Light harvesting using photonic crystals for photovoltaic applications
Photosynthetic Light-Harvesting SystemsPhotosynthetic Light-Harvesting Systems Organization and...
Transcript of Photosynthetic Light-Harvesting SystemsPhotosynthetic Light-Harvesting Systems Organization and...
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Photosynthetic Light-Harvesting Systems Organization and Function Proceedings of an International Workshop October 12-16,1987 Freising, Fed Rep. of Germany Editors Hugo Scheer · Siegfried Schneider
W DE G
Walter de Gruyter · Berlin · New York 1988
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CONTENTS
L i s t of P a r t i c i p a n t s X I I I
SECTION I . ORGANIZATION: BIOCHEMICAL METHODS
I n t r o d u c t i o n : The Biochemistry of Light-Harvesting Complexes by R.J. Cogdell 1
Phycobilisome-Thylakoid I n t e r a c t i o n : The Nature of High Molecular Weight Polypeptides by E. Gantt C.A. L i p s c h u l t z and F.X. Cunningham Jr 11
On the S t r u c t u r e of Photosystem II-Phycobilisome Complexes of Cyanobacteria by E. Mörschel and G.-H. Schatz 21
Stru c t u r e of Cryptophyte Photosynthetic Membranes by W. Wehrmeyer 35
S t r u c t u r a l and Phylogenetic Relationships of Phycoerythrins from Cyanobacteria, Red Algae and Cryptophyceae by W. S i d l e r and H. Zuber 49
I s o l a t i o n and C h a r a c t e r i z a t i o n of the Components of the Phycobilisome from Mastigocladus laminosus and Cross-l i n k i n g Experiments by R. Rümbeli and H. Zuber 61
C-Phycocyanin from Mastigocladus laminosus: Chromophore Assignment i n Higher Aggregates by Cystein M o d i f i c a t i o n by R. Fischer, S. Siebzehnrlibl and H. Scheer 71
Photochromic Prope r t i e s of C-Phycocyanin by G. Schmidt, S. Siebzehnrübl, R. Fischer and H. Scheer 77
Concerning the Relat i o n s h i p of L i g h t Harvesting B i l i -p r o t e i n s t o Phycochromes i n Cyanobacteria by W. Kufer 89
Subunit S t r u c t u r e and Reassembly of the Light-Harvesting Complex from Rhodospiri Hum rubrum G9+ by R. Ghosh, ThT~Rosatzin ancTR. Baclofen 93
Primary S t r u c t u r e Analyses of B a c t e r i a l Antenna Polypeptides - C o r r e l a t i o n of Aromatic Amino Acids w i t h Spectral Properties - S t r u c t u r a l S i m i l a r i t i e s w i t h Reaction Center Polypeptides by R.A. Brunisholz and H. Zuber 103
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VIII
The S t r u c t u r e o f the "Core1* of the Purple B a c t e r i a l Photos y n t h e t i c Unit by D.J. Dawkins, L.A. Ferguson and R.J. Cogdell 115
A Comparison o f the Ba c t e r i o c h l o r o p h y l 1 C--Binding Proteins of Chlorobium and Chloroflexus by P.D. Gerola, P. Hpjrup and J.M. Olson 129
I n t e r a c t i o n s between B a c t e r i o c h l o r o p h y l 1 c Molecules i n Oligomers and i n Chlorosomes of Green Photosynthetic Bacteria by D.C. Brune, G.H. King and R.E. Blankenship 141
Ligh t - H a r v e s t i n g Complexes of Chlorophyll c-Containing Algae by A.W.D. Larkum and R.G. H i l l e r 153
I s o l a t i o n and C h a r a c t e r i z a t i o n of a Chloro p h y l l a/c-Hetero-xanthin/Diadinoxanthin Light-Harvesting Complex from P l e u r o c h l o r i s meiringensis (Xanthophyceae) by C. Wilhelm, C. Bliche I and B. Rousseau 167
The Antenna Components of Photosystem I I w i t h Emphasis on the Major Pigment-Protein, LHC l i b by G.F. Peter and P. Thornber 175
SECTION I I : ORGANIZATION: MOLECULAR GENETICS AND CRYSTALLOGRAPHY
Molecular Biology of Antennas by G. Drews 187
High-Resolution C r y s t a l S t r u c t u r e of C-Phycocyanin and Polarized O p t i c a l Spectra of Single C r y s t a l s by T. Schirmer, W. Bode and R. Huber 195
C r y s t a l l i z a t i o n and Spectroscopic I n v e s t i g a t i o n of Purple B a c t e r i a l B800-850 and RC-B875 Complexes by W. Welte, Τ. Wacker and A. Becker 201
S t r u c t u r e of the Light-Harvesting Chlorophyll a/b-Protein Complex from Chloroplast Membranes by W. Kühlbrandt 211
PhycobiIi somes of Synchechococcus Sp. PCC 7002, Pseudanabaena Sp. PCC 7409, and Cyanopnora paracioxa: An "Analysis by" HoTecular Genetics by D.A. Bryant 217
Organization and Assembly of B a c t e r i a l Antenna Complexes by G. Drews 233
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IX
The Use of Mutants t o Inv e s t i g a t e the Organization of the Photosynthetic Apparatus of Rhodobacter sphaeroides by C.N. Hunter and R. van Grondel l e 77.77 247
Mechanisms of P l a s t i d and Nuclear Gene Expression During Thylakoid Membrane Biogenesis i n Higher Plants by P. Westhoff, Η. Grüne, Η. Schrubar, Α. Oswald, Μ. Streubel, U. Ljungberg and R.G. Herrmann 261
SECTION I I I : ORGANIZATION: SPECIAL SPECTROSCOPY TECHNIQUES AND MODELS
Assigment o f Spectral Forms i n the Photosynthetic Antennas to Chemically Defined Chromophores by A. Scherz 277
Linear Dichroism and Or i e n t a t i o n of Pigments i n Phycobilisomes and t h e i r Subunits by L. Juszcak, N.E. Geacintov, B.A. Z i l i n s k a s and J. Breton 281
Low Temperature Spectroscopy of Cyanobacteria! Antenna Pigments by W. Köhler, J. F r i e d r i c h , R. Fischer and H. Scheer 293
Chromophore Conformations i n Phycocyanin and Allophycocyanin as Studied by Resonance Raman Spectroscopy by B. S z a l o n t a i , V. Csizmadia, Z. Gombos, K. Csatorday and M. Lutz 307
Coherent Anti-Stokes Raman Spectroscopy of Phycobi1isomes, Phycocyanin and Allophycocyanin from Mastigocladus laminosus by S. Schneider, F. Baumann, W. Steiner, R. Fischer, S. Siebzehnrlibl and H. Scheer 317
Op t i c a l Absorption and C i r c u l a r Dichroism of Bacteriochlorophyl1 Oligomers i n T r i t o n X-100 and i n the Light-Harvesting-Complex B850; A Comparative Study by V. Rozenbach-Belkin, P. Braun, P. Kovatch and A.Scherz 323
Absorption Detected Magnetic Resonance i n Zero Magnetic F i e l d on Antenna Complexes from Rps. acidophila 7050 - The Temperature Dependence of tfie CarotenoTiTTrTpTet State Prop e r t i e s by J. U l l r i c h , J.U. v. Schütz and H.C. Wolf 339
E f f e c t o f Li t h i u m Dodecyl S u l f a t e on Β 800-850 Antenna Complexes from Rhodopseudomonas acidophila: A Resonance Raman Study by B. Robert and H. Frank 349
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χ
B a c t e r i o c h l o r o p h y l 1 a/b i n Antenna Complexes of Purple Bacteria by Β. Robert, A. Vermeglio, R. Steiner, Η. Scheer and Μ. Lutz 355
B a c t e r i o c h l o r o p h y l l c Aggregates i n Carbon T e t r a c h l o r i d e as Models f o r Chlo r o p h y l l Organization i n Green Photos y n t h e t i c B acteria by J.M. Olson and J.P. Pedersen 365
O r i e n t a t i o n o f t he Pigments i n the Reaction Center and the Core Antenna o f Photosystem I I by J. Breton, J. Duranton and K. Satoh 375
Non-Linear Absorption Spectroscopy of Antenna Chlorophyll a in Higher Plants by D. Leupold, H. S t i e l and P. Hoffmann 387
SECTION IV: FUNCTION: ELECTRONIC EXCITATION AND ENERGY TRANSFER
E x c i t a t i o n Energy Transfer i n Photosynthesis by R. van Grondelle and V. Sundström 403
Fluorescence Spectroscopy of Allophycocyanin Complexes from Synechococcus 6301 S t r a i n AN112 by P.Maxson, KTTauer and A.N. Glazer 439
Picosecond Energy Transfer K i n e t i c s i n Allophycocyanin Aggregates from Mastigocladus laminosus by E. Bittersmann, W. Reuter, W. Wehrmeyer and A.R. Holzwarth 451
Picosecond Time-Resolved Energy Transfer K i n e t i c s w i t h i n C-Phycocyanin and Allophycocyanin Aggregates by T. G i l l b r o , A. Sandström, V. Sundström, R. Fischer and H. Scheer 457
Energy Transfer i n "Native" and Chemically Modified C-Phycocyanin Trimers and the Constituent Subunits by S. Schneider, P. Gei s e l h a r t , F. Baumann, S. Siebzehnrübl, R. Fischer and Η. Scheer 469
E f f e c t of P r o t e i n Environment and Exc i t o n i c Coupling on the Excited-State Pro p e r t i e s of the Bilinchromophores i n C-Phycocyanin by S. Schneider, Ch. Scharnagl, M. Dürring, T. Schirmer and W. Bode 483
E x c i t a t i o n Energy M i g r a t i o n i n C-Phycocyanin Aggregates I s o l a t e d from Phormidium luridum: P r e d i c t i o n s from the Förster's I n d u c t i v e Resonance Theory by J. Grabowski and G.S. Björn 491
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XI
Energy Tra n s f e r Calculations f o r two C-Phycocyanins Based on Refined X-Ray Crystal S t r u c t u r e Coordinates of Chromophores by K. Sauer and H. Scheer 507
Energy Transfer i n Light-Harvesting Antenna o f Purple B a c t e r i a Studied by Picosecond Spectroscopy by V. Sundström, H. Bergström, Τ. G i l l b r o , R. van Grondelle, W. Westerhuis, R.A. Niederman and R.J. Cogdell 513
E x c i t a t i o n Energy Transfer i n the Light-Harvesting Antenna o f Photosynthetic Purple B a c t e r i a : The Role of the Long-Wave-Length Absorbing Pigment B896 by R. van Grondelle, H. Bergström, V. Sundström, R.J. van Dorssen, M. Vos and C.N. Hunter 519
The Function o f Chlorosomes i n Energy Transfer in Green Photos y n t h e t i c Bacteria by R.J. van Dorssen, M. Vos and J. Amesz 531
Energy Transfer i n Chloroflexus aurantiacus: E f f e c t s of Temperature and TSaerobic^onönHons by B.P. Wittmershaus, D.C. Brune and R.E. Blankenship 543
I n t e r p r e t a t i o n o f Optical Spectra of Bacteriochlorophyl 1 Antenna Complexes by R.M. P e a r l s t e i n 555
Time Resolution and K i n e t i c s o f "F680" a t Low Temperatures i n Spinach Chloroplasts by R. Knox and S. L i n 567
Picosecond Studies of Fluorescence and Absorbance Changes i n Photosystem I I P a r t i c l e s from Synechococcus Sp. by A.R. Holzwarth, G.H. Schatz and H. Brock 579
Analysis of E x c i t a t i o n Energy Transfer i n Thylakoid Membranes by the Time-Resolved Fluorescence Spectra by M. Mimuro 589
V. CONCLUDING REMARKS
Future Problems on Antenna Systems and Summary Remarks
by E. Gantt 601
Author Index 605
Subject Index 609
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LOW TEMPERATURE SPECTROSCOPY OF CYANOBACTERI AL ANTENNA
PIGMENTS
W. Köh le r , J . F r i ed r i ch
Phys ika l isches I n s t i t u t und Bay reu the r I n s t i t u t f ü r Makromolekü l fo rschung ( B I M F ) , Un i ve rs i t ä t B a y r e u t h , D-8580 B a y r e u t h , F . R . G .
R. F ischer , H. Scheer
Botanisches I n s t i t u t , Un i ve rs i t ä t München D-8000 München 19, F . R . G .
A b s t r a c t
D iso rder on a microscopic scale leads to inhomogeneous l ine b roaden ing of
the opt ical spect ra of ch romophores , wh ich p reven ts h igh reso lu t ion spec
t roscopy in a s t r a i g h t f o r w a r d manner . In th is paper emphasis is pu t on
hole b u r n i n g exper iments on C-phycocyan in (PC) and phycobi l isomes (PBS)
of Mast icogladus laminosus. Th is techn ique is capable of reso lv ing the zero
phonon f ine s t r u c t u r e in sp i te of d i s o r d e r . From the measured hole pro f i les
energy t r a n s f e r times w i t h i n a broad f requency range of the phycobi l isome
absorp t ion could be es t imated. From f luorescence l ine na r row ing e x p e r i
ments combined w i t h hole b u r n i n g , deta i ls about the level s t r u c t u r e and
the loss of co r re la t ion in ene rgy t r a n s f e r processes could be e luc ida ted .
From tempera tu re dependent hole f i l l i ng exper iments the d i s t r i b u t i o n of
conformat ional b a r r i e r s of the chromophore at tached p ro te ins could be
measured.
I n t r oduc t i on
In the case of antenna p igments the knowledge of the deta i ls of the elec-
t r o n i c - v i b r a t i o n a l level s t r u c t u r e is a p re requ i s i t e fo r unde rs tand ing the
A b b r e v i a t i o n s : PE p h y c o e r y t h r i n , PC p h y c o c y a n i n , APC a l l ophycocyan in ,
PBS phycobi l isome
Photosynthetic Light-Harvesting Systems © 1988 Walter de Gruyter & Co., Berlin · New York
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energy t r a n s f e r mechanisms a n d , hence, the f u n c t i o n i n g of these p igments
in the pho tosyn the t i c process ( 1 , 2 ) . Spect roscopy at l i qu id Helium tempe
ra tu res can usua l ly p rov ide th is in fo rmat ion in o rgan ic molecular systems.
Th is resu l ts f rom two fac ts : F i r s t , the l i new id ths may na r row dramat ica l ly
as the tempera tu re is l owered , and second, most o f the i n t ens i t y may be
conf ined to the so-cal led 'zero phonon t r a n s i t i o n s ' . Hence, low t e m p e r a t u
res p r i nc i pa l l y allow fo r h i g h l y resolved spect ra w i t h v e r y good s ignal to
noise rat io ( 3 ) .
U n f o r t u n a t e l y , d i so rde r on a microscopic scale p r e v e n t s s t r a i g h t f o r w a r d
h igh reso lu t ion spect roscopy in the p igments s t u d i e d , e . g . PC and PBS
from M. laminosus, because o f the concommitant inhomogeneous l ine b roa
d e n i n g . Inhomogeneous l ine b roaden ing not on ly obscures all the i n f o r
mation conta ined in the homogeneous l ine shape func t i on bu t also wipes out
the v ib ra t iona l pa t t e rn to a h i gh deg ree . Here we wish to r e p o r t how some
of these d rawbacks can be overcome by spect ra l hole b u r n i n g and f l u o r e
scence l ine na r row ing techn iques .
How d i so rde r on a microscopic scale obscures spect roscopic in fo rmat ion
To unde rs tand the k ind of in format ion one can obta in f rom a homogeneous
l ine shape func t i on of guest molecules in a host mat r i x we assume tha t the
hos t -gues t system is pe r fec t l y o rde red and the concen t ra t ion is low enough
so tha t each guest molecule has the same microscopic e n v i r o n m e n t . In th i s
case the absorp t ion l ine shape has the t yp ica l form shown in F i g . 1 . It
consists of a nar row so-cal led zero phonon l ine and a broad so-cal led
phonon side b a n d . The re la t i ve i n tens i t y in the nar row zero phonon l ine
determines the Debye-Wa l le r - fac to r , a charac te r i s t i c parameter o f the
cons idered hos t -gues t sys tem, which i s , fo r many systems on the o r d e r of
0 .4 -0 .8 for temperatures a round 4K. Above 40 K, or so, i t becomes
ext remely small so tha t the zero phonon s t r u c t u r e tends to v a n i s h . The
phonon side band is an outcome of the F r a n c k - C o n d o n - p r i n c i p l e app l ied to
the v i b ra t i ons of the host mater ia l : The exc i ted guest molecule has a
d i f f e r e n t charge d i s t r i b u t i o n . Hence, the mat r ix is po lar ized and tends to
assume a new equ i l i b r i um con f i gu ra t i on whi le the guest molecule is in the
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— Υ
F i g . 1 . Zero phonon l ine w i th homogeneous w id th γ and phonon s ideband. Δ is hal f the S tokes-sh i f t
exc i ted s ta te . Th is is equ iva lent to say that the mat r i x v i b ra tes as a
consequence of the guest exc i ta t i on . Mat r ix v i b r a t i o n s are usua l ly cal led
phonons . I f the mat r ix o r , s t r i c t l y speak ing , the immediate env i ronment of
the exc i ted chromophore is v e r y r i g i d , the coup l i ng of the e lect ron ic
exc i ta t ion to the mat r ix env i ronment ( to the la t t i ce) is small a n d , hence,
the phonon side band car r ies l i t t le osci l lator s t r e n g t h and the Debye-
Wal le r - fac to r wi l l be close to 1 . The spectrum is then dominated by the
na r row and intense zero phonon l ines.
The zero phonon l ine is a pu re l y e lect ronic t r a n s i t i o n , i . e . the v ib ra t iona l
s ta te of the lat t ice does not change d u r i n g th is k i n d of exc i t a t i on . Since
the l i fet ime of an e lect ronic level is o rde rs of magn i tude longer than tha t
of a v ib ra t iona l l eve l , i ts w id th γ i s , accord ing to the u n c e r t a i n t y re la
t i o n , much na r rower , a n d , hence, i ts peak i n t e n s i t y may be o rde rs of
magni tude h igher than the phonon side band . Hence, we have a t yp ica l
abso rp t i on line shape as depic ted in F ig . 1 . A long these l ines of reasoning
i t seems to be clear tha t one can determine the l i fet ime of an e lect ron ic
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level in case one succeeds in measur ing i ts homogeneous zero phonon l ine
shape. T h i s , however , is not an easy task because of severa l reasons:
F i r s t , on ly at ex t remely low tempera tures on the o rde r of 1 Κ is the w i d t h
determined by the t r u e l i fet ime of the e lec t ron ic level e x c i t e d . With i n
creas ing temperatures the dynamics of the host ma t r i x b roaden the l ine
w i thou t chang ing i ts l i fe t ime. Second, many dye molecules have l i fet imes on
the o rde r of 1 nsec, hence the w id th wi l l be on the o rde r of 10 to 100 MHz
(0.0003 - 0.003 cm Ί ) and the r equ i r ed reso lu t ion of the spect rometer has
to be on the o rde r of 1 0 7 . T h i r d , and most i m p o r t a n t , the presence o f
microscopic d i so rde r on a molecular level obscures the homogeneous l ine
shape f u n c t i o n . Hence, a s t r a i g h t f o r w a r d spec t roscopy is not possible ( f o r
a rev iew , see ( 1 ) ) . How d i so rde r changes the spect ra l p rope r t i es o f a
chromophore in a host mat r i x is d iscussed in the fo l low ing p a r a g r a p h .
F ig . 2 symbolizes a d i so rde red m a t r i x . The so lvent cage of molecule 1 is
d i f f e ren t f rom tha t of molecule 2 and 3. Hence, as schemat ical ly shown
a b amorphous lattice inhomogeneous line
F i g . 2. Schematic represen ta t ion of inhomogeneous l ine b roaden ing (b ) as a resu l t of microscopic d i so rde r (a)
in F i g , 2b , these molecules absorb at d i f f e r e n t f r equenc ies . As a conse
quence, spat ial d i so rde r leads to a spread of absorp t i on f r e q u e n c i e s , wh ich
is cal led inhomogeneous l ine b r o a d e n i n g . A t low t e m p e r a t u r e s , the inhomo-
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geneous w id th may be la rger than the homogeneous w i d t h by more than 4
o r d e r s of magn i tudes . Hence, all detai ls of the homogeneous molecular l ine
shape are wiped out due to d i so rde r .
F luorescence l ine na r row ing (FLN) and hole b u r n i n g (HB) spect roscopy
Both FLN and HB are h igh resolut ion techn iques des igned to overcome i n -
homogeneous l ine b roaden ing . The basic idea of FLN is sketched in F i g .
3a. A nar row bandw id th laser w i th f requency exc i tes on ly those
cen te rs in the inhomogeneous band which are acc identa l ly t uned w i th the i r
abso rp t i on f reuqency to ω^ . T h e n , on ly molecules abso rb ing in a f r e
quency range on the o rder o f the homogeneous w id th a round ω ^ can emit
f l uo rescence . I f the f luorescence is detected w i th a h igh reso lu t ion spec
t r ome te r , the l ines wi l l show a s t r u c t u r e as shown in F i g . 3a. A drawback
of FLN is the fact tha t i t is v e r y d i f f i c u l t to observe resonance f l u o r e s
cence a n d , hence, the l ines are broadened by v ib ra t iona l re laxat ion and
o ther processes.
Hole b u r n i n g is shown in F i g . 3b . It a lways works in case the guest
molecules are pho to reac t i ve , and the zero phonon l ine ca r r ies enough
osc i l la tor s t r e n g t h . T h e n , those molecules exc i ted w i t h i n a f r equency range
α b
Fluorescence Line Narrowing Spectral Hole Burning
frequency selected J
ω ω
F i g . 3. Schematic representa t ion of f l u o r e scence l ine na r row ing (a) and spect ra l hole b u r n i n g ( b ) .
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of the homogeneous l inewid th γ a round ω ^ are t rans fo rmed to a p r o d u c t ,
hence, the number of molecular absorbers at ω is d imin ished and a hole
appears in the spec t rum. C o n t r a r y to F L N , HB allows fo r a resonant
detect ion at the laser f r e q u e n c y ; t h e r e f o r e , th i s techn ique has the capa
b i l i t y of measur ing the homogeneous l i n e w i d t h . One has , however , to be
care fu l to ru le out all slow re laxat ion processes such as spect ra l d i f f u s i o n ,
because the time scale of HB-exper imen ts is slow and a lot o f processes
may occur wh ich broaden the ho le .
Hole b u r n i n g exper iments on phycobi l isomes and spec t ra l l y reso lved e n e r g y
t r ans fe r times
F i g . 4 shows an absorp t ion spect rum of phycobi l isomes of M. laminosus at
a tempera tu re of 4 Κ in a saccharose/phosphate b u f f e r so lu t i on . The
1.0
6000 6400 6800
MÄ) F i g . 4. Hole b u r n i n g in PBS of M. lamino
sus . B u r n i n g posi t ions are ind icated by a r r o w s . Typ ica l hole shapes obta ined in the PC and APC-peaks are shown on an en la rged scale. PBS p repa ra t i on , modif ied f rom Nies and Wehrmeyer ( 4 ) , was made in 0.9 Μ phosphate bu f fe r (pH 6 ) . C e n t r i f u -gat ion was repeated tw ice . Isolated PBS solut ion was sa tu ra ted w i t h saccharose, to ensure coup l i ng in a low tempera ture g lass .
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s p e c t r u m is charac ter ized by a c lear ly resolved s t r u c t u r e wh ich o r ig ina tes
f r om the var ious pigments which bu i ld the h i gh l y o rgan ized phycobi l isome
assembly APC, PC, PE. In sp i te of the h i gh l y o rgan ized s t r u c t u r e , t he re
is a remarkable amount of d i so rder as documented by the inhomogeneous
l i n e w i d t h s . It is a quest ion of great i n te res t whe the r the observed d i s
o r d e r is an i n t r i ns i c p r o p e r t y of the chromopro te in or of the prote inaceous
e n v i r o n m e n t . In case of the PBS, most of the chromophores are known to
i n t e r a c t s t r ong l y w i th the p r o t e i n . Consequen t l y , we bel ieve tha t d i so rde r
is i n t r i n s i c and re f lec ts the d i f f e ren t conformat ional states of the la t te r
( 5 ) . The ar rows in F i g . 4 indicate the posi t ions where hole b u r n i n g was
p e r f o r m e d . Typ ica l holes are shown in the same f i g u r e as inse ts . The i r
w i d t h is on the o rde r of 0.4 cm \ It is i n t e res t i ng to compare th is w id th
w i t h tha t measured for isolated phycocyan ine ( 6 ) . Th i s is done in F i g . 5
f o r phycobi l isome and PC in saccharose /bu f fe r so l u t i on . The hole w id th in
phycocyanine
c m -1
F i g . 5. Comparison of spect ra l holes in isolated PC and phycobi l isomes ( P C - p e a k ) .
the phycobi l isome system is much larger than in the isolated PC. We i n t e r -
pre te th i s f i nd ing in the fo l lowing way : Hole b u r n i n g on the PC- t r imer
occurs e f fec t ive ly on ly on the red edge of the v i s ib le b a n d . Hence, i t
seems tha t i t is on ly the f luoresc ing ch romophore (s ) (or chromo-
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phore states) wh ich are e f fec t i ve ly b u r n t . In these chromophobes, the
f luorescence l i fet ime is mainly gove rned by in t ramolecu lar decay p rocesses ,
which occur on the o rde r of nanoseconds. A t s u f f i c i e n t l y low t e m p e r a t u r e s ,
where dephas ing is smal l , these decay processes determine the w i d t h o f
the hole. In the phycobi l isomes t he re i s , apar t f rom these decay p r o c e s
ses, a fast energy t r ans fe r to acceptor ch romopro te ins , l ike APC. In case
these energy t r a n s f e r processes dominate the dynamics of t he exc i ted
leve l , the t r a n s f e r times can be de te rm ined f rom the measured hole w i d t h .
From our resu l ts we would est imate t r a n s f e r t imes on the o r d e r o f 30 p s .
It is i n te res t i ng to note tha t these resu l t s do not depend v e r y much on
the posi t ion of exc i ta t ion (see F i g . 4 ) .
We conclude th is sect ion w i t h a few remarks on the advantages and d i s a d
vantages of the hole b u r n i n g method in de te rm in ing l i fe t imes. Un l i ke
f luorescence detect ion methods, wh ich su f f e r f rom ove r l app ing c o n t r i
but ions f rom the var ious p igmen ts , hole b u r n i n g is a resonant method
which d i r e c t l y y ie lds in format ion on t he b u r n t s ta te . No k ine t i c model is
necessary in eva lua t ing th i s i n fo rma t i on . However , the w id th of the hole is
not on ly determined by the l i fet ime of the state cons ide red , bu t depends
also on the pu re dephasing t ime. It is d i f f i c u l t to determine the l a t te r one
separate ly a n d , hence, the hole b u r n i n g resu l ts have to be cons idered as
lower bounds fo r the t r a n s f e r t imes.
Spectra l d i s t r i b u t i o n of the pho top roduc t
The na tu re of the hole b u r n i n g react ion i s , as of y e t , not c lea r . Bas ica l ly
one d isce rns between photochemical - and pho tophys ica l reac t ions . In the
f i r s t case i t is assumed tha t photochemical changes of the dye molecule
i tse l f ( i . e . of the chromophore) occur ( e . g . bond b r e a k i n g , p r o t o n t r a n s
fer reac t ions , e t c . ) . In the second case it is assumed tha t the dye mole
cule i t se l f is unchanged whereas the s u r r o u n d i n g changes ( e . g . a change
in the conformat ional state of the p r o t e i n ) . Photochemical react ions usua l l y
lead to large spect ra l s h i f t s , whi le pho tophys ica l react ions lead to small
spect ra l sh i f t s ( 7 ) . In many cases ( i n c l u d i n g ch romopro te ins ) i t may be
d i f f i c u l t to d i s t i ngu i sh both types of react ions in a clear cu t way . F i g . 6
shows ( toge ther w i th the absorp t ion spec t rum) the d i f f e rence in opt ica l
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301
0.05
α ο <
-0.05
0
6800 6000 6400
x(Ä)
F i g . 6. D i f fe rence in opt ical dens i t y (AOD) be fo re and a f te r b u r n i n g the p h y c o bi l isome spect rum a round 6370 A .
d e n s i t y be fore and a f te r b u r n i n g . Note, tha t most o f the p roduc t occurs in
a r a the r nar row range around the educt wh ich favou rs a photophys ica l
hole b u r n i n g reac t ion . A n i n t e r e s t i n g observa t ion is the pho to t rans fo rma-
t i on wh ich occurs in the A P C - r a n g e a round 6560 A . Note, tha t laser
i r r a d i a t i o n is per fo rmed at 6370 A in the P C - r a n g e . As obv ious , the spec
t r a l range b u r n t in the APC-spec t rum is almost as broad as the inhomoge
neous b a n d . Hence, f requency select ion is lost in t h i s case. I n t e r e s t i n g l y ,
on ly the long wave length edge of t he APC- band is b u r n t . I t seems, tha t
on ly t h i s long wavelength range of the band is pho to reac t i ve .
Conformat ional b a r r i e r s
In case the react ion p roduc t is determined by the in te rac t ion between
probe molecule ( i . e . chromophore) and env i r onmen t , we expect t h a t , due
to the d i so rde r of the sys tem, the react ion b a r r i e r s are not well d e f i n e d ,
bu t are ra the r charac ter ized by a broad d i s t r i b u t i o n . Hole b u r n i n g o f fe rs
a poss ib i l i t y fo r d i r e c t l y measur ing th is d i s t r i b u t i o n of b a r r i e r s ( 5 , 7 ) . To
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th is end we make use of tempera tu re c y c l i n g expe r imen ts : A hole is b u r n t
at a low tempera ture ( • b u r n 1 ) . T h e n , the tempera tu re o f the sample is
raised to some value Τ ( the 'excurs ion t empera tu re 1 ) and cyc led back to
T ^ , where the hole is measured aga in .
In our case b u r n i n g was per fo rmed at the APC-peak , near 6550 A (see
F ig . 4 ) . T ^ was 4 K. The changes of the hole area d u r i n g such a cyc le is
a measure for the number of p r o d u c t molecules hav ing r e t u r n e d to t h e i r
educt s ta te . The exper iment is per fo rmed as a f u n c t i o n of the e x c u r s i o n
tempera ture T . For a f i xed excurs ion tempera tu re T , al l those cen te rs
r e t u r n to the educt state charac te r ized by b a r r i e r s
R q is the at tempt f requency and τ the exper imenta l t ime needed to d r i v e
the system t h r o u g h one cyc le . In R q t is on the o r d e r o f 30. Cen te rs w i t h
b a r r i e r s h igher than V-j- s tay una f f ec ted . The va r i a t i on o f t he e x c u r s i o n
tempera ture enables a sampling of the d i s t r i b u t i o n of b a r r i e r h e i g h t s . F i g .
7a shows the resu l t for PBS. For compar ison , the resu l ts ob ta ined fo r a
series of organ ic glasses are also shown .
V X = kT In R τ Τ ο (1)
F i g . 7. Annea l ing of a spec t ra l
< 0.5
<
ο
0 b
hole b u r n t in to the PBS abso rp t i on ( a ) . For compar ison the same behav io r fo r o rgan ic glasses is also s h o w n . The d i s t r i b u t i o n o f react ion b a r r i e r s can be well desc r i bed by a supe rpos i t i on of g lass l ike states and a d i s c re te f ea tu re d i s t r i bu ted in a Gaussian fashion ( b ) .
glass-like
0.8 Τ/Γ
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The exper iments on the p ro te in sample can be p e r f e c t l y f i t t ed by assuming
tha t the d i s t r i b u t i o n of ba r r i e r he ights cons is ts o f a superpos i t i on of a
g l ass - l i ke d i s t r i b u t i o n and a Gaussian d i s t r i b u t i o n wh ich is centered
a round 650 cm 1 and which has a w id th of 210 cm Ί . We i n t e r p r e t e these
f i n d i n g s in the fo l lowing way : We assume tha t hole b u r n i n g is due to a
conformat ional change of the pro te in env i ronment a round the i r r ad ia ted
chromophore (photophys ica l H B - mechanism). On one h a n d , a p ro te in is
v e r y much l ike a g lass , i . e . it is d i so rde red on a microscopic scale and
can ex is t in many conformational states w i t h d i f f e r e n t b a r r i e r s . We have
recen t l y shown tha t in a glass the p robab i l i t y of f i n d i n g a b a r r i e r w i th
he igh t V is p ropor t iona l to 1 / / V ( 5 , 7 ) . On the o the r hand a p ro te in has
also v e r y speci f ic conformat ional s ta tes , i . e . ' f unc t i ona l l y impor tan t s ta tes ' ,
wh ich are re lated to well def ined mot ions, say of a la rge pa r t of the pep t i d
chain ( 8 ) . I t is p lausib le tha t the ba r r i e r he igh t of such a speci f ic c o n
formational state depends on the actual subs ta te o f the g lass - l i ke states
which the p ro te in occupies, a n d , hence the b a r r i e r is spread a round some
mean va lue , which in our case is 650 cm 1 . The decomposit ion of the
exper imenta l resu l ts in a g lass- l i ke d i s t r i b u t i o n and a symmetr ica l ly b r o a
dened d isc re te fea ture is shown in F i g . 7b .
Fluorescence line na r row ing exper iments and level s t r u c t u r e in C - p h y c o -
cyan in
The hole b u r n i n g exper iments document well the zero phonon na tu re of the
opt ical t rans i t ions in chromoprote ins (9-11) in con t ras t to the resu l ts
obta ined for react ion centers of Rhodopseudomonas v i r i d i s and Rhodopseu-
domonas sphaeroides (12 -14 ) . However, the f luorescence emission is broad
i r respec t i ve of the mode of exc i ta t ion and d e t e c t i o n . A simple exp lanat ion
of t h i s phenomenon is the assumption of a loss of ene rgy cor re la t ion due to
t r ans fe r processes, tha t i s , a specif ic level w i t h a sharp f r equency in a
sens i t i z ing state populates the whole inhomogeneous band of the f l uo resc ing
s ta te. The idea was to nar row the f luorescence by resonant exc i ta t ion of
the f luo resc ing s ta te . F i g . 8 shows that th i s idea d id not work ou t . The re
is no sha rp l y s t r u c t u r e d f luorescence even in case exc i ta t ion is ca r r i ed out
far in the red edge of the PC-abso rp t i on . Th is phenomenon can be u n d e r -
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stood on the basis of the special level s t r u c t u r e i nvo l ved (15) and the loss
of ene rgy c o r r e l a t i o n . As to the special level s t r u c t u r e we have to assume
that the f l uo resc ing state consists in rea l i t y of (at least) two states w i th
s t r o n g l y ove r l app ing inhomogeneous bands . The loss of ene rgy c o r r e -
F i g . 8. Se lect ive ly exc i ted f luorescence of PC- t r ime r as a f unc t i on o f e x c i t a t ion w a v e l e n g t h .
lat ion means t h a t , though the inhomogeneous bands are s t r o n g l y o v e r
l a p p i n g , the energy spacing between the two states in an i n d i v i d u a l mole
cule can have any value between zero and ( r o u g h l y ) the inhomogeneous
w i d t h . Since these states are coupled v ia energy t r a n s f e r processes the
maximum of the f luorescence does not sh i f t w i th exc i ta t ion f r e q u e n c y a n d ,
as the f r equency is scanned into the r e d , the f luorescence shows a cha
rac te r i s t i c cu t o f f at the laser f r e q u e n c y . Ano the r consequence of th is
special level s t r u c t u r e is a wave leng th dependent hole b u r n i n g e f f i c i ency :
As long as the exc i ta t ion f r equency is far enough to the b lue of t he states
i n v o l v e d , the re is a h igh p r o b a b i l i t y tha t the second level is lower in
e n e r g y . Hence, the p robab i l i t y fo r energy t r a n s f e r is h igh a n d , c o r r e s -
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p o n d i n g l y , the p robab i l i t y fo r hole b u r n i n g or resonance f luorescence is
low. As the laser f requency is t u r n e d to the r e d , the p robab i l i t y of f i n
d i n g an acceptor state w i th lower energy decreases, hence, the e f f i c iency
for hole b u r n i n g and resonance emission inc reases . Both phenomena can
indeed be o b s e r v e d .
Acknowledgement
The au tho rs acknowledge g ran ts f rom the Deutsche Forschungsgemein
scha f t , SFB 213 ( B a y r e u t h ) and SFB 143-A1 ( M u n i c h ) .
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