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Isolation of an intrinsic antenna chlorophyll a-protein from the photosystem I reaction center complex of the thermophilic cyanobacterium Synechococcus sp
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 244, No. 1, January, pp. 254-260,1986
Isolation of an Intrinsic Antenna Chlorophyll a-Protein from the Photosystem I Reaction Center Complex of the Thermophilic Cyanobacterium Synechococcus sp.’ KINTAKE
SONOIKE
AND
SAKAE
KATOH
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Komaba, Megureku, Tokyo, 153, Japan Received July 151985,
and in revised form September
181985
A chlorophyll-protein was isolated from a Synechococcus P700-chlorophyll a-protein complex free from small subunits (CPl-e) by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis after treatment with 2% 2-mercaptoethanol and 2% SDS. In contrast to CPl-e which, when electrophoresed under denaturating conditions, showed two polypeptide bands of 62 and 60 kDa, the chlorophyll-protein contained only the 60kDa polypeptide and hence is called CP60. The yield of CP60 was maximal with l-2% SDS and 2-4% sulfhydryl reagents because the chlorophyll-protein was denatured at higher concentrations of the reagents. The absorption spectrum of CP60, which retained more than half of the chlorophyll a molecules originally associated with the 60-kDa subunit of the photosystem I reaction center complex, showed a red band maximum at 672 nm and a small absorption band around 700 nm at liquid nitrogen temperature. CP60 emitted a fluorescence band at 717 to 725 nm at 77’K. The temperature dependence of the far red band of CP60 was essentially the same as that of CPl-e between 77 and 273°K. No photoresponse of P700 was detected in CP60. The results suggest that the two polypeptides resolved by SDS-gel electrophoresis from CPl-e are apoproteins of two distinct chlorophyll-proteins and that CP60 represents a chlorophyll-bearing 60kDa subunit functioning as an intrinsic antenna protein of the photosystem I reaction center complex. It will also be shown that the temperature dependence of the far red fluorescence band is not related to the photosystem I photochemistry. o lsse Academic Press.Inc.
The reaction center complex of PS I2 is a supramolecular complex consisting of large subunits of 60-70 kDa and several small subunits of lo-25 kDa molecular mass classes (l-9). The large subunits carry the primary electron donorP700 and acceptor Ai or AO, together with antenna chlorophyll a and P-carotene, while the small subunits represent the iron-sulfur 1 The present work was supported in part by grants for Scientific Research from the Ministry of Education, Science and Culture, Japan. ‘Abbreviations used:PS I and PS II, photosystems I and II, respectively; SDS, sodium dodecyl sulfate. 0003-9861/86 $3.00 Copyright 0 1986by AcademicPress,Inc. All rights
of reproduction
in any form
reserved.
proteins serving as secondary electron acceptors. There is a concensus that the PS I reaction center complex contains more than one large subunit, although there are considerable discrepancies regarding the number and size of the large subunits present in the complex (1, 3, 5, 6, 8). Recently, our understanding of the roles of the two large subunits present in the PS II reaction center complex has been considerably advanced (10-13). Isolation and characterization of the two chlorophyllbinding large subunits led to the conclusion that the 47- to 50-kDa subunit carries the PS II reaction center, whereas the 40- to 254
AN
INTRINSIC
ANTENNA
CHLOROPHYLL-PROTEIN
43-kDa polypeptide serves as an intrinsic antenna protein of the reaction center complex. An analogous functional heterogeity of the large subunits should exist in the PS I reaction center complex that has more than one large polypeptide per P700: P’700 would be located on a specific large subunit, while the rest may function as antenna chlorophyll a proteins in the PS I reaction center complex. However, the resolution of the PS I reaction center complex into functionally different large chlorophyll-binding subunits has not yet been reported. Takahashi et al. have shown that SDSpolyacrylamide gel electrophoresis of the thylakoid membranes or digitonin PS I preparations from the thermophilic cyanobacterium Synechococcus sp. resolves five PS I reaction center complexes which are named CPl-a, -b, -c, -d, -e in the order of electrophoretic mobility (6,7). CPl-e has the simplest subunit structure and totally lacks small polypeptides which carry the iron-sulfur centers. P700, Al (or A,), 70 chlorophyll a, and 10 ,&carotene molecules are associated with the large subunits of CPl-e which are usually resolved into two polypeptide bands of 62 and 60 kDa on SDS-gel electrophoresis under denaturing conditions (6, 9). Here we report the isolation and characterization of a chlorophyll a-protein from CPl-e. The polypeptide composition, photochemical activity, and low-temperature absorption and fluorescence spectra indicate that the isolated chlorophyll-protein is an intrinsic antenna chlorophyllprotein of the PS I reaction center complex. The origin of the far-red fluorescence emission band of PS I at liquid nitrogen temperature will be discussed. MATERIALS
AND
METHODS
Synechococcus sp. was grown at 55°C in the light (5000 lx) with continuous bubbling with air containing 5% COP (14, 15). The thylakoid membranes and digitonin PS I particles were prepared from 2-day-grown cells by the procedures described previously (6). CPl-e was isolated by SDS-polyacrylamide gel electrophoresis from membranes or digitonin particles which had been solubilized with SDS at a SDWchlorophyll weight ratio of 50 or 100, respectively, at room
OF PHOTOSYSTEM
I
255
temperature for 1 h (6). The chlorophyll-protein was extracted from gels with 50 mM Tris/HCl (pH 7.5) and 10 mM NaCl, pelleted by centrifugation at 260,OOOg for 2 h, and resuspended in 0.5 M sucrose containing 50 mM Tris/HCl (pH 7.5) and 10 mM NaCl. Polypeptide compositions of chlorophyll-proteins were determined by SDS-polyacrylamide gel electrophoresis according to Laemmli (16). Chlorophyll-proteins were denatured by incubating with 10% SDS, 2.5% 2-mercaptoethanol, and 8 M urea at room temperature for 1 h. After electrophoresis, gels were stained with Coomassie brilliant blue R-250. Chlorophyll and polypeptides were scanned at 675 and 560 nm, respectively, with a Shimadzu dual-wavelength ehromatoscanner CS-910. Absorption spectra were recorded at liquid nitrogen temperature with a Hitachi 320 spectrophotometer with a low-temperature attachment. Gel slices containing chlorophyll-proteins were soaked for 1 h at 4°C in 50 mM Tris/HCl (pH 7.5), 10 mM NaCl, and 60% glycerol prior to measurement. Fluorescence emission spectra of chlorophyll-proteins in gels were determined at 77°K with a laboratory-made apparatus as described previously (17). To determine the temperature dependence of the emission intensity, the sample was first cooled to 77”K, then gradually warmed by allowing the liquid nitrogen to boil. A thermocouple was placed in the sample to monitor the temperature. The gel slices were soaked for 1 h at 4°C in 50 mM Tris/HCl (pH 7.5) and 10 mM NaCl prior to measurement. P700 photooxidation was measured at 700 nm with a Hitachi 356 spectrophotometer as described previously (18). The basal reaction mixture contained 50 mM Tris/HCl (pH 7.5), 10 mM NaCI, 1 mM ascorbate, 2 pM dichlorophenolindophenol, 1 mM methyl viologen, and about 3 fig chlorophyll a/ml. Chlorophyll a was determined by the method of MacKinney (19). RESULTS
Electrophoresis of the five PS I reaction center complexes isolated from Syneche coccus sp. under nondenaturing conditions has been described previously (6). When subjected to SDS-polyacrylamide gel electrophoresis after treatments with SDS at a detergent to chlorophyll ratio of 10, CPle was produced from CPl-a to CPl-d, whereas no faster moving chlorophyllprotein was resolved from CPl-e because CPl-e has the simplest subunit structure among the CPl complexes. When CPl-e was electrophoresed after treatment with 2% 2-mercaptoethanol and 2% SDS at room temperature for 1 h, how-
256
SONOIKE
ever, a small chlorophyll-containing band was found to migrate before the major CPl-e band and after the fast-moving freepigment band (Fig. 1, upper trace). That the band contains a chlorophyll-protein is shown in the lower trace: A polypeptide band was detected at the corresponding position when the gel had been stained with Coomassie brilliant blue. Two chlorophyllfree protein bands resolved between the CPl-e band and the new chlorophyll band were the large subunits of CPl-e. The eukaryotic PS I reaction center complex is associated with a specific antenna chlorophyll a/b-protein which has apoproteins of 25-20 kDa (20). The new chlorophyll-protein resolved from CPl-e, however, cannot be attributed to the peripheral antenna protein because the cyanobacterium totally lacks the chlorophyll a/b-protein and CPl-e has no small polypeptides. Another possibility would be that the fast-moving chlorophyll-protein is a modCPI-e
neybard
Protein 8 2
L-L h
(+I
FIG. 1. Separation of a new cholorophyll-protein band from CPl-e by gel electrophoresis. CPl-e was incubated with 2% 2-mercaptoethanol and 2% SDS at room temperature for 1 h and run on 7.5% acrylamide gel at 4°C for 2 h. The gel was scanned for chlorophyll at 675 nm (upper trace), and then stained with Coomassie brilliant blue and scanned for protein at 566 nm (lower trace). Two chlorophyll-free protein bands resolved between the CPl-e band and the new chlorophyll-protein band were the 62-kDa (slow-moving) and the 60-kDa (fast-moving) subunits of CPl-e.
AND
KATOH
a
FIG. 2. Polypeptide compositions of the chlorophyllprotein separated from CPl-e. Samples were incubated with 10% SDS, 2.5% 2-mercaptoethanol, and 8 M urea for 1 h at room temperature and electrophoresed on gels containing 10% acrylamide. Lane a, CPl-e; lane b, the chlorophyll-protein separated from CPl-e; lane c, reelectrophoresis of the 60-kDa band from the chlorophyll-protein.
ified form of CPl-e. Treatment of CPl-e with the sulfhydryl reagent and SDS might have resulted in a partial denaturation of the protein conformation upon which the electrophoretic mobility strongly depends. The polypeptide composition of the chlorophyll-protein showed, however, that this is not the case (Fig. 2). As described previously (6), the large subunits of CPl-e were resolved into two polypeptide bands by denaturing electrophoresis (lane a). They are called the 62- and 60-kDa polypeptides, although the molecular masses of the two polypeptides vary to some extent with electrophoretic conditions. The fastmoving chlorophyll-protein showed a different polypeptide pattern: The main polypeptide resolved was the 60-kDa polypeptide and there was only a trace of the 62-kDa polypeptide (lane b). The small 62kDa band can be ascribed to contamination because reelectrophoresis of the 60-kDa polypeptide resolved from the fast-moving chlorophyll-protein produced only a diffuse 60-kDa band (lane c). The results strongly suggest that the chlorophyll-protein iso-
AN
INTRINSIC
ANTENNA
CHLOROPHYLL-PROTEIN
lated here is the chlorophyll-bearing 60kDa subunit of the PS I reaction center complex. For simplicity, the chlorophyllprotein is hereafter referred to as CP60. Because the yield of CP60 was very low (see Fig. l), attempts have been made to increase the amount of chlorophyll-protein resolved by gel electrophoresis from CPl-e. Figure 3A shows the yields of CP60 as a function of SDS concentration. When CPl-e had been treated with increasing concentrations of SDS but with a constant concentration (2%) of dithiothreitol, the amount of chlorophyll associated with CP60 reached a maximum at l-2% SDS and decreased at higher detergent concentrations, whereas the amount of the protein in the CP60 band remained constant at SDS concentrations above 1% . Thus, chlorophyll seems to be solubilized from CP60 at higher SDS concentrations. In Fig. 3B, the dithiothreitol concentration was varied, while the SDS concentration was kept at 2%. There were parallel changes in the amounts of chlorophyll and protein in the CP60 band, showing a maximum at a dithiothreitol concentration of 2-4%. This indicates that high concentrations of the sulfhydryl reagent cause denaturation of the chlorophyll-protein. Thus the yield of CP60 is limited by the instability of the chlorophyll-protein at the reagent concentrations required to dissociate CPl-e.
OTT 2% ;
A
‘0 5
I. OO
a 2
I . 4 6 SDS 1%)
,I* 80
. 2
4
, 6
,I 6
DTT t%.)
FIG. 3. Yields of CP60 as functions of (A) SDS and (B) dithiothreitol concentrations. CPI-e was treated with the indicated concentrations of SDS and dithiothreitol at room temperature for 1 h and electrophoresed on 7.5% acrylamide gel at 4°C for 2 h. Heights of chlorophyll peaks and protein peaks were determined at 675 and 560 nm, respectively.
OF PHOTOSYSTEM TABLE
I
257
I
PEAK HEIGHT RATIO OF CHLOROPHYLL BAND TO PROTEIN BAND OF CPl-e AND CP60 Chlorophyll-protein
Chlorophyll/Protein
CPl-e CP60 DPercentages
0.34 (100) 0.21 (61) in parentheses.
Figure 3B also shows that a small amount of CP60 was separated in the absence of dithiothreitol. This is evidence that no disulfide bond is involved in the binding between the 62- and 60-kDa subunits in CPl-e. It is suggested therefore that the sulfhydryl reagent promotes the dissociation of the subunits by affecting the conformation of the subunits through the reduction of intramolecular disulfide bonds. Various attempts have been made to isolate a chlorophyll-protein containing only the 62-kDa subunit but so far without success. The 62-kDa subunit may be more sensitive to the treatment with SDS and a sulfhydryl reagent than the 60-kDa subunit. In fact, a larger amount of chlorophyll-free 62-kDa subunit relative to chlorophyll-free 60-kDa subunit was produced during the isolation of CP60 (see Fig. 1). A possibility remains, however, that a small amount of the 62-kDa polypeptide resolved from the CP60 band (see Fig. 2, lane b) is due to the comigration of a chlorophyllbearing 62-kDa subunit with CP60. The amount of chlorophyll a associated with CP60 was estimated by comparing the peak height ratio of the chlorophyll band (in an unstained gel) and the protein band (in the same gel but after staining) of CP60 with those of CPl-e which had been run in parallel. The chlorophyll a to protein ratio of CP60 thus determined was approximately 60% of the ratio of CPl-e (Table I). It appears, therefore, that CP60 still retains more than a half of the chlorophyll a molecules originally associated with the 60-kDa subunit of CPl-e. Figure 4 compares the absorption spectra in the red band region of CPl-e and CP60 determined at liquid nitrogen tem-
258
SONOIKE
AND
KATOH
I..
77K
1 660
690
720
I 750
WAiJELENGTH ( nm 1
FIG. 5. Fluorescence CP60 at 7’7 K.
emission spectra of CPl-e and
WAVELENGTH hml
FIG. 4. Absorption 77 K.
spectra of CPl-e
and CP60 at
A major part of the P700 photoresponse survived the incubation of CPl-e with 2% 2-mercaptoethanol and 2% SDS for 1 h (not shown). However, no P700 photooxidation was detected in isolated CP60, whether the photoresponse was determined in gels or after the extraction from gels. It was impossible to determine P700 by measuring chemically oxidized minus reduced difference spectra of CP60 because intensive bleaching of bulk chlorophyll a takes place on addition of ferricyanide.
perature. The low-temperature spectrum of CP60 is similar to that of CPl-e but shifted to shorter wavelength: The absorption maximum of the red band was 675 nm for CPl-e and 672 nm for CP60. Note that the two spectra have an appreciable absorption band at 700-710 nm. About 10 ficarotene molecules per P700 are present in CPl-e (9). The blue region of the spectrum of CP60 indicates that the chlorophyllDISCUSSION protein still contains p-carotene but in a reduced amount (not shown). PS I reaction center preparations from The low-temperature fluorescence emisvarious plants and algae often showed two sion spectra of CPl-e and CP60 are illuspolypeptide bands in the molecular mass trated in Fig. 5. CPl-e shows a strong classes 60-70 kDa, when subjected to SDSemission band in the far red region at 77°K (6). The band maximum occurred at 725 nm in the CPl-e preparation used here. CP60 was found to emit the far red fluorescence band, although the emission maximum varied from 717 to 725 nm with preparations. The emission intensity of CPl-e and CP60 decreased in parallel as the temperature was raised (Fig. 6). It is concluded therefore that a specific PS I antenna which emits the far red fluorescence band TEMfERATURE (‘C) at low temperatures is associated with the 60-kDa subunit. FIG. 6. Temperature dependence of the fluorescence On illumination, CPl-e showed a rapid emission intensity of CPl-e and CP60. Fluorescence were norabsorption decrease at 700 nm, which is intensities of the two chlorophyll-proteins ascribed to photooxidation of P700 (Fig. 7). malized at -196°C.
4,
AN
INTRINSIC
ANTENNA
CHLOROPHYLL-PROTEIN
CPI-a on
I
AA700 = 0.001
Off I 30s
CP60
FIG. 7. P700 photooxidation in CPl-e and CP60. Chlorophyll concentrations were about 3 pg/ml for the two samples. For other conditions, see Materials and Methods.
polyacrylamide gel electrophoresis under denaturating conditions (4,5,8,21-24). The two polypeptide bands are generally considered to originate from a single polypeptide as a result of proteolytic digestion during the isolation (22, 23), post-translational modification (4), or differential denaturation (5, 8), because they showed similar amino acid compositions (5) and, on partial proteolysis, similar peptide maps (4,5,8). Models of the PS I reaction center complexes containing two to six copies of the single homologous polypeptide have been proposed (2,8,25). Recently, Fish et al. (26) have shown that the maize chloroplast DNA contains two photoinducible genes that code for apoproteins of the P700-chlorophyll u-proteins. The two genes are separated by only a short distance (25 bp) and hence most likely are transcribed into a single mRNA. The polypeptide sequences predicted from the genes are about 45% identical and 9% more of the amino acids are conservative replacements. These important findings urge the reevaluation of the two polypeptide bands resolved from PS I complexes because the amino acid compositions and the peptide maps should be similar, though not identical, between the two highly homologous apoproteins.
OF PHOTOSYSTEM
I
259
The present work provides direct evidence for the heterogeneity of the large subunits in the PS I reaction center complex. CP60 isolated from CPl-e, a Synechococcus PS I complex, has a simpler polypeptide composition than the parent complex: The denaturating SDS-polyacrylamide gel electrophoresis of CPl-e resolves two distinct polypeptide bands in the region 60-62 kDa, whereas only one of the two polypeptides with an apparent molecular mass of 60 kDa was found in CP60. Although a chlorophyll-protein containing the 62-kDa subunit remains to be isolated, the finding strongly suggests that the two polypeptides separated in SDS-gels are apoproteins of two different chlorophyllproteins and that the CP60 we have isolated here is a chlorphyll-bearing 60-kDa subunit. The PS II reaction center complex contains the two types of chlorphyll-binding large subunits which are different in the function (10-13). By analogy, a large subunit of the PS I complex would carry the reaction center components, while the rest would function only in light harvesting. A chlorophyll-protein having a photoactive P700 has yet to be isolated. However, the absence of P700 photoresponse from CP60 suggests an antenna nature of the chlorophyll-protein. It seems less likely that P’700 photooxidation was totally inactivated during the isolation of CP60 because P700 is not necessarily more sensitive to treatments with detergent or organic solvent than antenna chlorophyll. In fact, the photoresponse is considerably resistant to the treatment of CPl-e with 2-mercaptoethano1 and SDS. Furthermore, the low-temperature absorption and fluorescence spectra of CP60, similar to those of CPl-e, indicate that the original chlorophyll-protein linkages or microenvironments surrounding chlorophyll a molecules are largely preserved in the isolated chlorophyll-protein. We may conclude that CP60 is an intrinsic antenna chlorophyll-protein of the PS I reaction center complex. Characteristic features of the PS I antenna are the long-wavelength fluorescence emission bands which are intensified as the ambient temperature is lowered. The pe-
260
SONOIKE
AND
ripheral light-harvesting chlorophyll a/bprotein emits the fluorescence band at about 735 nm (ZO),while the reaction center complex per se has the emission band at 725 nm at liquid nitrogen temperature. The present work shows that the 725~nm fluorescence band is emitted from a chlorophyll a species located on the intrinsic antenna proteins. Butler has proposed that the emitter of the far red band is C-705, a small amount of chlorophyll a which has absoption at a longer wavelength (27). The effect of lowering the temperature was related to the competition between the far red fluorescing energy trap and P700 for the excitation energy in the antenna chlorophyll of PS I (28). Our results are compatible with the proposal that the far red band originates from a long-wavelength chlorophyll a form because CP60 shows an absorption band at 705-710 nm at liquid nitrogen temperature. However, the temperature dependence of the far red band was found to be essentially the same between the CPl-e which has P700 and CP60 which lacks P700. Thus, the low-temperature effect cannot be related to the PS I photochemistry. It is more likely that the far red band develops because the excitation energy transfer from the far red fluorescing traps to nonfluorescing traps is inhibited at low temperature. ACKNOWLEDGMENT The authors thank Miss Fumiko lent technical assistance.
Idei for her excei-
REFERENCES 1. BENGIS, C., AND NELSON, N. (1975) J. BioL Chem. 250,2783-2’788. 2. BENGIS, C., AND NELSON, N. (1977) .I BioL Chem. 252,4564-4569. 3. THORNBER, J. P., ALBERTE, R. S., HUNTER, R. S., SHIOZAWA, J. A., AND KAN, K.-S. (1977) Brookhaven Symp. Biol. 28.138-152. 4. NECHUSHTAI, R., AND NELSON, N. (1981) J. BioL Chem. 256.11624-11628.
KATOH
5. LAGOUTTE, B., SETIF, P., AND DURANTON, J. (1981) in Proceedings of the 5th International Congress (Akoyunoglou, G., ed.), Vol. 3, pp. 237243, Balaban Int. Sci. Services, Philadelphia. 6. TAKAHASHI, Y., KOIKE, H., AND KATOH, S. (1982) Arch. B&hem. Biuphys. 219,209-218. 7. TAKAHASHI, Y., AND KATOH, S. (1982) Arch. Biochem Biophys. 219,219-227. 8. VIERLING, E., AND ALBERTE, R. S. (1983) Plant PhysioL 72.625-633. 9. TAKAHASHI, Y., HIROTA, K., AND KATOH, S. (1985) Photosynth Res. 6, 183-192. 10. YAMAGISHI, A., AND KATOH, S. (1983) Arch Biochem Biophys. 225.836-846. 11. YAMAGISHI, A., AND KATOH, S. (1984) B&him Biophys. Acta 765,118-B?& 12. NAKATANI, H. Y., KE, B., DOLAN, E., AND ARNTZEN, C. J. (1984) B&him. Biqphys. Acta 765, 347352. 13. YAMAGISHI, A., AND KATOH, S. (1985) Biochim. Biophys. Acta 807, 74-80. 14. YAMAOKA, T., SATOH, K., AND KATOH, S. (1978) Plant Cell Physiol. 19,943-954. 15. HIRANO, M., SATOH, K., AND KATOH, S. (1980) Phe tosynth. Res. 1,163-170. 16. LAEMMLI, U. K. (1970) Nature (London) 227,680685. 17. NAKAYAMA, K., YAMAOKA, T., AND KAMH, S. (1979) Plant Cell PhysioL 20,1565-1576. 18. KOIKE, H., SATOH, K., AND KATOH, S. (1982) Plant Cell PhysioL 23, 293-299. 19. MACKINNEY, G. (1941) .I BioL Chem. 140.315-322. 20. MULLET, J. E., BURKE, J. J., AND ARNTZEN, C. J. (1980) Plant PhysioL 65,814-822. 21. ANDERSON, J. M., AND LEVINE, R. P. (1974) Biochim Biophys. Acta 357,118-126. 22. CHUA, N.-H., MALTIN, K., AND BENNOUN, P. (1975) J. Cell BioL 67,361-377. 23. MACHOLD, 0. (1975) B&him. Biophys. Acta 281, 103-122. 24. LAGOUTTE, B., SETIF, P., AND DURANTON, J. (1980) Photosynth. Res. 1, 3-16. 25. THORNBER, J. P., AND ALBERTE, R. S. (1977) in Encyclopedia of Plant Physiology, New Series (A. Trebst and M. Avron, eds.), Vol. 5, pp. 574-582, Springer-Verlag, Berlin. 26. FISH, L. E., KOCK, U., AND BOGORAD, L. (19%) J BioL Chem 260,1413-1421. 27. BUTLER, W. L. (1961) Arch. B&hem. Biophys. 93, 413-422. 28. SATOH, K., AND BUTLER, W. L. (1978) B&him Biophys. Acta 502,103-110.
Isolation of an Intrinsic Antenna Chlorophyll a-Protein from the Photosystem I Reaction Center Complex of the Thermophilic Cyanobacterium Synechococcus sp.’ KINTAKE
SONOIKE
AND
SAKAE
KATOH
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Komaba, Megureku, Tokyo, 153, Japan Received July 151985,
and in revised form September
181985
A chlorophyll-protein was isolated from a Synechococcus P700-chlorophyll a-protein complex free from small subunits (CPl-e) by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis after treatment with 2% 2-mercaptoethanol and 2% SDS. In contrast to CPl-e which, when electrophoresed under denaturating conditions, showed two polypeptide bands of 62 and 60 kDa, the chlorophyll-protein contained only the 60kDa polypeptide and hence is called CP60. The yield of CP60 was maximal with l-2% SDS and 2-4% sulfhydryl reagents because the chlorophyll-protein was denatured at higher concentrations of the reagents. The absorption spectrum of CP60, which retained more than half of the chlorophyll a molecules originally associated with the 60-kDa subunit of the photosystem I reaction center complex, showed a red band maximum at 672 nm and a small absorption band around 700 nm at liquid nitrogen temperature. CP60 emitted a fluorescence band at 717 to 725 nm at 77’K. The temperature dependence of the far red band of CP60 was essentially the same as that of CPl-e between 77 and 273°K. No photoresponse of P700 was detected in CP60. The results suggest that the two polypeptides resolved by SDS-gel electrophoresis from CPl-e are apoproteins of two distinct chlorophyll-proteins and that CP60 represents a chlorophyll-bearing 60kDa subunit functioning as an intrinsic antenna protein of the photosystem I reaction center complex. It will also be shown that the temperature dependence of the far red fluorescence band is not related to the photosystem I photochemistry. o lsse Academic Press.Inc.
The reaction center complex of PS I2 is a supramolecular complex consisting of large subunits of 60-70 kDa and several small subunits of lo-25 kDa molecular mass classes (l-9). The large subunits carry the primary electron donorP700 and acceptor Ai or AO, together with antenna chlorophyll a and P-carotene, while the small subunits represent the iron-sulfur 1 The present work was supported in part by grants for Scientific Research from the Ministry of Education, Science and Culture, Japan. ‘Abbreviations used:PS I and PS II, photosystems I and II, respectively; SDS, sodium dodecyl sulfate. 0003-9861/86 $3.00 Copyright 0 1986by AcademicPress,Inc. All rights
of reproduction
in any form
reserved.
proteins serving as secondary electron acceptors. There is a concensus that the PS I reaction center complex contains more than one large subunit, although there are considerable discrepancies regarding the number and size of the large subunits present in the complex (1, 3, 5, 6, 8). Recently, our understanding of the roles of the two large subunits present in the PS II reaction center complex has been considerably advanced (10-13). Isolation and characterization of the two chlorophyllbinding large subunits led to the conclusion that the 47- to 50-kDa subunit carries the PS II reaction center, whereas the 40- to 254
AN
INTRINSIC
ANTENNA
CHLOROPHYLL-PROTEIN
43-kDa polypeptide serves as an intrinsic antenna protein of the reaction center complex. An analogous functional heterogeity of the large subunits should exist in the PS I reaction center complex that has more than one large polypeptide per P700: P’700 would be located on a specific large subunit, while the rest may function as antenna chlorophyll a proteins in the PS I reaction center complex. However, the resolution of the PS I reaction center complex into functionally different large chlorophyll-binding subunits has not yet been reported. Takahashi et al. have shown that SDSpolyacrylamide gel electrophoresis of the thylakoid membranes or digitonin PS I preparations from the thermophilic cyanobacterium Synechococcus sp. resolves five PS I reaction center complexes which are named CPl-a, -b, -c, -d, -e in the order of electrophoretic mobility (6,7). CPl-e has the simplest subunit structure and totally lacks small polypeptides which carry the iron-sulfur centers. P700, Al (or A,), 70 chlorophyll a, and 10 ,&carotene molecules are associated with the large subunits of CPl-e which are usually resolved into two polypeptide bands of 62 and 60 kDa on SDS-gel electrophoresis under denaturing conditions (6, 9). Here we report the isolation and characterization of a chlorophyll a-protein from CPl-e. The polypeptide composition, photochemical activity, and low-temperature absorption and fluorescence spectra indicate that the isolated chlorophyll-protein is an intrinsic antenna chlorophyllprotein of the PS I reaction center complex. The origin of the far-red fluorescence emission band of PS I at liquid nitrogen temperature will be discussed. MATERIALS
AND
METHODS
Synechococcus sp. was grown at 55°C in the light (5000 lx) with continuous bubbling with air containing 5% COP (14, 15). The thylakoid membranes and digitonin PS I particles were prepared from 2-day-grown cells by the procedures described previously (6). CPl-e was isolated by SDS-polyacrylamide gel electrophoresis from membranes or digitonin particles which had been solubilized with SDS at a SDWchlorophyll weight ratio of 50 or 100, respectively, at room
OF PHOTOSYSTEM
I
255
temperature for 1 h (6). The chlorophyll-protein was extracted from gels with 50 mM Tris/HCl (pH 7.5) and 10 mM NaCl, pelleted by centrifugation at 260,OOOg for 2 h, and resuspended in 0.5 M sucrose containing 50 mM Tris/HCl (pH 7.5) and 10 mM NaCl. Polypeptide compositions of chlorophyll-proteins were determined by SDS-polyacrylamide gel electrophoresis according to Laemmli (16). Chlorophyll-proteins were denatured by incubating with 10% SDS, 2.5% 2-mercaptoethanol, and 8 M urea at room temperature for 1 h. After electrophoresis, gels were stained with Coomassie brilliant blue R-250. Chlorophyll and polypeptides were scanned at 675 and 560 nm, respectively, with a Shimadzu dual-wavelength ehromatoscanner CS-910. Absorption spectra were recorded at liquid nitrogen temperature with a Hitachi 320 spectrophotometer with a low-temperature attachment. Gel slices containing chlorophyll-proteins were soaked for 1 h at 4°C in 50 mM Tris/HCl (pH 7.5), 10 mM NaCl, and 60% glycerol prior to measurement. Fluorescence emission spectra of chlorophyll-proteins in gels were determined at 77°K with a laboratory-made apparatus as described previously (17). To determine the temperature dependence of the emission intensity, the sample was first cooled to 77”K, then gradually warmed by allowing the liquid nitrogen to boil. A thermocouple was placed in the sample to monitor the temperature. The gel slices were soaked for 1 h at 4°C in 50 mM Tris/HCl (pH 7.5) and 10 mM NaCl prior to measurement. P700 photooxidation was measured at 700 nm with a Hitachi 356 spectrophotometer as described previously (18). The basal reaction mixture contained 50 mM Tris/HCl (pH 7.5), 10 mM NaCI, 1 mM ascorbate, 2 pM dichlorophenolindophenol, 1 mM methyl viologen, and about 3 fig chlorophyll a/ml. Chlorophyll a was determined by the method of MacKinney (19). RESULTS
Electrophoresis of the five PS I reaction center complexes isolated from Syneche coccus sp. under nondenaturing conditions has been described previously (6). When subjected to SDS-polyacrylamide gel electrophoresis after treatments with SDS at a detergent to chlorophyll ratio of 10, CPle was produced from CPl-a to CPl-d, whereas no faster moving chlorophyllprotein was resolved from CPl-e because CPl-e has the simplest subunit structure among the CPl complexes. When CPl-e was electrophoresed after treatment with 2% 2-mercaptoethanol and 2% SDS at room temperature for 1 h, how-
256
SONOIKE
ever, a small chlorophyll-containing band was found to migrate before the major CPl-e band and after the fast-moving freepigment band (Fig. 1, upper trace). That the band contains a chlorophyll-protein is shown in the lower trace: A polypeptide band was detected at the corresponding position when the gel had been stained with Coomassie brilliant blue. Two chlorophyllfree protein bands resolved between the CPl-e band and the new chlorophyll band were the large subunits of CPl-e. The eukaryotic PS I reaction center complex is associated with a specific antenna chlorophyll a/b-protein which has apoproteins of 25-20 kDa (20). The new chlorophyll-protein resolved from CPl-e, however, cannot be attributed to the peripheral antenna protein because the cyanobacterium totally lacks the chlorophyll a/b-protein and CPl-e has no small polypeptides. Another possibility would be that the fast-moving chlorophyll-protein is a modCPI-e
neybard
Protein 8 2
L-L h
(+I
FIG. 1. Separation of a new cholorophyll-protein band from CPl-e by gel electrophoresis. CPl-e was incubated with 2% 2-mercaptoethanol and 2% SDS at room temperature for 1 h and run on 7.5% acrylamide gel at 4°C for 2 h. The gel was scanned for chlorophyll at 675 nm (upper trace), and then stained with Coomassie brilliant blue and scanned for protein at 566 nm (lower trace). Two chlorophyll-free protein bands resolved between the CPl-e band and the new chlorophyll-protein band were the 62-kDa (slow-moving) and the 60-kDa (fast-moving) subunits of CPl-e.
AND
KATOH
a
FIG. 2. Polypeptide compositions of the chlorophyllprotein separated from CPl-e. Samples were incubated with 10% SDS, 2.5% 2-mercaptoethanol, and 8 M urea for 1 h at room temperature and electrophoresed on gels containing 10% acrylamide. Lane a, CPl-e; lane b, the chlorophyll-protein separated from CPl-e; lane c, reelectrophoresis of the 60-kDa band from the chlorophyll-protein.
ified form of CPl-e. Treatment of CPl-e with the sulfhydryl reagent and SDS might have resulted in a partial denaturation of the protein conformation upon which the electrophoretic mobility strongly depends. The polypeptide composition of the chlorophyll-protein showed, however, that this is not the case (Fig. 2). As described previously (6), the large subunits of CPl-e were resolved into two polypeptide bands by denaturing electrophoresis (lane a). They are called the 62- and 60-kDa polypeptides, although the molecular masses of the two polypeptides vary to some extent with electrophoretic conditions. The fastmoving chlorophyll-protein showed a different polypeptide pattern: The main polypeptide resolved was the 60-kDa polypeptide and there was only a trace of the 62-kDa polypeptide (lane b). The small 62kDa band can be ascribed to contamination because reelectrophoresis of the 60-kDa polypeptide resolved from the fast-moving chlorophyll-protein produced only a diffuse 60-kDa band (lane c). The results strongly suggest that the chlorophyll-protein iso-
AN
INTRINSIC
ANTENNA
CHLOROPHYLL-PROTEIN
lated here is the chlorophyll-bearing 60kDa subunit of the PS I reaction center complex. For simplicity, the chlorophyllprotein is hereafter referred to as CP60. Because the yield of CP60 was very low (see Fig. l), attempts have been made to increase the amount of chlorophyll-protein resolved by gel electrophoresis from CPl-e. Figure 3A shows the yields of CP60 as a function of SDS concentration. When CPl-e had been treated with increasing concentrations of SDS but with a constant concentration (2%) of dithiothreitol, the amount of chlorophyll associated with CP60 reached a maximum at l-2% SDS and decreased at higher detergent concentrations, whereas the amount of the protein in the CP60 band remained constant at SDS concentrations above 1% . Thus, chlorophyll seems to be solubilized from CP60 at higher SDS concentrations. In Fig. 3B, the dithiothreitol concentration was varied, while the SDS concentration was kept at 2%. There were parallel changes in the amounts of chlorophyll and protein in the CP60 band, showing a maximum at a dithiothreitol concentration of 2-4%. This indicates that high concentrations of the sulfhydryl reagent cause denaturation of the chlorophyll-protein. Thus the yield of CP60 is limited by the instability of the chlorophyll-protein at the reagent concentrations required to dissociate CPl-e.
OTT 2% ;
A
‘0 5
I. OO
a 2
I . 4 6 SDS 1%)
,I* 80
. 2
4
, 6
,I 6
DTT t%.)
FIG. 3. Yields of CP60 as functions of (A) SDS and (B) dithiothreitol concentrations. CPI-e was treated with the indicated concentrations of SDS and dithiothreitol at room temperature for 1 h and electrophoresed on 7.5% acrylamide gel at 4°C for 2 h. Heights of chlorophyll peaks and protein peaks were determined at 675 and 560 nm, respectively.
OF PHOTOSYSTEM TABLE
I
257
I
PEAK HEIGHT RATIO OF CHLOROPHYLL BAND TO PROTEIN BAND OF CPl-e AND CP60 Chlorophyll-protein
Chlorophyll/Protein
CPl-e CP60 DPercentages
0.34 (100) 0.21 (61) in parentheses.
Figure 3B also shows that a small amount of CP60 was separated in the absence of dithiothreitol. This is evidence that no disulfide bond is involved in the binding between the 62- and 60-kDa subunits in CPl-e. It is suggested therefore that the sulfhydryl reagent promotes the dissociation of the subunits by affecting the conformation of the subunits through the reduction of intramolecular disulfide bonds. Various attempts have been made to isolate a chlorophyll-protein containing only the 62-kDa subunit but so far without success. The 62-kDa subunit may be more sensitive to the treatment with SDS and a sulfhydryl reagent than the 60-kDa subunit. In fact, a larger amount of chlorophyll-free 62-kDa subunit relative to chlorophyll-free 60-kDa subunit was produced during the isolation of CP60 (see Fig. 1). A possibility remains, however, that a small amount of the 62-kDa polypeptide resolved from the CP60 band (see Fig. 2, lane b) is due to the comigration of a chlorophyllbearing 62-kDa subunit with CP60. The amount of chlorophyll a associated with CP60 was estimated by comparing the peak height ratio of the chlorophyll band (in an unstained gel) and the protein band (in the same gel but after staining) of CP60 with those of CPl-e which had been run in parallel. The chlorophyll a to protein ratio of CP60 thus determined was approximately 60% of the ratio of CPl-e (Table I). It appears, therefore, that CP60 still retains more than a half of the chlorophyll a molecules originally associated with the 60-kDa subunit of CPl-e. Figure 4 compares the absorption spectra in the red band region of CPl-e and CP60 determined at liquid nitrogen tem-
258
SONOIKE
AND
KATOH
I..
77K
1 660
690
720
I 750
WAiJELENGTH ( nm 1
FIG. 5. Fluorescence CP60 at 7’7 K.
emission spectra of CPl-e and
WAVELENGTH hml
FIG. 4. Absorption 77 K.
spectra of CPl-e
and CP60 at
A major part of the P700 photoresponse survived the incubation of CPl-e with 2% 2-mercaptoethanol and 2% SDS for 1 h (not shown). However, no P700 photooxidation was detected in isolated CP60, whether the photoresponse was determined in gels or after the extraction from gels. It was impossible to determine P700 by measuring chemically oxidized minus reduced difference spectra of CP60 because intensive bleaching of bulk chlorophyll a takes place on addition of ferricyanide.
perature. The low-temperature spectrum of CP60 is similar to that of CPl-e but shifted to shorter wavelength: The absorption maximum of the red band was 675 nm for CPl-e and 672 nm for CP60. Note that the two spectra have an appreciable absorption band at 700-710 nm. About 10 ficarotene molecules per P700 are present in CPl-e (9). The blue region of the spectrum of CP60 indicates that the chlorophyllDISCUSSION protein still contains p-carotene but in a reduced amount (not shown). PS I reaction center preparations from The low-temperature fluorescence emisvarious plants and algae often showed two sion spectra of CPl-e and CP60 are illuspolypeptide bands in the molecular mass trated in Fig. 5. CPl-e shows a strong classes 60-70 kDa, when subjected to SDSemission band in the far red region at 77°K (6). The band maximum occurred at 725 nm in the CPl-e preparation used here. CP60 was found to emit the far red fluorescence band, although the emission maximum varied from 717 to 725 nm with preparations. The emission intensity of CPl-e and CP60 decreased in parallel as the temperature was raised (Fig. 6). It is concluded therefore that a specific PS I antenna which emits the far red fluorescence band TEMfERATURE (‘C) at low temperatures is associated with the 60-kDa subunit. FIG. 6. Temperature dependence of the fluorescence On illumination, CPl-e showed a rapid emission intensity of CPl-e and CP60. Fluorescence were norabsorption decrease at 700 nm, which is intensities of the two chlorophyll-proteins ascribed to photooxidation of P700 (Fig. 7). malized at -196°C.
4,
AN
INTRINSIC
ANTENNA
CHLOROPHYLL-PROTEIN
CPI-a on
I
AA700 = 0.001
Off I 30s
CP60
FIG. 7. P700 photooxidation in CPl-e and CP60. Chlorophyll concentrations were about 3 pg/ml for the two samples. For other conditions, see Materials and Methods.
polyacrylamide gel electrophoresis under denaturating conditions (4,5,8,21-24). The two polypeptide bands are generally considered to originate from a single polypeptide as a result of proteolytic digestion during the isolation (22, 23), post-translational modification (4), or differential denaturation (5, 8), because they showed similar amino acid compositions (5) and, on partial proteolysis, similar peptide maps (4,5,8). Models of the PS I reaction center complexes containing two to six copies of the single homologous polypeptide have been proposed (2,8,25). Recently, Fish et al. (26) have shown that the maize chloroplast DNA contains two photoinducible genes that code for apoproteins of the P700-chlorophyll u-proteins. The two genes are separated by only a short distance (25 bp) and hence most likely are transcribed into a single mRNA. The polypeptide sequences predicted from the genes are about 45% identical and 9% more of the amino acids are conservative replacements. These important findings urge the reevaluation of the two polypeptide bands resolved from PS I complexes because the amino acid compositions and the peptide maps should be similar, though not identical, between the two highly homologous apoproteins.
OF PHOTOSYSTEM
I
259
The present work provides direct evidence for the heterogeneity of the large subunits in the PS I reaction center complex. CP60 isolated from CPl-e, a Synechococcus PS I complex, has a simpler polypeptide composition than the parent complex: The denaturating SDS-polyacrylamide gel electrophoresis of CPl-e resolves two distinct polypeptide bands in the region 60-62 kDa, whereas only one of the two polypeptides with an apparent molecular mass of 60 kDa was found in CP60. Although a chlorophyll-protein containing the 62-kDa subunit remains to be isolated, the finding strongly suggests that the two polypeptides separated in SDS-gels are apoproteins of two different chlorophyllproteins and that the CP60 we have isolated here is a chlorphyll-bearing 60-kDa subunit. The PS II reaction center complex contains the two types of chlorphyll-binding large subunits which are different in the function (10-13). By analogy, a large subunit of the PS I complex would carry the reaction center components, while the rest would function only in light harvesting. A chlorophyll-protein having a photoactive P700 has yet to be isolated. However, the absence of P700 photoresponse from CP60 suggests an antenna nature of the chlorophyll-protein. It seems less likely that P’700 photooxidation was totally inactivated during the isolation of CP60 because P700 is not necessarily more sensitive to treatments with detergent or organic solvent than antenna chlorophyll. In fact, the photoresponse is considerably resistant to the treatment of CPl-e with 2-mercaptoethano1 and SDS. Furthermore, the low-temperature absorption and fluorescence spectra of CP60, similar to those of CPl-e, indicate that the original chlorophyll-protein linkages or microenvironments surrounding chlorophyll a molecules are largely preserved in the isolated chlorophyll-protein. We may conclude that CP60 is an intrinsic antenna chlorophyll-protein of the PS I reaction center complex. Characteristic features of the PS I antenna are the long-wavelength fluorescence emission bands which are intensified as the ambient temperature is lowered. The pe-
260
SONOIKE
AND
ripheral light-harvesting chlorophyll a/bprotein emits the fluorescence band at about 735 nm (ZO),while the reaction center complex per se has the emission band at 725 nm at liquid nitrogen temperature. The present work shows that the 725~nm fluorescence band is emitted from a chlorophyll a species located on the intrinsic antenna proteins. Butler has proposed that the emitter of the far red band is C-705, a small amount of chlorophyll a which has absoption at a longer wavelength (27). The effect of lowering the temperature was related to the competition between the far red fluorescing energy trap and P700 for the excitation energy in the antenna chlorophyll of PS I (28). Our results are compatible with the proposal that the far red band originates from a long-wavelength chlorophyll a form because CP60 shows an absorption band at 705-710 nm at liquid nitrogen temperature. However, the temperature dependence of the far red band was found to be essentially the same between the CPl-e which has P700 and CP60 which lacks P700. Thus, the low-temperature effect cannot be related to the PS I photochemistry. It is more likely that the far red band develops because the excitation energy transfer from the far red fluorescing traps to nonfluorescing traps is inhibited at low temperature. ACKNOWLEDGMENT The authors thank Miss Fumiko lent technical assistance.
Idei for her excei-
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KATOH
5. LAGOUTTE, B., SETIF, P., AND DURANTON, J. (1981) in Proceedings of the 5th International Congress (Akoyunoglou, G., ed.), Vol. 3, pp. 237243, Balaban Int. Sci. Services, Philadelphia. 6. TAKAHASHI, Y., KOIKE, H., AND KATOH, S. (1982) Arch. B&hem. Biuphys. 219,209-218. 7. TAKAHASHI, Y., AND KATOH, S. (1982) Arch. Biochem Biophys. 219,219-227. 8. VIERLING, E., AND ALBERTE, R. S. (1983) Plant PhysioL 72.625-633. 9. TAKAHASHI, Y., HIROTA, K., AND KATOH, S. (1985) Photosynth Res. 6, 183-192. 10. YAMAGISHI, A., AND KATOH, S. (1983) Arch Biochem Biophys. 225.836-846. 11. YAMAGISHI, A., AND KATOH, S. (1984) B&him Biophys. Acta 765,118-B?& 12. NAKATANI, H. Y., KE, B., DOLAN, E., AND ARNTZEN, C. J. (1984) B&him. Biqphys. Acta 765, 347352. 13. YAMAGISHI, A., AND KATOH, S. (1985) Biochim. Biophys. Acta 807, 74-80. 14. YAMAOKA, T., SATOH, K., AND KATOH, S. (1978) Plant Cell Physiol. 19,943-954. 15. HIRANO, M., SATOH, K., AND KATOH, S. (1980) Phe tosynth. Res. 1,163-170. 16. LAEMMLI, U. K. (1970) Nature (London) 227,680685. 17. NAKAYAMA, K., YAMAOKA, T., AND KAMH, S. (1979) Plant Cell PhysioL 20,1565-1576. 18. KOIKE, H., SATOH, K., AND KATOH, S. (1982) Plant Cell PhysioL 23, 293-299. 19. MACKINNEY, G. (1941) .I BioL Chem. 140.315-322. 20. MULLET, J. E., BURKE, J. J., AND ARNTZEN, C. J. (1980) Plant PhysioL 65,814-822. 21. ANDERSON, J. M., AND LEVINE, R. P. (1974) Biochim Biophys. Acta 357,118-126. 22. CHUA, N.-H., MALTIN, K., AND BENNOUN, P. (1975) J. Cell BioL 67,361-377. 23. MACHOLD, 0. (1975) B&him. Biophys. Acta 281, 103-122. 24. LAGOUTTE, B., SETIF, P., AND DURANTON, J. (1980) Photosynth. Res. 1, 3-16. 25. THORNBER, J. P., AND ALBERTE, R. S. (1977) in Encyclopedia of Plant Physiology, New Series (A. Trebst and M. Avron, eds.), Vol. 5, pp. 574-582, Springer-Verlag, Berlin. 26. FISH, L. E., KOCK, U., AND BOGORAD, L. (19%) J BioL Chem 260,1413-1421. 27. BUTLER, W. L. (1961) Arch. B&hem. Biophys. 93, 413-422. 28. SATOH, K., AND BUTLER, W. L. (1978) B&him Biophys. Acta 502,103-110.