Photosynthesis Research 10: 209-215(1986) © Martinus N i j h o f f Publishers, Dordrecht - Printed in the Netherlands
209
EXCITON INTERACTIONS IN PHYCOERYTHRIN
KAROLY CSATORDAY, SHARON CAMPBELL, AND BARBARA A. ZILINSKAS Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, NJ 08903, U.S.A.
ABSTRACT Upon assembly of the phycoerythrin trimer into hexamer and the hexamer into dodecamer, marked spectral changes are observed. The absorption and circular dichroism spectra of the various phycoerythrin aggregates were resolved into Gaussian components representing individual electronic transitions of phycoerythrobilin chromophoreswlthin these proteins. While the contribution of a broad, sensitizing band (at 525 nm) is constant, with increasing aggregate size, a short-wavelength pair of bands centered at 555 r~m decreases concomitantly with a dramatic increase in the intensity of a lonE-wavelength pair of ehromophore transitions centered at 563 rmL The implications of these spectral changes for efficient energy transfer in the phycobilisome are discussed. INTRODUCTION Light energy is harvested in the cyanobacterla and red algae by speclal multlprotein aggregates called phycobillsomes (PBscmes). These contain phycobillprotelns that absorb radiation and transfer its energy in the form of electronic excitation to the photochemical reaction center. A complete transfer sequence involves phyeoerythrln (PE), phycocyanln (PC) and allophycocyanln (APC) [11]. Since this hlghly efficient transfer is dependent on the distance between donor and accepter molecules, as well as on the spectral overlap between their respective emission and absorption spectra, the spectral properties as well as the posslbillty of their delegation to particular chremophore transitions are of interest. Chromophores with short-wavelength absorption bands sensitize the fluorescence of those absorbing at longer wavelengths, and thus, the terms "sensitizing" ('s') and "fluorescent" ('f') chr~zzophores were introduced by Teale and Dale (8). In the present study, we attempt to correlate spectral characteristics of PE with individual electronic transitions of chromophores within the protein. Phycoerythrln in vlvo is assembled into so-called rods that serve as constituents of the llght-harvestlng PBsome. Within the rods of the cyanobacteri~-,, Nostoc sp., grown in cool-whlte fluorescent light, PE exists as a stack of four disks each consisting of a PE trimer (~B)~Each trlmer contains 15 phycoerythrobilln chr~mophores. The four tflmers comprising a dodecamer of PE are attached to a PC hexamer, also in the form of a double disk. The assembly is governed and facilltated by linker proteins that determine the final structure of the PBsome [12]. The spectral properties of isolated PE ngg~-egates closely resemble those they manifest within the PBsome [10], thus allowlng the study of electronlc transitions in a simpler system.
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210
MATERIALS AND METHODS Phycoerythrln in the trimeric and hexameric forms were isolated from PBsomes of the cyanobacterium Nostoc sp. as described earlier [12]. Subsequent to dialysis of the PE hexamer against 0.75M K-phosphate, pH 7.0, the PE dodecamer was obtained by sedimentation in linear gradients of sucrose (0.15M-0.8M) containing 0.75M K-phosphate, pH 7.0, for 16h at 40,000 rpm in a SW40 Beckman rotor. The PE trimer and hexamer each contained approximately 5% PC, and the dodecamer contained approximately 7% PC; however, the PC was not physically associated with PE as demonstrated by the absence of fluorescence emission from PC fol lowir~ PE excitation. The curve-fitting analysis included a component to account for the contribution of the contaminating PC. Aggregate size and polypeptide composition were determined respectively by sedimentation in linear gradients of sucrose and by sodium dodecyl sulfate polyacrylamide gel electrophoresis as described earlier [12]. Absorption and circular dichroism (CD) were measured as previously described [13] wlth a Cary 17D and a Cary 60 spectrometer, respectively. The circular dichrolsm and absorption spectra were resolved into Gausslan components using a curve-fitting program written for the Apple II computer [I] with the blue edge of the spectra approximated by a wide bandwidth chimeric component attributed to sensitizing chromophores. RESULTS AND DISCUSSION The absorption and CD spectra of PE trimers, hexamers and dodecamers were resolved into Gaussian components representing individual electronic transitions of phycoerythrobilin chrcmophores within these protein aggregates. Figure I (left panel) shows the computer-generated fits of both 1.0
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FIGURE I. Left panel. Absorption and CD spectra of PE aggregates. The experlmental and fitted curves are plotted with dots and crosses respectlvely. Only every third point in the fitted curve is shown for clarity. The absorption spectra were normalized for unit area. FIGURE I. Right panel. The resolved components of the absorption and CD spectra of the three PE aggregates.
t h e a b s o r p t i o n and CD s p e o t r a f o r eaoh o f t h e a g g r e g a t e s . Only t h e e x p e r i m o n t a l om~re and t h e sun o f t h e o a l o u l a t e d oomponenta a r e shown f o r o l a r t t y and t o d e m o n s t r a t e t h e e r r o r o f t h e f i t . The e v o l u t i o n o f a band i n b o t h t h e a b s o r p t i o n and CD s p e o t r a a t 570 em As s e e n . A l s o , t h e n e g a t t v ~ b a n d i n t h e CD speotrum i s s h i f t e d toward the b l u e as a f~notton o f ngg~egate s i z e . These ohanges h a v e p r e v i o u s l y been i n t e r p r e t e d i n t e r m s o f o h ~ m o p h o r e - o h r ~ m o p h o r e i n t e ~ a o t t o n s [10]. A reasonably olosenatohwas found between t h e a b s o r p t i o n and CD o c ~ p o n e n t s with regard to the essential parameters of peakwavelengthand halfbandwidth ( s e e T a b l e I ) . F i g u r e I ( r i g h t p a n e l ) shows t h e i n d i v i d u a l oomponent bands a n d a l l o w s a q u a n t i t a t i v e t r e a t m e n t o f t h e s e ohanges. The r e s o l v e d oomponents a r e p l o t t e d on t h e same s o a l e as t h e e x p e r i m e n t a l and f i t t e d o u r v e s i n Fig. 1 (left). The c o r r e s p o n d i n g s p e o t r a l p a r a n e t e r s a r e g i v e n below. TABLE 1. S p e o t r a l p a r a m e t e r s o f t h e r e s o l v e d o o n p o n e n t s o f t h e p h y o o e r y t h r i n a b s o r p t i o n and o t r o u l a r d t o h r o i o m s p e o t r a .
Abs
L 524 W ~5 A .50
5~6 29 .25
56~ 28 .21
563 17 .02
568 16 .02
CD
L 525 W 43 A .~8
5~7 25 .19
56~ 25 .23
563 17 .05
57~ 17 .05
Abs
L 526 W ~5 A .51
5~7 28 .27
562 28 .05
558 17 .08
570 16 .12
CD
L 524 W 42 A .38
5~8 26 .23
563 25 .17
562 16 .11
570 17 .12
Abs
L 527 W ~9 A .51
5~4 26 .14
563 26 .02
554 21 .14
572 18 .20
600 66
E=2.5~
Triter E=6.1~
593 66
E=1.8~
Hsxomor
E=7.8~ 601 58
B=2.61l
Dodeoamer
L 517
548 56~ 553 568 26 28 20 14 E=I0.75 .13 .16 .26 .25 L=wavelength maximum, W=half-band~rldl;h, A=area under the oomponent ourve CD
W ~9 A .19
a s t h e p e r ~ o n t n g e o f t o t a l a r e a , and E = e r r o r o f t h e f i t . All numerloal d a t a a r e rounded t o t h e l a s t d i g i t shown. The oemponent a t "600nn i s a t t r i b u t e d t o e o n t a n t n a t t n g PC. N o r m a l i z i n g t h e a b s o r p t i o n s p e o t r a w i t h r e s p e o t t o t h e a r e a under t h e s p e o t r a l o u r v e a l l o w s t h e i r ocmparison b ased on ohanges i n t h e r e l a t i v e a r e a s u n d e r eaoh oomponent o r e ' r e , a q u a n t i t y p r o p o r t i o n a l t o t h e o s c i l lator strength of a given transition. All the absorption spectra feature a b r o s d ~5 un wide band i n t h e h£Kh e n e r g y r~Kton. T h i s o~etponon~, oenter,Dd around 525 nm, r e m a i n s o o n s t a n t i n a r e a , o o m p r i s t n g a b o u t 50~ o f the t o t a l absorbanoe. The o o r r e s p o n d i n g band i n t h e CD s p e o t r a has t h e s a n e f e a t u r e s w i t h t h e e x o e p t i o n t h a t i n g o i n g from t h e t r i m e r t o t h e dodeoamer i t d e o r e a s e s i n I n t e n s i t y and i s b l u e - s h i f t e d t o 517 nm. I n £ a e t , f i t t i n g two wide o h i m e r i o o~mponents a t 513 and 532 nm, i n s t e a d o f
212
[661
t h e one a t 525 mR, b e t t e r a o o o u n t e d f o r t h e CD f e a t u r e s i n t h e dodeoamer s p e c t r u m b u t d~d n o t ehanEe t h e t o t a l a r e a a s c r i b e d t o ~he h i g h e n e r g y band n o r d i d i t s i g n i f i c a n t l y i n f l u e n c e t h e p a r a m e t e r s o r t h e d i s c u s s i o n which f o l l o w s f o r t h e two e x o i t o n band p a i r s . A p p l y i n g Oecam'a r a z o r , we r e s t r i c t e d t h e r e s o l u t i o n s t o one component i n t h i s r e g i o n a l t h o u g h two wide bands r e p r e s e n t i n g N n a i t i n t n g chrcmophores may be p r e s e n t . A l t h o u g h t h e r e s u l t s d i s c u s s e d i n t h i s p a p e r p e r t a i n s o l e l y t o PE a ~ r e g a t e s , we c o n s i d e r t h e c o n c l u s i o n s r e l e v a n t t o t h e n a t i v e PBsome s i n c e a p a o t r o s Q o p i e a l l y t h e a x e i t o n f e a t u r e s a r e e s s e n t i a l l y t h e same f o r t h e dodecamer o f EE, t h e i s o l a t e d PBsome r o d which i s co m p r i sed o f a PE dodeeamer a s s o c i a t e d w i t h a PC hexamer, and t h e i s o l a t e d PBsome. The o n l y d i f f e r e n c e s e e n i n comparing t h e PE dodecamer w i t h t h e PBsome o r t h e PBsoma rod i s t h e d i m i n i s h e d h i g h e n e r g y CD band a t t r i b u t e d t o s e n s i t i z i n g PE ohromophores. I n t h e PBsome o r i n t h e r o d , t h i s CD band r e m a i n s a t t h e l e v e l o b s e r v e d f o r t h e i s o l a t e d PE t r i m e r . There may be a s l i g h t l y d i f f e r e n t c o n f o r m a t i o n o f t h e s e n s i t i z i n g ohromophores i n t h e i s o l a t e d PE dodecamer which a f f e c t s i t s o p t i c a l a c t i v i t y b u t n o t i t s a b s o r p t i o n . The t r i m e r s p e c t r u m a l s o e n v e l o p s a p a i r o f bands c e n t e r e d around 555 na w i t h maxima a t 5q6 and 56q r~ and bandwidths o f 27+2 na. B a r e l y d i s t i n g u i s h a b l e in the f i t of the t r t m e r a b s o r p t i o n spectrum i s another s e t o f bands w i t h peaks a t 56B and 568 urn. T h e i r t o t a l c o n t r i b u t i o n t o the t r i m e r absorption spectrum i s almost w i t h i n the e r r o r of the f i t . On t h e o t h e r hand, t h e hexamer a b s o r p t i o n and CD s p e c t r a show t h a t t h e c o n t r i b u t i o n from t h i s low e n e r ~ p a i r o f t r a n s i t i o n s i s s i g n i f i c a n t . It c o n t i n u e s t o i n c r e a s e as a g g r e g a t i o n p r o c e e d s ; t h e dodecumer a b s o r p t i o n and CD s p e c t r a a r e dominated by t h i s l o n g - w a v e l e n E t h p a i r . At t h e same t i m e , t h e g r a d u a l and c o n c o m i t a n t d e c l i n e i n t h e c o n t r i b u t i o n from t h e p a i r o f bands c e n t e r e d around 555 nm i s e v i d e n t from t h e r e s o l u t i o n s . Whereas t h e b i s i g n a t e bands i n t h e CD s p e c t r a a r e c l o s e t o b e i n g c o n s e r v a t i v e , i t i s i n t e r e s t i n g t o n o t e t h a t t h e two low e n e r g y compon e n t s , r e p r e s e n t i n g a chrCmophore direst i n t h e l o n g - w a v e l e n g t h r e E i o n o f t h e s p e c t r a ( c e n t e r e d around 56B-56q nm), i n c r e a s e i n i n t e n s i t y p r i m a r i l y a t t h e expense o f t h e low e n e r g y (56B am) t r a n s i t i o n o f t h e chrcmophore dimer whose a b s o r p t i o n i s c e n t e r e d around 555 nm. I n t er m s o f r o d a s s e m b l y , t h i s i m p l i e s t h a t t h e same ehromophores t a k e p a r t i n b o t h i n t e r a c t i o n s , t h e l o n g - w a v e l e n g t h d im er e m e r g i n g a s a r e s u l t o f i n t e r a c t i o n e i t h e r a t t h e i n t e r f a c e o f two p r o x i m a l d i s k s o r due t o t h e change i n t h e l o c a l c o n c e n t r a t i o n and o r i e n t a t i o n o f chrcmophores w i t h i n t h e p r o t e i n . I t i s r e a d i l y s e e n t h a t w h i l e t h e c o n t r i b u t i o n from t h e broad band i s c o n s t a n t , t h a t o f t h e s h o r t - w a v e l e n g t h p a i r o f bands c e n t e r e d a b o u t 555 nm decreases. C o n c u r r e n t l y , the. l o n g - w a v e l e n g t h p a i r o f chrcmophore t r a n s i tions increases dramatically in intensity. F i g u r e 2 shows a r e p r e s e n t a t i o n o f t h e change i n a r e a under t h e b r o ad band a s w e l l a s under t h e s h o r t - and l o n g - w a v e l e n g t h p a i r o f bands as a f ~ m c t i o n o f a g g r e g a t e s i z e , i.e~, t h e nunber o f p h y c o e r y t h r o b l l i n ehromophores i n each a g ~ r e ~ t e . A l i n e a r r e g r e s s i o n a n a l y s i s f o r each of the s e t s o f t h r e e d a t a P o i n t s , r e p r e s e n t i n g t h e change i n a r e a under t h e a b s o r p t i o n bands a t t r i b u t e d t o t h e s h o r t - and l o n g - ~ a v e l e n g t h ehromophore d i a e r , g i v e s s l o p e s o f - 0 ~ 6 and 0 ~ 3 , r e s p e c t i v e l y , s u b s t a n t i a t i n g t h e co m p l e m e n t a r y r e l a t i o n s h i p between t h e two c ~ o m o p h o r e i n t e r a c t i o n s . In a c o m p a r a b l e p r e s e n t a t i o n o f t h e CD d a t a , t h e t r e n d i s s i m i l a r w i t h t h a t f o r a b s o r p t i o n . However, due t o t h e d i m t h i s h e d o p t i c a l a c t i v i t y o f t h e s e n s i t i z i n g chrcmophore band i n t h e dodecamer, t h e r e l a t i v e magnitude o f t h e e x c i t o n bands i s e x a g e r r a t e d and c o n s e q u e n t l y t h e a b s o r p t i o n and CD d a t a quantitatively do n o t c o f n o i d e .
I671
213 60
~
o
~
~
x
x
4o.
~ao.
L NUMBER OF CHROMOPHORES
Fibre 2.
Relative areas of the absorption band at 525 r~ (x), the short-
wavelength pair of bands of 27+_2 n m b a l f - w l d t h (s), and the longwaveler~th pair of bands of 17±2 nm hal f-wldth (L), plotted as a funQtlon of the number of chromophores in the three PE aggreEates. A curious feature of the complementary behavior of the two pairs of exolton interactions is the chanEe in the spectral half-bandwidth parameter. The broad, presumably non-lnteractlng, band is approximately 45 tea in width, whereas the half-wldths of the dlmer bands are 27±2 nm and 17+_3 nm respeotlvely. This series of values is olose to that which is expeoted upon chromophore-ohromophore Interaotion where the half-wldths decrease by a factor of I/~-M, M being the number of ohrumophores taking part in the interaotlon [4]. The faot that this relationship holds for the chromophore-ohrumophore interaetlons in PE means that the chromophore confIEur"atlon responsible for the lonE-wavelenEth split may either be a quatromer or that the disappearing 564 n m b a n d of the short-wavelength dlmer interaotion aoquired in part propePtles of a monomer. This would imply that the excited state is not shared equally between the interactlng ohromophores. Suoh an idea has been invoked as a posslbillty to explain the behavior of the 870 nm band in baoterloohlorophy11 [5] based on dlfferenoes in the "environmental interactions" which "reduoe the amount of mixing of the exoited states localized on slngle moleoules". The 545 band retains a oonslderable portion of its intensity; therefore, it may well be less a fleeted by the attaohment of another disk of PE. In terms of transfer of electronio exoitation enerEy, a oasoade of energy levels among ehrnmophores of preoise spatial orEanlzatlon ensures a direotlonal flow toward ever longer wavelenEth ohromophores. The ohromophores with short-wavelength absorption bands sensitize the fluoresoence of those absorbing at longer waveler~ths, and thus, the terms "sensitizing" ('s ~) and "fluorescent" ('f~) ohromophores were introduoed by Teale and Dale [8]. The wide, 525 um band absorption in PE may therefore be attributed to sensitizing ohrnmophores. Sines its contribution to the total absorption is constant regardless of the state of aEgreEation, the amount of light energy harvested inoreases linearly with the number of disks in the PE rod oonstituents of the PBsome.
214
[68]
The Larger the Pod, however, the further away the sensitizing chrc~ophores will be from PC (the next pigment in the energy transfer sequence). The same is true fop the fluorescent ehromophores with transitions at longer wavelengths. Upon assembly of two trimers into a hexamer, the low energy exciton split becomes conspicuous. Were this the result of exciton interaction at the interface of the two trimer disks assembled into a hexamer, there would be little evolutionary advantage in assembling even larger aggregates because the intradisk transfer of electronic excitation energy quickly ends up on the lowest energy exciton band via resonance transfer and internal conversion from the higher energy exciton band. Subsequent i n t e ~ i s k transfer would necessitate uphill transfer to the next layer of sensitizing chremophores, in effect abolishing any directional flow of excitation energy toward the core of the PBseme. However, if the lowest energy level is distributed within the aggregate or spans across the stack of disks, then the disadvantage stemming from increased distances within the light-harvesting apparatus is balanced by an increase in the spectral overlap between the fluorescence from the lowest, 572 nm exoiton level at 580 nm, and the absorption band of the sensitizing phycoeyanobilin chremophore at 586-600 ran. In fact, in the dodecemer, at least 35% of the total absorption of PE is due to the chromophores participating in the long-wavelength exciton interaction centered around 561 nm; that is, about 20-24 chromophcres are able to create a conduit for delooalized excitation. The two-fold narrowing of the spectral bands already indicates that the excitation energy is delooalized, albeit only partially, over at least four chromophores. In PBsemes of Syneohooystis 6701, it was shown [2] that compared to a fast excitation transfer between sensitizing and fluorescent chremophoPes within the disk, disk-to-disk transfer is the rate-limiting step in PBsome Pods. In the scheme of chromophore-ohromophore interactions discussed above, symmetry considerations would warrant that if at least four chromophores interact as the disks are assembled into a hexamer and subsequently into a rod and if there is no total mixing of excited states, internal conversion to the lowest level will not always coincide with the vectorial transfer toward one particular end of the Pod. In an in rive assembly involving dodecameric PE, this would result in a slowdown of exoiton transfer across the disk interfaces. This slowdown may not be critical since recently [5] time-resolved fluorescence spectra of chromatically adapted Tolypothrix tenuis showed that excitation energy is transferred faster in PE-Pich, rE-excited PBsomes than in PBsomes whose Pods contained only PC [6]. The difference was attributed to slower transfer among the "f" chromophoPes of PC in the rE-less system [6], and we suggest that it may be due to the fact that the delocalized excitation on interacting chromophores in PE is able to span greater distances with fewer transfer steps than would be involved in transfer via fluorescent non-interacting chPemophoPes as is the case fop PC. With the advent of tunable dye-lasers in picosecond spectroscopy, these ideas may be tested since individual transitions can be excited and the fate of excitation monitored on the timeseale of chremophore-chromophore transfers (see, fop example, reference [9]). Internal conversion between exciton components should give faster fluorescence rise-times than "s" to "f" transfer. In addition, x-Pay crystallographic analysis has already yielded information concerning possible chremophcre arrangements in PC [7], and work on the other biliproteins is sure to follow.
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215
ACKNOWLEDGMENTS New Jerse 7 Agricultural Experiment Station, Publication No. D-011043-85, supported in part by State funds and by the United States Hatch Act. This work was also supported by the Science and Education Administration of the United States Department of Agriculture under Grant 85-CRCR-I-1562 from the Competitive Research Grants Office. We are grateful to Dr. Peter Kahn and Dr. Jozef Grabowski for helpful discussions. R~ERENCES I. Csatorday K, MacColl R, Csizmadia V, Grabowski J and Bagyinka C (1984) Biochemistry 23:6466-6470 2. Glazer AN, Yeh SW, Webb SP and Clark JH (1985) Science 227:419-423 3. Glick RE and Zilinskas BA (1982) Plant Physiol 69:991-997 4. Hemenger RP (1977) J Chem Phys 67:262-264 5. Mar T and Gingras G (1984) Biochim Biophys AcOa 765:125-132 6. Mimuro M, Yamazaki I, Yamazaki T and FuJita Y (1985) Photoohem Photcbicl 41:597-603 7. Schirmer T, Bode W, Huber R, Sidler W and Zuber H (1985) J Mol Bicl 184:257-277 8. Teale FWJ and Dale RE (1970) Biochem J 116:161-169 9. Wehrmeyer W, Wendler J and Holzwarth AR (1985) Eur J Cell Biol 36: 17-23 10. Zilinskas BA, Campbell S and Grabowski J (1984) In: Advances in Photosynthesis Research, (Sybesma,C,ed.) Vol. II, pp. 687-690, Martinus NiJhoff Dr. W. Junk Publishers, the Hague 11. Zilinskas BA and Greenwald LS (1985) Photosynthesis Res: in press 12. Zilinskas BA and Howell DA (1983) Plant Physiol 71:379-387
209
EXCITON INTERACTIONS IN PHYCOERYTHRIN
KAROLY CSATORDAY, SHARON CAMPBELL, AND BARBARA A. ZILINSKAS Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, NJ 08903, U.S.A.
ABSTRACT Upon assembly of the phycoerythrin trimer into hexamer and the hexamer into dodecamer, marked spectral changes are observed. The absorption and circular dichroism spectra of the various phycoerythrin aggregates were resolved into Gaussian components representing individual electronic transitions of phycoerythrobilin chromophoreswlthin these proteins. While the contribution of a broad, sensitizing band (at 525 nm) is constant, with increasing aggregate size, a short-wavelength pair of bands centered at 555 r~m decreases concomitantly with a dramatic increase in the intensity of a lonE-wavelength pair of ehromophore transitions centered at 563 rmL The implications of these spectral changes for efficient energy transfer in the phycobilisome are discussed. INTRODUCTION Light energy is harvested in the cyanobacterla and red algae by speclal multlprotein aggregates called phycobillsomes (PBscmes). These contain phycobillprotelns that absorb radiation and transfer its energy in the form of electronic excitation to the photochemical reaction center. A complete transfer sequence involves phyeoerythrln (PE), phycocyanln (PC) and allophycocyanln (APC) [11]. Since this hlghly efficient transfer is dependent on the distance between donor and accepter molecules, as well as on the spectral overlap between their respective emission and absorption spectra, the spectral properties as well as the posslbillty of their delegation to particular chremophore transitions are of interest. Chromophores with short-wavelength absorption bands sensitize the fluorescence of those absorbing at longer wavelengths, and thus, the terms "sensitizing" ('s') and "fluorescent" ('f') chr~zzophores were introduced by Teale and Dale (8). In the present study, we attempt to correlate spectral characteristics of PE with individual electronic transitions of chromophores within the protein. Phycoerythrln in vlvo is assembled into so-called rods that serve as constituents of the llght-harvestlng PBsome. Within the rods of the cyanobacteri~-,, Nostoc sp., grown in cool-whlte fluorescent light, PE exists as a stack of four disks each consisting of a PE trimer (~B)~Each trlmer contains 15 phycoerythrobilln chr~mophores. The four tflmers comprising a dodecamer of PE are attached to a PC hexamer, also in the form of a double disk. The assembly is governed and facilltated by linker proteins that determine the final structure of the PBsome [12]. The spectral properties of isolated PE ngg~-egates closely resemble those they manifest within the PBsome [10], thus allowlng the study of electronlc transitions in a simpler system.
[6~]
[64]
210
MATERIALS AND METHODS Phycoerythrln in the trimeric and hexameric forms were isolated from PBsomes of the cyanobacterium Nostoc sp. as described earlier [12]. Subsequent to dialysis of the PE hexamer against 0.75M K-phosphate, pH 7.0, the PE dodecamer was obtained by sedimentation in linear gradients of sucrose (0.15M-0.8M) containing 0.75M K-phosphate, pH 7.0, for 16h at 40,000 rpm in a SW40 Beckman rotor. The PE trimer and hexamer each contained approximately 5% PC, and the dodecamer contained approximately 7% PC; however, the PC was not physically associated with PE as demonstrated by the absence of fluorescence emission from PC fol lowir~ PE excitation. The curve-fitting analysis included a component to account for the contribution of the contaminating PC. Aggregate size and polypeptide composition were determined respectively by sedimentation in linear gradients of sucrose and by sodium dodecyl sulfate polyacrylamide gel electrophoresis as described earlier [12]. Absorption and circular dichroism (CD) were measured as previously described [13] wlth a Cary 17D and a Cary 60 spectrometer, respectively. The circular dichrolsm and absorption spectra were resolved into Gausslan components using a curve-fitting program written for the Apple II computer [I] with the blue edge of the spectra approximated by a wide bandwidth chimeric component attributed to sensitizing chromophores. RESULTS AND DISCUSSION The absorption and CD spectra of PE trimers, hexamers and dodecamers were resolved into Gaussian components representing individual electronic transitions of phycoerythrobilin chrcmophores within these protein aggregates. Figure I (left panel) shows the computer-generated fits of both 1.0
TRIMER
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I
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1.0 4EXAMER z
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DODECAMER
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0.5 ....., .
-0.5 I I
450
I
5OO ~ WAVELENGTH (nnl)
:"
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FIGURE I. Left panel. Absorption and CD spectra of PE aggregates. The experlmental and fitted curves are plotted with dots and crosses respectlvely. Only every third point in the fitted curve is shown for clarity. The absorption spectra were normalized for unit area. FIGURE I. Right panel. The resolved components of the absorption and CD spectra of the three PE aggregates.
t h e a b s o r p t i o n and CD s p e o t r a f o r eaoh o f t h e a g g r e g a t e s . Only t h e e x p e r i m o n t a l om~re and t h e sun o f t h e o a l o u l a t e d oomponenta a r e shown f o r o l a r t t y and t o d e m o n s t r a t e t h e e r r o r o f t h e f i t . The e v o l u t i o n o f a band i n b o t h t h e a b s o r p t i o n and CD s p e o t r a a t 570 em As s e e n . A l s o , t h e n e g a t t v ~ b a n d i n t h e CD speotrum i s s h i f t e d toward the b l u e as a f~notton o f ngg~egate s i z e . These ohanges h a v e p r e v i o u s l y been i n t e r p r e t e d i n t e r m s o f o h ~ m o p h o r e - o h r ~ m o p h o r e i n t e ~ a o t t o n s [10]. A reasonably olosenatohwas found between t h e a b s o r p t i o n and CD o c ~ p o n e n t s with regard to the essential parameters of peakwavelengthand halfbandwidth ( s e e T a b l e I ) . F i g u r e I ( r i g h t p a n e l ) shows t h e i n d i v i d u a l oomponent bands a n d a l l o w s a q u a n t i t a t i v e t r e a t m e n t o f t h e s e ohanges. The r e s o l v e d oomponents a r e p l o t t e d on t h e same s o a l e as t h e e x p e r i m e n t a l and f i t t e d o u r v e s i n Fig. 1 (left). The c o r r e s p o n d i n g s p e o t r a l p a r a n e t e r s a r e g i v e n below. TABLE 1. S p e o t r a l p a r a m e t e r s o f t h e r e s o l v e d o o n p o n e n t s o f t h e p h y o o e r y t h r i n a b s o r p t i o n and o t r o u l a r d t o h r o i o m s p e o t r a .
Abs
L 524 W ~5 A .50
5~6 29 .25
56~ 28 .21
563 17 .02
568 16 .02
CD
L 525 W 43 A .~8
5~7 25 .19
56~ 25 .23
563 17 .05
57~ 17 .05
Abs
L 526 W ~5 A .51
5~7 28 .27
562 28 .05
558 17 .08
570 16 .12
CD
L 524 W 42 A .38
5~8 26 .23
563 25 .17
562 16 .11
570 17 .12
Abs
L 527 W ~9 A .51
5~4 26 .14
563 26 .02
554 21 .14
572 18 .20
600 66
E=2.5~
Triter E=6.1~
593 66
E=1.8~
Hsxomor
E=7.8~ 601 58
B=2.61l
Dodeoamer
L 517
548 56~ 553 568 26 28 20 14 E=I0.75 .13 .16 .26 .25 L=wavelength maximum, W=half-band~rldl;h, A=area under the oomponent ourve CD
W ~9 A .19
a s t h e p e r ~ o n t n g e o f t o t a l a r e a , and E = e r r o r o f t h e f i t . All numerloal d a t a a r e rounded t o t h e l a s t d i g i t shown. The oemponent a t "600nn i s a t t r i b u t e d t o e o n t a n t n a t t n g PC. N o r m a l i z i n g t h e a b s o r p t i o n s p e o t r a w i t h r e s p e o t t o t h e a r e a under t h e s p e o t r a l o u r v e a l l o w s t h e i r ocmparison b ased on ohanges i n t h e r e l a t i v e a r e a s u n d e r eaoh oomponent o r e ' r e , a q u a n t i t y p r o p o r t i o n a l t o t h e o s c i l lator strength of a given transition. All the absorption spectra feature a b r o s d ~5 un wide band i n t h e h£Kh e n e r g y r~Kton. T h i s o~etponon~, oenter,Dd around 525 nm, r e m a i n s o o n s t a n t i n a r e a , o o m p r i s t n g a b o u t 50~ o f the t o t a l absorbanoe. The o o r r e s p o n d i n g band i n t h e CD s p e o t r a has t h e s a n e f e a t u r e s w i t h t h e e x o e p t i o n t h a t i n g o i n g from t h e t r i m e r t o t h e dodeoamer i t d e o r e a s e s i n I n t e n s i t y and i s b l u e - s h i f t e d t o 517 nm. I n £ a e t , f i t t i n g two wide o h i m e r i o o~mponents a t 513 and 532 nm, i n s t e a d o f
212
[661
t h e one a t 525 mR, b e t t e r a o o o u n t e d f o r t h e CD f e a t u r e s i n t h e dodeoamer s p e c t r u m b u t d~d n o t ehanEe t h e t o t a l a r e a a s c r i b e d t o ~he h i g h e n e r g y band n o r d i d i t s i g n i f i c a n t l y i n f l u e n c e t h e p a r a m e t e r s o r t h e d i s c u s s i o n which f o l l o w s f o r t h e two e x o i t o n band p a i r s . A p p l y i n g Oecam'a r a z o r , we r e s t r i c t e d t h e r e s o l u t i o n s t o one component i n t h i s r e g i o n a l t h o u g h two wide bands r e p r e s e n t i n g N n a i t i n t n g chrcmophores may be p r e s e n t . A l t h o u g h t h e r e s u l t s d i s c u s s e d i n t h i s p a p e r p e r t a i n s o l e l y t o PE a ~ r e g a t e s , we c o n s i d e r t h e c o n c l u s i o n s r e l e v a n t t o t h e n a t i v e PBsome s i n c e a p a o t r o s Q o p i e a l l y t h e a x e i t o n f e a t u r e s a r e e s s e n t i a l l y t h e same f o r t h e dodecamer o f EE, t h e i s o l a t e d PBsome r o d which i s co m p r i sed o f a PE dodeeamer a s s o c i a t e d w i t h a PC hexamer, and t h e i s o l a t e d PBsome. The o n l y d i f f e r e n c e s e e n i n comparing t h e PE dodecamer w i t h t h e PBsome o r t h e PBsoma rod i s t h e d i m i n i s h e d h i g h e n e r g y CD band a t t r i b u t e d t o s e n s i t i z i n g PE ohromophores. I n t h e PBsome o r i n t h e r o d , t h i s CD band r e m a i n s a t t h e l e v e l o b s e r v e d f o r t h e i s o l a t e d PE t r i m e r . There may be a s l i g h t l y d i f f e r e n t c o n f o r m a t i o n o f t h e s e n s i t i z i n g ohromophores i n t h e i s o l a t e d PE dodecamer which a f f e c t s i t s o p t i c a l a c t i v i t y b u t n o t i t s a b s o r p t i o n . The t r i m e r s p e c t r u m a l s o e n v e l o p s a p a i r o f bands c e n t e r e d around 555 na w i t h maxima a t 5q6 and 56q r~ and bandwidths o f 27+2 na. B a r e l y d i s t i n g u i s h a b l e in the f i t of the t r t m e r a b s o r p t i o n spectrum i s another s e t o f bands w i t h peaks a t 56B and 568 urn. T h e i r t o t a l c o n t r i b u t i o n t o the t r i m e r absorption spectrum i s almost w i t h i n the e r r o r of the f i t . On t h e o t h e r hand, t h e hexamer a b s o r p t i o n and CD s p e c t r a show t h a t t h e c o n t r i b u t i o n from t h i s low e n e r ~ p a i r o f t r a n s i t i o n s i s s i g n i f i c a n t . It c o n t i n u e s t o i n c r e a s e as a g g r e g a t i o n p r o c e e d s ; t h e dodecumer a b s o r p t i o n and CD s p e c t r a a r e dominated by t h i s l o n g - w a v e l e n E t h p a i r . At t h e same t i m e , t h e g r a d u a l and c o n c o m i t a n t d e c l i n e i n t h e c o n t r i b u t i o n from t h e p a i r o f bands c e n t e r e d around 555 nm i s e v i d e n t from t h e r e s o l u t i o n s . Whereas t h e b i s i g n a t e bands i n t h e CD s p e c t r a a r e c l o s e t o b e i n g c o n s e r v a t i v e , i t i s i n t e r e s t i n g t o n o t e t h a t t h e two low e n e r g y compon e n t s , r e p r e s e n t i n g a chrCmophore direst i n t h e l o n g - w a v e l e n g t h r e E i o n o f t h e s p e c t r a ( c e n t e r e d around 56B-56q nm), i n c r e a s e i n i n t e n s i t y p r i m a r i l y a t t h e expense o f t h e low e n e r g y (56B am) t r a n s i t i o n o f t h e chrcmophore dimer whose a b s o r p t i o n i s c e n t e r e d around 555 nm. I n t er m s o f r o d a s s e m b l y , t h i s i m p l i e s t h a t t h e same ehromophores t a k e p a r t i n b o t h i n t e r a c t i o n s , t h e l o n g - w a v e l e n g t h d im er e m e r g i n g a s a r e s u l t o f i n t e r a c t i o n e i t h e r a t t h e i n t e r f a c e o f two p r o x i m a l d i s k s o r due t o t h e change i n t h e l o c a l c o n c e n t r a t i o n and o r i e n t a t i o n o f chrcmophores w i t h i n t h e p r o t e i n . I t i s r e a d i l y s e e n t h a t w h i l e t h e c o n t r i b u t i o n from t h e broad band i s c o n s t a n t , t h a t o f t h e s h o r t - w a v e l e n g t h p a i r o f bands c e n t e r e d a b o u t 555 nm decreases. C o n c u r r e n t l y , the. l o n g - w a v e l e n g t h p a i r o f chrcmophore t r a n s i tions increases dramatically in intensity. F i g u r e 2 shows a r e p r e s e n t a t i o n o f t h e change i n a r e a under t h e b r o ad band a s w e l l a s under t h e s h o r t - and l o n g - w a v e l e n g t h p a i r o f bands as a f ~ m c t i o n o f a g g r e g a t e s i z e , i.e~, t h e nunber o f p h y c o e r y t h r o b l l i n ehromophores i n each a g ~ r e ~ t e . A l i n e a r r e g r e s s i o n a n a l y s i s f o r each of the s e t s o f t h r e e d a t a P o i n t s , r e p r e s e n t i n g t h e change i n a r e a under t h e a b s o r p t i o n bands a t t r i b u t e d t o t h e s h o r t - and l o n g - ~ a v e l e n g t h ehromophore d i a e r , g i v e s s l o p e s o f - 0 ~ 6 and 0 ~ 3 , r e s p e c t i v e l y , s u b s t a n t i a t i n g t h e co m p l e m e n t a r y r e l a t i o n s h i p between t h e two c ~ o m o p h o r e i n t e r a c t i o n s . In a c o m p a r a b l e p r e s e n t a t i o n o f t h e CD d a t a , t h e t r e n d i s s i m i l a r w i t h t h a t f o r a b s o r p t i o n . However, due t o t h e d i m t h i s h e d o p t i c a l a c t i v i t y o f t h e s e n s i t i z i n g chrcmophore band i n t h e dodecamer, t h e r e l a t i v e magnitude o f t h e e x c i t o n bands i s e x a g e r r a t e d and c o n s e q u e n t l y t h e a b s o r p t i o n and CD d a t a quantitatively do n o t c o f n o i d e .
I671
213 60
~
o
~
~
x
x
4o.
~ao.
L NUMBER OF CHROMOPHORES
Fibre 2.
Relative areas of the absorption band at 525 r~ (x), the short-
wavelength pair of bands of 27+_2 n m b a l f - w l d t h (s), and the longwaveler~th pair of bands of 17±2 nm hal f-wldth (L), plotted as a funQtlon of the number of chromophores in the three PE aggreEates. A curious feature of the complementary behavior of the two pairs of exolton interactions is the chanEe in the spectral half-bandwidth parameter. The broad, presumably non-lnteractlng, band is approximately 45 tea in width, whereas the half-wldths of the dlmer bands are 27±2 nm and 17+_3 nm respeotlvely. This series of values is olose to that which is expeoted upon chromophore-ohromophore Interaotion where the half-wldths decrease by a factor of I/~-M, M being the number of ohrumophores taking part in the interaotlon [4]. The faot that this relationship holds for the chromophore-ohrumophore interaetlons in PE means that the chromophore confIEur"atlon responsible for the lonE-wavelenEth split may either be a quatromer or that the disappearing 564 n m b a n d of the short-wavelength dlmer interaotion aoquired in part propePtles of a monomer. This would imply that the excited state is not shared equally between the interactlng ohromophores. Suoh an idea has been invoked as a posslbillty to explain the behavior of the 870 nm band in baoterloohlorophy11 [5] based on dlfferenoes in the "environmental interactions" which "reduoe the amount of mixing of the exoited states localized on slngle moleoules". The 545 band retains a oonslderable portion of its intensity; therefore, it may well be less a fleeted by the attaohment of another disk of PE. In terms of transfer of electronio exoitation enerEy, a oasoade of energy levels among ehrnmophores of preoise spatial orEanlzatlon ensures a direotlonal flow toward ever longer wavelenEth ohromophores. The ohromophores with short-wavelength absorption bands sensitize the fluoresoence of those absorbing at longer waveler~ths, and thus, the terms "sensitizing" ('s ~) and "fluorescent" ('f~) ohromophores were introduoed by Teale and Dale [8]. The wide, 525 um band absorption in PE may therefore be attributed to sensitizing ohrnmophores. Sines its contribution to the total absorption is constant regardless of the state of aEgreEation, the amount of light energy harvested inoreases linearly with the number of disks in the PE rod oonstituents of the PBsome.
214
[68]
The Larger the Pod, however, the further away the sensitizing chrc~ophores will be from PC (the next pigment in the energy transfer sequence). The same is true fop the fluorescent ehromophores with transitions at longer wavelengths. Upon assembly of two trimers into a hexamer, the low energy exciton split becomes conspicuous. Were this the result of exciton interaction at the interface of the two trimer disks assembled into a hexamer, there would be little evolutionary advantage in assembling even larger aggregates because the intradisk transfer of electronic excitation energy quickly ends up on the lowest energy exciton band via resonance transfer and internal conversion from the higher energy exciton band. Subsequent i n t e ~ i s k transfer would necessitate uphill transfer to the next layer of sensitizing chremophores, in effect abolishing any directional flow of excitation energy toward the core of the PBseme. However, if the lowest energy level is distributed within the aggregate or spans across the stack of disks, then the disadvantage stemming from increased distances within the light-harvesting apparatus is balanced by an increase in the spectral overlap between the fluorescence from the lowest, 572 nm exoiton level at 580 nm, and the absorption band of the sensitizing phycoeyanobilin chremophore at 586-600 ran. In fact, in the dodecemer, at least 35% of the total absorption of PE is due to the chromophores participating in the long-wavelength exciton interaction centered around 561 nm; that is, about 20-24 chromophcres are able to create a conduit for delooalized excitation. The two-fold narrowing of the spectral bands already indicates that the excitation energy is delooalized, albeit only partially, over at least four chromophores. In PBsemes of Syneohooystis 6701, it was shown [2] that compared to a fast excitation transfer between sensitizing and fluorescent chremophoPes within the disk, disk-to-disk transfer is the rate-limiting step in PBsome Pods. In the scheme of chromophore-ohromophore interactions discussed above, symmetry considerations would warrant that if at least four chromophores interact as the disks are assembled into a hexamer and subsequently into a rod and if there is no total mixing of excited states, internal conversion to the lowest level will not always coincide with the vectorial transfer toward one particular end of the Pod. In an in rive assembly involving dodecameric PE, this would result in a slowdown of exoiton transfer across the disk interfaces. This slowdown may not be critical since recently [5] time-resolved fluorescence spectra of chromatically adapted Tolypothrix tenuis showed that excitation energy is transferred faster in PE-Pich, rE-excited PBsomes than in PBsomes whose Pods contained only PC [6]. The difference was attributed to slower transfer among the "f" chromophoPes of PC in the rE-less system [6], and we suggest that it may be due to the fact that the delocalized excitation on interacting chromophores in PE is able to span greater distances with fewer transfer steps than would be involved in transfer via fluorescent non-interacting chPemophoPes as is the case fop PC. With the advent of tunable dye-lasers in picosecond spectroscopy, these ideas may be tested since individual transitions can be excited and the fate of excitation monitored on the timeseale of chremophore-chromophore transfers (see, fop example, reference [9]). Internal conversion between exciton components should give faster fluorescence rise-times than "s" to "f" transfer. In addition, x-Pay crystallographic analysis has already yielded information concerning possible chremophcre arrangements in PC [7], and work on the other biliproteins is sure to follow.
[69]
215
ACKNOWLEDGMENTS New Jerse 7 Agricultural Experiment Station, Publication No. D-011043-85, supported in part by State funds and by the United States Hatch Act. This work was also supported by the Science and Education Administration of the United States Department of Agriculture under Grant 85-CRCR-I-1562 from the Competitive Research Grants Office. We are grateful to Dr. Peter Kahn and Dr. Jozef Grabowski for helpful discussions. R~ERENCES I. Csatorday K, MacColl R, Csizmadia V, Grabowski J and Bagyinka C (1984) Biochemistry 23:6466-6470 2. Glazer AN, Yeh SW, Webb SP and Clark JH (1985) Science 227:419-423 3. Glick RE and Zilinskas BA (1982) Plant Physiol 69:991-997 4. Hemenger RP (1977) J Chem Phys 67:262-264 5. Mar T and Gingras G (1984) Biochim Biophys AcOa 765:125-132 6. Mimuro M, Yamazaki I, Yamazaki T and FuJita Y (1985) Photoohem Photcbicl 41:597-603 7. Schirmer T, Bode W, Huber R, Sidler W and Zuber H (1985) J Mol Bicl 184:257-277 8. Teale FWJ and Dale RE (1970) Biochem J 116:161-169 9. Wehrmeyer W, Wendler J and Holzwarth AR (1985) Eur J Cell Biol 36: 17-23 10. Zilinskas BA, Campbell S and Grabowski J (1984) In: Advances in Photosynthesis Research, (Sybesma,C,ed.) Vol. II, pp. 687-690, Martinus NiJhoff Dr. W. Junk Publishers, the Hague 11. Zilinskas BA and Greenwald LS (1985) Photosynthesis Res: in press 12. Zilinskas BA and Howell DA (1983) Plant Physiol 71:379-387
