TmaJounxu OF BIOLOGICAL CH~MIBTBI Vol. 249, No. S,Imm of April 26, pp. 27S2-2788,197S

Printed in U.S.A.

Purification and Properties of the Photosystem I Reaction Center from Chloroplasts

(Received for publication, July 29, 1974)

CARMELA BENGIS AND NATHAN NELSON

From the Department of Biology, Technion-Israel Institute of Technology, Haija, Israel

SUMMARY 1. A reaction center from chloroplasts was purified by

means of detergent treatment, difIerential centrifugation, column chromatography, and sucrose gradient.

2. The reaction center is active in NADP photoreduction by ascorbate. Ferredoxin, ferredoxin-NADP-reductase, and plastocyanin were required for the reaction.

3. The preparation contains five classes of polypeptide chains with apparent molecular weights of 70,000, 25,000, 20,000, 18,000, and 16,000 as determined by gel electro- phoresis in sodium dodecyl sulfate.

4. Treatment with 0.5% sodium dodecyl sulfate abolished the NADP photoreduction activity and released the low molecular weight subunits, which were removed by sucrose gradient centrifugation from the high molecular weight ones. The PToO signal is associated with the 70,000 molecular weight polypeptide.

5. Antibody, prepared against the active reaction center, inhibited NADP photoreduction catalyzed by the purified reaction center as well as by isolated chloroplasts. The anti- body interacted on immunodiffusion plates with any sub- chloroplast preparation capable of NADP photoreduction. It also interacted with the purified 70,000 molecular weight polypeptide.

6. It is concluded that both the primary oxidation and the primary reduction in Photosystem I are associated with the 70,000 molecular weight polypeptide.

Two photosystems of the photosynthetic electron transport chain from chloroplasts were detected and separated (1, 2).

It was established that Pro0 is the photochemical reaction cen- ter of Photosystem I, and it was suggested that the Pro0 signal is the expression of oxidized chlorophyll a, namely the primary elec- tron donor of the reaction center (3). Many components have been proposed for the primary electron acceptor of Photosystem I including pteridines (4), FMN-chlorophyll complex (5), ferre- doxin-reducing substance (6, 7), Pdae (8), and ferredoxin (9). Recently Malkin and Bearden (10) observed a light-induced EPR signal at 25 K, which was characteristic of reduced ferre- doxin. This observation was confirmed and extended by others (11-13). Because the reduction occurred at liquid nitrogen tem- perature, they suggested that bound ferredoxin is the primary

electron acceptor of Photosystem I. Subsequently the resem- blence between the Pdao signal and the difference spectrum of oxidized minus reduced ferredoxin was pointed out by Ke (14), and it was suggested that the P4ao signal might be an expression of the oxidation or reduction of bound ferredoxin. Later it was suggested by Siedow et al. (15) that the ferredoxin-reducing sub- stance might serve at a secondary electron acceptor which medi- ates electron transport between bound and soluble ferredoxins.

Reaction center particles from photosynthetic bacteria have been isolated and purified extensively (16-23). The preparations consist of three polypeptides with molecular weights of 27,000, 22,000, and 19,000, non-heme iron, and 4 molecules of bacterio- chlorophyll per reaction center (18, 19). They are photochemi- tally active in the oxidation-reduction of PsrO and in photooxida- tion of reduced cytochrome c (22).

Treatment with digitonin or Triton X-100 and differential or sucrose gradient centrifugations yielded puriffed Photosystem I particles from chloroplasts (24-26). Chlorophyll a to b or P~OO to chlorophyll ratios were used as criteria for purification (1, 2). Recently, using digitonin treatment and sucrose gradient cen- trifugation, Wessels (27, 28) separated two photosystems which were both low in chlorophyll 5. In these preparations the protein profiles were not characterized. The polypeptide composition of photochemically active Photosystems I and II-enriched particles was analyzed recently in several laboratories (29-35). Multiple bands were observed on the sodium dodecyl sulfate gels, and the participation of certain polypeptides in the photosystems was deduced from their relative intensity on the gels. Takamiya (36) described purification procedures yielding chlorophyll-protein complexes with molecular weight of about 70,000 from several sources. Thornber (37) purified a chlorophyll a-protein complex with molecular weight of about 150,000 from blue-green algae which contained one P&80 chlorophyll a molecules. However, the purified protein-chlorophyll complex was not active in NADP photoreduction.

It is the purpose of this communication to describe a prepara- tion of reaction centers from chloroplasts having NADP photo- reduction activity and of a single polypeptide having P~OO signal.

EXPERIMENTAL PROCEDURE

Materials-Swiss chard leaves were purchased from a local market. DEAE-cellulose DE11 was obtained from BDH Chemi- cals and was washed and equilibrated as previously described (38). Digitonin, NADP, and Triciner were obtained from Sigma.

l The abbreviations used are: Tricine, N-tris(hydroxymethyl)- methylglycine; CFr, chloroplast coupling factor 1.

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Sodium dodecyl sulfate, acrylamide, and methylenebisacrylamide were purchased from Bio-Rad. Freund’s Bacto-adjuvant (com- plete form) was obtained from Difco Laboratories.

Preparations-Chloroplasts were prepared as described pre- viously (39). Spinach plastocyanin was prepared according to Anderson and McCarty (49).

Analytical Methods-The Ouchterlony immunodiffusion reac- tion (41), NADP photoreduction (42), protein (43), and chloro- phyll concentration (44) were assayed by published procedures. Gel electrophoresis in the presence of sodium dodecyl sulfate was performed as described by Weber and Osborn (45). The gels were fixed, stained, and destained as previously described (38). Non- heme iron was determined according to Brumby and Massey (46).

Pigments were extracted with 99% acetone. To 26 ml of the acetone extract were added 4 ml of petroleum ether (b.p. 49-60”) and it was mixed on a vortex shaker. Four milliliters of water and 2 ml of 4 M NaCl were added and mixed and the upper layer, which was formed, was transferred to a test tube. Four milliliters of water were added and the test tube was shaken on a vortex. The upper layer was transferred and solid Na,SOs was added to it. The volume of the pigment extract was reduced to the desired concentration under reduced pressure at 20”.

The pigments were separated by thin layer chromatography with kieselgur according to Randerath (47). Quinones were sep- arated by thin layer chromatography with silica gel according to Dilley (48). The separated pigments were dissolved in appropriate solvent and their absorption spectra were recorded with a Cary 14 spectrophotometer. The pigment concentrations were calcu- lated using absorption coefficients reported by Davies (49) for carotenoids, Strain et al. (59) for chlorophylls, and Barr and Crane

RESULTS

Preparation of Reaction Centers Active in NADP Photoreduction

Step I: Extraction with Digz’tonin-Chloroplasts were prepared from Swiss Chard leaves as described previously (39) and were suspended in a medium containing 0.4 M sucrose, 0.01 M Tricine (pH 8), 0.01 M NaCl, and 5 mu MgClr at a chlorophyll concentra- tion of 1 mg per ml. Digitonin was added as a 10% solution in water to give a final concentration of 1 To. After incubation at 0” for 1 hour the mixture was centrifuged at 30,000 x g for 10 min and the pellet was discarded. Solid NaCl and 10% digitonin were added to the supernatant to give final concentrations of 0.1 Y and 1.5oj,, respectively. After incubation overnight in the cold room the mixture was centrifuged at 30,000 x g for 10 min, the pellet was discarded, and the supernatant was centrifuged at 150,009 x g for 2 hours. The supernatant of the high speed cen- trifugation was saved for use in the preparation of cytochrome j

TABLE I Purification of the Photosystem Z reaction center from chloroplasts

The reaction mixture contained in a final volume of 1 ml, 29 pmol of 2-(N-morpholino)ethanesulfonic acid-Tricine (pH S), 69 pm01 of NaCl, 0.5 amol of NADP, 3 nmol of ferredoxin, 0.05 nmol of ferredoxin-NADP-reductase, 2.5 nmol of plsstocyanin, 0.1% Triton X-199, and particles equivalent to 10 to 26 pg of chloro- phyll. The reaction mixture was illuminated by white light (1.6 _ -

(51) for quinones. The amount of Pro0 was measured by placing X 10’ ergs per cm* per s) for 1 min. equal samples in identical cuvettes. After the base-line was re- corded by a Gary 118C one of the two samples was then treated Preparation with ferricyanide (ml mM) and the other with ascorbate (~2 mM)

“gir

and allowed to equilibrate prior to recording a difference spec- trum. A molar extinction coefficient of 64,969 at 799 nm was used m.8 (14). The light-induced Pro0 signal at 430 nm was recorded by Chloroplasts.. 1,696 an Aminco-Chance dual wavelength spectrophotometer. The Photosystem I.. . 696 solution contained 50 mM Tricine (pH 8), 10 PM N-methylphena- After DEAE-cellulose . 64 zonium methosulfate, and 166 PM ascorbate. A molar extinction After sucrose gradient. . 21.3 coefficient of 45,996 was used with the reference beam at 468 nm.

Protein NADP

72,696 3,266

160 68

wwm chllhr

60 80

180 400

Total units

80,000 48,996 11,599 f&m

FIQ. 1. Absorption spectrum of the purified reaction center. The spec- trum was recorded on a Cary 118C spectrophotometer using l-cm quartz cuvettes.

WAVELENGTH (nm)

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or plastocyanine. The pellet was homogenized in a medium con- taining 0.4 M sucrose, 0.01 M Tricine (pH 8), and 0.01 M NaCl at a chlorophyll concentration of 3 mg per ml and was kept frozen at -70°C for as long as six months.

Step II: Triton Treatment and DEAE-cellulose Chromatography -Photosystem I particles (30 ml) were thawed and Triton X-100 was added as a 20% solution to give a final concentration of 4%. After incubation overnight in the cold room (at 4’) the solution was applied to a DEAE-cellulose column (2 x 25 cm) which was equilibrated with a solution containing 50 mM Tris-Cl (pH 8) and 0.2% Triton X-100. The column was washed with 100 ml of the same buffer and the reaction center was eluted with a linear NaCl gradient from 0 to 400 mM (200 ml in each chamber) in the same buffer.

TABLE II

Chemical composition of the Photosystem I reaction center and of the active jractions after DEAE-cellulose

Chlorophyll a. . . Chlorophyll b. &Carotene. Lutein. . . . Violaxantin . Neoxantin. . Iron...... . . . . . . . . .

-- After DEAE-celIulose Reaction center

nmol/lOO nmol chl

82 98 18 2 9.2 12.8 2.5 0 1.0 0 0 0 1.7 3.5

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Step ZZZ: Sucrose Gradient CentrifugatGm-The most activ- fractions were applied (0.5 ml to each tube) on gradients of 5 to 25% sucrose in 50 rnr+f Tris-Cl (pH 8) and 0.2% Triton and cen- trifuged for 15 hours in an SW 50 Spinco rotor at 35,000 rpm. Fractions of 0.5 ml were collected and the active fractions (the lower green band) were kept at 0” in the dark for up to 3 days.

Table I summarizes the purification procedure for Photosystem I reaction centers. It can be seen that the specific activity of the purified preparation is increased about &fold on a chlorophyll basis and about lo-fold on a protein basis. Its spectrum, given in Fig. 1, shows an absorption maximum at 673.5 nm and limited amounts of carotenoids. The chemical composition of the reaction center is summarized in Table II. The reaction center is almost free of chlorophyll b and the ratio of chlorophyll a to chlorophyll b is higher than 40. Neoxanthin, lutein, and violaxanthin are missing,‘and the amount of p-carotene is larger than in chloro- plasts. Quinones could not be detected in the purified reaction center. One Pro,, per about 100 chlorophyll molecules was found in the preparation. Some of the chlorophyll molecules were prob- ably. solubil$l since the preparation gave rather high fluores- cence and rapid ascorbate photooxidation without the addition of an electron acceptor. A similar value was obtained by Vernon and Shaw (52) for the purified Photosystem I particles (TSF-1) which were prepared by Triton treatment. Unlike TSF-1 particles, cytochromes could not be detected in the reaction center either by spectrophotometry or by antibody against cytochrome f (53).

The reaction center migrates nearly as a single band on acryl- amide gel electrophoresis in the presence of Triton X-100, and the chlorophyll accompanies the protein band. On sodium dodecyl

FIG. 2. Sodium dodecyl sulfate gel electrophoresis pattern of the puri- fied reaction center. Experimental conditions were as described under “Experimental Procedure.”

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sulfate gels the reaction center dissociates to five distinct bands (Fig. 2)) with molecular weights of 70,000,25,000,20,000, 18,000, and 16,000 (Fig. 3).

Antibody against the active reaction center was obtained by mixing it (0.5 mg protein) on a Vortex mixer with 1.5 parts of complete Freund’s adjuvant and injecting the mixture into the footpads of two rabbits and into the skin at sites on each side of the back, as previously described (38). After 4 weeks the rabbits were boosted intravenously with about 0.5 mg of the purified reaction center. The rabbits were bled as described previously (38) and boosted every month. Both rabbits gave identical anti-

2-

. I-

I I

, ,

0.1 0.2 03 0.4 0.5 06 07 OB Relative mabillty

Fxo. 3. Determination of the molecular weight of the reaction center subunits by sodium dodecyl sulfate gel electrophoresis. Phosphorylase a (MW 94,000), bovine albumin (MW 68,990), pyruvate kinase (MW 57,OOO), ovalbumin (MW 43,000), pepsin (MW 35,690), and ol-chymotrypsin, and its subunits (MW 25,009, 13,099, and 11,999) were used as standards.

FIG. 4. Interaction between the reaction center and its anti- body in immunodiffusion experiments. 1,5 pl of serum from rabbit I injected with the reaction center; b, 5 ~1 of serum from rabbit II injected with the reaction center; d, control serum; 4, anti-y sub- unit of CF1; a, purified reaction center (0.6 pg of chlorophyll). b, active fraction after DEAE-cellulose column (2 /*g of chloro- phyll); c, photosystem I particles (12 rg of chlorophyll); d, in- active fraction from the sucrose gradient (0.3 rg of chlorophyll); e, lettuce chloroplasts dissolved in 4% Triton (6 rg of chloro- phyll); f, chloroplast pigments extracted by 80% acetone and dissolved in 4% Triton (10 rg of chlorophyll). The immunodiffu- sion plates contained 0.5y0 Triton X-100.

bodies even after 6 months of repeated boosting. On Ouchterlony immunodiffusion plates precipitation bands without any spur were obtained with any one of the chloroplast preparations cap- able of NADP photoreduction (Fig. 4). No positive reaction could be detected between the purified reaction center and antibodies against CFi and its five individual subunits (38), cytochrome f (53)) plastocyanin (54)) or ferredoxin-NADP-reductase (42). The antibody inhibited NADP photoreduction not only by the purified reaction center but also by isolated chloroplasts from lettuce leaves (Fig. 5). Photophosphorylation in the presence of NADP was inhibited to the same extent as was NADP photore- duction. None of the other reactions tested including photo- reduction of methyl viologen were inhibited.

Treatment of the active reaction center with 0.5% sodium dodecyl sulfate for 20 min at 0” abolished the NADP photoreduc- tion activity and shifted the P700 peak to 697 nm. After dilution with an equal volume of water, portions of 0.5 ml were applied on sucrose gradients as in the last step in the purification of the ac- tive reaction centers. Two green bands were formed and collected separately. The upper yellowish green band contained most of the /3 carotene and the four low molecular weight subunits. The lower bluish-green band contained only the 70,000 molecular weight polypeptide (Fig. 6) and one PToo per 40 to 50 chlorophyll a molecules. The antibody against the active reaction centers interacted exclusively with the fraction containing the 70,000 molecular weight polypeptide.

DISCUSSION

A purification procedure for the Photosystem I reaction center has been described which yields a preparation consisting of five polypeptide subunits, and which is active in NADP photoreduc- tion. The source for the reaction center was a Photosystem I particle depleted of cytochromes and prepared by digitonin treatment and differential centrifugation (55). The DEAE-cellu- lose column that followed the Triton treatment removed residual cytochromes and membrane fragments which were retained on the column under the conditions described (56).

03 J 5 IO 15 20 25 30

JA Serum

FIG. 5. Effect of antibody against Swiss chard reaction center on NADP nhotoreduction bv lettuce chloronlasts. The reaction mixture contained, in a final “volume of 1 ml : -%Cl fig mol of Tricine- NaOH (pH 8), 50 pmol of NaCl, 5 pmol of NH&l, 0.5 pmol of NADP, 0.993 rmol of ferredoxin, chloroplasts equivalent to 17 pg of chlorophyll, and the volume of antibody indicated in the figure. After 10 min of incubation at room temperature it was illuminated by white light (1.5 X lo6 ergs per cm2 per s) for 1 min.

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FIG. 6. Sodium dodecyl sulfate gel electrophoresis pattern of the blu- ish-green fraction after 0.5y0 dodecyl sulfate treatment and sucrose gra- dient centrifugation. Experimental conditions were as described under “Experimental Procedure.”

The antibody we obtained against the purified reaction center interfered with the reduction of soluble ferredoxin by chloroplasts and it might be directed toward the primary electron acceptor or toward the binding site for ferredoxin. A similar antibody was ob- tained by Regitz et al. (57) by injecting broken chloroplasts. However among several injected rabbits, two gave antibodies directed against ferredoxin-reducing substance and the serum also contained antibodies against CF1 and ferredoxin-NADP re- ductase which had to be neutralized by adding an excess of the reductase in the NADP reduction experiments (57). One of these rabbits (6 Cls) gave antibodies similar to those described in this paper, the other one (11 Cl,) gave antibodies that inhibited not only ferredoxin-dependent reactions but also anthraquinone photoreduction (57). The antibody described in this paper was specific for subchloroplast preparations having a Pro0 signal and did not interact with CFi or with ferredoxin-NADP reductase.

Using a molar extinction coefficient of 64,000 at 700 nm (14), one Pro0 was found per two 70,000 molecular weight polypeptides. Cooperation of two subunits might be necessary for the formation of Proo, or some of the P700 molecules were lost during the purifica- tion procedure and every 70,000 molecular weight polypeptide originally had one PToO. We cannot conclude yet if all of the sub- units are an integral part of the reaction center; however, it seems that the 70,000 molecular weight polypeptide is necessary for NADP photoreduction. The fact that after sodium dodecyl sulfate treatment the low molecular weight subunits are missing and the P700 signal is retained means that they are not required for the primary photoreaction. It might be suggested that one or some of them are required for NADP photoreduction either to mediate electron transport from PToO to ferredoxin (acting as sug-

gested for ferredoxin-reducing substance (6, 7)) or they induce a proper conformation of the large subunit suitable for transferring electrons directly to ferredoxin. The third possibility is that they are not a part of the reaction center, and sodium dodecyl sulfate treatment partially denatured the functional subunit.

After removal of the low molecular weight subunits the anti- body still interacted with the fraction which contained the 70,000 molecular weight polypeptide and P,OO signal. This might suggest that both the oxidized site of Photosystem I and the primary acceptor, or at least the binding site, for ferredoxin are located on the same polypeptide.

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C Bengis and N NelsonPurification and properties of the photosystem I reaction center from chloroplasts.

1975, 250:2783-2788.J. Biol. Chem. 

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