How carotenoids function in photosynthetic bacteria

Biochimica et Biophysica Acta, 895 (1987) 63-79

63

Elsevier BBA 86145

How carotenoids function in photosynthetic bacteria Richard J. Cogdell a and Harry A. Frank b a Department of Botany, University of Glasgow, Glasgow (U.K.) and b Department of Chemistry, University of Connecticut, Storrs, CT (U.S.A.) (Received 25 November 1987)

Contents Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.

-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

•. . . . . . .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The problem of understanding how carotenoids function in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

... ....

63 64 64

IL The photochemistry of carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i .............. A. The use of model systems to investigate how carotenoids function in photosynthesis . . . . . . . . . . . . . . . . . . . . . . . .

66 67

III. Carotenoids in reaction centers and antenna complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Carotenoids in bacterial reaction centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Carotenoids in bacterial antenna complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The roles of carotenoids in exciton processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

68 68 73 76

IV. General conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

77

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

77

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. ...............

77

C a r o t e n o i d s a r e e s s e n t i a l f o r t h e s u r v i v a l o f p h o t o s y n t h e t i c o r g a n i s m s . T h e y f u n c t i o n as l i g h t - h a r v e s t i n g m o l e c u l e s a n d p r o v i d e p h o t o p r o t e c t i o n . In this review, the m o l e c u l a r features w h i c h d e t e r m i n e the efficiencies of the various p h o t o p h y s i c a l a n d p h o t o c h e m i c a l processes of c a r o t e n o i d s are discussed. T h e b e h a v i o r o f c a r o t e n o i d s i n p h o t o s y n t h e t i c b a c t e r i a l r e a c t i o n c e n t e r s a n d l i g h t - h a r v e s t i n g c o m p l e x e s is c o r r e l a t e d with d a t a f r o m e x p e r i m e n t s carried out on c a r o t e n o i d s a n d m o d e l systems in vitro. T h e status of t h e c a r o t e n o i d s t r u c t u r a l d e t e r m i n a t i o n s i n v i v o is r e v i e w e d .

Abbreviations: Chl, chlorophyll; BChl, bacteriochlorophyll; Car, carotenoid; ESR, electron spin resonance. Correspondence: H.A. Frank, Department of Chemistry, University of Connecticut, U-60, 215 Glenbrook Road, Storrs, CT 06268, U.S.A. 0304-4173/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

64

I. Introduction

Carotenoids are a class of unsaturated hydrocarbons and their oxygenated derivatives which consist of eight isoprenoid units [1]. They are essentially hydrophobic molecules and are typically found associated with photosynthetic membranes. They are thought not to be freely mobile within the lipid interior of these membranes, but rather noncovalently bound to specific pigmentprotein complexes [2-5]. These complexes also usually contain chlorophylls or bacteriochlorophylls [2-5]. Carotenoids have been shown to perform two major functions in photosynthesis [6-9]: photoprotection and light harvesting. If photosynthetic organisms lacking carotenoids (e.g., the wellknown carotenoidless mutant of Rhodobacter (formerly Rhodopseudomonas) sphaeroides R26) are illuminated in the presence of oxygen they sensitize their own death [6]. This reaction is called the 'photodynamic reaction' (or photo-oxidative killing), and is probably the result of triplet excited state chlorophyll or bacteriochlorophyll donating its energy to molecular oxygen to produce singlet oxygen (1A~ 02). Singlet oxygen is a powerful enough oxidant to kill the organism. The first, and arguably the essential function of carotenoids is to prevent this harmful photooxidative reaction [6,7]. The importance of this primary role of carotenoids is often not fully appreciated. There would be no photosynthesis as we now recognize it were it not for the presence of carotenoids! The second function of carotenoids is to act as accessory lightharvesting pigments, that is to absorb light-energy in the 450-570 nm region, where the chlorophyllous pigments do not, and to transfer the energy to the chlorophylls. If only light absorbed by the chlorophylls and bacteriochlorophylls were to be used to drive photosynthesis then the efficiency of light usage in this wavelength range would be very poor. Carotenoids expand the wavelength range over which light is able to be harnessed for driving photosynthesis. The aim of this short review is to discuss, especially in view of recent experimental data, the details of the mechanisms whereby carotenoids are able on the one hand to act as photoprotectors and on the other hand to act as light-harvesters.

I-A. The problem of understanding how carotenoids function in vivo The light-harvesting role of carotenoids is easily demonstrated by measuring sensitized fluorescence (for example, see Refs. 9-11). Typically, a light-harvesting complex is excited in the wavelength region where the carotenoids absorb, and energy transfer to the chlorophylls is detected by monitoring their fluorescence in the red or near infrared spectral region; i.e. C a r + h g -~ 1 C a r * 1 C a r * + C h l ( o r BChl) -~ C a r + 1Chl * 1 Chl * ~ Chl + h ~ (fluorescence)

The antenna or light-harvesting function of carotenoids is a singlet-singlet energy-transfer process, but by what mechanism does it take place? In vitro caronetoids are essentially non-fluorescent having typical fluorescence yields of less than 10 .6 [12]. Picosecond absorption spectroscopic experiments [13] have indicated that the excited singlet state lifetime of B-carotene is 8.4 + 0.6 ps. This combination of a low fluorescence yield and an extremely short excited singlet state lifetime means that a s~inglet-singlet energy-transfer process according to the F/Srster (long range) dipole-dipole mechanism (this is the mechanism Usually invoked to explain singlet-singlet energy transfer between chlorophyll molecules in vivo) would be very unfavorable. It is not surprising, therefore, that when carotenoids and chlorophylls (or bacteriochlorophylls) are mixed together and frozen in organic solvent matrices singlet-singlet energy transfer from the carotenoid to the chlorophyll does not occur. In vivo, however, singletsinglet energy transfer from the carotenoid to the chlorophyll can be extremely efficient even when the molecules are tightly held in place by the protein. For example, in the case of the peridininchlorophyll a antenna complex from dinoflagellares [11] the efficiency of the carotenoid-to-chlorophyll singlet-singlet energy transfer is close to 100%. If the F~Srster mechanism is ruled out, what other possibilities are there? It has been suggested that if the edge-to-edge distance between the carotenoid and the chlorophyll (or bacteriochloro-

65 25 ? Car

/ -

BCh[

115~ Rb. sphaeroides Rps. viridis
-

20 -

BChl 2

-

02 b

Ag

15 o o o

s[

10E [~]

T,~

--

~,*

T,*

5--

O--

Fig. 1. Schematic representation of the singlet and triplet energies of carotenoids, bacteriochlorophylls and oxygen. Car represents a generic carotenoid having a high-lying 1B~u excited singlet state and a low-lying 1Ag. singlet. The singlet energies were based on those of fl-carotene [15]. Because the triplet energy of carotenoid polyenes have never been measured directly, the wavy box represents their approximate energy (for carotenoids having 9 or 10 carbon-carbon double bonds) from the extrapolated values of Bensasson et al. [261.The singlet and triplet energies of the primary donors (BChl2) of Rb. sphaeroides and Rps. viridis and the monomeric BChl a and b are taken from Takiff and Boxer [67]. The energy of 1A.g 02 is well known [21,22]. phyll) is small enough (say less than 5 A) an electron exchange (or Dexter) mechanism could operate [14]. This m a y be mediated b y energy transfer not from the excited singlet state into which absorption is s y m m e t r y allowed (the socalled 1B~u state), but rather f r o m a lower excited singlet state [15] into which absorption is symmetry forbidden (the lag. state) (see Fig. 1) ¶. Both mechanisms (FiSrster or Dexter) have different geometric and proximity constraints for energy transfer. The F6rster mechanism involves a through-space d i p o l e - d i p o l e interaction, whereas the Dexter mechanism requires electron exchange and hence is facilitated b y orbital overlap between the d o n o r and acceptor. Thus, in principle the appropriateness of one of these mechanisms to

For nonlinear polyenes and carotenoids it is customary to refer to the excited states by their C2h symmetry designations. This is because the excited singlet states of these molecules retain many of the characteristics of the linear systems [16].

explain a given experimental circumstance m a y be experimentally verified. Clearly, the key to understanding the in vivo efficiencies of singlet-singlet energy transfer lies in u n d e r s t a n d i n g the detailed structure of the caroteno-chlorophyll(-bacteriochlorophyll) p i g m e n t - p r o t e i n complexes. This aspect is being actively pursued in n u m e r o u s laboratories. W h e n chlorophylls or bacteriochlorophylls are irradiated a p r o p o r t i o n of the molecules undergo intersystem crossing to p r o d u c e excited triplet states. These triplets are sufficiently long-lived (from tens of microseconds to a few milliseconds) and are energetic e n o u g h to interact with g r o u n d state oxygen to p r o d u c e singlet oxygen ( 17~g . 02 ) [17,18]. This triplet-triplet exchange reaction can be summarized as follows: Chl (or BChl) + h v ~ lChl* 1Ch]* ---)3Ch]* 3Chl* +302 ~ Chl+lA~ 02 I n principle, carotenoids can quench this reaction in two ways [19,20]. First, carotenoids can interact with singlet oxygen directly and quench it, i.e., scavenging:

1A~ 02 +Car ~ 3Car* +302 3Car * -~ Car + heat I n organic solution this reaction proceeds at diffusion-controlled rates. The major requirement for scavenging to proceed in solution is that the energy of the carotenoid triplet state is equal to or below that of single oxygen, i.e., less than 94 k J . m o l - I (or 7855 c m -1) [21,22] (see Fig. 1,). Carotenoids having m o r e than seven conjugated c a r b o n - c a r b o n double b o n d s are capable of this reaction [21,22]. Second, carotenoids can quench chlorophyll triplet states directly before they can interact with molecular oxygen, thereby preventing singlet oxygen production, i.e., trapping. The trapping reaction can be illustrated as follows: Chl (or BChl) + h v ~ 1Chl* ~Chl* ~ 3Chl* 3Chl* +Car ~ Chl+3Car * 3Car * ~ Car + heat We shall discuss in detail below which of these reactions predominates in vivo and why.

66

II. The photochemistry of carotenoids In general, carotenoids are capable of participating in a wide range of photochemical processes (e.g., isomerization, oxidation-reduction, energy transfer, etc.) in vitro [7], and the mechanisms which govern these processes continue to be an area of active research. Which of the possible reactions take place in vivo is controlled by the various pigment-pigment and pigment-protein interactions which exist within the different caroteno-chlorophyll (-bacteriochlorophyll) pigment-protein complexes [5]. Perhaps the most striking and functionally important feature of the photochemistry of carotenoids is illustrated in Fig. 1. Carotenoids are thought to have high energy excited singlet states and a rather low-lying first excited triplet state. It should be emphasized, however, that the lowest excited triplet energies of carotenoid polyenes have never been directly measured. The concept of a low lying triplet has derived from three observations. (i) Several carotenoids are capable of trapping the triplet states of donors whose energies are well defined, e.g., naphthalene, anthracene and Chl a [23-26]. Thus, an upper limit has been placed on these carotenoid triplet energies. (ii) Some carotenoids (e.g., B-carotene) are capable of scavenging singlet oxygen [19,22]. This indicates that the triplet energies of these molecules must be less than 94 kJ.mo1-1 (or 7855 cm-1). (iii) Bensasson et al. [26] presented an extrapolation to longer carotenoids of the singlet-triplet absorption data of Evans [27,28] taken from ethylene, butadiene, hexatriene and octatetraene. This resulted in qualitative estimates of the absolute triplet energies of several carotenoids. From a knowledge of the 1B~u energy, which is easily obtained from the absorption spectrum, a lower limit to the singlet-triplet energy gap may be obtained. In B-carotene, for example, the energy gap between these excited states is calculated to be high; i.e. more than 150 k J . m o l -a (or 12500 cm-~). The high-energy excited singlet state allows the carotenoid to be an energy donor to the chlorophyll (or bacteriochlorophyll), while the low-lying triplet state enables the carotenoid to be an acceptor f o r energy from the lowest-lying triplet state of chlorophyll (or bacteriochlorophyll).

The energies of the excited singlet (1B~u) and most probably also the triplet states of carotenoids depend upon the degree of 7r-electron conjugation in the carotenoid. In general, the more double bonds in the polyene chain the lower the spectroscopic energies of both states [23]. Most, it not all, wild-type photosynthetic organisms only accumulate carotenoids having more than seven conjugated double bonds [4]. It is thought that these carotenoids have the energy of their lowest lying excited triplet state less than 94 kJ. mo1-1 (or 7855 cm -1) (see Fig. 1). This is because mutants of purple photosynthetic bacteria can be obtained which accumulate carotenoids having seven or less conjugated double bonds [29], but these are very sensitive to photo-oxidative killing. It is clear that there is a well-defined triplet state energy requirement that the carotenoid must meet for it to be able to prevent the photodynamic reaction. When a carotenoid in solution is excited by fight into its 1B~u state, essentially all of the energy is very rapidly (in approx. 10 ps [13]) lost by radiationless internal conversion. As a consequence of this, intersystem crossing to produce triplets of carotenoids is a kinetically unfavorable process. Recently, there have been some reports of direct excitation of carotenoids to produce triplets [30]. However, these triplets were produced by a singlet fission mechanism rather than by intersystern crossing (see below). The triplet states of carotenoids have been extensively studied following their generation in triplet-triplet transfer reactions (for example, see Ref. 31). Fig. 2 shows the triplet-triplet absorption spectrum of spheroidene in reaction centers of Rb. sphaeroides. This spectrum is typical of carotenoid triplets. There is a bleaching in the ground state absorption bands and the appearance of a strong new absorption band at 545 nm, i.e., to the red of ground state bleachings. The position of the low-lying lag* singlet state of B-carotene has been deduced from Raman excitation profile in the preresonance region to be 17 230 + 100 cm 1 (i.e., approx. 580 nm) [15]. This state has also been examined both theoretically and experimentally in several shorter polyenes [32,33]. The energy of the 1Ag. state taken together with the likelihood that internal conversion from the 1B~u to the 1Ag. is extremely rapid are highly

67

II-A. The use of model systems to investigate how carotenoids function in photosynthesis

I

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480

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520

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Gust and Moore [34-36] have reported the synthesis of a variety of carotenoids linked to meso-tetraaryl porphyrins. These model carotenoporphyrins were synthesized with the object of trying to understand the structural requirements for the photosynthetic functions of carotenoids. Mimicry of both the photoprotective and antenna functions was achieved in those models where the ~r-electron systems of the porphyrin and the carotenoid were in very close proximity [34,35]. In these systems (for example, see Fig. 3A) the carotenoid quenched the porphyrin triplet state and so prevented it from sensitizing 1Ag 02 production. Similar results have also been obtained with a caroteno-pyropheophorbide, which more closely resembles a chlorophyll-derived chlorin macrocycle [37]. Mimicry of the photoprotective function of carotenoids can also be achieved in models where the edge-to-edge distance between the chlorin and the carotenoid is quite large [38]

I

560

Wavelength (nm) Fig. 2. The light-induced difference spectrum from splieroidene reconstituted into Rb. sphaeroides R26 and excited into its triplet state. The Y axis is expressed in arbitrary units. Data from Frank et al. [43].

zR suggestive of the 1Ag being the donor state responsible for the light-harvesting role of carotenoids. At the present time, however, there is no experimental evidence to support this hypothesis.

R2

2

A, R I :--C~

0

~

j

,

-

~ ,R 2 =H

B, R2 =--O~CH2CH2CH210--

, R1 = H

Fig. 3. Examples of covalently linked models for energy-transfer studies. (A) Carotenoporphyrin structure with short link. (B) Carotenoporphyrin structure with long link. Figure taken from Moore et al. [36].

68 (e.g., see Fig. 3B). However, molecular motion is required to allow the carotenoid to fold back over the macrocycle and come into close proximity with it. Mimicry of the antenna has much more stringent structural requirements. The lifetime of the carotenoid's excited singlet state is so short (i.e., 8.4 + 0.6 ps for/~-carotene in organic solvent [13]) that there is little time for a significant degree of molecular reorientation, or 'flipping' of the carotenoporphyrin structure into a stacked conformation, before deactivation of the singlet state by nonradiative processes. If the molecule is not already in a stacked conformation before the carotenoid is excited, no significant degree of singlet-singlet energy transfer to the porphyrin is measured [371. The marked distance dependence of the efficiency of the antenna function of the carotenoid in these models has been exemplified in a very recent picosecond absorption study of two caroteno-pyropheophorbides and a carotenoidacetamide by Wasielewski et al. [39]. In each case the carotenoid moiety was the same. The lifetime of the excited singlet state was determined for all three cases by picosecond flash photolysis. The measured values were 16.2 +_ 0.8 ps for the carotenoid-acetamide, 15.9 _+ 0.8 ps for one of the caroteno-pyropheophorbides and 7.6 __ 0.8 ps for the other. From a consideration of the lifetimes of the carotenoid's excited singlet state in the three molecules one can calculate the efficiency of energy transfer from the carotenoid to the pyropheophorbide (%t = ket/(ket + kd), where ket is the energy-transfer rate constant and and k a is the intrinsic carotenoid singlet-state decay constant obtained from the carotenoid-acetamide). The efficiency of energy transfer from the carotenoid to the macrocycle was determined for the two caroteno-pyropheophorbides to be 53 _+ 5% and less than 5%. N M R analysis of these two caroteno-pyropheophorbide models showed that in the one with the high-transfer efficiency the distance between the 7r systems of the carotenoid and the pyropheophorbide was 2 ,~, while in the one with the low-transfer efficiency, this distance was 5 A. The authors conclude that " v e r y strong electronic coupling between the system of the carotenoid donor and the chlorophyll-derivative acceptor is necessary to achieve efficient singlet

energy transfer rates". In effect they are suggesting that this energy-transfer reaction operates by an electron exchange (Dexter) mechanism. III. Carotenoids in reaction centers and antenna complexes

Most reaction center and antenna complexes from green plants and bacteria contain carotenoids. For the purpose of this review we shall confine our comments to the bacterial system, because reaction centers and antenna pigmentprotein complexes are much better characterized in bacteria than in plants [3,4]. It is very likely that the main conclusions drawn from the bacterial studies are also relevant to green plants. III-A. Carotenoids in bacterial reaction centers Carotenoids are not essential for the primary electron-transfer reactions which occur in bacterial reaction centers [2,3,40]. Indeed, the majority of experiments which have been carried out to investigate reaction-center structure and function have used reaction centers isolated from the carotenoidless mutants of Rb. sphaeroides R26 or R26.1 (a spontaneous partial revertant of R26) (for example, see Ref. 41). To our knowledge, however, all reaction centers isolated from wild-type species contain one, specifically bound carotenoid molecule per reaction center unit [4], and this carotenoid most probably plays an important photo-protective role in vivo. Carotenoids bound to reaction centers have absorption spectra which are red-shifted from their spectra in organic solvents (for example, see Refs. 42 or 43). Also, the carotenoids are optically active when bound to reaction centers, but not when they are in solution [42]. This is illustrated in Fig. 4 for reaction centers from Rb. sphaeroides 2.4.1. The major carotenoid (90% of the total) bound to these reaction centers is spheroidene. In organic solvents (e.g., pentane) its visible absorption maxima are at 475, 447 and 421 nm [43] and these absorption bands show no optical activity [42]. In the reaction centers the absorption maxima of the spheroidene are shifted to 499, 470 and 438 nm [43] and these absorption bands show a strong induced CD spectrum [42]. Carotenoids can be

69 A T

150K

0 . 5 0.4

c~ 0.3

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0.2 0.1 0 400

500

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B

600

700 Arlm

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860

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900

(1

8

center carotenoid is located in a unique and specific binding site. This binding site has been shown to be on the L a n d / o r M subunits of the reaction center [42]. Reaction centers can be partially dissociated with the addition of detergents such as SDS into LM dimers and free H subunits. All the pigments including the carotenoid remain associated with the LM dimer upon removal of the H subunit [42]. There have been several recent reports on the use of spectroscopic techniques to analyze the structure o f t h e reaction center-bound carotenoid. Frank et al. [47-54] used electron spin resonance (ESR) of the photoexcited triplet states of carotenoids to obtain the structural information. The structures were deduced from measurements of

0.021

TABLE I

500 Wovelength

600 nm

Fig. 4. The absorption and CD spectra of reaction centers of Rhodobacter sphaeroides strain 2.4.1. (A) The 150 K absorption spectrum of the reaction centers. At 150 K the absorptions due to the carotenoid in the 420-520 n m region are sharpened and more clearly visible than in the room temperature spectrum. Data from Verm6glio et al. [44]. (B) The C D spectrum of reaction centers from the carotenoid-containing strain 2.4.1 and the carotenoidless mutant R26. Data from Cogdell and Scheer [86]. (a) 2.4.1 reaction centers; (b) R26 reaction centers; and (c) baseline.

incorporated back into reaction centers isolated from carotenoidless mutants and have all of their in vivo characteristics fully restored [45-47]. The reconstitution approach has been used to study carotenoid function in reaction centers from both Rb. sphaeroides and Rhodospirillum rubrum [45-47]. The results are summarized in Table I. One of the major conclusions of these studies has been that reaction centers show a pronounced selectivity as to which carotenoids can be bound. Rb. sphaeroides reaction centers, for example, show a marked preference for carotenoids with terminal polar functional groups (e.g., methoxy-groups). This finding coupled with the fact that the carotenoid binds in a 1 : 1 stoichiometry with the primary electron donor suggests that the reaction

SOME PROPERTIES OF CAROTENOIDS RECONSTITUTED INTO CAROTENOIDLESS REACTION CENTERS OF RHODOSPIRILLUM RUBRUM AND

RHODOBACTER SPHAEROIDES How successful the incorporation was, was monitored by restoration of correct spectral properties. For the R. rubrum G9 properties, see Ref. 45; for the Rb. sphaeroides R26.1 properties, see Ref. 47. Species and carotenoid

Successful incorporation

Carotenoid triplet formation

no no yes yes yes yes

no no yes yes yes yes

yes yes yes yes no yes no no very small very small no

yes yes yes yes no yes no no not not no

R. rubrum G9 B-carotene lutein spirilloxanthin spheroidene spheroidenone chloroxanthin

a a a a

Rb. sphaeroides R26.1 hydroxyneurosporene methoxyneurosporene spheroidene spheroidenone rhodopsin spirilloxanthin zeaxanthin

apo-fl-carotenol neurosporene 1,2-dihydrolycopene fl-carotene

b b b b b

tested tested

Measured by recovery of photoprotection. Assayed by the appearance of an ESR-detected carotenoid triplet state.

70

the triplet state zero-field splitting parameters from carotenoids contained in native bacterial systems and from a series of carotenoids reconstituted into Rb. sphaeroides R26.1. These authors suggested that the native conformation of the carotenoid in the reaction center involves twists of the molecule at the C6-C 7 or higher carbon positions. Moreover, the ESR spectrum of spheroidene reconstituted into reaction centers of Rb. sphaeroides R26.1 is identical to the ESR spectrum of this same carotenoid in wild-type strain 2.4.1 reaction centers [47]. This indicates that the carotenoid assumes the same structure in both preparations, because the ESR spectrum is a very sensitive probe of the carotenoid's molecular features [47,48]. In addition to deducing the structures of carotenoids, ESR has been used to probe the orientation of the reaction center bound carotenoid in the membrane and with respect to the other reaction center pigments [52]. Further work is in progress to tie together the magnetic properties of carotenoids with their molecular structures. The conformations (single-bond rotation isomers) and configurations (double-bond rotation isomers) of carotenoids have also been investigated in vivo using resonance Raman spectroscopy [55-58] and comparing the frequencies of the resonances in the spectrum with those of carotenoids in solution. In this manner it has been possible to identify those resonances which are sensitive to the isomeric state of the carotenoid. It is important to emphasize that the interpretation of the in vivo spectroscopic results depends critically on a comparison with bonafide standards. It is possible that the protein induces a conformation of the carotenoid which is not able to be produced in solution. This would preclude a precise assignment of the structure of the reaction center carotenoid on the basis of correlative studies. Nevertheless, the results of such studies from several laboratories have indicated that the reaction center carotenoid adopts some form of a cis-conformation when it is bound to the reaction center [55]. There has been considerable argument as to the exact nature of the cis isomer. Lutz et al. [55] proposed that carotenoids bound to reaction centers adopt strained, di-cis configurations. Other groups have favored a central 15-15"-mono-cis configuration [45]. In a very recent report Lutz et

al. [59] now seem to favor the 15-15'-mono-cis configuration after having analyzed the protonN M R spectrum of spheroidene extracted from reaction centers of Rb. sphaeroides strain 2.4.1. They found that a stable cis isomer could be extracted and purified from the reaction centers and that this cis isomer had an NMR-determined 15-15'-mono-cis configuration. Moreover, a comparison of the resonance Raman spectra of the reaction center-bound carotenoid with that of the 15-15'-cis extract suggested that in the reaction center, the 15-15'-cis spheroidene is distorted into a nonplanar configuration by twists affecting the C 7 - C l l regions of the molecule. This is consistent with the ESR conclusions of Chadwick and Frank [47] previously mentioned. Thus, at present, most spectroscopists favor a 'strained' 15-15'-mono-cis isomer for the reaction center carotenoid configuration. Soon, however, these spectroscopic assignments will be confronted with X-ray structural determinations. The coordinate refinements carried out on the Rhodopseudomonas viridis reaction center crystal structure have located the reaction center carotenoid (1,2-dihydroneurosporene) in the M subunit in close proximity to the BChl monomer on the M side and approx. 10.5 A from the primary donor [60]. The X-ray-determined conformation is 13-cis with an out-of plane twist at the C13 carbon position. This is not in agreement with the previously mentioned resonance Raman-determined 'strained' 15-15'-cis isomeric structure. There is some degree of uncertainty in the X-ray assignment, however, because the ends of the carotenoid molecule were not resolved in the electron density map. Thus, one cannot be absolutely sure of the configurational assignment. Also, in attempting to understand the structural features which control the efficiency of triplet energy transfer in the reaction center, the structural studies on Rps. viridis are of limited value. This is because there is no carotenoid triplet formation in the Rps. viridis reaction center [48]. Very recently, crystallized reaction centers from two different strains of Rb. sphaeroides wild type have been reported. These are strain Y1 reported by Ducruix and Reiss-Husson [61], and strain 2.4.1 reported by Frank et al. [62] and Allen et al. [63]. X-ray diffraction data has been presented in

71 all cases. Spheroidene is the primary carotenoid in these strains. Because spheroidene has been shown to be strongly bound to the protein owing to its attached polar function group [47] - unlike 1,2-dihydroneurosporene in Rps. viridis which has no attached polar functional group - it was hoped that the carotenoid would be held more rigidly in the protein and that an X-ray analysis of these strains would yield a more precise structural picture of the location and c o n f o r m a t i o n / configuration of the reaction center carotenoid than was offered b y the Rps. viridis structure. The most recent coordinate refinement of the X-ray data carried out by Allen et al. [63] on reaction centers from the 2.4.1 strain have revealed the structure shown in Fig. 5. The carotenoid was found to lie between the B and C helices of the M subunit and near the associated monomeric accessory BChl which is between the carotenoid and

the primary donor. The carotenoid was found to lie in a plane with the two-fold symmetry axis of the reaction center perpendicular to this plane. The structure is not yet sufficiently resolved to reveal the stereochemistry of the carotenoid, but the data clearly show a bent carotenoid structure consistent with cis-isomerization. The data also show that spheroidene is positioned in reaction centers of Rb. sphaeroides 2.4.1 in essentially the same place as 1,2-dihydroneurosporene is in Rps. viridis. In reaction centers of Rb. sphaeroides 2.4.1 the carotenoid, spheroidene, quenches the primary donor with almost unity quantum yield at temperatures above 35 K [64]. Thus, two questions remain: (i) Given that the two different carotenoids are in essentially the same position relative to the primary donor, why is spheroidene an efficient triplet trap while 1,2-dihydroneurosporene is not? (ii) If spheroidene in the reaction center of Rb.

Fig. 5. The structure of the chromophores in the reaction center of Rb. sphaeroides wild type strain 2.4.1. C, the carotenoid represented as a simple polyene chain; DA and DB, the bacteriochlorophyll primary donor "special pair'; BA and BB, monomeric bacteriochlorophylls; ~A and ~B, monomeric bacteriopheophytins; QA and QB, quinones; Fe, ferrous iron. This picture was taken from Allen et al. [631.

72

sphaeroides 2.4.1 is approx. 11 A away from the primary donor, then what is the mechanism of triplet energy transfer, and how is this reconciled with the model system studies which demonstrate a close proximity requirement for this process? Before we can sensibly embark upon a discussion of this topic we must review briefly what is currently known about the mechanism of the primary photochemical reactions within the reaction center. The organization of the reaction center pigments including the carotenoid has been determined by Deisenhofer et al. for Rps. viridis [60,70,71] and by Allen et al. for Rb. sphaeroides strain 2.4.1 [63]. The latter structure is shown in Fig. 5. When the reaction center bacteriochlorophyll-dimer (the 'special pair') is excited by light electron transfer takes place from its first excited singlet state [72]. In 2-3 ps the electron moves from the 'special pair' to the bacteriopheophytin which is located on the 'L' branch of the reaction center. Subsequently, the electron moves from the bacteriopheophytin to the 'primary' quinone. This electron-transfer step, takes approx. 200 ps [72]. If the system is excited with light when the 'primary' quinone is absent or chemically reduced, forward electron transfer from the bacteriopheophytin is prevented [73]. Under these conditions a back reaction takes place between the oxidized 'special pair' and the reduced bacteriopheophytin. One product of this back reaction is the triplet state of the 'special pair' [73]. At room temperature in the absence of carotenoids; e.g., in reaction centers from Rb. sphaeroides R26.1, this triplet state of the 'special pair' subsequently decays in approx. 10 microseconds. Were this triplet state to be produced in vivo in the presence of oxygen, then it could potentially sensitize the formation of singlet oxygen. This potentially harmful series of events is apparently averted in carotenoid-containing reaction centers [74]. When a carotenoid is present the triplet of the reaction center's 'special pair' is rapidly quenched [74]. Instead of lasting for several microseconds it is now quenched almost as quickly as it is formed, and a carotenoid triplet is produced in high yield [74]. The rise-time of the carotenoid triplet state is between 10 and 30 ns [74]. This then reduces the lifetime of the triplet state of the 'special pair' by

about three orders of magnitude and so effectively prevents the generation of singlet oxygen. In a purified LM subunit preparation the yield of carotenoid triplets was found to be even higher than in the native reaction centers [75]. There did not seem to be a correlation between this and the fact that the LM subunit structure was considerably more sensitive to photooxidative damage in the presence of oxygen, however. The rapid quenching of the triplet state of the 'special pair' is the most efficient way of preventing the photodynamic reaction. Exactly how this energy is transferred from the primary donor to the carotenoid, and what energy states are involved in the process, is still open to debate. Parson and Monger [64] invoked a temperature-induced shift of the carotenoid triplet state energy to explain the fact that the transfer was inhibited at temperatures below 35 K. Schenck et al. [68] suggested that the transfer goes via a triplet charge-transfer state purported to be higher in energy than the triplet of the 'special pair'. Frank et al. [51] favored a model whereby energy transfer is promoted by a special vibrational mode which also induces spinlattice relaxation between the triplet spin sublevels of the special pair triplet state. Hoff and coworkers [76,77] have presented ESR and magnetooptical difference spectroscopic data on the triplet of the special pair in Rb. sphaeroides R26, Rb. sphaeroides 2.4.1, R. rubrum and Rps. viridis but were not able to elucidate further the mechanism of the transfer. Kolaczkowski et al. [78] addressed this question using optical detection of magnetic resonance applied ,to reaction centers of Rb. sphaeroides 2.4.1. They deduced that the triplet radical pair state was not being directly quenched by the spheroidene triplet. Their most recent ESR work [79] using deuterated and undeuterated spheroidene reconstituted into deuterated and undeuterated reaction centers of Rb. sphaeroides R26 suggests that the rate of triplet energy transfer is dependent on a low-frequency liberational mode of the carotenoid. Regardless of the precise mechanism of triplet trapping, if carotenoids were only able to photoprotect by scavenging singlet oxygen, the photoprotection would be only partial. This is because: (i) the efficiency would depend on the proximity of molecular oxygen to the carotenoid; and (ii) the carotenoid would be competing for the

73 singlet oxygen energy with other sensitive, photooxidizable substrates. Direct quenching of the triplet state which generates singlet oxygen is the most efficient strategy because, in this case, photoprotection can be nearly complete. At least two possibilities exist to explain the difference between the extremely efficient triplet energy transfer process which takes place in Rb. sphaeroides and the lack thereof in Rps. viridis. (i) The location of the accessory BChl monomer between the primary donor and the carotenoid on the M subunit side is highly suggestive of an involvement of the BChl molecule as an intermediate in the triplet transfer process. The mechanism could involve a model with stepwise transfer of the triplet energy from primary donor to monomeric BChl to carotenoid. Alternatively, a superexchange model (analogous to that presented to explain the participation of the L side monomeric BChl in the primary electron-transfer reaction [65]) with a coherent mixing of wavefunctions contributed from the primary donor, monomeric BChl and carotenoid could be the more appropriate description. If the accessory BChl is involved in the mechanism, spheroidene may lie slightly closer or be coupled more strongly to this molecule (or to the primary donor) in reaction centers of Rb. sphaeroides 2.4.1 than 1,2-dihydroneurosporene in Rps. viridis; i.e., structural differences may exist between the complexes which are too small to be discerned in the current level of X-ray coordinate resolution. In fact, the primary donor triplet state in Rps. viridis is asymmetric and localized on the L side of the BChl special pair; i.e., away from the carotenoid [65]. On the other hand the primary donor triplet state in Rb. sphaeroides is symmetric and delocalized over the pair of BChl molecules [651. (ii) The triplet energy of the primary donor or the accessory BChl in Rps. viridis (a BChl b-containing organism) may lie below that of 1,2-dihydroneurosporene, whereas the corresponding triplet energies in Rb. sphaeroides 2.4.1 (a BChl a-containing organism) may fie above that of spheroidene; i.e., energy differences may exist between the complexes which result in favorable energy transfer in one species but not in the other. As previously mentioned, the triplet energies of several carotenoids have been estimated by extrapolation

from the known triplet energies of short polyenes [26]. The extrapolated values are 6560 cm 1 for spheroidene (ten carbon-carbon double bonds) and 7285 c m - 1 for n e u r o s p o r e n e (nine carbon-carbon double bonds). Also, the triplet energies of monomeric BChl a and BChl b and the primary donors in Rb. sphaeroides and Rps. viridis have recently been measured by phosphorescence techniques [66,67]. The values (based on the positions of the phosphorescence maxima) are: 7590 cm -1 for the primary donor triplet energy in Rb. sphaeroides, and 8157 cm -1 for BChl a; 6680 cm -1 for the primary donor triplet energy in Rps. viridis, and 7970 cm -a for BChl b (see Fig. 1.) These numbers are consistent with this second interpretation. A similar energetics argument was recently advanced by Schenck et al. [68] to explain the low (less than 10%) carotenoid triplet yield in the methoxy-, hydroxy- and neurosporene-containing GA strain of Rb. sphaeroides. However, the uncertainties in the carotenoid values (which have not been experimentally determined) coupled with the fact that these energies are likely to be inappropriate considering the twisted structures of the carotenoids present in the bacterial reaction centers, makes this interpretation in need of further verification. Alternatively, Takiff and Boxer [67] have recently suggested that owing to the approximate 1000 cm -1 energy gap between the (higher energy) monomeric BChl b triplet and the (lower energy) primary donor triplet in Rps. viridis (Fig. 1), triplet energy transfer is inhibited at all temperatures. The corresponding energy gap in Rb. sphaeroides is approx. 200 cm-~, which explains the absence of energy transfer only at low temperatures in this species. Singlet-singlet energy transfer from the reaction center carotenoid to the reaction center bacteriochlorophyll does take place [421. It is probably of little functional significance though, because reaction centers in vivo come attached to a large antenna array in which most of the light absorption takes place and where singlet-singlet energy transfer from carotenoids to bactefiochlorophyll is a very important process.

III-B. Carotenoids in bacterial antenna complexes There are two main types of antenna complexes found in purple bacteria [80,81]. One type is closely

74 associated with the reaction centers and forms the 'core' of the photosynthetic unit. An example of this is the B-875-complex from Rb. sphaeroides. The second type is arranged more peripherally and represents the variable component of the photosynthetic unit. An example of this is the B800-850-complex from Rb. sphaeroides. Both types of complexes are built on the same structural principle, whereby in their native, pigmented forms they are constructed by the aggregation of simple 'modules' [81,82]. Each 'module' consists of a small number of bacteriochlorophyll (two or three) and carotenoid molecules (one or two) nonconvalently bound to two very hydrophobic, lowmolecular-weight apoproteins (the a- and flapoproteins), which typically have about 50-60 amino acids [82]. Perhaps the best studied complex, so far, is the B800-850-complex from Rb. sphaeroides and so we shall use this complex in the following discussion as the prototypical bacterial antenna complex. The B800-850-complex from Rb. sphaeroides contains bacteriochlorophyll and carotenoid in the molar ratio of 2 : 1 (the previous estimate of 3 : 1 was incorrect (Evans, M.B. and Cogdell, R.J., unpublished observations)). The absorption spectrum of this complex is shown in Fig. 6. As in reaction centers, the carotenoid bound to

<1 o

7-----

~

<

150

L_J_.__ 350

550

750

950

nm

Fig. 6. The absorptionspectrum(bottom) and the CD-spectrum (top) of the B800-850-complex from Rhodobacter sphaeroides strain 2.4.1. The figure was adapted from data in Cogdell and Scheer [86]. The main carotenoid in this antenna complex is spheroidene. ,~A, absorbance difference, for the CD spectrum; A, absorbance.

this complex has an absorption spectrum which is red-shifted by approx. 20 nm compared to the same carotenoid in organic solution [83]. In the wild-type strain of Rb. sphaeroides 2.4.1 spheroidene is the major carotenoid present in the B800-850-complex. Also, as in the case of the reaction center, carotenoids bound to the antenna complex have strong optical activity, and show a pronounced CD spectrum (Fig. 6) [83]. It is worthwhile comparing the CD spectrum of spheroidene bound to the reaction center (Fig. 4) with its CD spectrum when it is bound to the B800-850-complex (Fig. 6). In the reaction center the CD spectrum mirrors the absorption spectrum while in the B800-850-complex the CD spectrum looks more like a first derivative of a gaussianshaped absorption spectrum [84]. Thus, the carotenoid binding sites in the two complexes must be quite different. Recently, the first carotenoidless B800-850 complex was reported [85]. It was shown that carotenoid pigments were not essential for the assembly of the B800-850 complex. Such a complex is likely to be valuable for carotenoid reconstitution experiments which investigate the nature of the binding of the carotenoid to the protein. Previously, Rb. sphaeroides R26.1 was shown to produce a carotenoidless antenna complex, the so-called B850 complex [5,87]. This complex lacks the 800 nm absorbing bacteriochlorophyll a molecule, but the primary structure of its apoproteins are identical to those of the wild-type B800-850 complex except at two amino acid positions [88], and this complex has proved to be useful for reconstitution studies. A range of carotenoids were tested for their ability to restore normal carotenoid function to the B850 complex. Reconstitution was achieved with spheroidene and neurosporene but not with fi-carotene, spirilloxanthin, lycopene or dihydrolycopene [83]; i.e., the binding site showed quite a high degree of selectivity. It appears, therefore, just as with reaction centers, that the antenna carotenoids are bound into the antenna complexes in a specific way, the precise details of which have not yet been elucidated. The conformation and the configuration of the antenna carotenoids have been probed by resonance Raman spectroscopy [57,89,90]. In contrast to the reaction center case, the antenna carotenoids

75

seem to be all-trans isomers [57,89,90]. This is consistent with the trends in the triplet state ESR spectra of several of the B800-850 complexes which are easily understood in terms of the extent of linear ~r-electron conjugation within the carotenoids contained in these complexes [91]. Some researchers, though, have suggested that in several cases these isomers may be distorted away from a planar all-trans configuration [90]. So far, structural models of purple bacterial antenna complexes have been deduced primarily from spectroscopic studies. Recently, however, several purple bacterial antenna complexes have been crystallized [92-95], and it is hoped that soon this will lead to a high-resolution structural determination of one such complex by X-ray crystallography. Singlet-singlet energy transfer from the carotenoid to the bacteriochlorophyll has been measured in a range of B800-850 complexes from Rb. sphaeroides which were different only in the type of carotenoid present [10]. The results are summarized in Table II. In all cases the efficiency of energy transfer is high (greater than 70%) and does not seem to be very dependent on the type of carotenoid present. It appears that once a carotenoid assumes its correct position in the complex it will participate in singlet-singlet energy transfer rather efficiently. The structure of the B800-850 complex from Rb. sphaeroides can be altered by preparing the complex by gel electrophoresis in the presence of lithium dodecylsulphate (LDS)

TABLE II THE EFFECT OF CAROTENOID-TYPE ON THE EFFICIENCY OF SINGLET-SINGLET ENERGY TRANSFER FROM THE CAROTENOID TO THE BACTERIOCHLOROPHYLL IN THE B800-850-COMPLEX FROM RHODOBA CTER SPHAER OIDES The energy transfer efficiency rates were calculated from the data presented in Ref. J0. Energy transfer was assayed by sensitized fluorescence. Carotenoid composition

Energy transfer efficiency (%)

Spheroidene Spheroidenone Neurosporene Methoxyneurosporene Chloroxanthin

I00 75 95 95 95

TABLE III A REPRESENTATIVE COMPILATION TO ILLUSTRATE THE VARIATION O F THE EFFICIENCY OF CAROTENOID-TO-BACTERIOCHLOROPHYLL S I N G L E T SINGLET ENERGY-TRANSFER EFFICIENCIES SEEN IN A RANGE OF PURPLE BACTERIAL ANTENNA COMPLEXES Species and type of Antenna complex

Energy transfer efficiency (%)

Rb. sphaeroides 2.4.1 B800-850 B875

100 60-70

R. rubrum B880

30

Rps. acidophila B880 B800-850 (type I) B800-820

25 50-55 70-75

Ref.

10 99 9 100 100 100

[96,97] or by the addition of small amounts of lithium dodecylsulphate [98]. Treatment with this detergent results in a loss of the 800 nm absorption band and a blue shift of the carotenoid absorption bands. The protein conformation of the complex appears to be altered (Robert, B and Frank, H.A, unpublished results), and under these conditions the efficiency of carotenoid-tobacteriochlorophyll singlet-singlet energy transfer drops by about 30% [97,98]. The efficiency of this energy-transfer process is therefore strongly dependent upon the precise structure of the antenna complex. Although the carotenoid-to-bacteriochlorophyll singlet-singlet energy-transfer efficiency is not strongly affected by carotenoid type in the same complex, it is quite variable depending upon the type of antenna complex. Measured efficiencies vary in the range of 20-30% up to nearly 100% (see Table III for a representative selection of measured efficiencies). It is also a characteristic of this energy-transfer process that its efficiency is independent of temperature down to liquid helium temperatures (i.e., 4 K) [99,1001. Recently, Wasielewski and co-workers [13,39] have investigated the absorption changes in carotenoids in solution induced by 4 ps exciting pulses. Using a wide range of monitoring wavelengths Wasielewski and Kispert [13] were able to measure

76

the lifetime of the excited singlet state of three carotenoids dissolved in toluene. The measured lifetimes were 8.4 + 0.6 ps for all-trans-fi-carotene, 5.2 + 0.6 ps for canthaxanthin and 25.4 _+ 0.2 ps for/~-8'-apocarotenal. These initial measurements suggest that it should be possible to time-resolve the singlet-singlet energy-transfer reactions of carotenoids. Wasielewski et al. have now done this for the B800-850 complex isolated from Rhodopseudomonas acidophila strain 7750 [101]. This antenna complex was excited at 510 nm (into the carotenoid's absorption bands) with a 4 ps pulse. The ground state bleaching of the carotenoid (indicative of formation of the 1B2"u first excited singlet state) occurred concomitantly with the laser flash and decayed with a time constant of 5.6 +_ 0.9 ps. The rise time of the bleaching at 860 nm (which indicates arrival of the energy at the bacteriochlorophyll and it entering its first excited singlet state) occurred with a time constant of 6.1 _+0.9 ps; Le., within experimental error the same as the decay time of the excited singlet state of the carotenoid. Angerhofer et al. [100] measured the efficiency of energy transfer from the carotenoid to the bacteriochlorophyll in the B800=850-complex from Rps. acidophila strain 7750 to be 50-55% (see Table III). It will be interesting when the lifetimes of the excited singlet states of the carotenoids extracted from this complex are determined in organic solvent. The efficiencies of the energy transfer process calculated from the picosecond dynamics experiments can then be compared to those determined from the fluorescence excitation data to see if the results of both experiments are correlated. Notwithstanding this, it is clear that the kinetics of the singlet e n e r g y - t r a n s f e r p r o c e s s f r o m the d o n o r carotenoid(s) to the acceptor bacteriochlorophyll(s) are now amenable to direct investigation. This represents a new era in carotenoid research. When the B850 complex from the carotenoidless mutant of Rb. sphaeroides R26.1 is excited by light, a proportion of the singlet excited bacteriochlorophyll a molecules undergo intersystern crossing to produce triplet excited states [83]. These bacteriochlorophyll a triplet states are f o r m e d within a few nanoseconds and decay, at room temperature, in 10-20/xs [83]. If these reactions occur in the presence of oxygen then the

complex sensitizes its own, irreversible photooxidation, and the presence of singlet oxygen can be detected by its reaction with the chemical trap 1,3-diphenylisobenzfuran [831. In the presence of bound carotenoids the bacteriochlorophyll a triplets are quenched in 20-30 ns with the concomitant formation of carotenoid triplets. The lifetime of the bacteriochlorophyll a triplet is reduced by about 103 and this undoubtedly is the main source of photoprotection. If isolated carotenoidless B850 complex is mixed with unreconstituted carotenoid molecules, then the bacteriochlorophyll a triplets are not trapped and almost no photoprotection is seen [83]. This clearly means that if singlet oxygen is produced at all then the complex will degrade. The photoprotective effect of carotenoids is to stop singlet oxygen generation in the first place!

III.-C. The roles of carotenoids in exciton processes The presence of carotenoids in whole cells, chromatophores and isolated antenna complexes has a dramatic effect on the observed magneticfield dependencies of fluorescence and triplet yields [102-105]. These effects have been localized in the antenna and found to arise only upon direct carotenoid excitation. They are not observed in reaction centers, are not temperature dependent, and do not depend on the redox state of the reaction center (in whole cells or chromatophores). These magnetic field effects are best understood in terms of the theory of cooperative excitonic processes [106]. Upon excitation of the antenna carotenoid the molecule can transfer its energy to bacteriochlorophyll or undergo singlet exciton fission [107]. U p o n formation of the triplet pair state, which may consist of either two carotenoid triplets (homofission) or one carotenoid triplet and one bacteriochiorophyll triplet (heterofission) several processes may take place. Which of these processes take place depends on the energies of states of all the participating molecules as well as the structural construction of the complex. For example, for fission to be energetically possible, the energy of the triplet exciton pair state must be less than or equal to the singlet exciton energy. Because of their large singlet-triplet energy gap, previously alluded to (Fig. 1), this requirement is usually met for carotenoids but probably not met

77

for bacteriochlorophyll. This work demonstrates that there is an alternative route to energy transfer for the dissipation of carotenoid excited singlet state energy in the photosynthetic apparatus-singlet exciton fission. At the present time, the physiological significance of the fission process is unclear. IV. General conclusions The experiments discussed above are only the beginnings of attempts to understand the molecular details of carotenoid photochemistry and photophysics, and how this behavior translates into effective function (antenna and photoprotection) in the native photosynthetic systems. We have discussed that what primarily determines the ability of a carotenoid to function in a photosynthetic system depends on a combination of factors including the carotenoid's structure, energetics, excited state dynamics, the nature of its binding to the protein and its proximity to other pigments. Although several conclusions have been drawn about carotenoid structure and function from studies on carotenoids in solution and on model systems, we may be faced with the inescapable conclusion that the photosynthetic proteins determine what the precise mixture of these factors will be for a particular carotenoid to be able to carry out a designated function. Some structures, particularly conformational isomeric forms of carotenoids, may not be able to be reproduced in solution. The fact that carotenoids possess an extended flexible polyene chain which lends itself well to tunability by the protein may be the ultimate property of carotenoids which holds the key to their diversified abilities. Acknowledgements This work has been supported by grants to H.A.F. from the National Science Foundation (PCM-8408201), the Competitive Research Grant Office of the U.S. Department of Agriculture (86CRCR-1-2016), the National Institutes of Health (GM-30353) and the University of Connecticut Research Foundation. R.J.C. wishes to thank the S.E.R.C. for financial support.

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