Baumann, F.

Fischer, R.

Scheer, Hugo

Schneider, Siegfried

Siebzehnrübl, S.

Steiner, R.

English

Universität München: Elektronischen Publikationen

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 
List of Participants X I I I 
SECTION I . ORGANIZATION: BIOCHEMICAL METHODS 
Introduction: The Biochemistry of Light-Harvesting Complexes 
by R.J. Cogdell 1 
Phycobilisome-Thylakoid Interaction: The Nature of High 
Molecular Weight Polypeptides 
by E. Gantt C.A. Lipschultz and F.X. Cunningham Jr 11 
On the Structure of Photosystem II-Phycobilisome Complexes 
of Cyanobacteria 
by E. Mörschel and G.-H. Schatz 21 
Structure of Cryptophyte Photosynthetic Membranes 
by W. Wehrmeyer 35 
Structural and Phylogenetic Relationships of Phycoerythrins 
from Cyanobacteria, Red Algae and Cryptophyceae 
by W. Sidler and H. Zuber 49 
Isolation and Characterization of the Components of the 
Phycobilisome from Mastigocladus laminosus and Cross-
linking Experiments 
by R. Rümbeli and H. Zuber 61 
C-Phycocyanin from Mastigocladus laminosus: Chromophore 
Assignment in Higher Aggregates by Cystein Modification 
by R. Fischer, S. Siebzehnrlibl and H. Scheer 71 
Photochromic Properties of C-Phycocyanin 
by G. Schmidt, S. Siebzehnrübl, R. Fischer and H. Scheer 77 
Concerning the Relationship of Light Harvesting B i l i -
proteins to Phycochromes in Cyanobacteria 
by W. Kufer 89 
Subunit Structure and Reassembly of the Light-Harvesting 
Complex from Rhodospiri Hum rubrum G9+ 
by R. Ghosh, ThT~Rosatzin ancTR. Baclofen 93 
Primary Structure Analyses of Bacterial Antenna Polypeptides 
- Correlation of Aromatic Amino Acids with Spectral 
Properties - Structural S i m i l a r i t i e s with Reaction Center 
Polypeptides 
by R.A. Brunisholz and H. Zuber 103 
VIII 
The Structure of the "Core
1
* of the Purple Bacterial Photo­
synthetic Unit 
by D.J. Dawkins, L.A. Ferguson and R.J. Cogdell 115 
A Comparison of the Bacteriochlorophyl1 C--Binding Proteins 
of Chlorobium and Chloroflexus 
by P.D. Gerola, P. Hpjrup and J.M. Olson 129 
Interactions between Bacteriochlorophyl1 c Molecules in 
Oligomers and in Chlorosomes of Green Photosynthetic 
Bacteria 
by D.C. Brune, G.H. King and R.E. Blankenship 141 
Light-Harvesting Complexes of Chlorophyll c-Containing Algae 
by A.W.D. Larkum and R.G. H i l l e r 153 
Isolation and Characterization of a Chlorophyll a/c-Hetero-
xanthin/Diadinoxanthin Light-Harvesting Complex from 
Pleurochloris meiringensis (Xanthophyceae) 
by C. Wilhelm, C. Bliche I and B. Rousseau 167 
The Antenna Components of Photosystem I I with 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 Crystal Structure of C-Phycocyanin and 
Polarized Optical Spectra of Single Crystals 
by T. Schirmer, W. Bode and R. Huber 195 
Crystallization and Spectroscopic Investigation of Purple 
Bacterial B800-850 and RC-B875 Complexes 
by W. Welte, Τ. Wacker and A. Becker 201 
Structure 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 Bacterial Antenna Complexes 
by G. Drews 233 
IX 
The Use of Mutants to Investigate the Organization of the 
Photosynthetic Apparatus of Rhodobacter sphaeroides 
by C.N. Hunter and R. van Grondel le 77.77 247 
Mechanisms of Plastid and Nuclear Gene Expression During 
Thylakoid Membrane Biogenesis in 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 of Spectral Forms in the Photosynthetic Antennas to 
Chemically Defined Chromophores 
by A. Scherz 277 
Linear Dichroism and Orientation of Pigments in Phycobilisomes 
and t h e i r Subunits 
by L. Juszcak, N.E. Geacintov, B.A. Zilinskas and J. Breton 281 
Low Temperature Spectroscopy of Cyanobacteria! Antenna Pigments 
by W. Köhler, J. Friedrich, R. Fischer and H. Scheer 293 
Chromophore Conformations in Phycocyanin and Allophycocyanin 
as Studied by Resonance Raman Spectroscopy 
by B. Szalontai, 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 
Optical Absorption and Circular Dichroism of Bacteriochlorophyl1 
Oligomers in Triton X-100 and in the Light-Harvesting-Complex 
B850; A Comparative Study 
by V. Rozenbach-Belkin, P. Braun, P. Kovatch and A.Scherz 323 
Absorption Detected Magnetic Resonance in Zero Magnetic Field 
on Antenna Complexes from Rps. acidophila 7050 - The 
Temperature Dependence of tfie CarotenoTiTTrTpTet State 
Properties 
by J. U l l r i c h , J.U. v. Schütz and H.C. Wolf 339 
Effect of Lithium Dodecyl Sulfate on Β 800-850 Antenna 
Complexes from Rhodopseudomonas acidophila: A Resonance 
Raman Study 
by B. Robert and H. Frank 349 
χ 
Bacteriochlorophyl 1 a/b in Antenna Complexes of Purple Bacteria 
by Β. Robert, A. Vermeglio, R. Steiner, Η. Scheer and Μ. Lutz 355 
Bacteriochlorophyll c Aggregates in Carbon Tetrachloride 
as Models for Chlorophyll Organization in Green Photo­
synthetic Bacteria 
by J.M. Olson and J.P. Pedersen 365 
Orientation of the Pigments in the Reaction Center and the 
Core Antenna of 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. St i e l and P. Hoffmann 387 
SECTION IV: FUNCTION: ELECTRONIC EXCITATION AND ENERGY TRANSFER 
Excitation Energy Transfer in Photosynthesis 
by R. van Grondelle and V. Sundström 403 
Fluorescence Spectroscopy of Allophycocyanin Complexes from 
Synechococcus 6301 Strain AN112 
by P.Maxson, KTTauer and A.N. Glazer 439 
Picosecond Energy Transfer Kinetics in Allophycocyanin 
Aggregates from Mastigocladus laminosus 
by E. Bittersmann, W. Reuter, W. Wehrmeyer and A.R. Holzwarth 451 
Picosecond Time-Resolved Energy Transfer Kinetics within 
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 in "Native" and Chemically Modified C-Phyco-
cyanin Trimers and the Constituent Subunits 
by S. Schneider, P. Geiselhart, F. Baumann, S. Siebzehnrübl, 
R. Fischer and Η. Scheer 469 
Effect of Protein Environment and Excitonic Coupling on the 
Excited-State Properties of the Bilinchromophores in 
C-Phycocyanin 
by S. Schneider, Ch. Scharnagl, M. Dürring, T. Schirmer and 
W. Bode 483 
Excitation Energy Migration in C-Phycocyanin Aggregates 
Isolated from Phormidium luridum: Predictions from the 
Förster's Inductive Resonance Theory 
by J. Grabowski and G.S. Björn 491 
XI 
Energy Transfer Calculations for two C-Phycocyanins Based on 
Refined X-Ray Crystal Structure Coordinates of Chromophores 
by K. Sauer and H. Scheer 507 
Energy Transfer in Light-Harvesting Antenna of Purple Bacteria 
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 
Excitation Energy Transfer in the Light-Harvesting Antenna of 
Photosynthetic Purple Bacteria: 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 of Chlorosomes in Energy Transfer in Green Photo-
synthetic Bacteria 
by R.J. van Dorssen, M. Vos and J. Amesz 531 
Energy Transfer in Chloroflexus aurantiacus: Effects of 
Temperature and TSaerobic^onTirfons 
by B.P. Wittmershaus, D.C. Brune and R.E. Blankenship 543 
Interpretation of Optical Spectra of Bacteriochlorophyl 1 
Antenna Complexes 
by R.M. Pearlstein 555 
Time Resolution and Kinetics of "F680" at Low Temperatures in 
Spinach Chloroplasts 
by R. Knox and S. Lin 567 
Picosecond Studies of Fluorescence and Absorbance Changes in 
Photosystem I I Particles from Synechococcus Sp. 
by A.R. Holzwarth, G.H. Schatz and H. Brock 579 
Analysis of Excitation Energy Transfer in 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 
COHERENT ΑΝΤΙ-STOKES RAMAN SPECTROSCOPY OF PHYCOBILISOMES, 
PHYCOCYANIN AND ALLOPHYCOCYANIN FROM MASTIGOCLADUS LAMINOSUS 
S. Schneider, F. Baumann, W. St e i n e r 
I n s t i t u t für P h y s i k a l i s c h e und Theoretische Chemie 
der TU-München, Licht e n b e r g s t r . 4, D-8046 Garching 
R. F i s c h e r , S. Siebzehnrübl, H. Scheer 
Botanisches I n s t i t u t der Ludwig-Maximilians-Universität 
Menzingerstr. 67, D-8000 München 19 
Introduction 
I n order to provide a large geometrical and s p e c t r a l c r o s s -
s e c t i o n f o r the absorption of l i g h t , red and blue-green algae 
produce the s o - c a l l e d phycobilisomes (PBS), which contain 
the chromoproteins phycoerythrin (or phycoerythrocyanin), 
phycocyanin (PC) and allophycocyanin (APC). Although PC and 
APC contain the same chromophore, phycocyanobilin, the s p e c t r a l 
p r o p e r t i e s of these b i l i p r o t e i n s ( t e t r a p y r r o l chromophore 
bound to apoprotein) are quite d i f f e r e n t . Their e x c i t a t i o n 
energies are modified according to t h e i r s p e c i a l function i n 
the l i g h t h a r vesting system (see e.g. 1,2) by chromophore-
protein i n t e r a c t i o n s (14). 
Resonance-enhanced Coherent Anti-Stokes Raman Spectroscopy 
(CARS) has proved to be a very s u i t a b l e technique to produce 
v i b r a t i o n a l spectra of highly f l u o r e s c i n g chromophores as 
e.g. l i g h t h arvesting pigments (3,4,5). I n t h i s c o n t ribution 
CARS-spectra of room-temperature s o l u t i o n s of phycobilisomes 
and of phycocyanin and allophycocyanin trimers from Mastigo-
cladus laminosus are presented and the implications to chromo-
phore s t r u c t u r e are discussed. 
Photosynthetic Light-Harvesting Systems 
© 1988 Walter de Gruyter & Co., Berlin · New York 
318 
Experimental 
Resonance-enhanced CARS spectra are obtained by focussing two 
l a s e r beams with d i f f e r e n t wavelengths λ pump (fixed) and 
X s t o k e s ( v a r i a b l e ) i n the sample, where by a non-linear e f f e c t 
the CARS beam i s generated, which holds the e q u a l i t y 
VCARS = V p u i p + (Vpump - V s t o k e s ) 
V C A R S > V p u a p > V s t o k e s , with V = 1/X [cm- 1] 
A plo t of the CARS i n t e n s i t y as function of V p u a p - V s t o k e s 
r e v e a l s a v i b r a t i o n a l spectrum of the chromophore. As the 
CARS frequency i s higher than e i t h e r pump and Stokes frequency, 
no flourescence problems as i n spontaneous Raman spectroscopy 
can a r i s e . (For more experimental d e t a i l s see r e f . 3 ) . 
Preparation 
PBS were i s o l a t e d according to the method published by Nies 
and Wehrmeyer ( 6 ) , except that the pH of the phosphate buffer 
was 6 and the p u r i f i c a t i o n on the sucrose gradient was done 
twice. APC was prepared very s i m i l a r to a procedure described 
by Füglistaller e t . a l . ( 7 ) . 
The aggregation s t a t e of PC and APC was determined by s e d i -
mentation runs according to Martin and Ames ( 8 ) . Both b i l i -
proteins were found to be t r i m e r i c . Linker peptides were not 
present as proved by absorption spec t r a , sedimentation co-
e f f i c i e n t s and SDS-gel e l e c t r o p h o r e s i s . 
Results 
As both the f i n g e r p r i n t (1100 - 1300 c u r 1 ) and the double 
bond s t r e t c h i n g region (1500 - 1750 cm - 1) have proved to be 
most s u i t a b l e for the i n v e s t i g a t i o n of chromophore geometry 
by CARS (3,4,5,9) or Resonance Raman Spectroscopy (10,11), 
both regions are considered i n the following. 
319 
The phycobilisome spectra (PBS) are dominated by the contribu­
t i o n of the cons t i t u e n t phycocyanin trimers (PC) and are there­
fore r a t h e r s i m i l a r to the spectra of the l a t t e r . Most of the 
v i b r a t i o n a l frequencies are reproduced w i t h i n experimental 
accuracy, though with d i f f e r e n t i n t e n s i t i e s ( e s p e c i a l l y between 
1200 and 1300 cm-1) ( f i g 1 ) . At 1623 cnr 1 an a d d i t i o n a l weak 
peak i s found i n PBS, which i s due to allophycocyanin (APC). 
A second i n t e r e s t i n g feature i s that for the pronounced bands 
a smaller width i s found i n PBS than i n PC. I t p o s s i b l y i n d i ­
cates a lower s t r u c t u r a l inhomogeneity i n PBS due to higher 
aggregation and the presence of l i n k e r peptides. 
1200 1220 1210 12I1H 1280 1500 1550 1600 IGS0 1/(HI 1 Λ.Π 
Wavenumber s Vevcnumbers 
F i g . 1: Resonance-enhanced CARS spectra of phycobilisomes, 
phycocyanin trimers and allophycocyanin trimers from 
Mastigocladus laminosus. Pump wavelength 640 nm. 
320 
APC spectra are very d i f f e r e n t from PC spectra (see also 
r e f . 10). The 1257 cm"1 and 1287 cm"1 bands observed i n PC 
are absent i n APC. I n the double bond s t r e t c h i n g region the 
dominant peak i n PC i s found at 1649 cm"1 with shoulders at 
«1631 and »1662 cm"1 . I n APC sp e c t r a , however, the dominant 
peak i s found at 1625 cm"1 with a strong shoulder at 
«1635 cm"1 and a weak one at «1650 cm"1. 
Discussion 
According to preliminary normal mode c a l c u l a t i o n s using the 
coordinates provided by X-ray a n a l y s i s (12,13), the bands 
found between 1200 and 1300 cm - 1 represent e x t e n s i v e l y mixed 
C-C s t r e t c h / C-H i n plane bending modes and can presently 
not be i n t e r p r e t e d i n a straight-forward manner. The band 
around 1585 cm"1 i s r e l a t e d to C=C s t r e t c h i n g v i b r a t i o n s i n 
the r i n g B/C fragment. That t h i s band e x h i b i t s n e a r l y no 
change i n frequency, most l i k e l y i n d i c a t e s a conservation of 
the geometry of t h i s part of the chromophore. 
The C=C double bonds of the methine bridges between r i n g A-B 
and r i n g C-D, on the other hand, should give r i s e to l o c a l i z e d 
modes with frequencies between 1650 and 1700 cm"1. The C=0 
s t r e t c h mixes with the C=C s t r e t c h i n r i n g D to produce v i b r a ­
t i o n a l frequencies around 1630 cm"1. The a c t u a l value of the 
c a l c u l a t e d frequencies i s found to be s e n s i t i v e to s t r u c t u r a l 
changes. 
From these t h e o r e t i c a l r e s u l t s and the comparison of CARS 
spectra of model compounds (9) with the presented CARS spectra 
a s i g n i f i c a n t d i f f e r e n c e i n chromophore geometry and/or 
Π-electron d i s t r i b u t i o n between PC and APC must be postulated. 
I n t e r p r e t a t i o n of e.g. fluorescence decay data of APC should 
therefore not be based on s t r u c t u r a l information gained from 
PC. 
321 
Additional measurements on antenna pigments and a more elabo-
r a t e normal mode a n a l y s i s are i n preparation. 
Acknowledgement 
F i n a n c i a l support by Deutsche Forschungsgemeinschaft 
(SFB 143) and Fonds der Chemie i s g r a t e f u l l y acknowledged. 
References 
(1) Gantt E. 1981. Phycobilisomes . 
Ann. Rev. Plant P h y s i o l . 32, 327 - 347. 
(2) Scheer H. 1982. I n : Light Reaction Path of Photosynthesis 
(F.K. Fong, ed.). Springer, B e r l i n , pp. 7 - 45. 
(3) Schneider S.,F. Baumann, U. Klüter, 1987. 
Ζ. Naturforsch. 42c(11/12). 
(4) Schneider S., F. Baumann, P. G e i s e l h a r t , S. Siebzehnrübl, 
R. F i s c h e r , H. Scheer, 1987. Proc. of the Sec. Europ. 
Conf. on the Spectr. of B i o l . Mol., J.Wiley, Chichester. 
(5) Schneider S., F. Baumann, P. G e i s e l h a r t , Η. Kayser, 
Η. Scheer, 1987. Photochem. Photobiol., submitted. 
(6) Nies Μ. and W. Wehrmeyer, 1980. 
Planta 150, 330 - 337. 
(7) Füglistaller P., Η. Widmer, G. Frank and H. Zuber, 1981. 
Arch. Microbiol. 129, 268 - 274. 
(8) Martin R.G. and B.N. Ames, 1961. 
J . B i o l . Chem. 236, 1372 - 1379 
(9) Schneider S., P. G e i s e l h a r t , F. Baumann, Η. Falk and 
W. Medinger, 1987. J . Biochem. Biophys., submitted. 
(10) Sz a l o n t a i B., Z. Gombos, V. Csizmadia, 1985. 
Biochem. Biophys. Res. Com. 130, 358-363. 
(11) Sz a l o n t a i Β., Z. Gombos, V. Csizmadia and M. Lutz, 1987. 
Biochim. Biophys. Acta 893, 296-304. 
322 
(12) Schirmer Τ., W. Bode and R. Huber, 1985. 
J . Mol. B i o l . 184, 257-277. 
(13) Schirmer T., W. Bode and R. Huber, 1987. 
J . Mol. B i o l . , 196, 677-695. 
(14) Schneider S., C. Scharnagl, M. Duerring, T. Schirmer 
and W. Bode, 1987. Contribution i n t h i s volume. 


Coherent anti-Stokes Raman spectroscopy of phycobilisomes, phycocyanin and allophycocyanin from Mastigocladus laminosus

Open Access LMU

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 List of Participants X I I I SECTION I . ORGANIZATION: BIOCHEMICAL METHODS Introduction: The Biochemistry of Light-Harvesting Complexes by R.J. Cogdell 1 Phycobilisome-Thylakoid Interaction: The Nature of High Molecular Weight Polypeptides by E. Gantt C.A. Lipschultz and F.X. Cunningham Jr 11 On the Structure of Photosystem II-Phycobilisome Complexes of Cyanobacteria by E. Mörschel and G.-H. Schatz 21 Structure of Cryptophyte Photosynthetic Membranes by W. Wehrmeyer 35 Structural and Phylogenetic Relationships of Phycoerythrins from Cyanobacteria, Red Algae and Cryptophyceae by W. Sidler and H. Zuber 49 Isolation and Characterization of the Components of the Phycobilisome from Mastigocladus laminosus and Cross-linking Experiments by R. Rümbeli and H. Zuber 61 C-Phycocyanin from Mastigocladus laminosus: Chromophore Assignment in Higher Aggregates by Cystein Modification by R. Fischer, S. Siebzehnrlibl and H. Scheer 71 Photochromic Properties of C-Phycocyanin by G. Schmidt, S. Siebzehnrübl, R. Fischer and H. Scheer 77 Concerning the Relationship of Light Harvesting B i l i -proteins to Phycochromes in Cyanobacteria by W. Kufer 89 Subunit Structure and Reassembly of the Light-Harvesting Complex from Rhodospiri Hum rubrum G9+ by R. Ghosh, ThT~Rosatzin ancTR. Baclofen 93 Primary Structure Analyses of Bacterial Antenna Polypeptides - Correlation of Aromatic Amino Acids with Spectral Properties - Structural S i m i l a r i t i e s with Reaction Center Polypeptides by R.A. Brunisholz and H. Zuber 103 VIII The Structure of the "Core1* of the Purple Bacterial Photo­synthetic Unit by D.J. Dawkins, L.A. Ferguson and R.J. Cogdell 115 A Comparison of the Bacteriochlorophyl1 C--Binding Proteins of Chlorobium and Chloroflexus by P.D. Gerola, P. Hpjrup and J.M. Olson 129 Interactions between Bacteriochlorophyl1 c Molecules in Oligomers and in Chlorosomes of Green Photosynthetic Bacteria by D.C. Brune, G.H. King and R.E. Blankenship 141 Light-Harvesting Complexes of Chlorophyll c-Containing Algae by A.W.D. Larkum and R.G. H i l l e r 153 Isolation and Characterization of a Chlorophyll a/c-Hetero-xanthin/Diadinoxanthin Light-Harvesting Complex from Pleurochloris meiringensis (Xanthophyceae) by C. Wilhelm, C. Bliche I and B. Rousseau 167 The Antenna Components of Photosystem I I with 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 Crystal Structure of C-Phycocyanin and Polarized Optical Spectra of Single Crystals by T. Schirmer, W. Bode and R. Huber 195 Crystallization and Spectroscopic Investigation of Purple Bacterial B800-850 and RC-B875 Complexes by W. Welte, Τ. Wacker and A. Becker 201 Structure 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 Bacterial Antenna Complexes by G. Drews 233 IX The Use of Mutants to Investigate the Organization of the Photosynthetic Apparatus of Rhodobacter sphaeroides by C.N. Hunter and R. van Grondel le 77.77 247 Mechanisms of Plastid and Nuclear Gene Expression During Thylakoid Membrane Biogenesis in 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 of Spectral Forms in the Photosynthetic Antennas to Chemically Defined Chromophores by A. Scherz 277 Linear Dichroism and Orientation of Pigments in Phycobilisomes and t h e i r Subunits by L. Juszcak, N.E. Geacintov, B.A. Zilinskas and J. Breton 281 Low Temperature Spectroscopy of Cyanobacteria! Antenna Pigments by W. Köhler, J. Friedrich, R. Fischer and H. Scheer 293 Chromophore Conformations in Phycocyanin and Allophycocyanin as Studied by Resonance Raman Spectroscopy by B. Szalontai, 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 Optical Absorption and Circular Dichroism of Bacteriochlorophyl1 Oligomers in Triton X-100 and in the Light-Harvesting-Complex B850; A Comparative Study by V. Rozenbach-Belkin, P. Braun, P. Kovatch and A.Scherz 323 Absorption Detected Magnetic Resonance in Zero Magnetic Field on Antenna Complexes from Rps. acidophila 7050 - The Temperature Dependence of tfie CarotenoTiTTrTpTet State Properties by J. U l l r i c h , J.U. v. Schütz and H.C. Wolf 339 Effect of Lithium Dodecyl Sulfate on Β 800-850 Antenna Complexes from Rhodopseudomonas acidophila: A Resonance Raman Study by B. Robert and H. Frank 349 χ Bacteriochlorophyl 1 a/b in Antenna Complexes of Purple Bacteria by Β. Robert, A. Vermeglio, R. Steiner, Η. Scheer and Μ. Lutz 355 Bacteriochlorophyll c Aggregates in Carbon Tetrachloride as Models for Chlorophyll Organization in Green Photo­synthetic Bacteria by J.M. Olson and J.P. Pedersen 365 Orientation of the Pigments in the Reaction Center and the Core Antenna of 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. St i e l and P. Hoffmann 387 SECTION IV: FUNCTION: ELECTRONIC EXCITATION AND ENERGY TRANSFER Excitation Energy Transfer in Photosynthesis by R. van Grondelle and V. Sundström 403 Fluorescence Spectroscopy of Allophycocyanin Complexes from Synechococcus 6301 Strain AN112 by P.Maxson, KTTauer and A.N. Glazer 439 Picosecond Energy Transfer Kinetics in Allophycocyanin Aggregates from Mastigocladus laminosus by E. Bittersmann, W. Reuter, W. Wehrmeyer and A.R. Holzwarth 451 Picosecond Time-Resolved Energy Transfer Kinetics within 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 in "Native" and Chemically Modified C-Phyco-cyanin Trimers and the Constituent Subunits by S. Schneider, P. Geiselhart, F. Baumann, S. Siebzehnrübl, R. Fischer and Η. Scheer 469 Effect of Protein Environment and Excitonic Coupling on the Excited-State Properties of the Bilinchromophores in C-Phycocyanin by S. Schneider, Ch. Scharnagl, M. Dürring, T. Schirmer and W. Bode 483 Excitation Energy Migration in C-Phycocyanin Aggregates Isolated from Phormidium luridum: Predictions from the Förster's Inductive Resonance Theory by J. Grabowski and G.S. Björn 491 XI Energy Transfer Calculations for two C-Phycocyanins Based on Refined X-Ray Crystal Structure Coordinates of Chromophores by K. Sauer and H. Scheer 507 Energy Transfer in Light-Harvesting Antenna of Purple Bacteria 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 Excitation Energy Transfer in the Light-Harvesting Antenna of Photosynthetic Purple Bacteria: 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 of Chlorosomes in Energy Transfer in Green Photo-synthetic Bacteria by R.J. van Dorssen, M. Vos and J. Amesz 531 Energy Transfer in Chloroflexus aurantiacus: Effects of Temperature and TSaerobic^onTirfons by B.P. Wittmershaus, D.C. Brune and R.E. Blankenship 543 Interpretation of Optical Spectra of Bacteriochlorophyl 1 Antenna Complexes by R.M. Pearlstein 555 Time Resolution and Kinetics of "F680" at Low Temperatures in Spinach Chloroplasts by R. Knox and S. Lin 567 Picosecond Studies of Fluorescence and Absorbance Changes in Photosystem I I Particles from Synechococcus Sp. by A.R. Holzwarth, G.H. Schatz and H. Brock 579 Analysis of Excitation Energy Transfer in 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 COHERENT ΑΝΤΙ-STOKES RAMAN SPECTROSCOPY OF PHYCOBILISOMES, PHYCOCYANIN AND ALLOPHYCOCYANIN FROM MASTIGOCLADUS LAMINOSUS S. Schneider, F. Baumann, W. St e i n e r I n s t i t u t für P h y s i k a l i s c h e und Theoretische Chemie der TU-München, Licht e n b e r g s t r . 4, D-8046 Garching R. F i s c h e r , S. Siebzehnrübl, H. Scheer Botanisches I n s t i t u t der Ludwig-Maximilians-Universität Menzingerstr. 67, D-8000 München 19 Introduction I n order to provide a large geometrical and s p e c t r a l c r o s s -s e c t i o n f o r the absorption of l i g h t , red and blue-green algae produce the s o - c a l l e d phycobilisomes (PBS), which contain the chromoproteins phycoerythrin (or phycoerythrocyanin), phycocyanin (PC) and allophycocyanin (APC). Although PC and APC contain the same chromophore, phycocyanobilin, the s p e c t r a l p r o p e r t i e s of these b i l i p r o t e i n s ( t e t r a p y r r o l chromophore bound to apoprotein) are quite d i f f e r e n t . Their e x c i t a t i o n energies are modified according to t h e i r s p e c i a l function i n the l i g h t h a r vesting system (see e.g. 1,2) by chromophore-protein i n t e r a c t i o n s (14). Resonance-enhanced Coherent Anti-Stokes Raman Spectroscopy (CARS) has proved to be a very s u i t a b l e technique to produce v i b r a t i o n a l spectra of highly f l u o r e s c i n g chromophores as e.g. l i g h t h arvesting pigments (3,4,5). I n t h i s c o n t ribution CARS-spectra of room-temperature s o l u t i o n s of phycobilisomes and of phycocyanin and allophycocyanin trimers from Mastigo-cladus laminosus are presented and the implications to chromo-phore s t r u c t u r e are discussed. Photosynthetic Light-Harvesting Systems © 1988 Walter de Gruyter & Co., Berlin · New York 318 Experimental Resonance-enhanced CARS spectra are obtained by focussing two l a s e r beams with d i f f e r e n t wavelengths λ pump (fixed) and X s t o k e s ( v a r i a b l e ) i n the sample, where by a non-linear e f f e c t the CARS beam i s generated, which holds the e q u a l i t y VCARS = V p u i p + (Vpump - V s t o k e s ) V C A R S > V p u a p > V s t o k e s , with V = 1/X [cm- 1] A plo t of the CARS i n t e n s i t y as function of V p u a p - V s t o k e s r e v e a l s a v i b r a t i o n a l spectrum of the chromophore. As the CARS frequency i s higher than e i t h e r pump and Stokes frequency, no flourescence problems as i n spontaneous Raman spectroscopy can a r i s e . (For more experimental d e t a i l s see r e f . 3 ) . Preparation PBS were i s o l a t e d according to the method published by Nies and Wehrmeyer ( 6 ) , except that the pH of the phosphate buffer was 6 and the p u r i f i c a t i o n on the sucrose gradient was done twice. APC was prepared very s i m i l a r to a procedure described by Füglistaller e t . a l . ( 7 ) . The aggregation s t a t e of PC and APC was determined by s e d i -mentation runs according to Martin and Ames ( 8 ) . Both b i l i -proteins were found to be t r i m e r i c . Linker peptides were not present as proved by absorption spec t r a , sedimentation co-e f f i c i e n t s and SDS-gel e l e c t r o p h o r e s i s . Results As both the f i n g e r p r i n t (1100 - 1300 c u r 1 ) and the double bond s t r e t c h i n g region (1500 - 1750 cm - 1) have proved to be most s u i t a b l e for the i n v e s t i g a t i o n of chromophore geometry by CARS (3,4,5,9) or Resonance Raman Spectroscopy (10,11), both regions are considered i n the following. 319 The phycobilisome spectra (PBS) are dominated by the contribu­t i o n of the cons t i t u e n t phycocyanin trimers (PC) and are there­fore r a t h e r s i m i l a r to the spectra of the l a t t e r . Most of the v i b r a t i o n a l frequencies are reproduced w i t h i n experimental accuracy, though with d i f f e r e n t i n t e n s i t i e s ( e s p e c i a l l y between 1200 and 1300 cm-1) ( f i g 1 ) . At 1623 cnr 1 an a d d i t i o n a l weak peak i s found i n PBS, which i s due to allophycocyanin (APC). A second i n t e r e s t i n g feature i s that for the pronounced bands a smaller width i s found i n PBS than i n PC. I t p o s s i b l y i n d i ­cates a lower s t r u c t u r a l inhomogeneity i n PBS due to higher aggregation and the presence of l i n k e r peptides. 1200 1220 1210 12I1H 1280 1500 1550 1600 IGS0 1/(HI 1 Λ.Π Wavenumber s Vevcnumbers F i g . 1: Resonance-enhanced CARS spectra of phycobilisomes, phycocyanin trimers and allophycocyanin trimers from Mastigocladus laminosus. Pump wavelength 640 nm. 320 APC spectra are very d i f f e r e n t from PC spectra (see also r e f . 10). The 1257 cm"1 and 1287 cm"1 bands observed i n PC are absent i n APC. I n the double bond s t r e t c h i n g region the dominant peak i n PC i s found at 1649 cm"1 with shoulders at «1631 and »1662 cm"1 . I n APC sp e c t r a , however, the dominant peak i s found at 1625 cm"1 with a strong shoulder at «1635 cm"1 and a weak one at «1650 cm"1. Discussion According to preliminary normal mode c a l c u l a t i o n s using the coordinates provided by X-ray a n a l y s i s (12,13), the bands found between 1200 and 1300 cm - 1 represent e x t e n s i v e l y mixed C-C s t r e t c h / C-H i n plane bending modes and can presently not be i n t e r p r e t e d i n a straight-forward manner. The band around 1585 cm"1 i s r e l a t e d to C=C s t r e t c h i n g v i b r a t i o n s i n the r i n g B/C fragment. That t h i s band e x h i b i t s n e a r l y no change i n frequency, most l i k e l y i n d i c a t e s a conservation of the geometry of t h i s part of the chromophore. The C=C double bonds of the methine bridges between r i n g A-B and r i n g C-D, on the other hand, should give r i s e to l o c a l i z e d modes with frequencies between 1650 and 1700 cm"1. The C=0 s t r e t c h mixes with the C=C s t r e t c h i n r i n g D to produce v i b r a ­t i o n a l frequencies around 1630 cm"1. The a c t u a l value of the c a l c u l a t e d frequencies i s found to be s e n s i t i v e to s t r u c t u r a l changes. From these t h e o r e t i c a l r e s u l t s and the comparison of CARS spectra of model compounds (9) with the presented CARS spectra a s i g n i f i c a n t d i f f e r e n c e i n chromophore geometry and/or Π-electron d i s t r i b u t i o n between PC and APC must be postulated. I n t e r p r e t a t i o n of e.g. fluorescence decay data of APC should therefore not be based on s t r u c t u r a l information gained from PC. 321 Additional measurements on antenna pigments and a more elabo-r a t e normal mode a n a l y s i s are i n preparation. Acknowledgement F i n a n c i a l support by Deutsche Forschungsgemeinschaft (SFB 143) and Fonds der Chemie i s g r a t e f u l l y acknowledged. References (1) Gantt E. 1981. Phycobilisomes . Ann. Rev. Plant P h y s i o l . 32, 327 - 347. (2) Scheer H. 1982. I n : Light Reaction Path of Photosynthesis (F.K. Fong, ed.). Springer, B e r l i n , pp. 7 - 45. (3) Schneider S.,F. Baumann, U. Klüter, 1987. Ζ. Naturforsch. 42c(11/12). (4) Schneider S., F. Baumann, P. G e i s e l h a r t , S. Siebzehnrübl, R. F i s c h e r , H. Scheer, 1987. Proc. of the Sec. Europ. Conf. on the Spectr. of B i o l . Mol., J.Wiley, Chichester. (5) Schneider S., F. Baumann, P. G e i s e l h a r t , Η. Kayser, Η. Scheer, 1987. Photochem. Photobiol., submitted. (6) Nies Μ. and W. Wehrmeyer, 1980. Planta 150, 330 - 337. (7) Füglistaller P., Η. Widmer, G. Frank and H. Zuber, 1981. Arch. Microbiol. 129, 268 - 274. (8) Martin R.G. and B.N. Ames, 1961. J . B i o l . Chem. 236, 1372 - 1379 (9) Schneider S., P. G e i s e l h a r t , F. Baumann, Η. Falk and W. Medinger, 1987. J . Biochem. Biophys., submitted. (10) Sz a l o n t a i B., Z. Gombos, V. Csizmadia, 1985. Biochem. Biophys. Res. Com. 130, 358-363. (11) Sz a l o n t a i Β., Z. Gombos, V. Csizmadia and M. Lutz, 1987. Biochim. Biophys. Acta 893, 296-304. 322 (12) Schirmer Τ., W. Bode and R. Huber, 1985. J . Mol. B i o l . 184, 257-277. (13) Schirmer T., W. Bode and R. Huber, 1987. J . Mol. B i o l . , 196, 677-695. (14) Schneider S., C. Scharnagl, M. Duerring, T. Schirmer and W. Bode, 1987. Contribution i n t h i s volume. 

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Coherent Anti-Stokes Raman Spectroscopy Of Phycobilisomes, Phycocyanin And Allophycocyanin From Mastigocladus Laminosus

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Coherent anti-Stokes Raman spectroscopy of phycobilisomes, phycocyanin and allophycocyanin from Mastigocladus laminosus

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