Chlorophyll Proteins of Photosystem I - NCBI - NIH

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JOHN E. MULLET2, JOHN J. BURKE"', AND CHARLES J. ARNTZEN. United States Department ofAgriculture, Science and Education Administration, ...
Plant Physiol. (1980) 65, 814-822 0032-0889/80/65/08 14/09/$00.50/0

Chlorophyll Proteins of Photosystem I' Received for publication June 4, 1979 and in revised form November 26, 1979

JOHN E. MULLET2, JOHN J. BURKE"', AND CHARLES J. ARNTZEN United States Department ofAgriculture, Science and Education Administration, Department of Botany, University of Illinois, Urbana, Illinois 61801 molecules in the absorption of quanta utilized during the photoData are presented which suggest the existence of a light-harvesting synthetic process. Their data provided evidence for the functional pigment-protein complex which is functionally and structurally associated interaction of approximately 2,400 Chl molecules in absorbing with photosystem I (PSI) reaction centers. These observations are based quanta necessary to fix one molecule of CO2 or in the evolution of one molecule of 02. Subsequent interpretations of these data on techniques which aflow isolation of PSI using minimal concentrations of Triton X-100. Properties of density and self aggregation allowed purifi- have led to the concept that approximately 600 Chl molecules are structurally and functionally organized to interact with the comcation of a "native" PSI complex. The isolated PSI particles appear as 106 A spherical subunits when ponents of one functional electron transport chain (18). This viewed by freeze fracture microscopy. When incorporated into phosphatidyl information can be related to three other generally accepted concepts concerning the photosynthetic membrane: (a) most, if choline vesicles, the particles lose self-aggregation properties and disperse not all, Chl are functionally associated with protein in pigmentwithin the membrane. uniformly lipid protein complexes (22); (b) the components of the two photosysThe isolated PSI preparation contains 110 ± 10 chlorophylls/P700 (Chi tems (enzymic constituents plus pigments) can be isolated sepaa/b ratio greater than 18); this represents a recovery of 27% of the original rately (7); and (c) the protein components of the two photosystems chloroplast membrane Chi. These particles were enriched in Chi a forms are structurally organized into morphologically distinct membrane absorbing at 701 to 710 nm. Chi fluorescence at room temperature exsubunits (3). hibited a maximum at 690 nm with a pronounced shoulder at 710 nm. At Many studies have reported isolation techniques which have 77 K, peak fluorescence emission was at 736 am; in the presence of dithionite an additional fluorescence maximum at 695 nm was obtained at yielded PSI preparations in various states of purity. Most early 77 K. This dual fluorescence emission peak for the PSI particles is evidence studies primarily attempted to show that sublamellar fragments for at least two Chl populations within the PSI membrane subunit. The could be obtained which retained enzymic activity for specific fluorescence emission observed at 695 nm was identified as arising from photosynthetic partial reactions. In general, these PSI preparations the core of PSI which contains 40 Chl/P700 (PSI40). This core complex, were enriched in Chl a (Chl a/b ratios were greater than 4) and derived from native PSI particles, was enriched in Chi a absorbing at 680 had Chl/P7004 ratios ranging from 80 to 150 (7, 32). In recent and 690 nm and fluorescing with maximal emission at 694 am at 77 K. PSI years, numerous attempts have been made to isolate "reaction particles consisting of the PSI core complex plus 20 to 25 Chi antennae center" preparations of PSI (6, 20, 28, 30). These had been depleted of antennae Chl, Chl/P700 ratios ranged from (65 Chl/P700) could also be derived from native PSI complexes. These extensively 7:1 (20), 15:1 (30) to 40:1 (6). Detailed characterization of the preparations were enriched in Chl a forms absorbing at 697 nm and enzymic activity and polypeptide composition of the reaction exhibited a 77 K fluorescence emission maximum at 722 am. A comparison of native PSI particles which contain 110 Chl/P700 (PSI- center preparations has been published (6). The spectral properties of PSI in situ have been characterized 110) and PSI particles containing 65 Chl/P700 (PSI-65) provides evidence (29, 31), as have been preparations of PSI reaction centers (10, 20, for the existence of a peripheral Chi-protein complex tightly associated in and PSI preparations retaining light-harvesting antennae (8, 30), the native PSI complex. The native PSI subunits contain polypeptides of 77 K, PSI is thought to have a major fluorescence emission At 27). 22,500 to 24,500 daltons which are not found in the PSI-65 or PSI-40 at peak 736 nm, with little fluorescence in the range 680-700 nm subfractions. It is suggested that these polypeptides function to bind 40 to We have characterized a PSI preparation, obtained by (27, 29). 45 Chi per structural complex, including the Chi which emits fluorescence mild detergent techniques, which retains native spectral properties. at 736 am. ABSTRACT

A model for the organization of Chli forms is presented in which the native PSI membrane subunit consists of a reaction center core complex plus two regions of associated light-harvesting antennae. The presence of energy "sinks" within the antennae is discussed.

Emerson and Arnold (16) investigated the involvement of Chl

Supported in part by National Science Foundation Grant PCM 77-

MATERIALS AND METHODS

Chloroplasts were isolated from 2-week-old pea (Pisum sativum) seedlings by grinding excised leaves in 0.4 M sorbitol and 50 mm Tricine-NaOH (pH 7.8), as previously described (4). Isolated chloroplasts were washed in 5 mm Na-EDTA (pH 7.8), then resuspended in H20 to 0.8 mg Chl/ml. Chl concentrations and Chl a/b ratios up to 6.0 were determined as described by Arnon (2). For samples with Chl a/b ratios greater than 6.0, the ratio of pigments was measured at 77 K in ethanol as described by Boardman and Thorne (9).

1 8953.

2 National Institutes of Health Predoctoral Fellows supported by NIH Grant GM 7283-1 to the University of Illinois. Present address: Plant Science Department, North Carolina State University, Raleigh, North Carolina 27607.

4Abbreviations: P700: chlorophyll absorbing maximally at 700 nm; reaction center of photosystem I; DCPIP: 2,6-dichlorophenol indophenol; C,..: chlorophyll form absorbing maximally at wavelength xxx (nm); F..,: chlorophyll form with fluorescence emission maximum at xxx (nm). 814

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PHOTOSYSTEM I CHLOROPHYLL PROTEINS

PSI-mediated electron transport from reduced DCPIP (0.3 mM) to methylviologen 0.5 mm (in the presence of saturating concentrations of plastocyanin) was measured by continuous recording of 02 uptake using a Clark-type 02 electrode and a YSI model 53 02 monitor (Yellow Springs Instrument Co.). Reaction mixtures contained I mm Na-ascorbate to reduce DCPIP. Light from a Unitron microscope illuminator was passed through a 1-75 Corning glass heat filter and a yellow Corning 3-69 fiter. The reaction mixture was stirred in a temperature-regulated vessel at 20 C. Absorption spectra, Cyt, and P700 determinations were obtained using an Aminco DW-2 spectrophotometer (American Instruments Co., Silver Spring, Md.). Cytochromes were assayed by chemically induced difference spectra as described by Bendall et al. (5). The amount of P7oo was determined by difference spectra. Samples of equal Chl concentration were placed in identical cuvettes. After a base line was recorded on the spectrophotometer, one of the two samples was treated with 1 mnm ferricyanide and the other with 2 mm ascorbate. The samples were allowed to equilibrate prior to recording a spectrum. Reversible A changes were then obtained by rereducing the oxidized sample with ascorbate. Reversible A changes at 700 nm were used to calculate the amount of P700 present using a mm extinction coefficient of 65 (23). Room temperature and 77 K fluorescence emission spectra were

815

ISOLATION SCHEME FOR "NATIVE" PHOTOSYSTEM I Homogenize leaves in 0.4 M Sorbitol, 0.05 M Tricine, pH 7.8, centrifuge at 1,000g for 5 min

Resuspend pellet in 0.05 M Sorbitol, 5 ill EDTA-NaOH, pH 7.8, centrifuge at 10,000g for 5 min Resuspend pellet in H20 to 0.8 mg Chl/ml, and Triton X-100 to a final concentration of approximately 0.7-0.8% (w/v) (see text and Table I for details) Incubate 30 min at 20CC with stirring; Centrifuge at 42,000g for 30 min; Discard pellet and retain supernatant Load 8 ml of supernatant on a 25 ml 0.1-1.0 M linear sucrose gradient (underlaid with a 5 ml, 2 M sucrose cushion) and centrifuged at 100,000g for 15 hr in a Beckman SW-27 rotor.

0. M

All sucrose solutions prepared with 0.02% Triton (see text and Table II for details).

d

si-.

Remove dark green band from above the 2 M

obtained by the dual channel ratiometric acquisition method using a System 4000 scanning polarization spectrofluorimeter (SLM Instruments, Urbana, Ill.). Multiple spectra (three to five for each sample) were recorded and averaged. Spectra were not corrected for photomultiplier sensitivity. Samples containing 0.5-2.0,ug Chl/ ml were assayed in 50%o glycerol at 77 K or in buffer at 20 C.

Analysis of membrane polypeptides using SDS-polyacrylamide gel electrophoresis was carried out as described previously (12) using a discontinuous buffer system. Electrophoresis was performed in a slab gel apparatus using a 5% (w/v) polyacrylamide stacking gel and a 6-16% gradient polyacrylamide running gel. Membranes or submembrane fractions were solubilized for 30 min in 65 mm Tris-Cl (pH 6.8) containing 10% (v/v) glycerol, 2% (v/ v) ,B-mercaptoethanol, and 2% (w/v) SDS. Electrophoresis was carried out at a constant current of 30 mamps. Gels were stained for 45 min in a solution containing 0.1% (w/v) Coomassie blue, 50% (v/v) methanol, and 7% (v/v) glacial acetic acid, and were destained in 20%1o methanol and 7% acetic acid. Mol wt determinations on the basis of electrophoretic mobility were made using the following mol wt standards: BSA (68,000), ovalbumen (45,000), carbonic anhydrase (29,000), /l-lactoglobulin (18,000), and lysozyme (14,500). Phosphatidyl choline vesicles were prepared by a procedure similar to that described by Brunner (11). Phosphatidyl choline was dispersed in 50 mm sodium cholate, loaded on a Sephadex G75 column and eluted with 50 mM Tricine-NaOH (pH 7.8). Vesicles were collected using a Brinkmann fractionator and stored under N2 until use. Freeze-fracture techniques were as described by Armond et al. (1). Ion exchange resin DE-52 was obtained from Bio-Rad. Ion exchange columns were run in the ascending direction at 4 C. RESULTS Isolation of a "Native" PSI. A flow diagram which outlines the procedure utilized to isolate PSI is shown in Figure 1. The first step in the isolation of PSI involved a titration of EDTA-washed chloroplast lamellae with Triton X-100 in order to disperse the membranes using a minimum concentration of detergent. Various amounts of Triton were added to a chloroplast solution containing 0.8 mg Chl/ml. This detergent-plastid mixture was stirred for 30 min at 20 C, and then centrifuged at 42,000g for 30 min. The amount of Chl found in the pellet after centrifugation was determined. This pellet represented undigested membrane material;

FIG. 1. Flow diagram which outlines the isolation of PSI-O 10 from pea chloroplasts. Solubilization of pea chloroplasts with concentrations of Triton X-100 ranging from 0.7 to 0.8% allowed good separation of PSI from other chloroplast complexes. The Triton-chloroplast slurry should be -pH 7.5. Further details of the procedure are enumerated in the text.

Table I. "Titration" of Chloroplast Membrane Solubilization Chloroplast isolation and EDTA washing were as described in Figure l. Membranes were resuspended to 0.8 mg Chl/ml in H20 containing the Triton X-100 concentration indicated, stirred 30 min at 20 C and then centrifuged at 42,000g for 30 min. Supernatant and pellet fractions were recovered; Chl concentrations of each were measured. Chl Recovered in a Pellet After Triton Concentration Used to Solubilize Chloroplasts Centrifugation at 42,000g % % total 0.4 30.3 0.6 0.7 0.8 0.9

24.1 14.4 6.0 2.7

increasing solubilization at increasing detergent to Chl ratios is shown in Table I. Based on preliminary experiments, we selected a detergent concentration that resulted in 90%o membrane disruption (10%1o Chl recovery in the 42,000g pellet of Table 1; 0.75% Triton X-100 in the experiment presented). This allowed maximal sample recovery with no detectable sample alterations. It was found necessary to repeat this detergent titration frequently to correct for differences in Triton stock solutions and/or differences in chloroplast samples. This precaution is very important since the use of excessive concentrations of Triton caused release of free Chl (monitored by increased fluorescence at 670-675 nm [18,

191) and disruption of the "native" PSI complex, and suboptimal concentrations decreased final sample yield. Once an appropriate detergent to Chl ratio was determined, and the initial centrifugation at 42,000g was completed, the green supernatant solution was loaded onto a 0.1-1.0 M sucrose gradient (Fig. 1). Inclusion of low concentrations of Triton X-100 (0.02%) in the sucrose increased the separation of two broad Chl-containing bands on the gradients. This gradient was fractionated into 20 equal volume aliquots which were analyzed for Chl a and b

MULLET, BURKE, AND ARNTZEN

816

content and for P700 (Fig. 2). The top of the tube (fractions 1-10) were enriched in Chl b; P700 could not be detected in these samples. Fractions 13-18 (just above the 2 M sucrose cushion), were deficient in Chl b and greatly enriched in P700 (Fig. 2). The specific property of PSI which allowed the gradient separation may involve both a change in density due to a decrease in detergent binding during centrifugation and self-aggregation of PSI. It was noted that centrifugation of solubilized chloroplasts on gradients containing higher amounts of Triton (0.5%) resulted in low yields of PSI. In contrast, omission of Triton on the gradients resulted in higher yields of Chl in the lower portion of the gradient but lower Chl a/b ratios of these fractions. This indicates contamination by the Chl b-containing light-harvesting complex of PSII (12). These data are presented in Table II. Characterization of the "Native" PSI (PSI-110). (a) Pigment content and photochemical properties. The "heavy" green band (fractions 13-18 from the gradient as described above) were collected in several experiments and characterized with respect to properties of intact membranes (Table IrI). These samples, hereafter referred to as PSI-1O, exhibited high rates of PSI-mediated electron transport, had no PSII activity, and were totally depleted in Cytfand b6. The samples had a Chl/P700 ratio of 1 10. The very low residual amount of Chl b in these samples probably represents slight contamination of the light-harvesting Chl a/b complex which comprises the major portion of the upper green band on these gradients (12). (b) Protein composition. Chloroplast membranes and PSI-110 samples were solubilized in SDS and subjected to electrophoresis on slab gels (Fig. 3). PSI- 110 was resolved into 11 clearly distinguishable bands (size estimates indicated at the right of Fig. 3): a doublet of 66 and 68 kdaltons, several individual bands ranging in size from 16.5 to 24.5 kdaltons, and three low mol wt peptides of 10.5, 11, and 11.5 kdaltons. In some gel preparations, the band migrating at 22.5 kdaltons was separated into two bands; this is indicated by two arrows at the right of the figure.

Plant Physiol. Vol. 65, 1980

Table III. Comparison of Isolated Chloroplast Membranes and Pooled Fractions 13-18from Sucrose Gradients These pooled fractions are designated PSI-I 10.

Chl a/b ratio

Chloroplast Membrane 2.9

PSI-O10 18-25 (range of observations for 13 experiments; average = 22)

Chl/P700

380

% of initial Chl recovered

Rate of PSII-mediated, DCMU-sensitive electron transport (,umol DCPIP reduced mg Chl' h-') H20 as electron donor Diphenylcarbazide as electron donor

350

320

1 10 ± 10 (average of 13 experiments) 25-30%37o (range for 13 experiments; average = 27%)

0

0 (minimal limit of detection in these experiments was a rate of 3)

Rate of PSI-mediated electron transport (DCPIPH2 to methyl-

viologen; UMol 02 consumed mg Chl'

h-1)

300

980 (rates were measured in the presence of purified

plastocyanin) Cyt content: nmolf/mg Chl nmol b6/mgChl

-11::. t3 -AZ

(1)

1.7 3.7

0 0 (minimal level of detection in these experiments was I Cytf/l,500 Chl or I

Cyt b6/1,500 Chl) ~Z O(c 0.. QC

Fraction Number FIG. 2. Fractionation pattern obtained after Triton-solubilized chloroplasts were centrifuged for 15 h at lOO,OOOg on 0.1-1.0 M sucrose gradients. The lower portion of the gradients contained P700 and had high Chl a/b ratios.

Table II. Effect of Varying Triton X-100 Concentration in Sucrose Gradient upon Recovery of Chl in Fractions 13-18 Chl in Pooled Fractions 13-18 of SuChl a/b in Pooled Triton on Gradient crose Gradient (see Fractions 13-18

Fig. 2) %

%

ratio

0 0.02 0.04 0.5

35 25

13 20 25 30

15 3

(c) Structural analysis of PSI-110. Fractions 13-18 collected from the gradients were dialyzed against 10 mm Tricine-NaOH (pH 7.8) for 6 h and then concentrated by centrifugation at 42,000g for 10 min. The green pellet which formed was examined by freeze-fracture electron microscopy (Fig. 4A). The sample consisted of aggregated particles with diameters of 100-130 A. The aggregation of these particles probably represents interaction of hydrophobic portions of each complex. To examine the structure of PSI-110 in a lipid environment, particles were reconstituted in phosphatidyl choline vesicles by mixing the particles with vesicles in the presence of 5 mM MgCl2 for 1 h at 20 C. The mixture was then loaded on 0.5-2.0 M sucrose gradients and centrifuged at 42,000g for 20 min. Examination of the resultant PSI-l 10/phosphatidyl choline vesicles by freeze-fracture revealed that the complexes dispersed uniformly in the lipid phase (Fig. 4B). These particles had an average diameter of 106 ± 8 A. (d) Spectral characteristics of PSI-110. The 20 C absorption spectrum of PSI-l10 revealed a red maximum at 680 nm. Fluorescence emission at 20 C was of low intensity. The emission spectrum had a maximum at 690 nm and contained a distinct shoulder at 710-740 nm (Fig. 5); this suggested that PSI contained at least two populations of Chl antennae. The 77 K absorption spectra of PSI- I O is shown in Figure 6. The first derivative of the

._,

Plant Physiol. Vol. 65, 1980

MEMB.

68-

PSI-110

PHOTOSYSTEM I CHLOROPHYLL PROTEINS

PSI- 65

-8

45_

66

_

45 -_

29-

:-245

o

24

-

=2 21

22.5

817

20-25% increase in total fluorescence yield. At 77 K the fluorescence yield was also increased under reducing conditions; it was noted that yield of fluorescence at 695 nm was enhanced relative to fluorescence emission at 736 nm. This is shown in Figure 7 where the spectra have been normalized at 736 nm. The two peaks observed in PSI-l lO under reducing conditions provide evidence for heterogeneity in the PSI Chl antennae. Excitation energy transferred from antennae to a long wavelength absorber which emits fluorescence at 736 nm appears to be relatively insulated (at 77 K) from changes in the redox state of P700. Excitation energy transferred directly to P700 (bypassing the long wavelength sink) reveals greater sensitivity to redox changes in the reaction center. This excitation energy is probably reemitted from Chl a antennae molecules in close proximity to P700. Analysis of PSI Antennae. (a) Spectral properties. The spectral evidence for at least two functionally distinct Chl antennae forms suggested that these may be associated with different Chl-proteins. To test the possibility of physically separating these forms, PSI110 preparations were further subfractionated (Fig. 8). The resultant material retained high PSI partial reactions (DCPIPH2 -* methylviologen electron transfer rates in the range of 1,500-1,700 tmol 02 consumed mg Chl-M h-') and was found to have a Chl/ P700 value of 65. This sample (hereafter referred to as PSI-65) was found to have altered absorption and fluorescence properties from PSI- 11O.

The 77 K absorption spectrum of PSI-65 and its first derivative, shown in Figure 9, reveals that C700 710 is depleted in these particles -,A compared to PSI-ll0 (Fig. 6). The PSI-65 particles retain C663 I8 17 (although the absorption band appears to be sharpened when 16.5 compared to PSI-llO) CQ0, C60, and a reduced amount of C697. The 20 C absorption spectra of PSI-65 and PSI-I 10 (Fig. lOA) were compared to gain further evidence for specific Chl a absorption forms which had been depleted during Triton subfractionation of the native PSI-l lO particle. The difference spectrum, shown 1 1.5 3 _ in Figure lOB, reveals that absorption at 648 nm, 683 nm, and 701-710 nm are more prominent in the PSI-ll0 particles. The 10.5 648-nm band may reflect minor contamination of PSI-l lO by Chl b of the light-harvesting Chl a/b complex (12) or a Chl a form which absorbs near 650 nm (27). The unique absorption at 701FIG. 3. SDS (6-16%)-polyacrylamide slab gel separation of pea mem- 710 nm in PSI-110 was of particular interest because of its brane proteins (MEMB), purified PSI containing 110 Chl/P700 (PSI-I IO) hypothesized role as the Chl a which has its fluorescence emission and PSI, depleted of 40-45 peripheral antennae, containing 65 Chl/P700 maximum at 736 nm (14). The depletion of Chl a forms absorbing at 701-7 10 nm (C701-710) (PSI-65). in the PSI-65 particles corresponded to a loss in long wavelength spectrum gave an indication of at least four major absorption fluorescence of this preparation at both room temperature (Fig. 5) bands in PSI-llO: C60, C6so, C690, C697, and a broad band at and at 77 K (compare Fig. 6 to Fig. 9). The 77 K fluorescence emission maximum of PSI-llO (736 nm) was red-shifted to 722 C700.710. At 77 K, PSI- I IO particles exhibited a major fluorescence emis- nm in the PSI-65 preparations. These data suggest that the pesion peak at 736 nm (Fig. 6). The lack of fluorescence at 670-675 ripheral antennae of PSI- l10, which may be removed by detergent, consists of 40-45 Chl/P700 including long wavelength-absorbing nm indicates the absence of free Chl in the preparation (18, 19). It was noted that the fluorescence yield of the far red-fluorescing forms of Chl a responsible for fluorescence emission at 710-740 Chl increased dramatically at low temperature when compared to nm (20 C) or at 736 nm (77 K). (b) Polypeptide composition. Preparations of PSI-65 were subChM a forms emitting at 695 nm. This observation has been previously related to the fact that at 20 C, P700 in vivo is an efficient jected to slab gel electrophoresis peptide analysis (Fig. 3). The quencher of fluorescence (20), which accounts for the low yield of sample was found to be specifically depleted in four proteins when fluorescence observed at this temperature. The increase in fluo- compared to PSI-l lO: the polypeptides of 24.0 and 24.5 kdaltons and two polypeptides which co-migrate in the 22.5 kdalton size rescence yield of long wavelength forms of Chl at low temperature class. All other polypeptides appeared relatively constant in suggests that energy transfer from these ChM to P700 is reduced (13, amount in comparisons of PSI- I1O and PSI-65. 26). Antennae-depleted PSI "Core". To examine the spectral comTests were made to see if changes in the redox state of P700X would alter the fluorescence yield of portions of the PSI antennae position of the PSI reaction center "core complex", PSI-65 partidifferentially. It has been reported that the primary acceptors of cles were depleted of antennae Chl as described in Figure 8. The PSI act as quenchers in their oxidized state; and that the reduction particles contained 40 Chl/P700 (hereafter referred to as PSI-40) of these acceptors requires illumination in the presence of dithio- indicating that depletion of 25 Chl/P700 had occurred by treatment nite (20). The fluorescence emission of PSI-IO was monitored in of the PSI-65. The absorption spectrum of PSI-40 was recorded the presence or absence of dithionite after preillumination. At and compared to PSI-65 (Fig. I lA). At 20 C, C675 and C686 were 20 C, samples incubated in the presence of dithionite exhibited a found preferentially in PSI-40 whereas C695 700 were found prefAlA.

818

MULLET, BURKE, AND ARNTZEN

Plant Physiol. Vol. 65, 1980

FIG. 4. A: electron micrograph of freeze-fractured PSI-I 10. The 100-130 A particles appear as aggregates. B: electron micrograph of freeze-fractured PSI-II0 incorporated in phosphatidyl choline vesicles. The PSI-I 10 particles were homogeneously distributed in the vesicles and averaged 106 A in diameter. Bars = 0.5 Lm.

LUJ z LUJ

LI,

q)

00

04 LI,-

mi

q,) CZ

550

700 750 Wavelength (nm)

800

FIG. 5. Fluorescence emission spectra (20 C) of PSI-IO0 (- --) and PSI-65 (-). Fluorescence was excited at 440 nm (8-nm slit) and emission collected using a 2-nm slit. Multiple fluorescence emission spectra were collected, averaged, and normalized at their maximum before plotting.

erentially in PSI-65 (Fig. 1 1B). The 77 K absorption spectrum of PSI-40 is shown in Figure 12. The first derivative of the absorption spectrum revealed the presence of three major absorption forms in PSI-40, C63, C678, and CQ. The 77 K fluorescence emission maximum of PSI-40 (Fig. 12), was blue shifted compared to PSI-65 (Fig. 9). It contained a peak at 694 nm, and a shoulder at 710 nm. The blue shift in emission exhibited by PSI-40 probably reflects the depletion of long wavelength-absorbing forms of Chl a in this particle. There is little fluorescence in the 670-680 nm region (indicating no free Chl). The PSI-40 particles have been examined by slab gel elec-

600

-0--8I 650 700 WAVELENGTH (nm)

750

800

FIG. 6. Fluorescence emission spectra (77 K) (- - -) of PSI- 1 10 samples were acquired and averaged using exciting light of 440 nm (8-nm slit). Fluorescence emission was collected using a 1-nm slit. When spectra were corrected for photomultiplier sensitivity, emission maxima were red shifted by 1-2 nm as compared to the data shown. Absorption spectra (77 K) of PSI-110 ( ) were acquired on 1-mm thick samples frozen in 701% glycerol. Samples contained 8-15 ,ug Chl/ml. The spectrum was acquired using a 1-nm slit. The first derivative of the absorption spectrum revealed the following absorption bands: C663, C680, C690, C697, and a broad band at

C701-710.

trophoresis. No significant differences to the polypeptide content of PSI-65 could be detected. DISCUSSION Most, if not all, ChM exists in chloroplast membranes bound in Chl-protein complexes (3, 22). Various lines of experimentation have provided evidence for the fact that the Chl-protein complexes and enzymic components of PSI and PSII exist as complexes of polypeptides embedded in the lipid phase of thylakoid membrane

Plant Physiol. Vol. 65, 1980

PHOTOSYSTEM I CHLOROPHYLL PROTEINS

819

ISOLATION OF PS I-65

Dialyze PS 1-110 vs 0.05 M Sorbitol for 8 hr; centrifuge at 41,(OOg for 10 min Resuspend pellet in H 0 to 0.2 mg Chl/ml; add Triton X-100 to 0.45% (w/v); Incgbate 30 min at 200C with stirring Load 8 ml of solution on a 30 ml 0.1-1.0 M sucrose gradient containing 0.35% (w/v) Triton X-100; Centrifuge in an SW-27 Beckman rotor for 9 hr at

z

100, 000g

w O.AM

LJ

:

-

-

Collect green bands, dilute 1:1 with H 0 centrifuge at 200,000g for 1 hr in fix d angle rotor Resuspend soft pellet in H20; repeat centrifugation at 200,0002 for 1 hr

z

LLJ (I)

cnLuI 0

l.OM

WI

C Col lect resul tant pel let

(P1CJ-65 )

UISOLATION OF PS I-40 Resuspend PS 1-65 to 0.2 mq Chl/ml in 25 mM Tris, pH 7.2, 1% Triton X-100, 10 FM 1-0-N octyl s-D glucopyranoside

Incubate 8 hr, 4C, with stirring

650

700

750

800 Absorb on DE-52 ion exchange column; wash 1 hr with 50 ml of 0.2% Digitonin, 25 mM Tris, pH 7.2

WAVELENGTH (NM) FIG. 7. Fluorescence emission of PSI-I 10 obtained as in Figure 6. PSI110 was incubated in the presence or absence of 1.5 mm dithionite in the dark at 20 C for 5 min, then illuminated for 3 min prior to freezing and recording an emission spectra. Fluorescence emission at 695 nm (relative to other wavelengths) was enhanced in the presence of dithionite.

Elute PS 1-40 using 0.4 M NaCl, 25 nM Tris, pH 7.2, 0.2% Digitonin

FIG. 8. Flow diagram which describes the techniques to isolate PSI-65 and PS140. Isolation of PS140 could be accomplished directly from PSI110 by solubilization in 2% Triton, incubation with stirring at 4 C for 10 h, and DE-52 chromatography as described under isolation of PSI-40.

(3).

Data from excitation spectra of partial reactions (31) and investigations of subchloroplast particles (15, 17) suggest that specific forms of Chl are associated with the membrane-bound complexes of PSI and PSII. Although there is much data concerning the distribution of Chl forms between the photosystems, little information is available which elucidates the organization of spectral forms within each photosystem. It has been reported (27) that PSI, isolated using Digitonin, contains a heterogeneous population of Chl a absorbers including C663, C679, Cw88, C699, and C705 nm. Absorption bands with similar maxima have been tentatively correlated with fluorescence bands as follows: C663 -- F674, C670 - F683, C677 -- F693, C683 -- F710, C692 -- F725, and C702 F738 nm (18, 19). To gain information concerning the structure, function and spectral organization of PSI, we have developed a procedure for the isolation of a "native" PSI particle which retains characteristics attributed to PSI in vivo. The use of minimal concentrations of Triton X- 100 and centrifugation on sucrose gradients allowed isolation of a purified PSI in high yield which retained its full compliment of ChM antennae, good activity and structural integrity. Spectral analysis revealed that free Chl was not present in the "native" PSI complex or in the subsequent antenna depleted particles. We have attempted to minimize artifacts generated by the use of detergents (ie. release of Chl, spectral band shifts, bandsharpening) by using low concentrations of detergents, and removing excess detergent prior to spectral analysis. We recognize that questions concerning isolation artifacts may still be raised. We have conducted studies on intact membranes of various mutant or developmental chloroplast systems (24) to corroborate evidence obtained from antennae depleted particles. Our observations concerning the organization of the antennae

550

600

650

WAVELENGTH

700

750

800

(nm)

FIG. 9. Fluorescence emission spectrum (77 K) of PSI-65 (---) and absorption spectrum ( ) were acquired as described in Figure 6. The first derivative of the A spectrum indicates the presence of the following absorption bands: C663, Ca8o, C6em, and C697.

Chl protein of PSI are diagrammed in the model shown in Figure 13. We suggest that the antennae Chl proteins of PSI are arranged in three domains which are characterized by the polypeptides involved in binding the antennae Chl, the spectral forms of Chl contained in each domain, and the physical and energetic distance of the Chl antennae from the reaction center, P700. To facilitate discussion of the various spectral forms of Chl a found in PSI we have assigned absorption forms (C1..) using bands observed in difference spectra (Figs. 10 and 11) or obtained by taking the first derivative of 77 K absorption spectra (Figs. 6, 9 and 12). Fluorescence bands (Fy..) are assigned directly from

Plant Physiol. Vol. 65, 1980

MULLET, BURKE, AND ARNTZEN

820 4

(4

3

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-2550

600

650

700

750

Wavelength (nm) FIG. 10. A: absorption spectra (20C) of PSI-110 (---) and PSI-65 ). Spectra were normalized at their A maximum and plotted with an offset base line for the spectrum of PSI-65 (for clarity of display). B: normalized spectra were subtracted (PSI- 1 10)-(PSI-65) and the difference spectrum (X5) was plotted. The absorption at 648 nm, 683 nm, and 7017 10 nm was preferentially found in PSI-I 10.

their maxima. These assignments must be considered tentative until deconvolution of the spectra are completed. We have used Triton X-100 to isolate PSI core complexes containing 40 Chl/P700 (PSI-40). These particles are enriched in Chl a forms absorbing at 675 nm and 686 nm at 20 C (Fig. 11) or at 680 nm (C680) and 690 nm (C690) at 77 K (Fig. 12). At 77 K, PSI-40 exhibits a fluorescence emission peak at 694 nm (F695) (Fig. 12). In earlier work, spectral analysis of PSI core preparations containing 8-37 Chl/P7oo revealed low temperature fluorescence emission peaks at 678-683 nm (F60w) and 692-697 nm (F695) (10, 20, 30). Chl a forms absorbing at 672-675 nm (C675) have been found to excite F90o and F695 (20). A Chl a form absorbing at 684 nm (Cm4) has also been reported to excite F695 (20). In the present work PSI-40 did not exhibit fluorescence emission at 678-683 nm although C675 was enriched in the core complex (Fig. 12). This suggests that fluorescence emission peaking at 678-683 nm, found in previously described core complexes, probably originated from Chl a forms absorbing at 672-675 nm which were energetically isolated due to disruption of the antennae bed by random pigment extraction during preparation. These data also imply that in intact PSI core complexes (i.e. PSI-40) excitation energy absorbed by C675o is efficiently tranferred to longer wavelength Chl a forms

Wavelength (nm) FIG. 11. A: absorption spectra (20 C) of PSI-65 and PSI-40 were obtained, normalized, and plotted as in Figure 5, with the base line for the PSI-65 sample offset for clarity. B: difference spectrum shown (PSI-65)(PSI-40) was obtained as described in Figure 9 and is plotted (X2.5) in Figure IIB.

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FIG. 12. Fluorescence emission spectrum (77 K) of PSI-40 (--)and absorption spectrum ( ) were acquired as described in Figure 6. The first derivative of the absorption spectrum revealed three major absorption

bands:C663, C678, and C69o.

Plant Physiol. Vol. 65, 1980

PHOTOSYSTEM I CHLOROPHYLL PROTEINS

821

wavelength Chl a, C705 (14). The first derivative of the 77 K absorption spectrum of PSI-110, which is enriched in F736 (Fig. 6), exhibits a broad absorption band at 701-710 nm which probably corresponds to C705 (Fig. 6). PSI-65 particles, which are deficient in F736, lack an absorption band at 701-710 nm (Fig. 9). This correlation supports the assignment C705 -* F736 (14). It has been suggested that C705 forms only at low temperature (27). A comparison of spectral features of PSI-l10 and PSI-65 reveals that PSI-65 is relatively deficient in F736 (Fig. 6 versus Fig. 9, 77 K), F710o740 (Fig. 5, 20 C), and C700710 (Fig. 10, 20 C) when compared to PSI-l10. This argues that C705 exists at 20 C as well as at 77 K. Recent measurements showing linearity between lifetime and yield of F736, both of which increase with decreasing temperature, also suggests that C705 exists at all temperatures studied (13). Data presented in this paper indicate that "native" PSI comCore Antennae contain a peripheral antennae of 40-45 Chl/P7o0 in addition plexes 40 Chl to 40 core Chl and 20-25 internal Chl antennae. We have provided C685-9o F694 evidence (Fig. 3) for the fact that the PSI peripheral Chl antennae Polypeptides: 66,000, exist as unique pigment proteins (polypeptides in the size may 68,000 daltons range 22,500-24,500 daltons). The fact that the C705 -* F736 is only detected in the PSI- 110 complex suggests that this pigment form Internal Antennae is physically located in the peripheral antennae of PSI. 20-25 Chi C696-97 It has been proposed (14) that C705 -+ F736 acts as a sink for excitation energy absorbed by the mixed population of Chl a Peripheral Antennae found in the system I antennae. Evidence for this point has been 40-45 Chl at 77 K where transfer from C705 to P700 is limited (13). gained C705-' F736 Under these conditions fluorescence at 736 nm predominates (Fig. Polypeptides; 21,0006) indicating that a large part of the excitation energy absorbed by 24,500 daltons PSI flows through C705 -- F736. Evidence which suggests that C705 FIG. 13. Model which describes the organization of antennae Chl -. F736 exists as a metastable sink at 20 C is shown in Figure 5. If protein in the PSI complex. Each layer of antennae is thought to contain excitation energy, absorbed by a mixed population of Chl a, is in equilibrium with no significant uphill transfer barriers present, a mixture of Chl a absorbers including the long wavelength absorption spectra of absorption and fluorescence emission should be mirror fluorescence pairs designated in the figure. images. The 20 C fluorescence emission spectra of PSI-i 10 is asymmetric from 710 to 740 nm (Fig. 5). This indicates that at (i.e. C690 F695). 20 C excitation energy transfer from C705 to shorter wavelength Two possibilities have been suggested to explain the origin of antennae is to some extent an uphill process. The existence of this F695 in PSI. The first, based on the work of Philipson et al. (25), uphill barrier helps concentrate excitation energy in C705 and may involves the formation of C690 -+ F695 by photooxidation of the facilitate energy transfer from peripheral antennae to P700. The P70O dimer; (Chl Chl)6,s697 (Chl Chl)6. This proposal is sup- usefulness of this weak PSI antennae "sink" in a physiological ported by observation of positive A changes at 686 nm when PSI context may relate to the relative distribution of Chl between PSI is oxidized (21). However, Ikegami (20) has reported increases in and PSII in fully developed chloroplast lamellae. Only the fluorescence yield of Fs5% when system I reaction centers are to one-third of the total absorbing pigment is directly one-fourth associated in the reduced state, P700X-. This leads to a second explanation F720

to

--

which identified F695 as originating from a Chl antennae absorbing at 684 nm (20). In this case the fluorescence yield of F695 is influenced by, but not dependent upon the redox state of P700X, and does not originate from the dimer Chl of P700. Data presented in this paper support Ikegami's interpretation since increases in F695 were observed when P700 and the primary acceptors of PSI were reduced (Fig. 7). To evaluate the spectral composition of Chl antennae external to Chl bound in the PSI core, PSI particles were isolated which contained core antennae plus 20-25 Chl (internal antennae, Fig. 13). These particles contained 65 Chl/P700 (PSI-65); when compared to PSI-40, PSI-65 particles were found to be enriched in Chl a forms absorbing at 696-700 nm (Fig. 1 1). The particles were also enriched in fluorescence which peaked at 722 nm at 77 K (Fig. 9). This suggests the assignment of C697 F722. Relatively small differences in the polypeptide content of PSI-40 and PSI-65 indicate that the internal antennae may bind directly to polypeptides of the PSI core complex. it is possible that one of the eight polypeptides of the PSI-65 complex (Fig. 3)

may

bind these

internal antennae, but that the PSI-40 preparation procedures selectively remove the bound Chl without removing an associated protein. In vivo, low temperature fluorescence emission at 736 been attributed to PSI and is thought to arise from

nm

a

has

long

with PSI. Energy absorbed by this photosystem would be most efficiently utilized if it remains localized in the PSI pigment bed; a long wavelength sink in the PSI antennae could enhance this localization. In addition, under conditions when PSI reaction centers are closed, transfer of excitation energy to a long wavelength Chl a form located at the periphery of PSI may aid in the nondestructive dissipation of excitation energy, and/or facilitate energy transfer to other PSI complexes.

Acknowledgments-We thank Drs. J.-M. Briantais, C. Vernotte, I. Moya, Govindjee, and L. Shipman for useful discussions. The excellent assistance of J. Watson and C. Ditto was very much appreciated. LITERATURE CITED 1. ARMOND PA, LA STAEHLIN, CJ ARNTZEN 1977 Spatial relationship of photosystem I, photosystem II and the light-harvesting complex in chloroplast membranes. J Cell Biol 73: 400-418 2. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol 24: 1-15 3. ARNTZEN CJ 1978 Dynamic structural features of chloroplast lamellae. In D Rao Sandai, LP Vernon, eds, Current Topics in Bioenergetics. Academic Press, New York 8: 112-155 4. ARNTZEN CJ, CL DiTTo 1976 Effects of cations upon chloroplast membrane subunit interaction and excitation energy distribution. Biochim Biophys Acta 449: 259-274 5. BENDALL DS, HE DAVENPORT, R HILL 1971 Cytochrome components in chlo-

822

MULLET, BURKE, AND ARNTZEN

roplasts of higher plants. Methods Enzymol 23: 327-344 6. BENGIS C, N NELSON 1977 Subunit structure of chloroplast photosystem I reaction center. J Biol Chem 252: 4564-4569 7. BOARDMAN NK, JM ANDERSON 1964 Isolation from spinach chloroplasts of particles containing different proportions of chlorophyll a and chlorophyll b and their possible role in the light reactions of photosynthesis. Nature 203: 166-167 8. BOARDMAN NK, SW THORNE, JM ANDERSON 1966 Fluorescence properties of particles obtained by digitonin fragmentation of spinach chloroplasts. Biochemistry 56: 586-593 9. BOARDMAN NK, SW THORNE 1971 Sensitive fluorescence method of the determination of chlorophyll a/chlorophyll b ratios. Biochim Biophys Acta 253: 222-231 10. BROWN JS 1977 Fluorescence spectroscopy of a P700-chlorophyll-protein complex. Photochem Photobiol 26: 519-525 11. BRUNNER J, P SKRABAL, H HAUSER 1976 Single layer bilayer vesicles prepared without sonication physico-chemical properties. Biochim Biophys Acta 455: 322-331 12. BuRKE JJ, CL DITTo, CJ ARNTZEN 1978 Involvement of the light-harvesting complex in cation regulation of excitation energy distribution in chloroplasts. Arch Biochem Biophys 187: 252-263 13. BUTLER WL, CJ TREDWELL, R MALKIN, J BARBER 1979 The relationship between the lifetime and yield of the 735 nm fluorescence of chloroplasts at low temperatures. Biochim Biophys Acta 545: 309-315 14. BUTLER WL 1961 A far red absorbing form of chlorophyll, in vivo. Arch Biochim Biophys 93: 413-422 15. ELGERSMA 0, G VOORN 1974 Deconvolution of absorption spectra at room temperature of chloroplast fragments. In M Avron, ed, Proc 3rd Int Congr Photosynthesis. pp 1943-1949 16. EMERSON R, W ARNOLD 1932 The photochemical reaction in photosynthesis. J Gen Physiol 16: 191-205 17. GASANOV RA, CS FRENCH 1973 Chlorophyll composition and photochemical activity of photosystems detached from chloroplast grana and stroma lamellae. Proc Nat Acad Sci USA 70: 2082-2085 18. Govindjee, R Govindjee 1975 In Govindjee, ed, Bioenergetics of Photosynthesis. Academic Press, New York, pp 1-43 19. HEATH, RL 1973 The energy state and structure of isolated chloroplasts: the

20. 21. 22.

23. 24. 25.

26.

27. 28. 29.

30. 31. 32.

Plant Physiol. Vol. 65, 1980

oxidative reactions involving the water splitting step in photosynthesis. Internat Rev Cytol 34: 49-101 IKEGAMI I 1976 Fluorescence changes related in the primary photochemical reaction in the P700-enriched particle isolated from spinach chloroplasts. Biochim Biophys Acta 449: 245-258 LOZIER RH, WL BUTLER 1974 Light induced absorbance changes in chloroplasts mediated by photosystem I and photosystem II at low temperature. Biochim Biophys Acta 333: 465-480 MARKWELL JP, JP THORNBER, RT BOGGS 1979 Higher plant chloroplasts: Evidence that all the chlorophyll exists as chlorophyll-protein complexes. Proc Nat Acad Sci USA 76: 1233-1235 MARSHO TV, B KOK 1971 Detection and isolation of P700. Methods Enzymol 23: 515-522 MULLET JE, JJ BuRKE, CJ ARNTZEN 1980 A developmental study of photosystem I peripheral chlorophyll proteins. Plant Physiol 65: 823-827 PHILIPSON KD, VL SATO, K SAUER 1972 Excitation interaction in the photosystem I reaction center from spinach chloroplasts. Absorption and circular dichroism difference spectra. Biochemistry 11: 4591-4595 RIJGERSBERG CP, A MELIS, J AMESZ, JA SWAGER 1979 Quenching of chlorophyll fluorescence and photochemical activity of chloroplasts at low temperature. In G. Wolstenholme, D. Fitzsimons, eds, Chlorophyll Organization and Energy Transfer in Photosynthesis 61: 305-319 SATOH K, WL BUTLER 1978 Low temperature spectral properties of subchloroplast fractions purified from spinach. Plant Physiol 61: 373-379 SHIOZAWA JA, RS ALBERTE, JP THORNBER 1974 The P700 chlorophyll a-protein. Arch Biochem Biophys 165: 388-397 STRASSER RJ, WL BUTLER 1977 Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants. Biochim Biophys Acta 462: 307-313 VACEK K, D WONG, GOVINDJEE 1977 Absorption and fluorescence properties of highly enriched reaction center particles of photosystem I and of artificial systems. Photochem Photobiol 26: 269-276 VAN GINKEL G 1975 Solar energy conversion in a green plant. PhD thesis. University of Utrecht WESSELS JSC, G VOORN 1971 Photochemical activities of chloroplast fragments obtained by the action of digitonin. 2nd Intern Congr Photosynthesis. Stresa 833-845