Photochemistry and Photobiology, Vol. 57, No.Printed in the United States. All rights reserved

pp. 49-55, 1993 0031-8655/93 $05.00+0.00@ 1993 American Society for Photobiology

CAROTENOID- TO-BACfERIOCHLOROPHYLL SINGLET ENERGYTRANSFER IN CAROTENOID-INCORPORATED B850

LIGHT -HARVESllNG COMPLEXES OFRhodobacter sphaeroides R-26.1

~

HARRY A. FRANK*!, ROYA FARHOOSHI, MILA L. ALDEMA!, BEVERLY DECOSTER2,RONALD L. CHRlSTENSEN2, RONALD GEB~ and JOHAN LUGTENBURO3

'Department of Chemistry, University of Connecticut, Storrs, CT 06269-3060, USA, 2Department of Chemistry,Bowdoin College, Brunswick, ME 040 II, USA, and 'Department of Chemistry, Gorlaeus Laboratories,

Leiden University, 2300 RA Leiden, The Netherlands

(Received 30 M4lrch 1992; Accepted 30 July 1992)

Abstract- Four carotenoids, 3,4,7 ,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidenea)1,d spheroidene, have been incorporated into the B850 light-harvesting complex of the carotenoidless mutant,photosynthetic bacterium, Rhodobacter sphaeroides R-26.1. The extent of 'If-electron conjugation in these moleculesin,creases from 7 to 10 carbon-<:arbon double bonds. Carotenoid-to-bacteriochlorophyll singlet state energy transferefliciencies were measured using steady-state fluorescence excitation spectroscopy to be 54 :t 2%, 66 :t 4%, 71 :t611& and 56 :t 3% for the carotenoid series. These results are discussed with respect to the position of the energy levelsand the magnitude of spectral overlap between the S, (2'AJ state emission from the isolated carotenoids and thebacteriochlorophyll absorption of the native complex. These studies provide a systematic approach to exploring theefl"ect of excited state energies, spectral overlap and excited state lifetimes on the efficiencies of carotenoid-to-bacteriochlorophyll singlet energy transfer in photosynthetic systems.

INTRODUCTION

One ofthe important roles carotenoids play in photosynthesisis that of light-harvesting pigmenV.2 Carotenoids absorb lightenergy in the region of the visible spectrum where chlorophylland bacteriochlorophyll (BChl)t are not efficient absorbersand then transfer this energy for use in driving the primaryphotochemic:a1 electron transfer events in the reaction cen-ters. In photosynthetic bacterial light-harvesting complexes,the efficiency of carotenoid-to-BChl singlet energy transfer ishighly variable among preparations from different bacterialspecies.3-1o To explain this, researchers have invoked differ-ences in sevc~ral factors including: the donor-acceptor dis-tance and geometry, the chemical and stereochemical struc-ture of the carotenoid, the energies of the donor and acceptorexcited electronic states and spectral overlap.

Based on extensive electronic spectroscopic studies of C40skeleton carotenoids and their shorter polyene model sys-tems, carotenoids are known to possess three spectroscopi-cally important low-energy electronic states. 1.2 The first is theground singlt:t state, denoted So or alternatively, 11~, usingthe notation for the irreducible representations of the ide-alized C2h point group. Strictly speaking, carotenoids do notpossess this high degree of symmetry. However, because manyof these molecules retain the spectral characteristics of short-er polyenes that do have C2h symmetry, it is customary touse the same electronic state notation. II The next state ofimportance is the first excited singlet state denoted SI or 21~.This is the state most often implicated as the donor state insinglet energy transfer. Direct excitation by one-photon ab-

sorption into this state is forbidden by symmetry, but somecarotenoids, particularly those having 7r-electron chain lengthsless than nine carbon-carbon double bonds, display fluores-cence from this state.12-18 The final state of interest is thesecond excited singlet state denoted 82 or I'Bu. The l'Ag -+IIBu transition has a very high oscillator strength and is

responsible for the major visible absorption profile of carot-enoids. Fluorescence from this state has also been ob-served.12-15.17-21 It is most pronounced from carotenoids hav-ing eight or more carbon-carbon double bonds. Also, it isgenerally assumed that internal conversion from the I'Bustate to the 2'Ag state is extremely rapid,1.2 occurring in 0.2and 0.34 ps for fJ-carotene and spheroidene, respectively.21.22Nevertheless, evidence has recently been presented, whichsuggests that carotenoid-to-BChl energy transfer may occuron a time-scale short enough to compete with I'Bu -+ 21Aginternal conversion.2J This raises the distinct possibility thatboth the 8, and 82 states of the carotenoid may transfer singletenergy directly to BChUJ.18,2J

There are two important excited states ofBChl, which canact as potential acceptors of the excitation energy.l.2 Theseare the states associated with the so-called BChl Qx and Qytransitions. In the B850 complex of Rhodobacter sphaeroidesR-26.1 the Qx transition appears near 590 nm (16900 cm-l)whereas the Qy transition is near 850 nm (11800 cm-I). Boththe excited states associated with these transitions have beenimplicated as acceptors of carotenoid singlet energy in thetransfer process.4.16

To address the controlling features of carotenoid-to-BChlenergy transfer, we have utilized fluorescence excitation spec-troscopy in tandem with absorption spectroscopy to inves-tigate the energy transfer efficiencies of four carotenoids,

3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroi-dene, 3,4-dihydrospheroidene and spheroidene (Fig. I) in-

*To whom correspondence should be addressed.tAbbreviations: BChl, bacteriochlorophyll; LOAD, N,N-dimethyl.

dodecylamine-N-oxide; LOS, lithium dodecylsulfate.

49

50 HARRy A. FRANK et at.

10QO-I:t

::;,I ""A"'/'/~,""-/'-('-"Y""""(/'~

spheroidene

00-1,~ 9

~/~A.,,~(""-"r'-~~~~3,4-dihydrospheroidene

"8

~

:;;.l'-/'""~~/'-/~'-./'(""""""I/'-"""I"" y3.4.5.6- te trahydrospheroi dene

oa-i3

~'/~'~~./'-/'!""""'--""'(""""'i/"""""r"'~'-"(3.4.7.8-tetrahydrospheroidene

Figure 1. The four carotenoids used in this study. The numbers onthe right indicate the number of conjugated carbon-carbon double

bonds.

Spheroidene and 3,4-dihydrospheroidene were extracted from thewhole cells of Rb. sphaeroideswild-type strain 2.4.1 and Rhodobactercapsulatus MTl131 as previously described. 12 Prior to incorporationinto the B850 complex these carotenoids were purified by thin layerchromatography. The synthesis and purification of 3,4,5,6-tetrahy-drospheroidene and 3,4,7 ,8-tetrahydrospheroidene have been pre-viously described.2-'.26

The incorporation of the carotenoids into the light-harvestingcomplex was carried out as follows: The light-harvesting complexwas dialyzed against 15 mM Tris buffer, pH 8.0, overnight. Then,10% sodium deoxycholate (5{J-cholan-24-oic acid-3a, 12a-diol) (wtIvol) in 15 mM Tris buffer, pH 8.0, was added to obtain a finalsolution of 2% of sodium deoxycholate. A 20-fold molar excess ofcarotenoids over BChl was added in petroleum ether to the light-harvesting complex. The petroleum ether was evaporated with astream of nitrogen. The mixture was then sonicated for 3a-45 minat 4"C in the dark. The solution was then dialyzed against 0.02%sodium deoxycholate, 15 mMTris buffer, pH 8, overnight. A sucrosedensity gradient using 2 M, 1.5 Mand 0.75 Min 15 mMTris buffer,pH 8.0, and spinning at 150000 g in a 55.2 Ti rotor at 4"C for 18h eliminated the excess carotenoids. The light-harvesting complexwas found at the interface of the 1.5 M and 2 M sucrose solutions.An absorbance ofO.4-0.45/cm was used for the fluorescence exper-iments.

fluorescence spectroscopy was performed on a Perkin-Elmer MPF-66 spectrometer using the following conditions: excitation slit, 5 nm;emission slit, 15 nm; A.m;,.;oo, 850 nm; response time, 2 s; sweepspeed, 120 nm/min; 780 nm cutoff filter used.

corporated into the B850 light-harvesting complex from thecarotenoidl(~ss mutant, photosynthetic bacterium, Rh. sphae-roides R-26.1. These molecules have variable extents of1r-electron conjugation ranging from 7 to 10 carbon-carbondouble bonds. In vitro these four carotenoid molecules ex-hibit a systematic crossover from 2'Ag -+ l'Ag to IlBu -+I lAg fluorescence with increasing chain length./z These so-lution fluorescence studies have revealed the positions of theexcited 21Ag and IIBu states relative to the BChl Q. and Qytransitions.1z Hence, upon incorporation of these four ca-rotenoids into the B850 light-harvesting complex, it is pos-sible to study systematically the effects of the position of theenergy levels of the carotenoids relative to those of the BChland of spectral overlap on the efficiency of carotenoid-to-BChl singlet energy transfer.

MATERIAlS AND METHODS

Cells of tht~ Rh. sphaeroides R-26.1 bacteria were grown as pre-viously described.z4 The B850 complex was prepared as follows: TheRb. sphaeroides R-26.1 cells were diluted with 15 mM Tris buffer,pH 8.0, in order to obtain an absorbance of 40-50/cm at 860 nm.A very small amount ofMgCl2 and DNAase was added to the cells.DNAase was used to break down DNA, and MgCl2 was used as acofactor of DNAase. The cells were then passed through a Frenchpressure cell three times at 20000 psi. During the press procedureboth broken and unbroken cells were kept on ice in the dark. Thebroken cells were then centrifuged in a 80rvall 8834 rotor at 15 000rpm (27000 g) at 4-15"C for 10 min. The supernatant was decantedinto a flask and kept on ice. The pellet-free supernatant was centri-fuged at 50000 rpm (250000 g) in a 55.2 Ti rotor at 4~fur 90 min.The yellow supernatant was discarded. At this stage the pellet hada bright shiny color and was gently suspended in 15 mM Tris buffer,containing 150 mMNaCl, pH 8.0. The final absorbance was 40-50/cm at 860 nm. While stirring in low light, N,N-dimethyldodecyl-amine-N-oxide (LDAO) was added dropwise to a final concentrationof 0.6% vol/vol. The mixture was stirred for 30 min at room tem-perature in the dark, then centrifuged at 250000 g for 90 min in the55.2 Ti rotor at 4"C. The supernatant contained mostly reactioncenters, and tile pellet contained the B850 light-harvesting complex.The latter was purified using a discontinuous sucrose density gradientconsisting of 0.3 M, 0.6 M and 1.2 M sucrose in 20 mMTris buffer,0.1 % LD8, pH 8.0. The pellet was added to the top of the 0.3 Msolution and then centrifuged at 150000 g in a 55.2 Ti rotor at 4°Cfor 18 h. The purified B850 light-harvesting complex was located inthe 1.2 Msu(;rose layer.

RESULTS AND DISCUSSION

Figure 2 shows the absorption and fluorescence excitationspectra of the four carotenoids, 3,4,7 ,8-tetrahydrospheroi-dene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroi-dene and spheroidene, incorporated into the B850 light-har-vesting complex from Rb. sphaeroides R-26.1. Thefluorescence excitation spectra were normalized to the BChlabsorption band near 590 nm and the efficiencies of energytransfer were calculated from the ratio of the intensity of thefluorescence excitation spectra to that of the carotenoid ab-sorption. In order to minimize the error resulting from back-ground BCW absorption, the values were obtained by aver-aging the efficiencies over comparable parts of the spectra;viz. the range spanned by the two red-most carotenoid spec-tral peaks. This was done for at least three separate samplesof each complex. The efficiencies for the four incorporatedcarotenoids were 54 :t 2% for 3,4,7 ,8-tetrahydrospheroi-dene,66 :t 4% for 3,4,5,6-tetrahydrospheroidene, 71 :t 6%for 3,4-dihydrospheroidene and 56 :t 3% for spheroidene,where the uncertainties represent the standard deviations ofthe variation in efficiencies over the carotenoid spectral fea-tures. These data are summarized in Table 1.

There are two primary electronic interactions that havebeen cited in the literature as mechanisms for singlet energytransfer between photosynthetic pigments in vivo. These arethe coulombic and exchange interactions. A special case ofthe coulombic interaction is the Forster mechanism27 where-by the electronic transition dipoles are proportional to theoscillator strengths for the donor and acceptor radiative tran-sitions. Energy transfer via the coulomb mechanism generallyrequires large transition dipoles (allowed transitions) for boththe donor and acceptor molecules and has an r-6 donor/acceptor distance dependence. The exchange interaction pro-motes a simultaneous transfer of two electrons between thedonor and acceptor species28 and therefore must operate overshorter distances than the coulombic interaction. The ex-

Carot~noid-to-BChI energy transfer 51

1(:) 3~4-dihydrospheroidene-B850

Wavelength (nm) Wavelength (nm)

d) spheroidene-B850

Wavelength (nm) j Wavelength (nm)i

Figure 2.spheroidene; (c) 3,4-dihydrospheroidene; and (d) spheroid~e incorporated into the B850 complex from Rhodobacter sphaeroides R-26. Theleft y-axis corresponds to the absorption (solid line) trace. The fluorescence excitation curves (in arbitrary units) were normalized to the Qx

transition at 590 nm.

change mechanism does not require large transition dipolesto be present on the donor and acceptor molecules. Thus, itis frequently invoked to account for triplet state energy trans-fer for which returning the donor molecule to its ground stateis a spin-forbidden process. Also, it is the most likel~ mech-anism for carotenoid-to-BChI energy transfer if the energydonor state is the 21~ state of the carotenoid, regardless ofwhether the accepting state is associated with the BChI Qxor Qy transitions. This is because of the extreme~ small dipole moment associated with the symmetry-forbidd n II ~

--+ 21Ag transition in the carotenoid. If the donor stat is theIIBu state of the carotenoid, then either coulomb or e hange

interactions may be active in promoting energy tra sfer toBChl. I

According to the exchange mechanism, the rate constantfor energy transfer29 is given by

(-2r )kET = K exp L JexChange (1)

where K depends on the specific orbitals involved, r describesthe donor-acceptor distance relative to their van der Waalsradii, L, and Jexchange is the overlap integral given by

i: Fd(p)Ea(P)p-4dvIexchange =. ~ (2)

£:

Fd(V)dv i: E.(v)dv

Table 1. The energy transfer efficiencies, En, spectral origins and relative overlap integrals for the four carotenoids, 3,4,7 ,8-tetrahydrospheroi-dene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene and spheroidene incorporated into the B850 light-harvesting complex from

Rhodobacter sphaeroides R-26.1

3,4,7,8- Tetrahydrospheroidene3,4,5,6-Tetrahydrospheroidene3,4-DihydrospheroideneSpheroidene

789

10

54 :t 266 :t 471:t 656:t 3

18400167001530014000

0.150.580.911.0

The calculated value of JexChange fOT the spheroidene-B850 complex is 2.45 x 10-31 S5.

52 HARRy A. FRANK et al.

Fd(lI) is the emission spectrallineshape function of the donor,f:a(lI) describes the absorption spectral lineshape for the ac-ceptor and II is the spectral frequency. The efficiency of energytransfer, f:ET, is related to the rate consant for energy transfer,kET' by the expression

(3)~ETkg

kET+};k;

where ~ ki represents the sum of the rate constants for allthe processes that compete with energy transfer. The ex-tremely small fluorescence efficielicies (IO-3-l0-S) from the21 Ag states of the carotenoids indicate that this sum is mostlikely dominated by the rate constant for radiationless in-ternal conversion, k;c, to the ground state.2 The 21 A. lifetimesof several carotenoids have been measured to be in the range5.2 ps to 25.4 pS.23,30,32 The 21A. lifetime of spheroidene is9.1 pS.23

Figure 3 displays the position of the spectral origins of thecarotenoid excited states overlaid with the absorption spec-trum of the B850 complex. The spectral origins of the llBustates were assigned from the lowest energy spectral featuresin the solution absorption spectra. Using the well-resolvedoptical spectra of model polyenes/3 as a guide, the 21Ag spec-tral origins wc~re assigned to the highest energy inflection inthe emission lineshapes in vitro./2 In order to see the con-nection between the value of the overlap integral, Jcxchang.,and the measllTed efficiencies, EET, of the carotenoid-to-BChlenergy transff:r, the magnitude of the spectral overlap wascalculated for each of the four carotenoid-incorporated sam-ples in the following manner: First, the absorption spectrumof the B850 complex and the in vitro 21A. emission spectrumfrom 3,4,7 ,8-tetrahydrospheroidene (seven carbon-carbondouble bonds) were converted from wavelength to energyunits (Fig. 4). The J..change value was then numerically eval-uated by summing the products of the intensities of the spec-tral traces weighted by their corresponding v-4 frequency termsand normaliz(~d by the product of the fluorescence and ab-sorption integrals. This yielded the value for the overlapintegral of the: 3,4,7 ,8-tetrahydrospheroidene-B850 system.The 21 A. spectral curve was then shifted to lower energy bythe energy difierence between the spectral origins of 3,4,7,8-tetrahydrospheroidene (seven carbon-carbon double bonds)and 3,4,5,6-tetrahydrospheroidene (eight carbon-carbondouble bonds) (Fig. 4), and the calculation repeated. Thisgave the value for the overlap integral of the 3,4,5,6-tetrahy-drospheroidene-B850 system. Similar shifts in the 21A.emission profile gave values for the overlap integrals for 3,4-dihydrospheroidene and spheroidene. The relative values ofthese overlap integrals are summarized in Table I. It isl im-portant to note that the fluorescence spectrum of the 3,4;7,8-tetrahydrospheroidene was used to calculate the overlap in-tegrals for all fourcarotenoid-B850 systems. Ideally, the actualSI emission curves for each of the four carotenoids shouldbe used. However, the present approach is justified by thefollowing arguments: (1) The 3,4,7 ,8-tetrahydrospheriodene21A. emission lineshape Was very similar (within the signal-to-noise) in width and in vibronic structure to the emissiontraces of the 3,4,5,6-tetrahydrospheroidene and 3,4-dihy-drospheroidene molecules. (2) The experimental lineshapeof3,4, 7 ,8-tetrahydrospheroidene had the best signal-to-noise

ratio for 2[~ emission of all of the carotenoids. (3) So farwe have been unable to detect 21~ emission from spheroi-dene in solution, and therefore had to use the 3,4,7 ,8-tetrahy-drospheroidene fluorescence lineshape for the analysis. Theassignment of the spectral origin for spheroidene was doneby extrapolation from the spectral origins of the other mol-ecules.12.18 Also, in this analysis, no attempt was made toadjust the spectral origins of the carotenoid emissions forany dispersive shifts in the spectra brought about by bindingthe carotenoid into the protein matrix. These shifts are knownto be proportional to the dipole strength of the transition!2For the symmetry-forbidden 1 [~ -., 21~ transition the sol-vent shifts are likely to be very small, at most a few hundredcm-1.18.32

The values of the relative overlap integrals are given inTable 1. As the 21 ~ emission profile is shifted to lower energyfrom the spectral origins of the 3,4,7 ,8-tetrahydrospheroi-dene (7 carbon-carbon double bonds) to spheroidene (10carbon-carbon double bonds) the overlap integral increasesin magnitude. This is directly attributable to enhanced over-lap with the strong Qy transition of the BChl (Fig. 4). Thisresult is paralleled by changes in the energy transfer efficien-cies of the B850 complex incorporated with 3,4,7 ,8-tetrahy-drospheroidene, 3j4,5,6-tetrahydrospheroidene and 3,4-di-hydrospheroidene. For these three cases the energy transferefficiencies increase with the extent of 1r-electron conjugationand suggest a correlation between the efficiencies and spectraloverlap. The energy transfer efficiency of the spheroidene-B850 complex, on the other hand, decreases in spite of thecorresponding increase in the overlap.

There are two possible explanations for the failure of thespheroidene system to follow the trend in spectral overlap.The first explanation is that our estimation of the spectralorigin of the spheroidene 21 ~ energy is too high. If the energyof the 21 ~ state of spheroidene were lower, its fluorescencewould have less overlap with the Qy of the BChl, giving alower energy transfer efficiency. This is unlikely because thespheroidene-containing B800-850 complex from Rh. sphae-raides shows nearly 100% efficient energy transfer.8.lo Thiswould not be the case if spheroidene had poor overlap withthe 800 and 850 nm Qy transitions in this spectral region.The second and more plausible explanation is that increasingthe number of carbon-carbon double bonds to 10 (for sphe-roidene) lowers the energy of the 21 ~ state, leading to anenhancement in the rate of SI -., So internal conversion. Forthe carotenoid-B850 complexes studied here, and particu-larly in the case of the spheroidene-B850 complex, radia-tionless decay to So successfully competes with energy trans-fer. Otherwise, the measured energy transfer efficiencies wouldbe 100%.

Figure 5 shows a series of plots of energy transfer efficiencyversus JexChange based on the equation given above for EET andassuming various values of k;c. The figure was derived in thefollowing manner: The efficiency of energy transfer was as-sumed to be governed by the equation

K' x JK ' L- (4)

x J exchange + ,,;c

where K' is an electronic factor that depends on the orbitalsinvolved and on the donor-acceptor distance. K' is assumed

fET

Carotenoid-to-BChl energy transfer 53

0.6

OJ(JCIn

.Q'-0(/)

.Q«

0.4

0.2

0.0300400500600700 $00 900

Wavelength (nm)wavenumbers (cm-1)Figure 3. The positions of the carotenoid spectral origins deduced

from previous studies/2 relative to the B850 BChI absorption spectralfeatures.

Figure 4. The carotenoid 2' A. emlission trace from 3,4,7 ,8-tetrahy-drospheroidene (7 denotes seven carbon-carbon double bonds forthis carotenoid) shifted to correspond to the spectral origins of3,4,5,6-tetrahydrospheroidene (8), 3,4-dibydrospheroidene (9) and spheroi-dene (10). These are overlaid with the B850 absorption spectral

line shape and plotted OD! an energy (cm-') scale.

of energy transfer occurs owing to the rapid, 9.1 ps, 21Aalifetime of spheroidene.

This analysis does not take into account that singlet energytransfer to BChI via the 11 Bu state of the carotenoid also isplausible.J3,J8.23 Evaluation of the overlap integrals betweenthe IIBu emi$sion and the BChl absorption shows that theoverlap is considerably less sensitive to the extent of carot-enoid conjugation than for the case of 2IAa-BChl energytransfer. This derives from the fact that the IIBu -+ IIAgfluorescence spectra are relatively insensitive to the lengthof conjugation. Also, similar IIBu energies suggest similarinternal conversion rates, which would lead to smaller changesin energy transfer efficiencies than those measured here. Al-though a significant amount of IIBu state-mediated energytransfer cannot account for the trend in the energy transfer

to be identical for the four carotenoid-B850 complexes stud-ied here. K' was calculated from the energy transfer efficiency(0.56) for the spheroidene-B850 complex, the measured val-ue (1/9.1 ps) for the 21A. internal conversion rate constant,kic,2] and the spheriodene, 21A. emission-B850 absorption,spectral overlap integral (2.45 x 10-31 S5), giving K' = 5.71X 1041 S-6. Using this value of K', a series of curves were

generated that describe the effect of spectral overlap andinternal conversion rates on the energy transfer efficiency.Overlaid with these plots are four points giving the measuredefficiencies and spectral overlaps of the (a) spheroidene-, (b)3,4-dihydrospheroidene-, (c) 3,4,5,6-tetrahydrospheroidene-and (d) 3,4,7 ,8-tetrahydrospheroidene-incorporated B850complexes.

A five-fold change in ~c is consistent with the trend in themeasured energy transfer efficiencies as illustrated in Figure5. Larger than five-fold changes also could explain the data,given the uncertainties in overlap integrals and the steepdependence of energy transfer efficiency for small overlaps.This magnitude change in the internal conversion rate forthis series of carotenoids is readily accounted for by the "en-ergy gap law,"]] which predicts ~c = constant x exp( -'YIlE!!l",~ where IlE is the SI -So energy difference, f!"'M is theenergy of the high frequency "acceptor" modes and where 'Ycan be related to the relative displacement of the potentialsurfaces in the two electronic states. For aromatics, the dom-inant acceptor modes usually are the C-H stretching vibra-tions (!l"'M -3000 cm-I). For linear polyenes, the C-Cstretching vibrations f!"'M -1300-1600 cm-1 may be moreappropriate.]! According to the energy gap model, the inter-nal conversion rate ofspheroidene (IlE -14000 cm-l) willbe five times faster than the rate of3,4,5,6-tetrahydrospheroi-dene (IlE -18400 cm-l) when f!"'M/'Y = 2700 cm-1. Thiswould require 0.5 < 'Y < 0.6, well within the range of cited'Y (0.5-1.5) for radiationless decay processes in aromatics.

The efficiencies of energy transfer for the four complexescan be rationalized from changes in spectral overlaps and theincrease in the rate of 21 As to II A. internal conversion withincreasing conjugation. The efficiencies will increase withincreasing overlap integral up to the point at which 21A. -+II A. internal conversion becomes dominant. This is the casefor the spheroidene-B850 complex where the overlap integralis the largest in the series, but a diminishing of the efficiency

k,

>,()cOJ

"Q'+-~W

J/ 1 0-3'85

Figure 5. The effect of spectral overlap, JexCbanoc (denoted J in thefigure), on the efficiency of singlet energy transfer from the 2'A.carotenoid electronic state to the BChl acceptor. Overlaid on theplot are four points that refer to the measured efficiencies and spectraloverlaps of the (a) spheroidene-, (b) 3,4-dihydrospheroidene-, (c)3,4,5,6-tetrahydrospheroidene- and (d) 3,4,7 ,8-tetrahydrospheroi-

dene-incorporated 8850 complexes.

HARRy A. FRANK et at.

efficiencies, one cannot rule out the possibility that somecarotenoids in these complexes may be transferring singletenergy via this route.

Finally, in these experiments, it is not possible to be com-pletely sure that all of the carotenoids are bound to the proteinin sites that result in singlet energy transfer. If caro~enoidswere bound in the protein but not capable oftransfe~ng thesinglet energy (e.g., are uncoupled from the BChl), ~e ob-served efficiencies would be systematically low. HQWever,because the incorporation of all four carotenoids was hfmdledidentically, any such effects should be present for ~l fourcomplexes and lead to a uniform reduction in their transferefficiencies. Assuming that similar binding positions and ori-entations of the four different carotenoids exist, the trend inthe data should remain the same. Experiments are underwayto explore the structural and stoichiometric relationships be-tween the carotenoids and BChl molecules in these com-plexes.

Acknowledgements- This work was supported by grants to H.A.F.from the National Institutes of Health (GM-30353) and the Uni-versity of Connecticut Research Foundation, and to J.L. from theNetherlands Foundation of Chemical Research (SON), which is fi-nanced by the Netherlands Organization for the Advancement ofPure Research (NWO).

REFERENCES

I. Cogdell, R. J. and H. A. Frank (1987) How carotenoids func-tion in photosynthetic bacteria. Biochim. Biophys. Acta 895,63-79.

2. Frank, H. A. and R. J. Cogdell (1992) Photochemistry of ca-rotenoids. In Carotenoids in Photosynthesis (Edited by A. Youngand G. Britton), Springer-Verlag, London. (In press)

3. Cogdell, R.J., M. F. Hipkins, W. MacDonald and T. G. Truscott(1981) Energy transfer between the carotenoid and bacterio-chlorophyll within the B800-850 light-harvesting pigment-pro-tein complex of Rps. sphaeroides. Biochim. Biophys. Acta 634,191-202.

4. Noguchi, T., H. Hayashi and T. Tasumi (1990) Factors con-trolling Ihe efficiency of energy transfer from carotenoids tobacteriochlorophyll in purple photosynthetic bacteria. Biochim.Biophys. Acta 1017,280-290.

5. Van Grondelle, R., H. J. M. Kramer and C. P. Rijgersberg(1982) Energy transfer in the B800-850 carotenoid light-har-vesting complex of various mutants of Rhodopseudomonassphaeroides and of Rhodopseudomonas capsulata. Biochim. Bio-phys. Acta 682,208-215.

6. Davidson, E. and R. J. Cogdell (1981) Reconstitution ofca-rotenoids into the light-harvesting pigment-protein complex fromthe carotenoidless mutant of Rhodopseudomonas sphaeroidesR-26. Biochim. Biophys. Acta 635, 295-303.

7. Hayashi, H., T. Noguchi and M. Tasumi (1989) Studies onthe interrelationship among the intensity of a Raman markerband of (:arotenoids, polyene chain structure, and efficiency onthe energy transfer from carotenoids to bacteriochlorophyll inphotosynthetic bacteria. Photochem. Photobiol. 49, 337-343.

8. Kramer, H. J. M., R. van Grondelle, C. N. Hunter, W. H. J.Westerhuis and J. Amesz (1984) Pigment organization of theB800-850 antenna complex of Rhodopseudomonas sph~roides.Biochim. Biophys. Acta 765, 156-165. :

9. Angerhofer, A., R. J. Cogdell and M. F. Hipkins (1986) Aspectral characterization of the light-harvesting pigment-proteincomplexes from Rhodopseudomonas acidophila. Biochim. Bio-phys. Acta 848,333-341.

10. Chadwick, B. W., C. Zhang, R. J. Cogdell and H. A. Frank(1987) The effects of lithium dodecyl sulfate and sodium bo-rohydrid!: on the absorption spectrum of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila 7750.Biochim. Biophys. Acta 893, 444-457.

11. Birge,R.R. (1986) Twophotonspectroscopyofprotein-boundchromophores. Accts. Chern. Res. 19, 138-146.

12. DeCoster, B., R. L. Christensen, R. Gebhard, J. Lugtenburg, R.Farhoosh and H. A. Frank (1992) Low-lying electronic statesof carotenoids. Biochim. Biophys. Acta. (In press)

13. Cosgrove, S. A., M. A. Guite, T. B. Burnell and R. L. Christensen(1990) Electronic relaxation in long polyenes. J. Phys. Chern.94,8118-8124.

14. Mimuro, M., U. Nagashima, S. Takaichi, ¥. Nishimura, I. ¥a-mazaka and T. Katoh (1992) Molecular structure and opticalproperties of carotenoids for the in vivo energy transfer functionin algal photosynthetic pigment system. Biochim. Biophys. Acta.(In press)

15. Mimuro, M., ¥. Nishimura, I. Yamazaki, T. Katoh and U.Nagashima (1991) Fluorescence properties of the allenic ca-rotenoid fucoxanthin: analysis of the effect of keto carbonylgroup by using a model compound, all-trans-beta-apo-8' -carot-enal. J. Lumin. 50, 1-10.

16. Katoh, T., U. Nagashima and M. Mimuro (1990) Fluorescenceproperties of the allenic carotenoid fucoxanthin: implication forenergy transfer in photosynthetic systems. Photosynth. Res. 27,221-226.

17. Andersson, P.O., T. Gillbro, A. E. Asato and R. S. H. Liu(1992) Dual singlet state emission in a series of mini -carotenes.J. Lumin. (In press)

18. Snyder, R., E. Arvidson, C. Foote, L. Harrigan and R. L. Chris-tensen (1985) Electronic energy levels in long polyenes: s, ~So emission in all-trans-l ,3,5,7,9,11 , 13-tetradecaheptaene. J. Am.Chern. Soc. 107,4117-4122.

19. Bondarev, S. L., S. M. Bachilo, S. S. Dvornikov and S. A.Tikhomirov (1989) S2 ~ So fluorescence and transient Sn +-S, absorption of all-trans-fJ-carotene in solid and liquid solu-tions. J. Photochem. Photobiol. A Chern. 46, 315-322.

20. Gillbro, T. and R. J. Cogdell (1989) Carotenoid fluorescence.Chern. Phys. Lett. 158,312-316.

21. Shreve, A. P., J. K. Trautman, T. G. Owens and A. C. Albrecht(1991) Determination of the S2 lifetime of fJ-carotene. Chern.Phys. Lett. 178, 89-96.

22. Shreve, A. P., J. K. Trautman, T. G. Owens and A. C. Albrecht(1991) A femtosecond study of in vivo and in vitro electronicstate dynamics of fucoxanthin and implications for photosyn-thetic carotenoid-to-chlorophyll energy transfer mechanisms.Chern. Phys. 154,171-178.

23. Shreve, A. P., J. K. Trautman, H. A. Frank, T. G. Owens andA. C. Albrecht (1991) Femtosecond energy-transfer proceSsesin the B800-850 light-harvesting complex of Rhodobactersphaeroides 2.4.1. Biochim. Biophys. Acta 1058, 280-288.

24. Chadwick, B. W. and H. A. Frank (1986) Electron-spin res-onance studies of carotenoids incorporated into reaction centersof Rhodobacter sphaeroides R-26.1 Biochim. Biophys. Acta 851,257-266.

25. Gebhard, R. (1991) Synthesis and application of isotopicallyand chemical modified spheroidenes. Ph.D. Thesis, Universityof Leiden.

26. Gebhard, R., K. van der Hoef, C. A. Violette, H. J. M. de Groot,H.A.FrankandJ.Lugtenburg (1991) 13CMASNMRevidencefor a 15,15'-Z configuration of the spheroidene chromophore inthe Rhodobacter sphaeroides reaction center. Pure Appl. Chern.63,115-122.

27. Forster, Th. (1968) Intermolecular energy transfer and fluo-rescence. Ann. Physik. Leipzig 2, 55-75.

28. Dexter, D. L. (1953) A theory of sensitized luminescence insolids. J. Chern. Phys. 21, 836-860.

29. Turro, N. J. (1978) Modern Molecular Photochemistry. Ben-jamin/Cummings, Menlo Park, CA.

30. Wasielewski, M. R. and L. D. Kispert (1986) Direct mea-surement of the lowest excited singlet state lifetime of all-trans-fJ-carotene and related carotenoids. Chern. Phys. Lett. 128,238-243.

31. Wasielewski, M. R., D. G. Johnson, E. G. Bradford and L. D.Kispert (1989) Temperature dependence of the lowest excitedsinglet-state lifetime of all-trans-fJ-carotene and fully deuteratedall-trans-fJ-carotene. J. Chern. Phys. 91, 6691-6697.

32. Hudson, B. S., B. E. Kohler and K. Shulten (1982) Linear

55Carotenoid-to-BChl energy transfer

33. Engiman, R. and J. Jortner (1970) The energy gap law forradiation1ess transitions in large molecules. Mol. Phys. 18, 145-164.

polyene electronic structure and potential surfaces. In ExcitedStates. Vol. 6 (Edited by E. C. Lim), pp. 22-95. Academic Press,New York.

1