100 mw; modulation amplitude, 32 G; receiver gain, 1 x 10 4 ; scan time, 100 s. (B) Heat-stabilities of light-reduced iron-sulfur centers. Open triangles, g= 1.92 signal ( F B ); Open circles, g= 1.88 signal (FA); Closed circles, g=1.76 signal (F x ). tensity of the F x signal sharply decreased when the complexes were treated at higher temperatures with an apparent half inactivation temperature of about 70 C. Discussion The reaction components of Photosystem I reaction center complex (CPl-a) from the thermophilic cyanobacterium Synechococcus elongatus were highly heat-stable. Half inactivation of P-700 occurred at temperature as high as 88 C. All the iron-sulfur centers F A, F B and F x were inactivated with the half inactivation temperatures of about 70 C. The thermostability of the PS I photoresponses in the CPl-a complexes are, however, somewhat lower than those reported in the thylakoid membranes (Koike et al. 1982). The difference may be ascribed to a detergent-effect. In addition, magnitude of absorption changes might be diminished to some extent due to aggregation of CPl-a complexes which became significant after treatment at high temperatures. In contrast, PS II reactions of the cyanobacterium are more sensitive to heat. The photosynthetic activity of cells was 50% inactivated during 5 minutes incubation at 65 C and the half inactivation temperature of the oxygen-evolving activity in the isolated thylakoid membranes was about 55 C (Yamaoka et al. 1978). Thus, PS I seems to have proteins which are more resistant to heat and detergents as compared to PS II. The heat-stabilities of PS I reactions in higher plants and algae have been studied by exposing samples to various temperatures for 5 minutes. The half inactivation temperatures of P-700 photooxidation are about 70 C and 68 C in PS I preparations from spinach (Takamiya and Nishimura 1972) and pea (Shuvalov 1976), respectively. Cytochrome c-552 photooxidation in Euglena thylakoids was half inactivated at about 58 C (Katoh and San Pietro 1967) and the magnitude of P-430 photoresponse decreased by 50% by treatment of pea thylakoid at about 60 C for 5 min (Shuvalov 1976). More recently, Hoshina et al. (1989) showed that the half inactivation temperatures for the F A / F B, F x and P-700 in spinach thylakoids and PS I particles were approximately 53 C, 65 C and 78 C, respectively, in the presence of 50% ethylene glycol. The difference in the thermal stabilities of F A /F B and F x was ascribed to the effect of ethylene glycol, which increases the heat-sensitivity of F A /F B. Thus, even in mesophilic organisms, PS I components are thermostable, although the half inactivation temperatures of P-700 and iron-sulfur centers in ordinary plants are 10 C-20 C lower as compared to those of the counterparts of the thermophilic cyanobacterium. It may be speculated that the PS I reaction center complex has well

5 Heat-stability of Photosystem I reaction center 869 conserved its heat stability during the evolution from a thermophilic ancestral prokaryotic organism to mesophilic eukaryotic organisms. The previous experiments with flash spectroscopy showed that the half inactivation temperature of photoreduction of P-430 (F A /FB) was similar to that of A 2 (Fx) in the cyanobacterial thylakoid membranes (Koike et al. 1982). This does not necessarily mean that F A /F B and F x have similar thermal stability because, as stated in Introduction, destruction of F x results in parallel decrease in magnitudes of photoreduction of F A /F B. EPR spectroscopy allows to determine thermostability of individual ironsulfur centers separately. In particular, thermal properties of chemically reduced F A and F B determined by this technique are independent of functioning of F x. The results obtained indicate that the inactivation curves of chemically reduced F A and F B are similar not only to each other but also to those of photochemically reduced F A and F B, which in turn have the half inactivation temperatures nearly identical to that of photochemically reduced F x. A, (phylloquinone) is much more resistant to heat than F x (Koike et al. 1982). Thus, it is concluded that the three iron-sulfur centers have similar heat-stabilities. F x is located in the chlorophyll-carrying large subunits encoded by psaa and psab genes, whereas both F A and F B bind to the 10 kda protein encoded by psac gene. Thus the result shows that, irrespective of the difference in proteins, environments surrounding the iron-sulfur centers have similar stabilities. The iron-sulfur centers of Synechococcus elongatus PS I preparations are different from those of higher plant in the following two aspects. First, F A and F B of the cyanobacterium are difficult to be chemically reduced in the dark even at an alkaline ph, where the centers of higher plants are readily reduced (Evans and Heathcote 1980). It was necessary to illuminate samples in the presence of the reductant and at ph 10 to achieve complete reduction of F A / F B. This suggests that the iron-sulfur center of the cyanobacterium have more negative redox potentials than the counterparts of higher plants. However, it is to be mentioned that experiments were carried out at room temperature where proteins from the thermophilic cyanobacterium have very solid conformations. The observation that F x could not fully be reduced even in the light unless the PS I complexes are heated at 65 C suggests that accumulation of reduced F x is enhanced by a conformational change of the constituent subunits of the complex. Thus, a possibility still remains that dithionite or reduced benzyl viologen has an limited accessibility to or reactivity with the iron-sulfur centers or P in the PS I complexes from the thermophilic cyanobacterium at room temperature. Second, in higher plants, F A has a more positive redox potential than F B so that, when ambient redox potential is lowered, F A is firstly reduced and then F B. In contrast, reduction of F B preceded that of F A in the CP1 -a preparation of Synechococcus elongatus (not shown). A similar order of reduction of F A and F B was reported with membranes from Phormidium laminosum (Cammack et al. 1979). In view of the close location of F A and F B in the 10 kda protein, it is likely that electrons can readily equilibrate between the two centers in the thermophilic PS I complexes. Thus a more positive redox potential of F B than that of F A appears to be a characteristic of PS I in the cyanobacterial species. The authors thank Miss M. Iwaki of N.I.B.B. and Dr. I. Ikegami of Teikyo University for their kind suggestions and technical help for the EPR measurements and Dr. Y. Fujita of N.I.B.B. for his support for this study. The work was supported in part by grants for Scientific Research from the Ministry of Education, Science and Culture, Japan to S. I. and S. K. References Boekema, E. J., Dekker, J.P., van Heel, M. G., Rogner, M., Saenger, W., Witt, I. and Witt, H.T. (1987) Evidence for a trimeric organization of the photosystem I complex from the thermophilic cyanobacterium Synechococcus sp. FEBS Lett. 217: Cammack, R., Ryan, M. D. and Stewart, A. C. (1979) The EPR spectrum of iron-sulphur centre B in Photosystem 1 of Phormidium laminosum. FEBS Lett. 107: Enami, I., Kaiho, H., Izumi, H., Katoh, S., Kotani, N., Jone, C. S., Kamo, M. and Tsugita, A. 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