Optical and optically detected magnetic resonance investigation on purple photosynthetic bacterial antenna complexes

Chemical Physics ELSEVIER

Chemical Physics 194 (1995) 259-274

Optical and optically detected magnetic resonance investigation on purple photosynthetic bacterial antenna complexes A. Angerhofer a,l,*, F. Bornhfiuser a, A. Gall b, R.J. Cogdell b,, a 3. Physikalisches Institut, Universitiit Stuttgart, D-70550 Stuttgart, Germany b Department of Botany, The University of Glasgow, Glasgow, G12 8QQ, UK Received 4 November 1994

Abstract

Low temperature ( 10 K) absorption, fluorescenceemission, fluorescence excitation, absorption detected magnetic resonance, and microwave induced absorption spectra were recorded together with temperature dependent light induced absorption transients of carotenoid triplet formation and decay in the B800-850 antenna complexes of Rb. sphaeroides G1C, Rb. sphaeroides 2.4.1, Rps. acidophila 10050, Rps. palustris I, and Rps. palustris II (low-light), in B800-820 antenna complexes of Rps. acidophila 7050, and Rps. cryptolactis, and in the B830 complex of Chr. purpuratum. From the spectra the zero field splitting parameters [ D I and I E [ were extracted as well as the energies of the main carotenoid triplet-triplet and singletsinglet transitions. They are linearly dependent on the inverse of the number of conjugated double bonds in the polyene chain. The rates of triplet carotenoid formation correlate with the relative intensity of the carotenoid-bacteriochlorophyll interaction bands observed in the 800-850 nm region in the microwave induced absorption spectra. Both, the triplet energy transfer rate and the interaction bands depend on the exchange interaction between the excited and ground state wavefunctions of the two pigments.

I. Introduction

The function of carotenoids (Car) in photosynthetic pigment-protein complexes is two-fold [ 1-7]: They

* Corresponding authors. I New address: Chemistry Department, University of Florida Gainesville, FL 32611, USA. FAX: (904) 392 0872. Abbreviations. ADMR: absorption detected magnetic resonance; AT: light-harvesting antenna complex; BChI: bacteriochlorophyll; BPhe: bacteriopheophytin; Car: carotenoid; Chr.: Chromatium; EPR: electron paramagnetic resonance; LD-ADMR: linear dichroism detected ADMR; LDAO: lauryi-dimethyl-amineoxide; LDM1A: linear dichroism detected MIA; LH: light harvesting; MIA: microwave induced absorption; MO: molecular orbital; OD: optical density; Rb.: Rhodobacter; RC: reaction centre; Rps.: Rhodopseudomonas; T-S: triplet-minus-singlet; zfs: zero field splitting.

serve as efficient antenna (AT) pigments to cover the green and blue region of the visible light spectrum and transfer their excitation energy to the chlorophyll (Chl) or bacteriochlorophyll (BChl) molecules for further usage in the primary light separation process in the photosynthetic reaction centre ( R C ) . Secondly, they are used as efficient quenchers of Chl and BChl triplet states to prevent singlet oxygen formation and subsequent harmful oxidation reactions in the organism. By the quenching reaction the effective lifetime of the Chl triplet excitation is reduced by more than 3 orders of magnitude. The Car triplet (3Car) itself lives of the order of 5 - 1 0 / z s , its energy level being close or below that of singlet oxygen.

0301-0104/95/$09.50 (~) 1995 Elsevier Science B.V. All rights reserved SSDI 0301 -01 0 4 ( 9 5 ) 0 0 0 2 2 - 4

260

A. Angerhoferet al./Chemical Physics 194 (1995) 259-274

AT complexes of purple photosynthetic bacteria are generally classified by their redmost absorption bands. B880, which is closely connected with the RC, B800-850, and B800-820 are the most common types encountered [8-10]. The AT complexes studied in this contribution were mostly from purple non-sulfur photosynthetic bacteria. We used B800850 complexes of two strains of Rhodobacter (Rb.) sphaeroides 2.4.1 and G1C, Rhodopseudomonas (Rps.) acidophila 10050, Rps. palustris type I and II, where type II denotes the complex isolated from bacteria grown under low-light conditions. The B8008 2 0 complexes studied were isolated from Rps. acidophila strain 7050, and Rps. cryptolactis. The main carotenoids contained in these strains are: Neurosporene (9 conjugated double bonds in the polyene chain) in Rb. sphaeroides G1C [ 11 ], spheroidene and spheroidenone (10 conjugated double bonds) in Rb. sphaeroides 2.4.1 [12], rhodopin and rhodopin analogues ( 11 conjugated double bonds) in Rps. acidophila [ 13], a mixture of carotenoids with 11 to 13 conjugated double bonds (lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin, OH-spirilloxanthin, and spirilloxanthin) in Rps. palustris [ 14], and similarly in Rps. cryptolactis [ 11 ]. Additionally, we investigated the B830 complex of Chromatium (Chr.) purpuratum, a member of the purple-sulfur type marin6 bacteria, first described in 1980 [ 15]. Its photosynthetic membranes contain the B870-RC complex next to an LH-II complex, the B830 pigment-protein complex [16] which was the subject of our study. It contains the rarely found ketocarotenoid okenone. From resonance Raman and NMR experiments it is well known that the carotenoids in light-harvesting proteins are in all-trans configuration [ 17-21]. We have previously performed absorption detected magnetic resonance (ADMR) measurements on a series of B880-RC complexes [22] and on the LH-II complexes of Rps. acidophila 7050 and Rb. sphaeroides 2.4.1 [23,24] to correlate the zero field splittings (zfs) of the carotenoid triplet states with the length of the respective polyene chain. The present contribution is an extension of these earlier studies in which we use the high sensitivity and selectivity provided by ADMR to correlate not only zfs values with chain lengths, but also to investigate the carotenoidBChl interactions which are so essential in the triplet

energy transfer process of photoprotection. In addition, we have used purely optical studies, i.e. transient absorption, and fluorescence and fluorescence excitation spectroscopy, to study the rates of triplettriplet energy transfer as well as the efficiencies of Car--~BChl singlet-singlet energy transfer.

2. Materials and methods

2.1. Sample preparation The light-harvesting complexes used in the present study were prepared as described by Cogdell et al. [25], by a combination of sucrose density centrifugation and ion exchange chromatography on DEAE cellulose. In all cases the complexes were finally dissolved in 20 mM Tris HCI pH8.0, 0.1% LDAO, and then stored at - 2 0 ° C until required for use. For ADMR and low temperature absorption measurements the samples were thawed and diluted with glycerol (50 : 50 vol/vol) to form a clear glass upon freezing. The optical density of the sample in the NIR bands was adjusted to about 0.7 at 2 mm path length. For fluorescence experiments the OD was adjusted to approximately 0.02 at the redmost bands at 2 mm path length in order to avoid reabsorption effects.

2.2. Spectroscopic methods Absorption spectra at cryogenic temperatures were taken with the ADMR set-up using it as a single-beam spectrometer. The reference sample was a buffer glycerol mixture which was measured under identical conditions. The block diagram of our home-built ADMR apparatus is shown in Fig. 1 (upper part) which deviates somewhat from a set-up described earlier [26,27]. We use a 150 W tungsten-halogen lamp as excitation and detection light source. It is driven by an EA-4021 regulated power supply and yields an intense light flux with very little noise, essential for the high sensitivity needed. The light is collected by a condensor lens, passes through a water filter (WF) and an appropriate colour filter combination (FI), and is finally focused on the sample cuvette (2 mm path length). It is placed in a copper coil with 3 windings (ca. 6 mm diameter) which is used to couple the RF into the sample. Cool-

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274

~

T

[~

RF source

PIN

& S O ~ RF load

pulse ener.

RF amplifier

coil

double monochromotor

CF 1204

Fi WF lamp

....

double monochromator

CF 1204

Fi WF lamp

Fig. 1. Upper part: ADMR set-up. Lower part: Experimental set-up for transient absorption measurements. For details see text.

ing is performed by an Oxford CF1204 helium flow cryostat with 4 quartz windows. The transmitted light is focused on the entrance slit of a 1/4 meter double monochromator (Spex, Spectramate 1680B) the stepping motor of which is controlled by a microprocessor (CPU). The dispersed light is finally focused on a silicon avalanche photodiode (APD, RCA, model C-30955 E) which is driven at a reverse bias of 289 V from a high voltage power supply (fug, MCN 142000). The detector signal is further amplified by a current-to-voltage converter and preamplifier (Ithaca, model 1642) the output of which is fed into the lockin amplifier (Ithaca, model Dynatrac 393). The reference for the lock-in is supplied by a Wavetek function generator (model 20) which is also used to drive the PIN diode (General Microwaves, model DM 8638, or Minicircuits, model ZSDA-230). Data acquisition is performed by a microprocessor (CPU, Atari Mega ST) via VME-bus (TVE VME 2). Data storage and processing is done on a HP 720 station. The micro-

261

computer sets the wavelength of the monochromator as well as the frequency of the RF-source (direct synthesizer, Ailtech model 360D11 ) the output of which is modulated by the PIN-diode and fed into an RFamplifier (SCD, model ARS 10.40.40, Amplifier Research, model 25W1000M7, Hughes C-Band TWT, model 1177H, or Minicircuits ZHL 2-12). The output of the amplifier is put through a circulator (Teledyne, model 24022, or UTE, model CT-2103-N) which protects the amplifier against back reflected power which is dissipated by an RF load at port 3. The port 2 output of the circulator is fed into an RF attenuator (Weinschel Engineering, 3 dB) to suppress unwanted standing waves, since the coil is not matched to 50 ~, and finally through a semi-rigid coaxial cable into the ODMR coil. Fig. 1 (lower part) shows the apparatus for the time-resolved absorption difference spectra. We use the same set-up as in the ADMR experiment which allows us to perform both experiments on the same sample without changing any parameters like temperature, etc. In order to avoid high light flux in the detection beam, we use appropriate interference filters to pre-select the wavelength out of the spectrum of the halogen lamp. Another intereference filter is used to remove unwanted spontaneous dye emission from the laser beam. Pulsed excitation is performed by an excimer pumped dye laser (Lambda Physik LPX240iCC, and LPD 3002) using Styryl 9. The excitation wavelength could be varied between 820 and 860 nm and was adjusted to give optimum BChl excitation in the NIR bands of the AT complexes. Pulse widths were slightly shorter than 20 ns; the energy per pulse was set to approximately 1 mJ to avoid saturating conditions. In order to avoid jitter between the firing of the laser and the data acquisition we used a photodiode which used a light reflex of the pumping laser beam to give an accurate trigger signal with which the transient recorder was started. Detection was performed by a blue-enhanced avalanche photodiode (Hamamatsu, model $5343 ). Amplification of the photodiode signal was performed with a large bandwidth low-noise trans-impedance amplifier (FEMTO Messtechnik, model HCA-200M-20K-DC, bandwidth DC-200 MHz). The transient recorder was a 125 MHz bandwidth digital oscilloscope (LeCroy 9400) which was connected to the VME-bus computer via an ICE interface.

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274

262

4-_

.. Rps. ~

Rb..sphoeroides G1C,

....

,_%2_ "~1. "~0.

_4 3E 2~ lZ-

<~

35. 30_ E 25. ~q20. ~15. ~,10_ "< 5 0 20.0 17.5 E 15.0 g 12.5 10.0 ,,~ 7.5 5.0 2.5 0 25

Rb. sphoeroides 2.4.1 xlO

.

hromotium purpurotum

Rps. cryptolactis xlO

Rps. acidophila 7050

20

0 0.150 0.125 0.100 0.075 0.050 w. 0.025 <3 0 -0.025 8

.20.0 17.5

15.o

12.5 o.

10.0 _7.5

~5_

_5.0 _2.5

O_

2;0

560

7;0

Frequency [MHz]

10'00 12'50 2;0

560

7;0

Frequency [MHz]

"=

10'00 12'50

Fig. 2. ADMR spectra of the carotenoid triplet states in the different AT complexes acquired at 10 K. Microwave power was 500 mW. The weak I D ] 4- ] E [ signals were amplified by a factor of 10 compared to the 21 E I signals for which the vertical scale applies.

Our fluorescence and fluorescence excitation spectrometer was described in Refs. [28-30]. It is integrated on the same optical table as the ADMR and transient absorption spectrometer. Sample cooling is performed in the same cryostat using a sample holder that holds cylindrical quartz tubes (2 mm inner diameter). Excitation is performed by a Xe arc lamp (XBO900) the light of which passes a 5 cm water filter and a 0.22 m monochromator (Spex doublemate) before it is focused on the sample in the cryostat. Intensity regulation of the exciting light is performed by a variable slit at the entrance of the excitation monochromator. The slit width is controlled by an analog circuit using the output of a monitoring photodiode as a reference. It measures a fraction of the light transmitted by the monochromator. The light flux incident at the sample position was controlled and measured with a bolometer to get reference spectra with which the fluorescence excitation spectra were corrected. Fluorescence emission was recorded at 90 ° to the excitation beam using the detection set-up described above. Detection was performed with suitable Peltier- or liq-

uid nitrogen-cooled photomultipliers, with $20 or S 1 characteristics (EMI 9658QB, and EMI 9684). The preamplifier in that case was a Keithley 414A picoamperemeter. The spectral transparency of the detection path was measured using a calibrated tungsten band lamp (Osram Wi 17/G, test certificate by the Physikalisch-Technische Bundesanstalt No. 3.1330374/70). All emission spectra were thus corrected to show intensity proportional to the actual fluorescence quantum flux for the given spectral resolution, usually a 4.5 nm bandwidth for 2.5 nm slits.

3. Results Fig. 2 shows the ADMR spectra taken on the main triplet-triplet absorption band of the carotenoids in the 8 preparations investigated. In some cases the signals were so weak that only the 2 I E I signal could be recorded. It has been found to be the most intense ODMR signal of the carotenoid triplet state in as diverse preparations as bacterial RCs [22,24,31 ], and ATs [22-24], as well as the AT-complexes of higher

A. Angerhofer et aL / Chemical Physics 194 (1995) 259-274

263

Table 1 Frequencies of the 2 I E I and I D I 4- I E I transitions (in MHz), and zfs values calculated from them (in 10 -4 cm -1 ) of the carotenoid triplets in the AT complexes studied, with the number of conjugated double bonds in the corresponding polyene chain Sample

21 EI

I D l-

Rb. sphaeroides G1C B800-850 Rb. sphaeroides 2.4.1 B800-850 Rps. acidophila 10050 B800-850 Rps. acidophila 7050 B800-820 Rps. palustris 1 B800-850 Rps. palustris 11 (low-light) B800-850 Rps. cryptolactis B800-820 CTlr. purpuratum B830

215.7 209.1 173.5 170.7 158.8 161.2 161.4 216.7

890 753 759 644 648 602

plants [32-34], and dinoflagellates [35]. The I D ] -4- I E [ signals are usually weaker in intensity by more than an order of magnitude. The I D l a n d l E ] values calculated from these spectra are given in Table i. Microwave induced absorption (MIA) and absorption spectra, together with fluorescence emission and fluorescence excitation spectra of the 8 different samples are shown in Figs. 3 and 4. The spectral location of the absorption, emission and MIA bands together with their intensities normalized to the redmost BChl band (for absorption and fluorescence excitation spectra), main fluorescence band (fluorescence emission spectra), and main 3Car triplet-triplet absorption band (MIA spectra) are given in Table 2. The main feature of the carotenoid MIA spectra in the visible part is the (positive) triplet-triplet absorption band which always appears slightly to the red side of the red-most So ---~$2 absorption band (compare with absorption spectra and Table 2). It is superimposed by the bleaching bands of the carotenoid singlet-singlet (So ---~$2) absorption which causes the succession of positive and negative MIA bands between 400 and 600 nm. In the BChl region additional bands are seen, which in most cases appear much weaker than the main Car triplet-triplet absorption band. Bleachings around 800 nm are visible in the MIA spectra of the two preparations of Rps. palustris. In the other AT complexes either no or only very weak bleachings around 800 nm are observed. Bleachings at 820 and 850 nm (depending on whether B800-820 or B800-850 complexes are investigated) are seen in all preparations with the only exception of Rps. acidophila 7050. These "interaction bands" (see Section

let

I D l+ IEI

I EI

]D[

n

1091 930 950 813 818 765

36.0 34.9 28.9 28.5 26.5 26.9 26.9 36.1

330.4 280.7 281.6 243.0 244.5 228.0

9 10 11 11 12 12 13 10

4.3) are especially strong in the case of the B830 complex of Chr. purpuratum. A strong positive feature at 825 nm with a strong negative one at 834 nm suggest the appearance of a blue shift o f a BChl Qy band from 834 to 825 nm. All BChl bleaching bands are accompanied by more or less well pronounced positive and broader MIA bands just to the red. The fluorescence emission spectra exhibit as the usually most pronounced peak the fluorescence band of the longest wavelength antenna chromophore, i.e. the B850 or B820 pigments. In the case of the B800-820 complexes (Rps. acidophila 7050 and Rps. cryptolactis) weak shoulders to the red side of the B820 emission indicate the presence of some impurity B800-850 complexes. Fluorescence of the B800 chromophore is directly seen as a separate emission band in Rb. sphaeroides 2.4.1 and the two palustris preparations, and as a shoulder on the "free" BChl emission (around 780 nm) in Rb. sphaeroides G1C. These assignments are based on the excitation spectra of these emission bands (data not shown), and indicate somewhat insufficient energy transfer between the B800 and B850 chromophores. Free BChl emission is especially pronounced in the case of Chr. purpuratum where it appears stronger than the emission of the B830 complex. The fluorescence excitation spectra of the red-most fluorescence bands correspond very well to the absorption spectra with respect to band positions (see Table 2) with the only exception of Chr. purpuratum. There the carotenoid excitation bands are at different spectral locations than in the absorption spectrum. This may reflect a heterogeneity in the carotenoid pool which in turn may lead to a wrong evaluation of the

264

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274

Table 2 Spectral locations of the absorption peaks, fluorescence excitation and emission bands, as well as the positive and negative MIA bands (in nm) together with their respective intensities (in parentheses), normalized to the redmost main BChl absorption band, the main BChl fluorescence band, and the main 3Car triplet-triplet absorption band (MIA) Sample

Absorption

Fluorescence excitation

Emission

MIA

Rb. sphaeroides GIC

433(46.4),459(55.6), 494(54.7), 588(15.7), 800(83.4), 855(100)

435(22.7),462(38.4), 495(46.0), 590(20.4), 799(96.0)

788(29.7), 833S(3.3), 888(100)

459(-53.5), 479(+30.8), 495(-46.3),516(+100), 856(-12.9)

Rb. sphaeroides 2 . 4 . 1

451(37.3), 480(51.1), 514(53.6), 586(18.8), 799(89.1), 852(100)

454(20.7), 481(32.5), 517(38.1), 588(18.0), 800(94.4), 849(100)

792(4.4), 884(100)

449(-14.8), 465(+0.8), 477(-41.7), 499(+35.2), 514(-44.9), 537(+100), 802(-2.0), 841(-3.1), 865(+7.5)

Rps. acidophi~ 10050

438(16.2),463(29.1), 492(43.2), 529(46.9), 591(23.3), 804(94.5), 870(100)

438(5.4), 466(8.8), 496(13.9), 531(16.1), 593(17.9), 804(87.6), 872(100)

890(100)

463(-31.1), 491(-33.6), 530(-24.5), 556(+100), 803(-8.1), 868(-13.7), 891(+7.7)

Rps. acidophi~7050

438(11.7), 462(35.6), 491(59.7), 528(65.0), 586(26.1), 800(140.3), 825(100)

436(7.1), 464(13.7), 493(21.5), 528(22.8), 585(15.7), 799(100.6), 827(100)

845(100)

459(-32.8), 491(-30.0), 527(-27.9), 555(+100), 802(-3.1), 839(+5.0)

Rps. palustris I

469(33.7), 489S(54.5), 501(62.3), 529S(53.4), 542(43.9), 590(33.8), 804(185.9), 868(100)

469(10.4), 490S(13.2), 501(15.1), 530S(15.1), 542(14.2), 592(24.2), 804(108.5), 869(100)

695(9.1), 772(5.5), 822(16.2), 890(100)

470(-22.6), 502(21.7), 541(-11.4), 567(+100), 801(-13.6),825(+4.4), 853(-3.0),884(+7.6)

Rps. palustris II (low-light)

466(57.4), 495(87.4), 530(84.8), 544S(47.4), 589(50.9), 805(350), 865(100)

467(16.0), 495(22.5), 530(25.1), 544S(17.8), 591(29.0), 804(170.1), 867(100)

700(2.4), 780(3.1), 821(36.6), 888(100)

465(-32.2), 498(-25.3), 536(-4.7), 551 (+64.9), 567( + 100), 801 ( - 19.1 ), 819(+8.4), 881 (+8.4)

Rps. cryptolactis

497S(32.5), 513(38.5), 524S(37.7), 554(33.5), 798(98.4), 825(100), 865(18.0)

496S(5.3), 520(7.1), 556(8.4), 582(10.5), 826(100)

708(0.8), 778(2.5), 843(100) 885(23.5)

490(-24.2), 524(-22.6), 561 (-3.3), 589(+ 100), 842(+13.5)

Chr. purpuratum

494S(34.9), 524(51.5), 549(51.8), 588(42.7), 680(3.3), 727(10.3), 798(38.0), 832(100)

540(37.1), 588(39.4), 771(34.6), 799(41.5), 833(100)

699(10.8), 778(180.6), 851(100)

510(-56.1), 574S(+26.2), 623(+100), 825(+151.4), 834(-206.6), 855(40.2)

A. Angerhofer et al./ Chemical Physics 194 (1995) 259-274

Wavelength [nm] 600 700 800

4OO 500 i

i

i

i

i

go0 i

1000 i

400 i

500 i

Wavelength [nm] 600 700 800 i

i

265

900

i

1000

i

i

8,75 7.50

1.50, 1.25,

:oo:

g

0.50. 0.25. 0 •~ -0.25, -0.50 _

-0.75.

6,25 5.00

Rb. sphoeroides 2.4.1 209 MHz

Rb. sphoeroides G1C 216 MHz

....tilL_

3,75 .2,50

"E

.1.25

S : -1.25 < -2.50 -3.75 1.125

0.6

1.000

J~

0.5

il i! ~

0,875 0.750

0.4_

0.625 c~ 0.500 o

o 0.5_

0,375 0.250 0.125 0

0,2_ 0.1 0 I

I

t

I

I

I

I

I

I

I

I

I

I 1.25

4 3

I

Rps.

ocidophilo

1.00

Rps. ocidophilo 10050 174 MHz

7050

22

0.75 0.50 0.25

1 .

.

.

.

.

o

.

-0.25

-1 0.7

L

1.125 _ 1.000

0.6

i

0.5 0.4

1

0.3

'I

_ 0.875

I I

_ 0.625 t~ .0.500 o

.0.750

_ 0.375 _ 0.250

0.2 0.1 i ,

.

,

400

500

600

.

700

,

800

,

.

,

900 1000 400

,

500

Wavelength [nm]

.

600

,

,

700

600

-_

_0.125

_o

,

900 1000

Wavelength [nm]

Fig. 3. Absorption (lower part, - - ) , fluorescence emission (lower part, - - - ) , fluorescence excitation (lower part, - - -), and MIA spectra (upper part, - - ) of four different AT complexes at 10 K. The RF frequencies at which the MIA spectra were taken are denoted next to the names of the respective bacterial strains. Table 3 I D ] and ] E ] values (in 10 - 4 cm - ] ) and wavelengths (in nm) of the MIA spectra of the different triplet states found in Rps. palustris and Rps. cryptolactis together with the number of conjugated double bonds n of the carotenoids to which these triplets are related. In the case of Rps. cryptolactis the ] D ] 4- I E [ signals could only be observed unequivocally for the most prominent carotenoid triplets (n = 13). ] D I could therefore not be determined for the carotenoid triplets with n = 10, 11. The identification of these polyene chain lengths depends solely on the wavelength of the triplet-triplet absorption band in the MIA spectrum recorded on the 21 E I signal Sample

IO I

[E I

MIA bands

n

Rps. palustris [

220.7 240.2 275.2

23.2 26.7 28.4

4 8 6 ( - 4 2 . 1 ) , 5 1 8 ( - 2 5 , 5 ) , 5 5 9 ( - 1 7 . 9 ) , 584(+100) 4 7 2 ( - 1 4 . 0 ) , 504( -17,1 ), 5 4 3 ( - 2 0 . 2 ) , 567(+100) 4 6 0 ( - 2 4 . 4 ) , 491 (26.1), 5 3 0 ( - 2 6 . 4 ) , 550(100)

13 12 11

Rps. cryptolactis

227.7

26.7 30.4 31.7

5 2 5 ( - 2 3 . 5 ) , 5 6 5 ( - 3 . 6 ) , 589(+100) 4 7 8 ( - 3 0 . 9 ) , 5 1 0 ( - 2 3 . 3 ) , 5 5 0 ( - ) , 578(+100) 4 7 3 ( - 4 6 . 4 ) , 5 0 2 ( - 4 3 . 7 ) , 5 4 2 ( - ) , 558(+100)

13 12 11

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274

266

400 i

500 i

Wavelength [nrn] 600 700 800 i i i

900 i•

1000 400 I

Wavelength Into] 600 700 800

500

l

I

I

I

900

1000

I

I

I

.2.5 .2.0 .1.5

8.75.

7.50. 6.25.

Rps. polustris II 161 MHz

Rps. polustris I

I

.~. 3.75. 2.50. " ~ - 11"205'.25.' ""

E

.1.0

.o-°+ . -0.5

--. - - - - - - - - - - - - ~ --r,'o'+'+.a"+"J" ......

.0.8 1,000 0,875 0,750 0.625. £3 0 0.500_ 0.375 0.250. 0.125 _ O-

.0.7 I

.0.6 .0.5

.~ i! ~

f

.0.4 .0.3

~

.0.2

•\

0.1

.~

I

I

t

I

I

I

I

I

I

I

I

I

Chromotiurn purpurotum

7-

I I

0

I 1.0

Rps. cryptoloctis 160 IdHz

....

:¢-

i

p~

0.8 0.7 0.6 0.5 0.4. 0.3. 0.2.

!ii !i i

!i~ I

o.1.

r-X / ~

! i i! !,,i Ii

-0.5 ~ -1.0 -1.5 -2.0 0.40

0.35 _o.3o .o.25 o2o~ .o15 " ...... o

..j~.._

O.

++0 5~0 8~0 7~0 8ao 9ao 10'00 +ao 5~0 660 7ao 8ao 9ao 1000 Wavelength [nm]

Wavelength [nm]

Fig. 4. Absorption (lower part, - - ) , fluorescence emission (lower part, - - - ) , fluorescence excitation (lower part, - - -), and MIA spectra (upper part, - - ) of 4 different AT complexes at 10 K. The RF frequencies at which the MIA spectra were taken are denoted next to the names of the respective bacterial strains.

singlet energy transfer yield (see Section 4.5). Due to this complication the 57% singlet-singlet energy transfer efficiency for okenone which we deduce from the spectra (see Table 4) are just a lower limit for the real energy transfer efficiency for the carotenoid pool that strongly interacts with BChl, while another carotenoid pool may exist which shows much less interaction and a lower yield of energy transfer, The relative band intensities of excitation and absorption spectra are partly different which reflects the less than 100% efficiency of excitation energy transfer between the various pigments within the complex, The absorption and excitation spectra are plotted normalized to equal intensity of their respective longest

wavelength bands. In this representation the intensities of the excitation bands relative to those of the absorption bands reflect the yield of energy transfer from that transition to the emitting pigment. 1Car* ---, IBChl* energy transfer is relatively strong in the case of Rb. sphaeroides G1C and Rb. sphaeroides2.4.1. The values evaluated from the absorption and excitation spectra of all preparations are given in Table 4. Excitation energy transfer from B800 to B850 is usually quite efficient with the marked exception of the two palustris preparations where it approaches almost 50%. However, this could in principle also reflect a higher instability of the B850 chromophore. If it is selectively destroyed dur-

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274

5_

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T

,

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1_

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_

_ _

1.25 1.00 0.75 0.50 0.25

_ _

_ 160 MHz/~ I

640 MHz

_

_

-1.25 _

! o_

0.25

0.875 0.750 0.625

&

<]

-0.25

--2-

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c~

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0.20

0.500 0.375 0.250 0.125 0

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o

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-0.05

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-0.10 i

/

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500

i

i

600 700 800 wovelength [nm]

i

900

460 560

6oo

wovelength [nm]

760

Fig. 5. MIA spectra of 3Car in the B800-850 complex of Rps. palustris 1 at 10 K. Left: spectra taken by pumping the 21 E I signal at 139, 160, and 170 MHz. Right: spectra taken by pumping the ] D ] - ] E I signal at 592, 640, and 740 MHz. The microwave power was set to 1 W.

ing preparation or sample handling its absorption would most likely shift to the blue towards that of free BChl, and thus the B850-peak decrease showing a wrong ratio of intensities between B800 and B850. In the fluorescence excitation spectra, however, only the intact complexes would be sampled since they retain their long-wavelength fluorescence band. A similar argument applies to the B800 band of Rb. sphaeroides G1C and Rb. sphaeroides 2.4.1. It is known [ 11 ] that it is much more instable then the B850 chromophore in these AT complexes. In fact, prolongued exposure to glycerol at room temperature almost completely destroys B800 but does not affect B850. Since we need to add glycerol to get a glassy matrix, our sample is prone to this degradative effect, even though the time between admixture of glycerol and freezing to very low temperatures was kept to a minimum. Because of these uncertainties we decided not to evaluate the energy transfer efficiencies between the B800 and B850 chromophores further.

Table 4 Efficiencies of singlet energy transfer ( i Car* + 1BChl ---*t Car + 1BChl* ), deduced from the absorption and fluorescence excitation spectra Sample

Quantum yields

Rb. sphaeroides GIC Rb. sphaeroides 2.4.1 Rps. acidophila 10050 Rps. acidophila 7050 Rps. palustris I Rps. palustris II (low-light) Rps. cryptolactis Chr. purpuraturn

88% 83% 38% 39% 26% 23% 33% 57%

In the cases of Rps. palustris and Rps. cryptolactis the carotenoid composition is rather heterogeneous with polyene lengths between n = 11 and n = 13. It had already been shown for the B880-RC complexes of Rps. palustris that the total carotenoid composition is not only built into the AT complexes but also par-

268

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274 Temperature [K] 20 10

300 I

I

5 I

I

108-:

~

I0-8

÷ 4v÷ vV A

÷

X

•

I +-f

10 7 "

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10-6 ~

,.~ 10 6. Od

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105 • ~ × × ×

+ ×

+

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~' 10 .5

palustris II (low-light)). Within the limits of experimental error, only the B800-850 complex of Rps. acidophila 10050 shows a temperature dependence of 3Car induction with an activation energy of 28 cm -1 . Generally, the rise times of the carotenoid triplet states, and thus the triplet energy transfer times lie between 20 ns (our spectrometer-limited time-resolution) (B800-850 of Rps. acidophila at higher temperatures and Chr. purpuratum) and 200 ns (Rb. sphaeroides 2.4.1). The population rates for the carotenoides in Rps. c~ptolactis and Chr. purpuratum were at the instrumental limit at all temperatures tested. Data are given in Fig. 6 only for selected temperature points. The lifetimes of the carotenoid triplet states vary between 3 and 15/.ts.

x

4. Discussion I

o.o

o. o

o. 5

0.20

I / l - e m p e r a t u r e [K - I ]

Fig. 6. Rates of 3Car formation (filled and bold symbols) and decay (open and light symbols) for Rb. sphaeroides G 1C ( x ), Rb. sphaeroides 2.4.1 ( O ), Rps. acidophila 10050 B800-850 complex (+), Rps. palustris type I (W), Rps. palustris type II (A), Rps. cryptolactis B800-820 complex ( 0 ) , and Chr. purpuratum B830 complex (Fq). A full temperature dependence of the 3Car population rates was only recorded for the B800-850 complexes of Rb. sphaeroides 2.4.1, Rb. sphaeroides G1C, Rps. acidophila 10050, and Rps. palustris I.

ticipates in the triplet energy transfer process [22]. Therefore at least three different carotenoid triplet states can be observed belonging to carotenoids with 11, 12, and 13 conjugated double bonds. We show in Fig. 5 that this is also the case for the B800-850 complexes of Rps. palustris. We found the same to be true for Rps. cryptolactis (data not shown). When pumping with RF at different frequencies in the 2 ] E I or ] D I - [ E [ region, the MIA spectra of three different carotenoids are clearly distinguished. The ] D [ and ] E I values taken from these "site-selection" experiments on Rps. palustris and Rps. cryptolactis are given in Table 3. The results of the transient absorption measurements are seen in Fig. 6. We have recorded the temperature dependence of the rise time of the carotenoid triplet-triplet absorption as well as its decay for the B800-850 complexes (with the exception of Rps.

4.1. Zero field splitting parameters The zfs parameters of the carotenoid triplet states measured in the B800-850 and B800-820 complexes show the same inverse dependence on the number of conjugated double bonds "n" as in the case of the B 880 complexes, and thus fully agree with our earlier observation [22]. Fig. 7 shows this dependence for the zfs values along with D and E values from carotenoids in other bacterial and plant photosynthetic AT complexes collected from the literature [22-24,33-35]. The straight lines through the data points are fits to D = --D1+ D ~ ,

(1)

n

E = E-! + Eoo. n

(2)

The fit parameters D I , ~ and E I , ~ are D1 = 4812(±124) × 10 -4 cm - l , D ~ = - 1 5 2 ( + 1 2 ) × 10 -4 cm -1, E] = 390(±17) × 10 - 4 cm -1, and Eoo = - 5 ( 4 - 2 ) × 10 -4 cm -1. The rationalization of this inverse behaviour of D and E on n was given by Ros et al. [ 36]. It is due to the approximate Gaussian distribution of the triplet state wavefunction along the polyene chain which is already predicted by a simple Hiickel MO model. The model implies that the triplet excitation is ~r~r* in nature, and the zfs depends only on the dipolar interaction betwen the two unpaired electron spins.

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274 n

13

n

11

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9

I

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200

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o.o8

o.o9

o. o

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Fig. 7. Chain length (n) dependence of the zfs parameters ] D [ and I E I for AT carotenoid triplet states. Data collected from the literature 122-24,33-35] ( x ) reflect carotenoids in bacterial and plant photosynthetic antenna complexes. The data obtained in this work is denoted with open circles. The lines through the data points are least squares fits to a linear dependency between I D I and ] E ] and the inverse of the number of conjugated double bonds 'n' in the polyene chain. Identification of the chain lengths of the three 3Car states in Rps. palustris I is indicated by broken lines.

The D values taken from the B800-850 and B800820 complexes in this work (marked with circles in Fig. 7) lie well on the fitted line, i.e. agree well with the trend observed from other carotenoid triplet states. The E-values scatter somewhat, probably due to the fact that the asymmetry of the fine structure tensor perpendicular to the polyene chain is much more influenced by small deformations of the chain possibly induced by the protein environment. Using this inverse dependence on n we can easily identify the carotenoids according to their zfs parameters in those cases where more than one species is present, i.e. in Rps. palustris and Rps. cryptolactis. By comparison of the measured I D I-values (see Figs. 2 and 5, and Table 3) with the fitted line in Fig. 7 (indicated by dotted lines), one easily can deduce the number of conjugated double bonds of the different carotenoids present in these preparations.

4.2. Optical absorption bands Just like the zfs parameters depend on the number of conjugated double bonds, so do the energies of the

optical 7r~-* transitions. From simple Hiickel theory the triplet-triplet energy gap AET as well as the singlet-singlet energy gap AEs are proportional to the inverse of (2n + 1) [37]. This approach models correctly the singlet-singlet and triplet-triplet absorption bands of polyenes of intermediate chain length (n = 4 to 20). However, it fails to explain the band gap in polymers (n ~ cx~). More advanced Pariser-Parr-Pople theories predict an energy gap for that case and give a triplet excitation band of AEI AET = - - + AEoo n

(3)

very similar as in the case of the zfs parameters (see Eqs. (1) and (2)) [38]. When we take the triplet-triplet and singlet-singlet absorption bands from our MIA-spectra (Figs. 3, 4, and 5, and Tables 2 and 3), and relevant data for the B880 AT-complexes and plant photosynthetic AT from the literature [22-24,32,33,35], we arrive at values for AEI = 68860(±1980)cm -I, and AE~ = 11810(-4-174)cm -1 (see Fig. 8). The corresponding values for the singlet-singlet transition are AEl = 65060(+2680)cm - t , and AE~ =

A. Angerhofer et al./ Chemical Physics 194 (1995) 259-274

270

n

n

13

11

10

9

13

11

10

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Fig. 8. Chain length (n) dependence of the triplet-triplet and singlet-singlet transition energies for carotenoids in bacterial and plant photosynthetic antenna complexes. Data collected from the literature [22-24,32,33,35] (x) is shown together with data from this work (o). The lines through the data points are least squares fits to a linear dependencybetween ~ETr and AEss and the inverse of the number of conjugated double bonds 'n' in the polyene chain for bacterial AT complexes. Most of the plant AT carotenoids (n = 9) deviate from this linear behaviour as well as the data for Chr. purpuratum (n = 10, AETr = 16050 cm-t, &Ess = 17000 cm-l). 1 2 9 6 0 ( + 2 4 0 ) c m - l . For the fit (straight lines in Fig. 8) we only used values for the bacterial carotenoids since the triplet-triplet transitions in the plant AT complexes did not match the trend observed for bacterial ATs. This may have to do with a possible 9-cis conformation of neoxanthin and lutein in the LHCII complex which was proposed by van der Vos et al. [32]. We also did not use the values obtained from Chr. purpuratum since they obviously greatly deviate from the expected behaviour. It seems that for okenone, the carotenoid present in Chr. purpuratum, there is a strong red-shift o f the optical bands (triplettriplet as well as singlet-singlet) as compared to other carotenoids o f the same polyene chain length. A similar effect is not observed for the zfs parameters which one would expect if the red-shift were due to an intrinsic effect of the molecule itself (i.e. the influence of the renieratene-ring or the carbonyl group, see Fig. 10). Band shifts due to the end groups of the polyene chain are usually rather small and may not explain the large shift observed. The reason for the red-shift of about 2500 cm -1 (for both singlet-singlet and triplet-triplet absorption bands) may rather be found in the particularly strong interaction of okenone with two BChl molecules in

this special complex, as seen in its M I A spectrum (see discussion of the Car-BChl interaction bands in Section 4.4).

4.3. Triplet energy transfer Carotenoid triplet states have been known to form in the photosynthetic membranes by triplet energy transfer from BChl triplet states which themselves form by intersystem crossing from the BChl excited singlet states. The rates of this triplet energy transfer process are usually quite fast, of the order of ( 5 - 1 0 ) × 107s - ! [ 39-42]. Our transient data (see Fig. 6) show that carotenoid triplet population proceeds with rates between 5 x 106 s -1 and /> 5 x 107 s - l (the instrumental limit for the time resolution of our set-up). The lowest formation rate was observed for Rb. sphaeroides 2.4.1, containing spheroidene, a carotenoid with 10 conjugated double bonds (around 5 x 10 6 s -1, ( e ) in Fig. 6). The carotenoid neurosporene with 9 conjugated double bonds (Rb. sphaeroides G1C) experiences a biexponential population with the faster rate of formation around 1-2 x 107 s -1 being somewhat larger than that of spheroidene. Distinction between the rates of triplet formation for the carotenoids with longer chain

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274 [/~I/I (BChl)]/[AI/I(Car)] 0.5 1.0 1.5 I

Z

~o~

I

~.

2.0

I

2.5

I

820 - 850 nrn

................................................. I

I

I

=......

I

I

%

~

802 nm

~o~ "E ~

...................................................... 101

(3

015

110 115 [~I/I( BChl)]/IAI/I(Cor)]

ergy of the accessory monomeric BChl on the electrontransfer inactive side of the RC [44]. Whether a similar mechanism is operative in this case needs to be investigated in further studies.

4.4. Carotenoid-bacteriochlorophyll interactions

"E I 101

271

210

2.5

Fig. 9. Correlation between the 3Car formation times in the different AT complexes at temperatures at or below 50 K (the symbols obey the definition given in Fig. 6) with the relative intensities of the MIA Car-BChl interaction bands (normalized to the main 3Car triplet-triplet absorption band) at 802 and 850 nm. The lines are least squares fits of the data points (with the exception of Chr. purpuratum) and indicate a linear correlation between the triplet transfer rates and the interaction band intensities. The dotted lines mark our experimental time resolution.

0

Okenone Fig. 10. Molecular structure of okenone [57]. lengths ( 1 1 - 1 3 ) is not possible. They pretty much show similar rates between 2 t o / > 5 x 107 s -1 . The special situation of Rb. sphaeroides G1C with a biexponential carotenoid triplet population will be considered in a future paper. We observe a slight temperature dependence for the population of rhodovibrin (n = 11, Rps. acidophila 10050, (+) in Fig. 6). The activation energy is 28 cm -~ . Thermally activated triplet energy transfer has been known from the photosynthetic reaction centres where a thermal activation of around 200 c m - l has been observed [ 31,43 ], possibly due to the triplet en-

Triplet energy transfer is generally thought to proceed via a Dexter type exchange interaction since Frrster type energy transfer strongly depends on the strength of the transition dipoles between ground and excited states. However, for ground state to triplet excitation the transition dipoles are rather weak since it is a spin-forbidden transition. We are therefore limited to the short range exchange mechanism which relies heavily on the overlap of wavefunctions of the excited state molecular orbitals and thus obeys an exponential law on intermolecular distance. The carotenoids and BChls have to be in van der Waals contact for this type of energy transfer to take place. It is therefore not surprising that we observe "interaction bands" in the 3Car MIA spectra, i.e. BChl bleachings and band shifts when 3Car is populated. Such interaction bands have been reported for the MIA spectra of neoxanthin and lutein in plant LHCII complexes [32], but are also seen in the MIA of the RC carotenoids [45,46]. Our MIA spectra also show such bands to a smaller or larger extend (see Figs. 3 and 4). If we plot the time of formation of 3Car in logarithmic scale against the relative intensity of these bands (see Fig. 9), we get a quasi-linear correlation. It seems as if the intensity of the interaction band is directly correlated with the rate of energy transfer. Chr. purpuratum falls out of the picture in so far as we observe a huge interaction band indicating a very strong Car-BChl interaction. Since the observed 3Car formation time is at the instrumental limit of time resolution we do not consider this case to be included in this discussion. The easiest way to interprete the interaction bands is to assume a small delocalization of the SCar triplet wavefunction over an adjacent BChl molecule. Thus, BChl absorption is bleached while the carotenoid is in its triplet state. It is interesting to note that BChl bleaching bands are observed both in the 800 nm region as well as in the 850 (820) nm region. In Rb. sphaeroides G1C, Rb. sphaeroides 2.4.1, Rps. acidophila 7050, and Rps. cryptolactis the main interac-

272

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274

tion band is the 850 (820) nm band indicating that the carotenoid which carries the triplet state interacts predominantly with the longer wavelength chromophore. In the case of the two Rps. palustris preparations the opposite is the case. The main interaction band is the 800 nm BChl absorption. Obviously, in this case the carotenoid interacts more strongly with the 800 nm chromophore. Note however, that in the case of Rps. palustris (both samples) an effect of glycerol on the relative intensities of B800 and B850 may not be completely excluded. A similar effect may have to be considered for the MIA spectra if glycerol alters the AT complex in some way to change the B800/B850 intensity ratio. A heterogeneity of the MIA spectra could then result giving the wrong intensity ratio also for the B800/B850 "interaction bands". The Car-BChl interaction does not seem to be selective for the polyene chain length, since all the carotenoids present (n = 11-13) show similar relative intensities of their interaction bands (see Fig. 5). In most cases the BChl bleaching bands are accompanied by a small absorption enhancement, usually at somewhat longer wavelengths. Although this could in principle be due to a fluorescence signal, i.e. the observation of FDMR in the ADMR experiment, the enhancement also occurs in the vicinity of the 800 nm bands (e.g. in Rps. palustris). Fluorescence emission there is rather weak and may not account for the observed effect. Obviously, other interactions have to be considered. In principle one can explain an absorption increase of a molecule in the vicinity of an excited 3Car state by at least two different mechanisms: Structural changes which lead to an increase in oscillator strength, and band shifts. In the latter case the absorption increase would be complemented by a bleaching at a different wavelength. This may be of importance where BChl dimers are encountered, and the carotenoid triplet breaks the excitonic interaction between them (the case of Chr. purpuratum). At present our experimental data do not allow us to distinguish between these possiblities. Further investigations will have to be undertaken, among the most important being the elucidation of the three-dimensional structure of these complexes. The case of Chr. purpuratum is different from the other preparations since the interaction bands are unusually strong. One can clearly discern a blue shift of a BChl pigment from 834 to 825 nm. It may be super-

imposed by an additional bleaching and enhancement feature similar to the other 3Car MIA spectra. Similar band shifts have been observed for the monomeric accessory BChl molecules in the photosynthetic RC when the primary donor triplet is populated [ 46-49 ]. A similar and easier case is also found in the B820 subunit form of the LH-I AT complex of Rsp. rubrum. There, the triplet-minus-singlet spectrum shows the B820 bleaching at 825 nm together with a strong enhancement of absorption at 802.5 nm [50]. This was interpreted as being due to the breaking of the exciton interaction within a dimer when the triplet state is localized on one part of it. The correlation between the relative intensity of the Car-BChl interaction bands and the rise time of 3Car formation clearly indicates that the exchange interaction which is responsible for the triplet energy transfer also is the cause for the interaction bands.

4.5. Singlet-singlet energy transfer Comparison of the fluorescence excitation spectra with the absorption spectra allows the calculation of the singlet-singlet Car---~BChl energy transfer efficiency [3,30,51-56]. The numbers obtained from our spectra are given in Table 4. Interestingly, they do not correlate well with the triplet energy transfer rates as one would expect. Some of the B800-850 and B800-820 complexes show rather low singlet energy transfer efficiencies (Rps. acidophila 10050, Rps. acidophila 7050, Rps. cryptolactis, Rps. palustris I, and Rps. palustris II (low-light)) between 20 and 40%, which is more typical for B870 AT complexes [30]. The values around 80%, obtained for the two strains of Rb. sphaeroides ( Rb. sphaeroides G1C and Rb. sphaeroides 2.4.1 ) are more typical for B800-850 complexes [ 30]. These two are the complexes which show the slowest rise times of the carotenoid triplet states. In the case of Chr. purpuratum the carotenoid bands in the absorption and fluorescence excitation spectra do not match. Clearly, there are two different types of carotenoids present, distinguishable from their carotenoid excitation bands, however not necessarily chemically distinct. Different protein and/or pigment environments could also be envisaged to induce band shifts in these chromophores. The quantum yield of approx. 57% obtained for the B830 complex

A. Angerhofer et al./Chemical Physics 194 (1995) 259-274 was calculated for all carotenoids present and thus does not distinguish between these two carotenoid pools. It may be envisaged that one type o f carotenoid, presumably the one responsible for the strong M I A interaction bands, has a rather large efficiency of singlet energy transfer (e.g. close to 100%) whereas the other does not show high quantum yields, thus reducing the overall yield to 57%. In any case, the two pools must have drastically different quantum yields of singlet energy transfer to the BChl. Otherwise their absorption and excitation spectra should show identical peaks, which would make them indistinguishable.

5. Conclusions Absorption, fluorescence, fluorescence excitation, A D M R , M I A spectra and light-induced absorption transients were measured on the B800-850 AT complexes of Rb. sphaeroides G1C, Rb. sphaeroides 2.4.1, Rps. acidophila 10050, Rps. palustris I and II, on the B800-820 AT complexes o f Rps. acidophila 7050 and Rps. cryptolactis, and the B830 complex o f

Chr. purpuratum. From these experimental data the following conclusions can be derived: ( i ) The zfs parameters follow the 1/n dependence on the number of conjugated double bonds (n) in the polyene chain of the carotenoid. Thus, the pigments are in all-trans configuration just like in the case o f the B870 AT complexes. The triplet-triplet and singletsinglet transition energies also follow the 1/n law with the only exception o f okenone in the B830 complex o f Chr. purpuratum. This may be due to the strong C a r - B C h l interaction present in this case. (ii) C a r - B C h l interaction bands are observed in the M I A spectra. They are due to BChl bleaching a n d / o r BChl band shifts. The interaction is very strong in the case o f Chr. purpuratum where exciton breaking between a BChl dimer is induced by the formation of the carotenoid triplet state. (iii) The intensities of the interaction bands correlate with the rates of formation o f the carotenoid triplet states. This can be qualitatively understood if one considers the overlap o f the triplet wavefunctions o f the Car and BChl molecules as determining both, triplet energy transfer, and BChl Qy bleaching.

273

Acknowledgement We thank Professor H.C. W o l f for his continued support. This work was financially supported by the Deutsche Forschungsgemeinschaft under W o 4 1 / 4 2 2 and A n l 6 0 / 6 - 1 , and the SERC. RJC would like to thank the Heraeus Stiftung for a travel grant. AG thanks the SERC for a postgraduate studentship.

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