with a buffer solution, .... tion center solution containing about 50 yg of protein were incubated ..... to Subunit I.4 Hence, Subunit V is positioned in contact with.
THE JOURNAL OF Bromo~ci\~ CHEMISTRY Vol. 252, No. 13, Issue of July 10, pp. 4564-4569, Prmted L,, U.S.A.
Subunit Center*
Structure
1917
of Chloroplast
Photosystem
I Reaction
(Received for publication,
January 5, 1977)
CARMELA BENGIS AND NATHAN NELSON From
the Department
of Biology,
Technion-Israel
Purified Photosystem I reaction center preparation catalyzed a plastocyanin-dependent cytochrome f photooxidation. Euglena cytochrome 552 replaced plastocyanin in all photochemical reactions catalyzed by the purified reaction center and was also photooxidized. A reaction center preparation lacking Subunit III was obtained by means of Triton X-100 treatment and DEAEcellulose chromatography or sucrose gradient centrifugation. This preparation was incapable of cytochrome f photooxidation or of NADP photoreduction in the presence of plastocyanin. However, the reaction center lacking Subunit III photoreduced NADP when plastocyanin was replaced by N-methylphenazonium-3-sulfonate, serving as direct electron donor for P,,,,,. It was concluded that Subunit III mediates reduction of P,,,+ by plastocyanin. Mild treatment with sodium dodecyl sulfate yielded reaction center preparations which were partially depleted in Subunits II, III, IV, V, and VI. The NADP photoreduction activity and EPR-detectable bound ferredoxin were correlated to the presence of Subunits IV, V, and VI in the depleted preparations. Subunits IV, V, and VI might mediate photoreduction of soluble ferredoxin by the primary electron acceptor, in the reducing site of Photosystem I. The P,,, reaction center (Subunit I) was further purified. Preparations with chlorophyll a to PToOratios below 40 were obtained with the use of higher concentrations of Triton X100, which caused antenna chlorophyll a to be partially solubilized. Removal of the sdlubilized chlorophyll by DEAE-cellulose chromatography resulted in preparations which contained 10 to 20 chlorophyll a molecules/P70,,. It is suggested that a unit of about 20 chlorophyll a molecules is specifically associated with the reaction center protein 02, = 70,000) in the vicinity of PToO.A model has been presented which depicts probable organization of the Photosystem I reaction center in the thylakoid membrane.
Institute
of Technology,
Haifa,
Israel
We previously purified the reaction center of Photosystem I from chloroplasts (6, 7). The purified reaction center consisted of six polypeptides that were designated as Subunits I, II, III, IV, V, and VI in the order of decreasing molecular weights of 70,000, 25,000, 20,000, 18,000, 16,000, and about 8,000, respectively (6, 7). We have also shown in the previous papers that P,,,,, is associated with the 70,000-dalton polypeptide (Subunit I), which was purified by treating the reaction center with SDS’ and by subsequent sucrose gradient centrifugation. The latter preparation, called the P ,“” reaction center, was active in the light-induced reversible bleaching of Pi,,,,. However, this preparation was incapable of NADP photoreduction or of cytochrome f photooxidation. At this stage of investigation, the functions of the low molecular weight polypeptides of the purified reaction center in NADP photoreduction were not understood. McIntosh et al. (8) and Evans et al. (9) have recently suggested that two membrane-bound iron-sulfur centers of ferredoxin type serve in chloroplasts as intermediates in the photosynthetic electron transport from the primary electron acceptor of Photosystem I to soluble ferredoxin. However, this is disputed by Bearden and Malkin (10) who suggested that bound ferredoxin is the primary electron acceptor of Photosystern I. These authors were first to discover bound ferredoxin in chloroplasts by low temperature EPR spectroscopy (11). We have reported previously (121, that bound ferredoxin was detected in the purified Photosystem I reaction center by EPR spectroscopy at 18 K. The P,,, reaction center, associated with the single 70,000-dalton polypeptide, did not contain any EPRdetectable bound ferredoxin. The present communication deals with the results of the further studies on the subunit structure, functions and arrangement of the Photosystem I reaction center in the thylakoid membrane. EXPERIMENTAL
PROCEDURES
-DEAJZ-cellulose (DE111 was obtained from Whatman Biochemicals Ltd. and was washed and equilibrated as previously described (13). Digitonin, Triton X-100, NADP, Tricine, Tris, PMS, and Mes were obtained from Sigma. Sodium dodecyl sulfate, acrylamide, and methylenebisacrylamide were obtained from Bio-Rad Laboratories. Sodium dithionite was obtained from BDH Chemicals. PMS-S was a generous gift from Dr. Gunter Hauska, Universitat Regensburg, Fachbereich Biologie und Vorklinische Medizin, Regensburg, Germany. Materials
There are numerous reports in the literature on the purification of subchloroplast preparations enriched in Photosystem I (1, 2, 3). However, those preparations which were active in NADP photoreduction contained multiple polypeptides and accessory pigments (2). On the other hand, a more purified preparation, namely the PTO,,chlorophyll a protein, isolated by Thornber and co-workers (4, 5) was incapable of NADP photoreduction or of cytochrome f photooxidation. * This work was supported by a grant from the Advancement of Mankind Foundation.
1 The abbreviations used are: SDS, sodium dodecyl sulfate; Tritine, N-tris(hydroxymethyl)methylglycine; Mes, 2-(N-morpholinojethanesulfonic acid, PMS, N-methylphenazonium methosulfate; PMS-S, N-methylphenazonium-3-sulfonate.
4564
Subunit
Structure
of Chloroplast
Preparations - Chloroplasts and Photosystem I digitonin particles from Swiss chard leaves were prepared as previously described (6,7, 14). The Photosystem I reaction center was prepared as previously described (6, 71, with a slight modification in Step III in order to obtain large scale preparations. The active fractions after DEAEcellulose column chromatography were layered (4 ml on each tube) onto gradients of 5 to 25% sucrose, containing 50 rnM Tris/Cl (pH 8) and 0.2% Triton X-100 and centrifuged for 20 h in the SW 27 Spinco Rotor at 25,000 rpm. The lower green band formed upon centrifugation contained the Photosystem I reaction center. Plastocyanin, ferredoxin, ferredoxin NADP reductase, cytochrome f and Euglena cytochrome 552 were prepared by published procedures (15-17). Analytical Methods -Gel electrophoresis in the presence of sodium dodecyl sulfate was performed as described by Weber and Osborn (18). The gels were fixed, stained, and destained as previously described (13). Cytochrome f photooxidation (141, Euglena cytochrome 552 photooxidation (14), and NADP photoreduction (6, 7) were assayed by the published procedures. All of the above photochemical reactions as well as the light-induced P,,, signal at 430 nm, were assayed spectrophotometrically by the illumination of the samples in the Cary 118C spectrophotometer cuvette with actinic beam and by recording the light-induced absorbance changes at the appropriate wavelength. The actinic beam was provided by a 150 W slide projector and passed through a red filter (Corning 2403). The phototube was protected from the actinic beam by a blue filter (Corning 9782). The light intensity was 5. lo5 ergs/cm2/s at the level of the cuvette. The specific activity of NADP photoreduction was calculated using a millimolar extinction coefficient of 5.85 at 350 nm. Cytochrome f photooxidation was recorded at 553.5 nm, and a millimolar extinction coefficient of 20.6 was used for calculation of the amount of photooxidized cytochrome f. Euglena cytochrome 552 photooxidation was recorded at 552 nm and amounts of photooxidized cytochrome were calculated using a differential millimolar extinction coefficient of 19.6 (17). The reduced-minus-oxidized difference spectrum of P,,, was recorded with the Cary 118C spectrophotometer as previously described (7). The reaction mixture for the light-induced P,,,, signal contained, in a final volume of 1 ml, 20 pmol of Tricine/Mes (pH 71, 2 pm01 of ascorbate, 1 nmol of PMS, and reaction center preparations, equivalent to 0.2 to 0.4 nmol of P,,,. An extinction coefficient of 45/mM/cm was used for calculation of the amount of P,,, (19).
Photosystem
I Reaction
Center
4565
can be obtained by the use of DEAE-cellulose column. Prno reaction center equivalent to about 2 mg of chlorophyll was applied to a DEAF-cellulose column (1 x 10 cm), which was equilibrated with a buffer solution, containing 50 mM TrislCl (pH 8) and 0.5% TrYton X-100. The column was washed with 200 to 300 ml of the same buffer for several hours in the dark at 4”. The P,,,,, reaction center was eluted with 50 ml of 0.2 M NaCl in the same buffer. The preparation contained one P,,,/lO to 20 chlorophyll a molecules, and its absorption spectrum showed a maximum at 676 nm. In order to achieve the chlorophyll a to P,,,, ratio of 10, the column had to be washed overnight but the recovery of P,,, was lower than 10%. Table I summarizes the purification of P,,, reaction center on the chlorophyll basis and recovery of Pro0 signal through the purification procedure. The described Pro0 reaction center, highly enriched in ProO, appeared to be quite similar in other properties to the P,,,, reaction center preparation described previously (6, 7). It migrated as a single band (M, = 70,000) on the SDS gels and was completely free of carotenoids. Partial Resolution of Photosystem I Reaction Center -As described previously (6, 7), the purified Photosystem I reaction center was active in NADP photoreduction. Fig. 2 shows that the purified preparation is also active in cytochrome f photooxidation, which is completely dependent upon plastocyanin. Another assay for the oxidizing side of the Photosystem I reaction center is photooxidation of Euglena cytochrome 552. This cytochrome replaces plastocyanin in plant chloroplasts (20). In accord with this, we have observed that cytochrome 552 replaces plastocyanin in NADP photoreduction by the Chl (1 -= bO0
Chla=p5 500
50
RESULTS
Further Purification of P,,,, Reaction Center on Chlorophyll Basis -In our previous communications (6, 71, a purification procedure was described which yielded the Pro0 reaction center (Subunit I) containing 40 to 50 chlorophyll a molecules/P,,,. Subunit I with about 30 chlorophyll a molecules/Pro, can be obtained by increasing the Triton concentration in the sucrose gradient to 1% and prolonging the centrifugation time to about 40 h. However, recovery of the light-induced P,,, signal was rather low under these conditions (20 to 40%). A “blue shift” of the chlorophyll a absorption band to 670 to 672 nm probably reflected partial solubilization of antennae chlorophyll a from the reaction center complex as a result of T&on X-100 treatment. The experiment that is depicted in Fig. 1 is in line with this assumption. The extent of P,,, photooxidation in control P,,, reaction center was similar in the presence of ascorbate or ascorbate plus PMS. After the Triton treatment, the reaction center was depleted of 50% of its chlorophyll a and maintained comparable extent of Pro0 photooxidation in the presence of ascorbate. However, the rate of Pro,, photooxidation was slower and the extent of P,,, photooxidation in the presence of PMS was markedly decreased. This was due to fast reduction of P,,,+ by reduced PMS and the slower rate of P,,, photooxidation. The rate of P,,,+ reduction by reduced PMS was not altered by the T&on treatment. This means that some of the chlorophyll a molecules in this preparation failed to transfer the light energy to the P,,, pigment. Further depletion of chlorophyll from P,,,, reaction center
+PMS
\
1
lmin
-
FIG. 1. Effect of chlorophyll depletion on P,,, signal of P,,, reaction center. The reaction mixture contained, in a final volume of 1 ml, 20 pmol of Tricine/Mes (pH 71, 2 pmol of sodium ascorbate, and 1 nmol of PMS when added. The absorbance changes were recorded at 430 nm as described under “Analytical Methods.” ChZ a, chlorophyll a; L, light; D, dark. TABLE
I
Purification of the P,,,, reaction center from the reducedThe concentrations of P,,, were determined minus-oxidized difference spectrum of P,,, as previously described (7). Chloropbll
Chlorophyll dP,oo
umn
Recovery of P,,, signal
A
% 100
32
5.3 3.2
18
1.8
w
DEAE-active fractions P ml reaction center after sucrose gradient in 1% Triton P 7”” reaction center DEAE-cellulose col-
Total P,,, signal
10.3 1.6
124
0.5
60
34
4566
Subunit Structure of Chloroplast Photosystem I Reaction Center D
0” Lo 18 0
I
A t Plostocyonin 9
I
i
I min
FIG. 2. Effect of plastocyanin on cytochrome f photooxidation by Photosystem I reaction center. The reaction mixture contained, in a final volume of 1 ml, 20 pmol of Tricine (pH 8), 0.7 nmol of cytochrome f, 0.2 pmol of sodium ascorbate, 0.5 nmol of plastocyanin when added, and reaction center equivalent to 10 pg of chlorophyll. L, light; D, dark.
purified reaction center. The specific activity was quite similar to that obtained with plastocyanin. In looking for treatment that will modify specifically the donor site of Photosystem I, it was found that this is sensitive to Triton treatment. Partial inhibition of NADP photoreduction and cytochrome 552 photooxidation can be obtained by increasing the Triton concentration during the sucrose gradient centrifugation to 1%. Complete inhibition of these reactions was obtained by the adsorption of the Photosystem I reaction center to a DEAE-cellulose column (1 x 10 cm), which was equilibrated with 50 mM Tris/Cl (pH 8), containing 0.5% Triton X-100. After the column was washed with the same buffer for several hours, the reaction center was eluted with 50 mM Tris/Cl (pH 81, 0.2% T&on, and 0.2 M NaCl. The SDS-gel electrophoresis pattern and relative amounts of the subunits of the eluted preparation are shown in Fig. 3 and in Table II, respectively, in comparison to those of the control reaction center. It is seen that the preparation eluted from the DEAEcellulose column was completely free of Subunit III. Relative amounts of the remaining reaction center subunits were unaltered. The preparation contained about 35 chlorophyll a molecules/P,,, and was almost free of p-carotene. This reaction center preparation completely lost both NADP photoreduction and cytochrome f photooxidation activities. Fig. 4 shows that the cytochrome 552 photooxidation was markedly decreased in the reaction center lacking Subunit III. If Subunit III participated in NADP photoreduction on the reducing side of Photosystem I, one would expect cytochrome f photooxidation to be uninhibited in preparations depleted in this subunit. Therefore, we have assumed that Subunit III probably mediates the electron transfer from plastocyanin to P 7”“. To test this assumption, we needed a suitable electron donor which could bypass plastocyanin and reduce P,,, directly at a rate sufficiently high to accomplish NADP photoreduction, by the Photosystem I reaction center. If under these conditions the reaction center, lacking Subunit III, was capable of NADP photoreduction, the latter assumption would be corroborated. The only compound, out of several electron donors tested by us so far, which gave satisfactory rates of NADP photoreduction by the Photosystem I reaction center in the absence of plastocyanin, was PMS-S (21). This compound is a hydrophilic derivative of PMS, carrying an additional charge (--SO,-). Table III shows that the reaction center lacking
B
FIG. 3. The SDS gel pattern of Photosystem I reaction center and the reaction center depleted of Subunit III. Fifty microliters of reac-
tion center solution containing about 50 yg of protein were incubated at room temperature for 2 h with 2% SDS and 2% mercaptoethanol. After the electrophoresis, the gels were stained with Coomassie blue and scanned at 600 nm. Photosystem I reaction center, A; Depleted reaction center, B. Roman numerals indicate subunit peaks. TABLE II in the Photosystem I reaction center and in the reaction center lacking Subunit III The relative amounts of the subunits were integrated from the scans of the SDS gels by dividing the band area by the subunit molecular weight (given in parenthesis). The number 2.0 for Subunit I was used as a reference. Subunits
Subunit
ratios
Photosystem I reaction center Reaction center lacking Subunit III
2.0
1.1
1.0
0.9
1.0
2.0
1.0
0.0
1.0
0.8
Subunit III photoreduced NADP when PMS-S replaced plastocyanin. The data presented in Table III confirmed the suggestion that Subunit III might be located between plastocyanin and P 700, mediating the electron transfer from the former to the latter. Preparation Depleted in
and Properties Low Molecular
of Reaction Center Partially Weight Subunits-Active frac-
Subunit Control
reaction
Structure
of Chloroplast
Photosystem
I Reaction
Center
4567
center
D
I \
Reaction
center D I
depleted
of subunit
m
0.2 0.4
i
L
FIG. 4. Photooxidation of cytochrome 552 by Photosystem I reaction center and the reaction center depleted of Subunit III. The reaction mixture contained, in a final volume of 1 ml, 20 pmol of Tricine/Mes (pH 71, 1 pmol of sodium ascorbate, 0.6 nmol ofEugZena cytochrome 552, and Photosystem I reaction center or reaction center depleted of Subunit III containing 0.7 nmol of P,,,. L, light; D, dark. TABLE III andphotooxidation of cytochrome 552 by the reaction center, lacking Subunit III Photochemical activities were assayed as described under “Experimental Procedures.” Reaction mixture for cytochrome 552 photooxidation was as described in the legend to Fig. 4. Reaction mixture for NADP photoreduction was as described in the legend to Fig. 5. PMSS (1 pM) was added when indicated in the Table. Cytochrome 552 photooxidation is expressed as the amount of oxidized cytochrome 552 in steadv state. NADP photoreduction Cytochrome 552JhooxiPlastocy-PlastocyPMS-S anin anin
Photoreduction
ofNADP
/mollmg
Photosystem I reaction center (control) Reaction center lacking Subunit III
06 %
chlorophyll/h
nmol
214
0
33
0.6
0
0
31
0.06
tions after DEAE-cellulose column chromatography, prepared as previously described (6), were treated with SDS in concentrations up to 1% for 15 min at 0”. The solutions were then layered (4 ml on each tube) onto gradients of 5 to 25% sucrose in 50 mM Tris/Cl (pH 8) and 1% Triton. The use of 1% Triton X100 in the purification procedure for the photosystem reaction center yields preparations partially depleted only in Subunit III. Fig. 5 (right) shows that treatment of the DEAE-fractions with the increasing SDS concentrations prior to sucrose gradient centrifugation progressively released also Subunits IV and V from the reaction center and caused further inhibition of the photochemical activities in the depleted prkparations. Subunit II appeared to be quite resistant to SDS treatment. The relative amounts of Subunit VI were not followed quantitatively. Also shown in Fig. 5 is the selective mode of action of Triton X-100 and SDS on the Photosystem I reaction’center. While Triton X-100 released only Subunit III from the reaction center, SDS caused depletion in all low molecular weight subunits. Table IV shows the results of an experiment similar
0.8 Triton
IO
02
0.4 06 0.8 % SDS
IO
FIG. 5. Photochemical activities of preparations that were partially depleted in the low molecular weight subunits by means of Triton X-100 and SDS treatments. The reaction mixture for NADP photoreduction contained in a final volume of 1 ml, 20 pmol of Tricine/Mes (pH 71, 2 pmol of sodium ascorbate, 0.1% Triton X-100, 0.5 pm01 of NADP, 3 nmol of ferredoxin, 0.5 nmol of ferredoxin NADP reductase, 2.5 nmol of plastocyanin, and reaction center preparations containing 0.7 nmol of P,,,. The activities and the subunit amounts are expressed as percentage of control reaction center that was obtained from the sucrose gradient containing 0.2% Triton X-100. The DEAE-fractions were treated with the specified SDS concentrations and then were centrifuged in sucrose gradients containing 1% Triton X-100 (right). The control rate of NADP photoreduction was 190 pmol NADP/mg of chlorophyll/h. TABLE
IV
The NADP photoreduction activities and relative amounts of the subunits in the reaction centerpreparations, obtained by treatment of the DEAE-fractions with SDS prior to sucrose gradient centrifugation The DEAE-fractions were treated with SDS in specific concentrations for 15 min at 0”. The NADP photoreduction activity and subunit ratios in SDS-treated preparations are expressed as percentage of those in the control reaction center. Conditions for the NADP photoreduction reaction were as described in the legend of Fig. 5; 1 PM PMS-S was added to the reaction mixture where indicated. The subunit ratios were integrated from the scans of the SDS gels. Electrophoresis in the presence of SDS was performed as described under “Experimental Procedures” and in the legend of Fig. 3. The control rate of NADP photoreduction with plastocyanin was 210 pmol of NADP/mg of chlorophyll/h and with PMS-S 30 ymol of NADP/mg of chlorophyll/h. NADP photoreducSubunits tion I
II
III
100
100
100 100 100
100 100 100
With plastacvanin
v
100
100
100
100
100
80 43 11
80 73 44
90 0 0
86 35 15
61 27 0
% Photosystem I reaction center (control) 0.1% SDS 0.3% SDS 0.5% SDS
With PMS-S
IV
%
to that described in Fig. 5 (right), except for the Triton X-100 concentration (0.2%) during sucrose gradient centrifugation of the DEAE-fractions. It may be seen that depletion in Subunits IV and V correlated with the inhibition of NADP photoreduction, when either reduced plastocyanin or PMS-S were the donors of
4568
Subunit
Structure
of Chloroplast
Photosystem
I Reaction
Center Ferredoxin
electrons for P,,,. Progressive depletion in Subunit VI was also observed in the SDS-treated preparations, but it was not followed quantitatively. On the basis of the results described in this section, it may be suggested that Subunits IV, V, and VI probably participate in NADP photoreduction on the reducing side of Photosystem I.
Outside
DISCUSSION
Reaction center preparations, selectively depleted in the low molecular weight subunits, were obtained by means of SDS and Triton X-100 treatments and DEAE-cellulose chromatography or centrifugation in the sucrose gradients. The photochemical properties of the depleted preparations gave further insight into possible functions of the individual subunits of the purified reaction center in the overall photochemistry of Photosystem I. We have observed that photooxidation of cytochrome f by the Photosystem I reaction center was completely dependent upon plastocyanin. This gives another piece of evidence in favor of the currently accepted scheme of the photosynthetic electron transport, in which plastocyanin is placed between cytochrome f and PTno (for reviews see Refs. 22 and 23). While most of the biochemical experimental evidence supports this scheme (14, 23, 241, some kinetic experiments indicate that both cytochrome f and plastocyanin, acting in parallel, may reduce P,,,, (25, 26). The ability of PMS-S to serve as the electron donor for NADP photoreduction, is probably due to its slow autooxidizability (20) and hydrophilic properties. We have observed that PMS-S concentrations of 1 to 2 PM were optimal for NADP photoreduction. At higher concentrations, the rates of reduction were progressively inhibited. This may indicate that PMS-S, when added in excess, became capable of trapping the electrons from the primary electron acceptor for Photosystem I. It was previously suggested that Subunit I spans the lipid core of the thylakoid membrane (6, 7). This was based on the assumption that P,,, pigment is situated in the internal side of the chloroplast membrane (21, 27) and that a specific antibody interacted with the same polypeptide on the external side of the membrane (6, 7). Purified Subunit I is free of bound ferredoxin but active in P,“,, photooxidation (6, 7, 12). Kinetic studies revealed that Subunit I contains the primary electron acceptor and its nature is under investigation.* Two Subunits I are probably required to form one P,,,,,pigment and each one of them contains about 20 chlorophyll a molecules as an intrinsic part of the light-harvesting antenna of Photosystem I. Photoselection studies indicated that part of these chlorophyll a molecules are organized in a semicrystalline form with P,“,, pigment.” Therefore, it seems that Subunit I suits the definition of minimal photochemical reaction center in which the light can be collected by light-harvesting antenna chlorophyll, to be trapped in P,,,,, pigment and to bring about the reduction of a primary electron acceptor. A much more complex system is required for a reaction center to be active in NADP photoreduction. Photosystem I reaction center contains six different polypeptides with two copies of Subunit I, one copy of each of the Subunits II, III, IV, V, and probably more than one copy of Subunit VI (see Table II and Refs. 6 and 7). Fig. 6 depicts a tentative model for the structure and possible arrangement of the Photosystem I reac2 N. Nelson, H. Schaffernich, tions. 3 H. Schaffernich, N. Nelson, tions.
and W. Junge,
unpublished
observa-
and W. Junge,
unpublished
observa-
Inside
FIG. 6. A proposed model for the subunit structure and function Photosystem I reaction center in the thylakoid membrane.
of
tion center in the chloroplast membrane. Subunit III was placed on the internal side of the thylakoid membrane near the P,,,, pigment. This is based on the observations that Photosystem I reaction center that lacks Subunit III was inactive in plastocyanin or cytochrome 552 mediated NADP photoreduction while it was still active when PMS-S was used as an electron donor. Two or three different clusters of bound ferredoxin were identified in chloroplasts Photosystem I .preparations (10, 11, 28-30). Mild treatment with SDS diminished the EPR signal of bound ferredoxin with parallel loss of NADP photoreduction activity (12) and disappearance of Subunits IV, V, and VI (Fig. 5). Therefore, it was assumed that Subunits IV, V, and VI might be the bound ferredoxins that mediate electron transport
from
the
primary
acceptor
to soluble
ferredoxin
in the
external side of the thylakoid membrane. Nearest neighbor analysis revealed that Subunits III and V are in close position to Subunit I.4 Hence, Subunit V is positioned in contact with Subunit I on the reducing side of Photosystem I. Further studies are required to establish the role of each subunit in NADP photoreduction. would like to thank
Acknowledgment-We
lian
for reading
this
Mr. Chaim Ju-
manuscript. REFERENCES
1. Anderson,
J. M., and Boardman, N. K. (1966)Biochim. 112, 403-421 2. Vernon, L. P., andshaw, E. R. (1971) MethodsEnzymol..23,277-
Biophys.
Acta
289 3. Wessels,
J. S. C. (1968) Biochim. Biophys. Acta 153, 497-500 J. P. (1975) Annu. Reu. PZant Physiol. 26, 127-158 J. A., Alberte, R. S., and Thornber, J. P. (1974)Arch. Biochem. Biophys. 165, 388-397 6. Nelson, N., and Bengis, C. (1975) in Proceedings of the Third
4. Thornber, 5. Shiozawa,
International Seotember
Congress
on
Photosynthesis,
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Israel,
2 to 6. 1974 (Avron, M.. ed) Vol. 1, pp. _. 609-620, Eisevier, Amsterdam 7. Bengis, C., and Nelson, N., (1975) J. Biol. Chem. 250,2783-2788 8. McIntosh. A. R.. Chu. M.. and Bolton. J. R. (1975) Biochim. Biophyk.
9. Evans, them.
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E. H.,
430, S. A.
368-314
R., and Evans, M. C. W. (1976) Bio68, 1212-1218 A. J., and Malkin. R. (1976) Biochim. Biophrs. Acta
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R., and Bearden,
A. J. (1971) Proc.
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12. Nelson, N., Bengis, C., Silver, B. L., Getz, D., and Evans, M. C. W. (1975) FEBS Lett. 58, 363-365 13. Nelson, N., Deters, D. W., Nelson, H., and Racker, E. (1973)J. Biol.
Chem.
4 N. Nelson,
248,
2049-2055
and B. Notsani,
unpublished
observations.
Subunit
Structure
of Chloroplast
14. Nelson, N., and Racker, E., (1972) J. Biol. Chem. 247.3848-3853 15. Yocum, C. F., Nelson, N., and Racker, E., (1975) Prep. Biochem 5, 305-317 16. Nelson, N., and Neumann, J., (1969) J. Biol. Chem. 244, 19261931 17. Perini, F., Kamen, M. D., and Schiff, J. A. (1964) Biochim. Biophvs. Acta 88. 74-90 18. Webe;, k., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 19. Ke, B. (1973) Biochim. Biophys. Acta 301, 1-13 20. Hauska, G. A., McCarty, R. E., Berzborn, R. J., and Racker, E. (1971) J. Biol. Chem. 246, 3524-3531 21. Hauska, G. A., (1972) FEBS Lett. 28, 217-220 22. Trebst, A., (1974) Annu. Rev. Plant Physiol. 25, 423-485 23. Avron, M., (1975) in Bioenergetics ofPhotosynthesis (Govindjee, ed) pp. 373-386, Academic Press, New York 24. Siedow, J. N., Curtis, A. V., and San Pietro, A. (1973) Arch.
Photosystem
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Center
4569
Biochem. Biophys. 158, 889-897 25. Kok, B., and Rurainski, H. J. (1965) Biochim. Biophys. Actu 94, 588-590 26. Haehnel, W. (1975) in Proceedings of the Third International Congress on Photosynthesis, Rehouot, Israel, September2 to 6, 1974 (Avron, M., ed) Vol. 1, pp. 557-568, Elsevier, Amsterdam 27. Junge, W. (1975) in Proceed& of the Third International Congress on Photosynthesis, Rehouot, Israel, September 2 to 6, 1974 (Avon, M., ed) Vol. 1 pp. 272-286, Elsevier, Amsterdam 28. Evans, M. C. W., Telfer, A., and Lord, A. V. (1972) Biochim. Biophys. Acta 267, 530-537 29. Ke, B. (1975) in Proceedings ofthe Third International Congress on Photosynthesis, Rehouot, Israel, September 2 to 6, 1974 (Avron, M., ed) Vol. 1, pp. 373-382, Elsevier, Amsterdam 30. Evans, M. C. W., Sihra, C. K., Bolton, J. R., and Cammack, R. (1975) Nature 256, 668-670
