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Light-Induced Charge Separation between Plastocyanin and the Iron–Sulfur Clusters FAand FBin the Complex of Plastocyanin and Photosystem I
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 330, No. 2, June 15, pp. 414–418, 1996 Article No. 0270
Light-Induced Charge Separation between Plastocyanin and the Iron–Sulfur Clusters FA and FB in the Complex of Plastocyanin and Photosystem I1 Michael Hippler,*,2 Astrid Riedel,† Uwe Schro¨er,* Wolfgang Nitschke,*,3 and Wolfgang Haehnel* *Lehrstuhl fu¨r Biochemie der Pflanzen, Institut fu¨r Biologie II, Albert-Ludwigs-Universita¨t, Scha¨nzlestr. 1, D-79104, Freiburg, Germany; and †Institut fu¨r Biophysik, Universita¨t Regensburg, Universita¨tsstr. 32, D-93053 Regensburg, Germany
Received October 18, 1995, and in revised form March 19, 1996
The light-induced electron transfer in a crosslinked complex between plastocyanin and photosystem I from spinach was studied by EPR at low temperature. Electron donation from reduced plastocyanin to P700/ was observed under illumination above a temperature of about 160 K, resulting in a second charge separation and an electron transfer from rereduced P700 to the terminal electron acceptors FA /FB . The charge-separated state PcoxP700/[FA /FB ]20 was found to be stable at 15 K. Implications of these results for the kinetic constants of the donation reaction and the backtransfer of electrons from reduced acceptors as well as for the structural models of the terminal acceptors are discussed. q 1996 Academic Press, Inc. Key Words: photosystem I; plastocyanin; iron – sulfur clusters FA and FB ; P700; electron transfer; EPR; spinach.
The photosystem I complex functions as light-driven oxidoreductase that transports electrons from plastocyanin to ferredoxin in higher plants, most algae, and cyanobacteria. The PSI4 reaction center is a membranebound complex and consists of several polypeptide subunits. The core of PSI is assembled from two partially 1 This work was supported by Deutsche Forschungsgemeinschaft Ha1084/5-2. 2 To whom correspondence should be addressed at De´partement de Biologie Mole´culaire, Universite´ de Gene`ve, 30 Quai Ernest Ansemet, CH-1211 Gene`ve, Switzerland. Fax: 0041 227026868. 3 Present address: BIP-CNRS, Chemin Joseph-Aiguier, F-13402 Marseille Cedex 20, France. 4 Abbreviations used: Chl, chlorophyll; EPR, electron paramagnetic resonance; FeS, iron–sulfur cluster; Mops, 3-(N-morpholino)propanesulfonic acid; PSI, photosystem I; TX-100, Triton X-100.
homologous large subunits, PsaA and PsaB, containing the primary donor chlorophyll dimer, P700, the electron acceptors A0 and A1 , and the [4Fe–4S] iron–sulfur cluster FX , as well as about 80 antenna chlorophyll molecules (see Refs. 1 and 2 for review). The terminal electron acceptors FA and FB are [4Fe–4S] clusters of the stromal PsaC subunit (3, 4). The three-dimensional structure of PSI (5) from the cyanobacterium Synechococcus sp. has been determined by X-ray crystallogra˚. phy at a resolution of 6 A In addition to PsaC, the PsaD and PsaE subunits were shown to be located at the reducing side of the PSI complex and to protrude into the stroma (6). PsaD is necessary for stable binding of PsaC to the core complex; it is involved in the assembly of the complex and in the docking of ferredoxin to PSI (7–10). PsaE seems to be important for efficient electron transfer to ferredoxin and for cyclic electron transfer (11–13). On the oxidizing side of the PSI complex the PsaF subunit is exposed to the lumen (14, 15). It is involved in docking of plastocyanin, as suggested by crosslinking experiments (16, 17). The orientation of the crosslinked complex between plastocyanin and PSI was shown to resemble the conformation in the native complex. This was concluded from the fast kinetics of reduction of P700/ (18) with a half-time of 13–15 ms, which is comparable to that found in intact thylakoids (19, 21), in digitonin–PSI particles (22, 23), and in PSI200 particles (24) at high plastocyanin concentrations. An open question about the function of the PSI reaction center concerns the precise pathway(s) of electron flow through these terminal electron acceptors. Some progress has been achieved by site-directed mutagenesis affecting the FeS clusters FA and FB (9) as well as center FX (9, 25). These results together with the crystal structure of PSI (5), which shows that the clusters
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ELECTRON TRANSFER FROM PLASTOCYANIN TO PsaC SUBUNIT OF PHOTOSYSTEM I
FA and FB are not equal with respect to their distance to FX as well as to the surface of the protein (26), allow first conclusions with respect to sequential electron transfer between these clusters. However, an attribution of spectroscopically inequivalent centers FA and FB to the FeS centers seen in the X-ray structure was not possible so far. During recent years, the thermodynamic characteristics of the electron donation reaction to the photooxidized pigments in photosynthetic reaction centers have been studied in detail at low temperatures in purple bacteria (27, 28). The temperature dependence of the rereduction of photooxidized P700/ by reduced plastocyanin has been reported for chloroplasts (22). In this study, the fraction of centers performing electron donation to P700/ was found to fall below detection at temperatures below 180 K. However, it could not be determined unambiguously whether this lack of rereduction at low temperatures was due to alteration of the electron transfer steps or to temperature-induced changes in the binding constant between plastocyanin and PSI. Here we present the results of a low-temperature EPR study on the light-induced charge separations produced within the crosslinked complex. These data demonstrate the presence of a threshold temperature for the onset of electron donation and allow conclusions with respect to electron transfer between the two terminal electron acceptors FA and FB of PSI. METHODS PSI particles (PSI200) were isolated from spinach leaves according to Wynn and Malkin (16). Chlorophyll (Chl) concentrations were determined as described (29). The concentration of P700 was determined from flash-induced absorbance changes at 704.5 nm, where the amplitude was 80% of that at the maximum of the difference spectrum at 701 nm (not shown), using an extinction coefficient at 701 nm of 64 mM01 cm01 (30). Plastocyanin was isolated from spinach leaves as described (31). Its concentration was determined spectroscopically using an extinction coefficient of 4.9 mM01 cm01 at 597 nm for the oxidized form (32). Purified plastocyanin had absorbance ratios A278 /A597 between 1.2 and 1.4. Plastocyanin was crosslinked to PSI in the presence of 0.05% (w/v) Triton X-100 as described (18) except that a pH value was adjusted with 30 mM Mops buffer and that PSI particles were used at 0.4 mg Chl/ml. EPR samples contained PSI particles at a P700 concentration of about 5 mM. For optical experiments, PSI particles crosslinked with plastocyanin were used at a concentration of 15 mg Chl/ml in 100 mM Mops, pH 7.0, 5 mM MgCl2 , 0.02% (w/v) Triton X-100. Absorbances changes of P700 were measured with a single beam spectrophotometer essentially as described (33). Flash excitation was provided by a frequency-doubled Nd:YAG laser (5 ns FWHM). Prior to the flash a photoshutter protected the sample from the measuring light at 704.5 nm (2.6 nm FWHM). The shutter was opened 5 ms before the recording was started. The cuvette containing 3 ml of the sample had an optical pathlength of 10 mm. The detecting photodiode (1 cm2) was placed at a distance of 35 cm from the cuvette and was protected by an interference filter at 703 nm. The output of the photodiode was amplified with an electrical bandwidth ranging from dc to 1 MHz, with dc offset compensation by 16 bit ADC and DAC within 4 ms immediately before the recording. Signal disturbance by
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FIG. 1. Time course of flash-induced DA at 703 nm in PSI particles crosslinked with plastocyanin. The concentration of the PSI particles was 15 mg/ml Chl. Cuvette was of 10-mm optical path at 257C. The crosslinked complex was suspended in 100 mM Mops, pH 7.0, 5 mM MgCl2 , 0.02% (w/v) TX-100. The trace is the result of one flash. Single exponential analysis of fast component DA Å 03.69, t1/2 Å 15 ms.
fluorescence was monitored in the absence of measuring light and subtracted when necessary. EPR measurements were performed at X-Band on a Bruker ER 300 spectrometer equipped with an Oxford Instruments temperature control system. Spectra were taken at 15 K using 6.7 mW microwave power and 1.6 mT modulation. Illumination in the cavity was provided by an 800-W tungsten projector yielding 16,000 mE m02 s01 of white light at the EPR cavity window after being filtered through 2 cm of water and a calflex filter to remove infrared radiation. The full reduction of the FeS centers FA and FB was achieved by addition of 20 mM dithionite at pH 10.3 (100 mM glycine) and subsequent illumination at 200 K for 5 min.
RESULTS
The experiments were performed with a crosslinked complex between plastocyanin and PSI (18, 24) competent in fast reduction of P700/. The efficiency of the crosslinking was assayed by laser-induced absorbance changes of P700 (Fig. 1). The fast reduction kinetics with a half-time of 15 ms amounts to 63% of the total amplitude. This fraction of PSI crosslinked with plastocyanin in an active state exceeds previous amounts considerably (18). The crosslinked PSI particles were frozen in darkness and illuminated at 4 K. The EPR spectrum of this sample recorded at 15 K is shown in Fig. 2A (dotted line). It is characterized by dominant signals at g Å 2.05, 1.94, 1.86 (resulting from reduced FA ), minor signals at g Å 2.07, 1.92, 1.88 (resulting from reduced FB ), and the P700/ radical at g Å 2 (1). The ratio between the reduced FA and FB resonances was determined to
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FIG. 2. EPR spectra of PSI particles with crosslinked plastocyanin. (A) Dotted line, sample was frozen in the dark, illuminated at 4 K for 2 min; solid line, the sample was subsequently annealed to 160 K under illumination within the cavity. The crosslinked complex was suspended in 100 mM Mops, pH 7.0, 5 mM MgCl2 , 0.02% (w/v) TX100. The difference spectrum of the continous line spectrum minus the dotted line spectrum is shown below. (B) Sample was chemically reduced with 20 mM dithionite, 0.1 M glycine, pH 10.3, and photoaccumulated at 200 K. Spectrometer conditions: temperature 15 K, microwave power 6.7 mW, microwave frequency 9.46 GHz, modulation amplitude 1.6 mT.
be about 80/20, in line with previous observations with PSI particles of spinach or cyanobacteria (1). No further components are detected, indicating that illumination at 4 K of the crosslinked complex results in charge separation between P700 and the FeS electron acceptors without further transfer of the positive charge from P700/ to the copper center of the bound plastocyanin. To check whether this transfer was possible at higher temperatures, the sample was annealed to temperatures between 60 and 180 K in darkness. Under none of these conditions was plastocyanin oxidized. However, above 100 K, charge recombination was observed as described previously (1). Previous optical measurements (22, 34) have shown that the electron transfer from plastocyanin to P700/ can occur at temperatures significantly below 07C and at least down to 200 K (M. Hippler et al., unpublished
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results). Therefore the sample was exposed within the EPR cavity to 40, 80, 120, 140, and 160 K under illumination for 30 s and cooled down again to the measuring temperature of 15 K under continuous illumination. Subsequently the spectra were run in the dark. Heating to temperatures lower than 140 K had no effect on the EPR spectrum. However, at 160 K the shape of the light-induced spectrum changed significantly (Fig. 2A, continuous line). The intensity of the resonance at g Å 1.86, which is characteristic of reduced cluster FA in centers where FB is oxidized, decreased by 40% compared to the sample illuminated at 4 K. In contrast, the resonance at g Å 1.88, resulting from reduced center FB , in the presence of reduced FA cluster (1) increased from about 20 to 63% of the maximal FA /FB signal in reduced PSI. In addition, the signal of oxidized plastocyanin (resulting from Cu2/) appeared as broad positive contributions above g Å 2.05 and a derivative signal at g Å 2.013 (35). These differences are manifested in the difference of both spectra, as shown in Fig. 2A, bottom. (continuous minus dotted line spectrum). Using a control PSI sample, it was found that the ratio of 80/20 of photoreduction of FA and FB is also observed after illumination at 160 K (not shown). The maximal amount of FA and FB , which is reflected by the signal at g Å 1.88, was determined by chemical reduction with dithionite at high pH and subsequent photoaccumulation at 200 K, after the series of experiments. The signal is shown in Fig. 2B. The results are summarized in Table I. They demonstrate that crosslinked plastocyanin is able to reduce photooxidized P700/ above 160 K in the light, leading TABLE I 0 Comparison of EPR Measured Resonances of F0 A , FB , and 0 [F0 A FB ] for PSI Particles Crosslinked with Plastocyanin under Different Conditions: (i) Frozen in the Dark, Illuminated at 4 K for 2 min and (ii) Annealed to 160 K under Illumination within the Cavity
Intensities of the gx resonances of centers F0 A (g Å 1.86) and F0 B (g Å 1.88) relative to the fully reduced control (g Å 1.88) obtained by reduction at high pH followed by 5 min of photoaccumulation at 200 K PSI particles with crosslinked 0 plastocyanin [F0 [F0 [F0 A] B] A FB ] Frozen in the dark, illuminated for 2 min at 4 K Warmed to 160 K under illumination
0.8
0.2
0
0.4
low
0.6
Note. EPR spectra were recorded at 15 K with the light switched off. Close to 100% of centers were seen to perform stable charge separation at 4 K during the first turnover.
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ELECTRON TRANSFER FROM PLASTOCYANIN TO PsaC SUBUNIT OF PHOTOSYSTEM I
to an additional charge transfer from P700 to the FeS electron acceptors. DISCUSSION
In this work we have performed an EPR characterization of the electron transfer reaction within the crosslinked complex between PSI and its physiological electron donor plastocyanin. The results demonstrate that electron transfer from plastocyanin to P700/ does occur at cryogenic temperatures, at least down to 160 K. The detailed optical study of the temperature dependence of the electron donation reaction to P700/ in the crosslinked complex will be reported elsewhere (M. Hippler et al., unpublished results). The sample offers the possibility of performing two sequential electron transfer reactions at low temperature within the electron acceptor chain of PSI. The crosslinked complex, illuminated at 4 K, shows the same pattern of stably photoreduced centers FA and FB as the control PSI, i.e., only 20% of the molecules performed charge separation between P700 and FB , whereas in 80% of the centers, the electron ejected from P700 ended up at center FA (see Ref. 1 for review). An electron donation from reduced plastocyanin to P700/ occuring during illumination at 4 K in the crosslinked complex could not be detected. When such a preilluminated sample (i.e., in the state PCred –P700/ –FeS0) was annealed to temperatures above 100 K, still no oxidation of plastocyanin by P700/ could be observed, but a charge recombination (P700/ – FeS0 r P700–FeS) took place. This was true even for temperatures above 160 K, where under illumination electron donation from reduced plastocyanin to PSI was observed. Two different explanations can be put forward. (i) Below 160 K, the rate constant for electron donation from reduced plastocyanin to P700/ is slower than that of recombination from the reduced acceptors. Extrapolating the temperature dependence of the rate constants of electron transfer from plastocyanin to P700/ (Ref. 22, 34) in fact predicts that at temperatures around 110 K the rate constant of the donation reaction becomes comparable to that of charge recombination from the terminal electron acceptors (36). (ii) The efficiency of electron donation from reduced plastocyanin to P700/ drops drastically below 220 K. Such an effect has been proposed by Venturoli et al. (28) for the analogous reaction between cytochrome c2 and the purple bacterial reaction center. Time-resolved optical experiments at temperatures lower than 200 K are required to distinguish these possibilities. However, illumination at temperatures at or above 160 K induced the oxidation of plastocyanin by P700/, and a second charge separation between P700 and the terminal acceptors occurred. For a quantitative analysis it must be taken into account that this reaction can only occur
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in 63% of total PSI, i.e., the fraction of crosslinked complex (cf. Fig. 1). The EPR spectrum obtained for 160 K illumination showed that about 40% of total PSI contained terminal acceptors in which FA was reduced while FB remained oxidized. From the amount of crosslinked complexes with active electron transfer from Pc to P700/ as determined optically (see Fig. 1) it follows that 37% of total PSI cannot perform a second electron transfer step, i.e., should yield the ratio of 80/20 corresponding to a fraction of 30 and 7% of total PSI for FAredFBox and FAoxFBred, respectively. We conclude (i) that after 160 K illumination, the centers showing FA reduced in the absence of reduced FB were largely those lacking a crosslinked plastocyanin and (ii) that the majority of PSI centers crosslinked to plastocyanin underwent a double turnover during 160 K illumination resulting in the reduction of both FA and FB . Two major conclusions can be drawn from these results. (i) The state (P700/ –FA0/FB0) is stable at low temperatures. So far, full reduction of FA and FB has only been achieved by trapping the electron on the electron acceptors through rapid reduction of P700/ from exogenous reductants. No data have been reported excluding that the negative charge on one FeS center could preclude stable low-temperature reduction of the other center. (ii) While after the first turnover, the stoichiometry of photoreduced FA and FB was 80/20 as usual, close to full reduction of FA and FB was detected in those centers having performed a second charge separation. The electrons reducing the residual 80% of FB are transfered either (1) directly from center FX or (2) via center FA . Since the cluster FA is already reduced in the respective fraction of centers, the latter sequence implies rapid equilibration between FA and FB even at low temperatures. In this picture the reduction with an 80:20 stoichiometry of the FeS centers would therefore indicate a redox equilibrium between two communicating clusters with slightly differing Em values. An attribution to an effect of the different distances of electron transfer toward these clusters with lacking intercluster electron transfer is considered to be an unlikely interpretation. It is of note that in the family of 8Fe–8S ferredoxins, which includes the FA /FB0 -carrying subunit PsaC, both the presence and the absence of intercluster electron transfer have been observed by NMR in different species (37, 38), possibly depending on the detailed localization of the mixed-valence pairs in both clusters. Similar studies on the FA /FB -binding PsaC subunit may provide a clearer picture with respect to this crucial question. ACKNOWLEDGMENT The authors thank Dr. A. W. Rutherford (Paris, France) for help with the initial measurements and for critical reading of the manuscript.
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REFERENCES 1. Golbeck, J. H. (1992) Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 293–324. 2. Se´tif, P. (1992) in The Photosystems: Structure, Function, and Molecular Biology (Barber, J., Ed.), pp. 471–499, Elsevier, Amsterdam. 3. Hoj, P. B., Svendsen, I., Scheller, H. V., and Moller, B. L. (1987) J. Biol. Chem. 262, 12676–12684. 4. Wynn, M. R., and Malkin, R. (1988) FEBS Lett. 229, 293–297. 5. Krauss, N., Hinrichs, W., Witt, I., Fromme, P., Pritzkow, W., Dauter, Z., Betzel, C., Wilson, K. S., Witt, H. T., and Saenger, W. (1993) Nature 361, 326–331. 6. Kruip, J., Boekema, E. J., Bald, D., Boonstra, A. F., and Ro¨gner, M. (1993) J. Biol. Chem. 268, 23533–23360. 7. Zanetti, G., and Merati, G. (1987) Eur. J. Biochem. 169, 143– 146. 8. Zilber, A., and Malkin, R. (1988) Plant Physiol. 88, 810–814. 9. Zhao, J., Li, N., Warren, P., Golbeck, J. H., and Bryant, D. A. (1992) Biochemistry 31(22), 5093–5099. 10. Li, N., Warren, P. V., Golbeck, J. H., Frank, G., Zuber, H., and Bryant, D. A. (1991) Biochim. Biophys. Acta 1059, 215–225. 11. Rousseau, F., Se´tif, P., and Lagoutte, B. (1993) EMBO J. 12, 1755–1765. 12. Sonoike, H., Hatanaka, H., and Katoh, S. (1993) Biochim. Biophys. Acta 1141, 52–57. 13. Yu, L., Zhao, J., Mu¨hlenhoff, U., Bryant, D. A., and Golbeck, J. H. (1993) Plant Physiol. 103, 171–180. 14. Franzen, L. G., Frank, G., Zuber, H., and Rochaix, J. D. (1989) Plant Mol. Biol. 12, 463–464. 15. Steppuhn, J., Hermans, J., Nechushtai, R., Ljungberg, U., Thu¨mmler, F., Lottspeich, F., and Herrmann, R. G. (1988) FEBS Lett. 237, 218–224. 16. Wynn, R. M., and Malkin, R. (1988) Biochemistry 27, 5863–5869. 17. Wynn, R. M., Luong, C., and Malkin, R. (1889) Plant Physiol. 91, 445–449. 18. Hippler, M., Ratajczak, R., and Haehnel, W. (1989) FEBS Lett. 250, 280–284.
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19. Haehnel, W., Do¨ring, G., and Witt, H. T. (1971) Z. Naturforsch. 26B, 1171–1174. 20. Haehnel, W., Pro¨pper, A., and Krause, H. (1980) Biochim. Biophys. Acta 193, 384–399. 21. Haehnel, W., Ratajczak, R., and Robenek, H. (1989) J. Cell Biol. 108, 1397–1405. 22. Bottin, H., and Mathis, P. (1985) Biochemistry 24, 6453–6460. 23. Bottin, H., and Mathis, P. (1987) Biochim. Biophys. Acta 892, 91–98. 24. Drepper, F., Hippler, M., Nitschke, W., and Haehnel, W. (1996) Biochemistry 35, 1282–1295. 25. Warren, P. V., Smart, L. B., McIntosh, L., and Golbeck, J. H. (1993) Biochemistry 32, 4411–4419. 26. Fromme, P., Schubert, W.-D., and Krauß, N. (1994) Biochim. Biophys. Acta 1187, 99–105. 27. Ortega, J. M., and Mathis, P. (1993) Biochemistry 32, 1141– 1151. 28. Venturoli, G., Mallardi, A., and Mathis, P. (1993) Biochemistry 32, 13245–13523. 29. Porra, R. J., Thompson, W. A., and Kriedemann, P. E. (1989) Biochim. Biophys. Acta 975, 384–394. 30. Hiyama, T., and Ke, B. (1972) Biochim. Biophys. Acta 267, 160– 171. 31. Ratajczak, R., Mitchell, R., and Haehnel, W. (1988) Biochim. Biophys. Acta 933, 306–318. 32. Katoh, S., Shiratori, I., and Takamija, A. (1962) J. Biochem. 51, 32–40. 33. Haehnel, W., Jansen, T., Gause, K., Klo¨sgen, R. B., Stahl, B., Michl, D., Huvermann, B., Karas, M., and Herrmann, R. G. (1994) EMBO J. 13, 1028–1038. 34. Hippler, M. (1994) Ph.D. dissertation, Universita¨t Freiburg, Germany. 35. Penfield, K. W., Gewirth, A. A., and Solomon, E. L. (1985) J. Am. Chem. Soc. 107, 4519–4529. 36. Golbeck, J. H., and Cornelius, J. M. (1986) Biochim. Biophys. Acta 849, 25–31. 37. Bertini, I., Capozzi, F., Luchinat, C., Piccioli, M., and Vita, A. J. (1994) J. Am. Chem. Soc. 116, 651–660. 38. Huber, J. G., Gaillard, J., and Moulis, J.-M (1995) Biochemistry 34, 194–205.
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Vol. 330, No. 2, June 15, pp. 414–418, 1996 Article No. 0270
Light-Induced Charge Separation between Plastocyanin and the Iron–Sulfur Clusters FA and FB in the Complex of Plastocyanin and Photosystem I1 Michael Hippler,*,2 Astrid Riedel,† Uwe Schro¨er,* Wolfgang Nitschke,*,3 and Wolfgang Haehnel* *Lehrstuhl fu¨r Biochemie der Pflanzen, Institut fu¨r Biologie II, Albert-Ludwigs-Universita¨t, Scha¨nzlestr. 1, D-79104, Freiburg, Germany; and †Institut fu¨r Biophysik, Universita¨t Regensburg, Universita¨tsstr. 32, D-93053 Regensburg, Germany
Received October 18, 1995, and in revised form March 19, 1996
The light-induced electron transfer in a crosslinked complex between plastocyanin and photosystem I from spinach was studied by EPR at low temperature. Electron donation from reduced plastocyanin to P700/ was observed under illumination above a temperature of about 160 K, resulting in a second charge separation and an electron transfer from rereduced P700 to the terminal electron acceptors FA /FB . The charge-separated state PcoxP700/[FA /FB ]20 was found to be stable at 15 K. Implications of these results for the kinetic constants of the donation reaction and the backtransfer of electrons from reduced acceptors as well as for the structural models of the terminal acceptors are discussed. q 1996 Academic Press, Inc. Key Words: photosystem I; plastocyanin; iron – sulfur clusters FA and FB ; P700; electron transfer; EPR; spinach.
The photosystem I complex functions as light-driven oxidoreductase that transports electrons from plastocyanin to ferredoxin in higher plants, most algae, and cyanobacteria. The PSI4 reaction center is a membranebound complex and consists of several polypeptide subunits. The core of PSI is assembled from two partially 1 This work was supported by Deutsche Forschungsgemeinschaft Ha1084/5-2. 2 To whom correspondence should be addressed at De´partement de Biologie Mole´culaire, Universite´ de Gene`ve, 30 Quai Ernest Ansemet, CH-1211 Gene`ve, Switzerland. Fax: 0041 227026868. 3 Present address: BIP-CNRS, Chemin Joseph-Aiguier, F-13402 Marseille Cedex 20, France. 4 Abbreviations used: Chl, chlorophyll; EPR, electron paramagnetic resonance; FeS, iron–sulfur cluster; Mops, 3-(N-morpholino)propanesulfonic acid; PSI, photosystem I; TX-100, Triton X-100.
homologous large subunits, PsaA and PsaB, containing the primary donor chlorophyll dimer, P700, the electron acceptors A0 and A1 , and the [4Fe–4S] iron–sulfur cluster FX , as well as about 80 antenna chlorophyll molecules (see Refs. 1 and 2 for review). The terminal electron acceptors FA and FB are [4Fe–4S] clusters of the stromal PsaC subunit (3, 4). The three-dimensional structure of PSI (5) from the cyanobacterium Synechococcus sp. has been determined by X-ray crystallogra˚. phy at a resolution of 6 A In addition to PsaC, the PsaD and PsaE subunits were shown to be located at the reducing side of the PSI complex and to protrude into the stroma (6). PsaD is necessary for stable binding of PsaC to the core complex; it is involved in the assembly of the complex and in the docking of ferredoxin to PSI (7–10). PsaE seems to be important for efficient electron transfer to ferredoxin and for cyclic electron transfer (11–13). On the oxidizing side of the PSI complex the PsaF subunit is exposed to the lumen (14, 15). It is involved in docking of plastocyanin, as suggested by crosslinking experiments (16, 17). The orientation of the crosslinked complex between plastocyanin and PSI was shown to resemble the conformation in the native complex. This was concluded from the fast kinetics of reduction of P700/ (18) with a half-time of 13–15 ms, which is comparable to that found in intact thylakoids (19, 21), in digitonin–PSI particles (22, 23), and in PSI200 particles (24) at high plastocyanin concentrations. An open question about the function of the PSI reaction center concerns the precise pathway(s) of electron flow through these terminal electron acceptors. Some progress has been achieved by site-directed mutagenesis affecting the FeS clusters FA and FB (9) as well as center FX (9, 25). These results together with the crystal structure of PSI (5), which shows that the clusters
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ELECTRON TRANSFER FROM PLASTOCYANIN TO PsaC SUBUNIT OF PHOTOSYSTEM I
FA and FB are not equal with respect to their distance to FX as well as to the surface of the protein (26), allow first conclusions with respect to sequential electron transfer between these clusters. However, an attribution of spectroscopically inequivalent centers FA and FB to the FeS centers seen in the X-ray structure was not possible so far. During recent years, the thermodynamic characteristics of the electron donation reaction to the photooxidized pigments in photosynthetic reaction centers have been studied in detail at low temperatures in purple bacteria (27, 28). The temperature dependence of the rereduction of photooxidized P700/ by reduced plastocyanin has been reported for chloroplasts (22). In this study, the fraction of centers performing electron donation to P700/ was found to fall below detection at temperatures below 180 K. However, it could not be determined unambiguously whether this lack of rereduction at low temperatures was due to alteration of the electron transfer steps or to temperature-induced changes in the binding constant between plastocyanin and PSI. Here we present the results of a low-temperature EPR study on the light-induced charge separations produced within the crosslinked complex. These data demonstrate the presence of a threshold temperature for the onset of electron donation and allow conclusions with respect to electron transfer between the two terminal electron acceptors FA and FB of PSI. METHODS PSI particles (PSI200) were isolated from spinach leaves according to Wynn and Malkin (16). Chlorophyll (Chl) concentrations were determined as described (29). The concentration of P700 was determined from flash-induced absorbance changes at 704.5 nm, where the amplitude was 80% of that at the maximum of the difference spectrum at 701 nm (not shown), using an extinction coefficient at 701 nm of 64 mM01 cm01 (30). Plastocyanin was isolated from spinach leaves as described (31). Its concentration was determined spectroscopically using an extinction coefficient of 4.9 mM01 cm01 at 597 nm for the oxidized form (32). Purified plastocyanin had absorbance ratios A278 /A597 between 1.2 and 1.4. Plastocyanin was crosslinked to PSI in the presence of 0.05% (w/v) Triton X-100 as described (18) except that a pH value was adjusted with 30 mM Mops buffer and that PSI particles were used at 0.4 mg Chl/ml. EPR samples contained PSI particles at a P700 concentration of about 5 mM. For optical experiments, PSI particles crosslinked with plastocyanin were used at a concentration of 15 mg Chl/ml in 100 mM Mops, pH 7.0, 5 mM MgCl2 , 0.02% (w/v) Triton X-100. Absorbances changes of P700 were measured with a single beam spectrophotometer essentially as described (33). Flash excitation was provided by a frequency-doubled Nd:YAG laser (5 ns FWHM). Prior to the flash a photoshutter protected the sample from the measuring light at 704.5 nm (2.6 nm FWHM). The shutter was opened 5 ms before the recording was started. The cuvette containing 3 ml of the sample had an optical pathlength of 10 mm. The detecting photodiode (1 cm2) was placed at a distance of 35 cm from the cuvette and was protected by an interference filter at 703 nm. The output of the photodiode was amplified with an electrical bandwidth ranging from dc to 1 MHz, with dc offset compensation by 16 bit ADC and DAC within 4 ms immediately before the recording. Signal disturbance by
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FIG. 1. Time course of flash-induced DA at 703 nm in PSI particles crosslinked with plastocyanin. The concentration of the PSI particles was 15 mg/ml Chl. Cuvette was of 10-mm optical path at 257C. The crosslinked complex was suspended in 100 mM Mops, pH 7.0, 5 mM MgCl2 , 0.02% (w/v) TX-100. The trace is the result of one flash. Single exponential analysis of fast component DA Å 03.69, t1/2 Å 15 ms.
fluorescence was monitored in the absence of measuring light and subtracted when necessary. EPR measurements were performed at X-Band on a Bruker ER 300 spectrometer equipped with an Oxford Instruments temperature control system. Spectra were taken at 15 K using 6.7 mW microwave power and 1.6 mT modulation. Illumination in the cavity was provided by an 800-W tungsten projector yielding 16,000 mE m02 s01 of white light at the EPR cavity window after being filtered through 2 cm of water and a calflex filter to remove infrared radiation. The full reduction of the FeS centers FA and FB was achieved by addition of 20 mM dithionite at pH 10.3 (100 mM glycine) and subsequent illumination at 200 K for 5 min.
RESULTS
The experiments were performed with a crosslinked complex between plastocyanin and PSI (18, 24) competent in fast reduction of P700/. The efficiency of the crosslinking was assayed by laser-induced absorbance changes of P700 (Fig. 1). The fast reduction kinetics with a half-time of 15 ms amounts to 63% of the total amplitude. This fraction of PSI crosslinked with plastocyanin in an active state exceeds previous amounts considerably (18). The crosslinked PSI particles were frozen in darkness and illuminated at 4 K. The EPR spectrum of this sample recorded at 15 K is shown in Fig. 2A (dotted line). It is characterized by dominant signals at g Å 2.05, 1.94, 1.86 (resulting from reduced FA ), minor signals at g Å 2.07, 1.92, 1.88 (resulting from reduced FB ), and the P700/ radical at g Å 2 (1). The ratio between the reduced FA and FB resonances was determined to
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FIG. 2. EPR spectra of PSI particles with crosslinked plastocyanin. (A) Dotted line, sample was frozen in the dark, illuminated at 4 K for 2 min; solid line, the sample was subsequently annealed to 160 K under illumination within the cavity. The crosslinked complex was suspended in 100 mM Mops, pH 7.0, 5 mM MgCl2 , 0.02% (w/v) TX100. The difference spectrum of the continous line spectrum minus the dotted line spectrum is shown below. (B) Sample was chemically reduced with 20 mM dithionite, 0.1 M glycine, pH 10.3, and photoaccumulated at 200 K. Spectrometer conditions: temperature 15 K, microwave power 6.7 mW, microwave frequency 9.46 GHz, modulation amplitude 1.6 mT.
be about 80/20, in line with previous observations with PSI particles of spinach or cyanobacteria (1). No further components are detected, indicating that illumination at 4 K of the crosslinked complex results in charge separation between P700 and the FeS electron acceptors without further transfer of the positive charge from P700/ to the copper center of the bound plastocyanin. To check whether this transfer was possible at higher temperatures, the sample was annealed to temperatures between 60 and 180 K in darkness. Under none of these conditions was plastocyanin oxidized. However, above 100 K, charge recombination was observed as described previously (1). Previous optical measurements (22, 34) have shown that the electron transfer from plastocyanin to P700/ can occur at temperatures significantly below 07C and at least down to 200 K (M. Hippler et al., unpublished
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results). Therefore the sample was exposed within the EPR cavity to 40, 80, 120, 140, and 160 K under illumination for 30 s and cooled down again to the measuring temperature of 15 K under continuous illumination. Subsequently the spectra were run in the dark. Heating to temperatures lower than 140 K had no effect on the EPR spectrum. However, at 160 K the shape of the light-induced spectrum changed significantly (Fig. 2A, continuous line). The intensity of the resonance at g Å 1.86, which is characteristic of reduced cluster FA in centers where FB is oxidized, decreased by 40% compared to the sample illuminated at 4 K. In contrast, the resonance at g Å 1.88, resulting from reduced center FB , in the presence of reduced FA cluster (1) increased from about 20 to 63% of the maximal FA /FB signal in reduced PSI. In addition, the signal of oxidized plastocyanin (resulting from Cu2/) appeared as broad positive contributions above g Å 2.05 and a derivative signal at g Å 2.013 (35). These differences are manifested in the difference of both spectra, as shown in Fig. 2A, bottom. (continuous minus dotted line spectrum). Using a control PSI sample, it was found that the ratio of 80/20 of photoreduction of FA and FB is also observed after illumination at 160 K (not shown). The maximal amount of FA and FB , which is reflected by the signal at g Å 1.88, was determined by chemical reduction with dithionite at high pH and subsequent photoaccumulation at 200 K, after the series of experiments. The signal is shown in Fig. 2B. The results are summarized in Table I. They demonstrate that crosslinked plastocyanin is able to reduce photooxidized P700/ above 160 K in the light, leading TABLE I 0 Comparison of EPR Measured Resonances of F0 A , FB , and 0 [F0 A FB ] for PSI Particles Crosslinked with Plastocyanin under Different Conditions: (i) Frozen in the Dark, Illuminated at 4 K for 2 min and (ii) Annealed to 160 K under Illumination within the Cavity
Intensities of the gx resonances of centers F0 A (g Å 1.86) and F0 B (g Å 1.88) relative to the fully reduced control (g Å 1.88) obtained by reduction at high pH followed by 5 min of photoaccumulation at 200 K PSI particles with crosslinked 0 plastocyanin [F0 [F0 [F0 A] B] A FB ] Frozen in the dark, illuminated for 2 min at 4 K Warmed to 160 K under illumination
0.8
0.2
0
0.4
low
0.6
Note. EPR spectra were recorded at 15 K with the light switched off. Close to 100% of centers were seen to perform stable charge separation at 4 K during the first turnover.
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ELECTRON TRANSFER FROM PLASTOCYANIN TO PsaC SUBUNIT OF PHOTOSYSTEM I
to an additional charge transfer from P700 to the FeS electron acceptors. DISCUSSION
In this work we have performed an EPR characterization of the electron transfer reaction within the crosslinked complex between PSI and its physiological electron donor plastocyanin. The results demonstrate that electron transfer from plastocyanin to P700/ does occur at cryogenic temperatures, at least down to 160 K. The detailed optical study of the temperature dependence of the electron donation reaction to P700/ in the crosslinked complex will be reported elsewhere (M. Hippler et al., unpublished results). The sample offers the possibility of performing two sequential electron transfer reactions at low temperature within the electron acceptor chain of PSI. The crosslinked complex, illuminated at 4 K, shows the same pattern of stably photoreduced centers FA and FB as the control PSI, i.e., only 20% of the molecules performed charge separation between P700 and FB , whereas in 80% of the centers, the electron ejected from P700 ended up at center FA (see Ref. 1 for review). An electron donation from reduced plastocyanin to P700/ occuring during illumination at 4 K in the crosslinked complex could not be detected. When such a preilluminated sample (i.e., in the state PCred –P700/ –FeS0) was annealed to temperatures above 100 K, still no oxidation of plastocyanin by P700/ could be observed, but a charge recombination (P700/ – FeS0 r P700–FeS) took place. This was true even for temperatures above 160 K, where under illumination electron donation from reduced plastocyanin to PSI was observed. Two different explanations can be put forward. (i) Below 160 K, the rate constant for electron donation from reduced plastocyanin to P700/ is slower than that of recombination from the reduced acceptors. Extrapolating the temperature dependence of the rate constants of electron transfer from plastocyanin to P700/ (Ref. 22, 34) in fact predicts that at temperatures around 110 K the rate constant of the donation reaction becomes comparable to that of charge recombination from the terminal electron acceptors (36). (ii) The efficiency of electron donation from reduced plastocyanin to P700/ drops drastically below 220 K. Such an effect has been proposed by Venturoli et al. (28) for the analogous reaction between cytochrome c2 and the purple bacterial reaction center. Time-resolved optical experiments at temperatures lower than 200 K are required to distinguish these possibilities. However, illumination at temperatures at or above 160 K induced the oxidation of plastocyanin by P700/, and a second charge separation between P700 and the terminal acceptors occurred. For a quantitative analysis it must be taken into account that this reaction can only occur
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in 63% of total PSI, i.e., the fraction of crosslinked complex (cf. Fig. 1). The EPR spectrum obtained for 160 K illumination showed that about 40% of total PSI contained terminal acceptors in which FA was reduced while FB remained oxidized. From the amount of crosslinked complexes with active electron transfer from Pc to P700/ as determined optically (see Fig. 1) it follows that 37% of total PSI cannot perform a second electron transfer step, i.e., should yield the ratio of 80/20 corresponding to a fraction of 30 and 7% of total PSI for FAredFBox and FAoxFBred, respectively. We conclude (i) that after 160 K illumination, the centers showing FA reduced in the absence of reduced FB were largely those lacking a crosslinked plastocyanin and (ii) that the majority of PSI centers crosslinked to plastocyanin underwent a double turnover during 160 K illumination resulting in the reduction of both FA and FB . Two major conclusions can be drawn from these results. (i) The state (P700/ –FA0/FB0) is stable at low temperatures. So far, full reduction of FA and FB has only been achieved by trapping the electron on the electron acceptors through rapid reduction of P700/ from exogenous reductants. No data have been reported excluding that the negative charge on one FeS center could preclude stable low-temperature reduction of the other center. (ii) While after the first turnover, the stoichiometry of photoreduced FA and FB was 80/20 as usual, close to full reduction of FA and FB was detected in those centers having performed a second charge separation. The electrons reducing the residual 80% of FB are transfered either (1) directly from center FX or (2) via center FA . Since the cluster FA is already reduced in the respective fraction of centers, the latter sequence implies rapid equilibration between FA and FB even at low temperatures. In this picture the reduction with an 80:20 stoichiometry of the FeS centers would therefore indicate a redox equilibrium between two communicating clusters with slightly differing Em values. An attribution to an effect of the different distances of electron transfer toward these clusters with lacking intercluster electron transfer is considered to be an unlikely interpretation. It is of note that in the family of 8Fe–8S ferredoxins, which includes the FA /FB0 -carrying subunit PsaC, both the presence and the absence of intercluster electron transfer have been observed by NMR in different species (37, 38), possibly depending on the detailed localization of the mixed-valence pairs in both clusters. Similar studies on the FA /FB -binding PsaC subunit may provide a clearer picture with respect to this crucial question. ACKNOWLEDGMENT The authors thank Dr. A. W. Rutherford (Paris, France) for help with the initial measurements and for critical reading of the manuscript.
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19. Haehnel, W., Do¨ring, G., and Witt, H. T. (1971) Z. Naturforsch. 26B, 1171–1174. 20. Haehnel, W., Pro¨pper, A., and Krause, H. (1980) Biochim. Biophys. Acta 193, 384–399. 21. Haehnel, W., Ratajczak, R., and Robenek, H. (1989) J. Cell Biol. 108, 1397–1405. 22. Bottin, H., and Mathis, P. (1985) Biochemistry 24, 6453–6460. 23. Bottin, H., and Mathis, P. (1987) Biochim. Biophys. Acta 892, 91–98. 24. Drepper, F., Hippler, M., Nitschke, W., and Haehnel, W. (1996) Biochemistry 35, 1282–1295. 25. Warren, P. V., Smart, L. B., McIntosh, L., and Golbeck, J. H. (1993) Biochemistry 32, 4411–4419. 26. Fromme, P., Schubert, W.-D., and Krauß, N. (1994) Biochim. Biophys. Acta 1187, 99–105. 27. Ortega, J. M., and Mathis, P. (1993) Biochemistry 32, 1141– 1151. 28. Venturoli, G., Mallardi, A., and Mathis, P. (1993) Biochemistry 32, 13245–13523. 29. Porra, R. J., Thompson, W. A., and Kriedemann, P. E. (1989) Biochim. Biophys. Acta 975, 384–394. 30. Hiyama, T., and Ke, B. (1972) Biochim. Biophys. Acta 267, 160– 171. 31. Ratajczak, R., Mitchell, R., and Haehnel, W. (1988) Biochim. Biophys. Acta 933, 306–318. 32. Katoh, S., Shiratori, I., and Takamija, A. (1962) J. Biochem. 51, 32–40. 33. Haehnel, W., Jansen, T., Gause, K., Klo¨sgen, R. B., Stahl, B., Michl, D., Huvermann, B., Karas, M., and Herrmann, R. G. (1994) EMBO J. 13, 1028–1038. 34. Hippler, M. (1994) Ph.D. dissertation, Universita¨t Freiburg, Germany. 35. Penfield, K. W., Gewirth, A. A., and Solomon, E. L. (1985) J. Am. Chem. Soc. 107, 4519–4529. 36. Golbeck, J. H., and Cornelius, J. M. (1986) Biochim. Biophys. Acta 849, 25–31. 37. Bertini, I., Capozzi, F., Luchinat, C., Piccioli, M., and Vita, A. J. (1994) J. Am. Chem. Soc. 116, 651–660. 38. Huber, J. G., Gaillard, J., and Moulis, J.-M (1995) Biochemistry 34, 194–205.
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