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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
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
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
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
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
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
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
294
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
2 9 5
— 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
2 9 6
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-
297
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 ) .
2 9 8
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 .
2 9 9
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-
3 0 0
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
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
3 0 2
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
3 0 3
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 -
3 0 4
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 -
3 0 5
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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