chlorophyll organization and function in green photosynthetic bacteria

Photochemistry and Photobiology, 1998, 67(1): 61-75

Invited Review

Chlorophyll Organization and Function in Green Photosynthetic Bacteria*

John M. Olsont Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, USA

Received 28 August 1997; accepted 15 October 1997

INTRODUCTION

The green photosynthetic bacteria are characterized by the presence of chlorosomes appressed to the cytoplasmic side of the cytoplasmic membrane. The chlorosomes are filled with bacteriochlorophyll (BChl)$ c, d or e molecules in a highly aggregated state. The truly “green” bacteria contain mainly BChl c or d ; while the others look orange or brown because of a high content of carotenoid. From a phylogenetic point of view the green bacteria are really two separate “phyla” based on 16s rRNA (1-3), reaction center (RC) type and physiology. It is truly remarkable that such different types of bacteria (green filamentous bacteria and green sulfur bacteria) contain such similar light-harvesting entities as chlorosomes.

Green filamentous bacteria (Chloroflexaceae) contain a quinone-type RC similar to those found in purple bacteria (Proteobacteria), whereas the green sulfur bacteria (Chloro- biaceae) contain an iron-sulfur-type RC similar to those found in heliobacteria and in photosystem I of cyanobacteria and chloroplasts. The filamentous bacteria live either as facultative photoautotrophs that grow in relatively bright light or as respiring chemoheterotrophs. They are found predom- inantly in hot springs, often in mixed population with cyanobacteria that provide organic carbon compounds for them. Most of our knowledge about the filamentous bacteria at the molecular level comes from one species, Chlorojlexus aurantiacus. A second species, Chlorojlexus aggregans, has recently been isolated and characterized by Hanada et al.

The sulfur bacteria are obligate photoautotrophs and strict anaerobes that grow in dim light in sulfide-rich environ-

14).

*Dedicated to the memory of Philip Thornber (1934-1996). tTo whom correspondence should be addressed at: Department of

Biochemistry and Molecular Biology, Lederle Graduate Research Center, University of Massachusetts, Box 34505, Amherst, MA 01003-4505, USA. Fax: 413-545-3291; e-mail: [email protected]

$AAbhreviarions: B, BChl, bacteriochlorophyll; BChlide, bacteriochlorophyllide; BPh, H, bacteriopheophytin; F, iron-sulfur center; F-chlorosome, chlorosome from a green filamentous bacterium; FMO, Fenna-Matthews-Olson; FT, Fourier transform; LD, linear dichroism; MQ, menaquinone; P, pigment; PSU, photosynthetic unit; RC, reaction center; S-chlorosome, chlorosome from a green sulfur bacterium.

0 1998 American Society for Photobiology 003 1-8655/98 $5.00+0.00

ments. These conditions are found in effluents of sulfur springs and in the chemocline of stratified lakes and in ma- rine habitats. In one extreme case green sulfur bacteria can be found living at a depth of 80 m in the Black Sea (5) . Our knowledge about the sulfur bacteria comes from several species including Chlorobium limicola, Chlorobium phaeovi- brioides, Chlorobium tepidum, Chlorobium vibrioforme, Pe- lodictyon luteolum and Prosthecochloris aestuarii.

CHLOROPHYLLS The chlorophylls found in green bacteria are of two types, the so-called chlorosome chlorophylls (called chlorobium chlorophylls in the older literature) and BChl a (see Fig. 1). Bacteriochlorophyll a is the same molecule found in most purple bacteria; it is found in all RCs of green bacteria, in a membrane-bound antenna complex (filamentous bacteria) and in the water-soluble Fenna-Matthews-Olson (FM0)- protein (sulfur bacteria). The main esterifying alcohol is phy- tol. Chlorosome chlorophylls are a family of chlorophylls (BChl c, d and e) each of which contains an hydroxyethyl group at position 3. Only BChl c is found in the filamentous bacteria where the main esterifying alcohol is stearol. The homologs of BChl c tend to have the same alkylation pattern but differ in the type of esterifying alcohol (6,7). Sulfur bacteria may contain BChl c and/or d or e (sometimes BChl c and d occur together in the same organism). The main esterifying alcohol is farnesol. In some green sulfur bacteria there are at least four chemically distinct homologs of BChl c that differ in the degree of alkylation on the chlorin ring at positions 8 and 12 but have the same monomer absorption spectrum. The homolog distribution depends on the illumination conditions during growth. As the light intensity is lowered, the degree of alkylation increases, and at the same time there is a red shift in the absorption spectrum of the oligomeric antenna system (8,s). The homologs appear to be distributed uniformly within the antenna (10).

The photosynthetic unit (PSU) of a typical green filamentous bacterium such as Cf: aurantiacus may contain 100- 200 BChl c molecules (in a chlorosome) and about 10 BChl a molecules feeding excitation energy to a single quinone- type RC embedded in the cytoplasmic membrane (1 1).

The PSU of a green sulfur bacterium may contain 1000- 2000 chlorosome chlorophyll molecules and about 100 BChl a molecules feeding excitation energy to a single iron-sulfur-type RC embedded in the cytoplasmic membrane. The

61

62 John M. Olson

Figure 1. Structures of (A) BChl a and (B) the chlorosome chlorophylls (BChl c, d and e ) (163). For BChl a R = phytyl. For BChl c from C' auruntiucus R , = R, = R, = methyl, R, = ethyl, and R, = stearyl (major), phytyl (minor), geranylgeraniol (minor), cetyl (minor) or oleyl (minor) (161,162). In green sulfur bacteria BChl c is a mixture of several homologs in which R, = R, = methyl, R, = ethyl, n-propyl or isobutyl, R, = ethyl or methyl and R, = farnesyl (91%), phytyl (3%), cetyl (3%) or tetrahydrogeranylgeranyl (2%) (169-171). Bacteriochlorophyll d is also a mixture in which R, = H, R, = methyl, R3 = ethyl, n-propyl, isobutyl or neopentyl, R, = methyl or ethyl and R, = farnesyl (major) (169,170). Bacter- iochlorophyll e is another mixture in which R , = methyl, R, = formyl, R, = ethyl, n-propyl or isobutyl, R4 = ethyl and R, = farnesyl (major) (166,171).

PSU of a sulfur bacterium can easily be 10 times as large as the PSU of a filamentous bacterium.

REACTION CENTERS Filamentous bacteria

The RC of green filamentous bacteria (e .g . Cf: aurantiacus) is quite similar to that of purple bacteria with two branches of cofactors labeled A (L) and B (M). The primary electron donor is called P865 and consists of a homodimer of BChl a as in purple bacteria. The intermediate electron carrier

m ( L r u / . / l / J kj L branch Leu

Figure 2. Schematic diagram showing the pigment-protein interactions of the structural model for the primary donor microenviron- ment of the Cf. uurantiacus RC and the assignments of the acetyl and keto carbonyl groups as deduced by FT Raman spectroscopy, the proposed protein sequence alignment (19) and assuming structural analogy with the primary donor of Rb. sphaeroides. Also shown are possible neighboring residues for the primary donor of Cf: auruntiacus, and those in italics correspond to those of R6. sphaeroides RC (20). Reproduced with permission from Ivancich el al. (20).

(HA) is a bacteriopheophytin (BPh) a molecule, and the two final acceptors ( Q A and QB) are menaquinone molecules. However in green filamentous bacteria one of the two accessory BChl a molecules found in purple bacteria is re- placed by a BPh a molecule. The overall stoichiometry of BCh1:BPh is therefore 3:3 instead of 4:2 as in purple bacterial RC. Unlike the RC of most purple bacteria, the RC of the filamentous bacteria contain no carotenoid (12-16).

The electron carriers (cofactors) are held in a fixed configuration in the RC by interactions with two different 35 kDa polypeptides (A and B) each having sequence identities of about 40% with the L and M subunits of purple bacteria (17-19). All these polypeptides appear to have five trans-

Table 1. bacteria

Redox potentials of cofactors in reaction centers of green

Filamentous bacteria Sulfur bacteria -

Cofactor Em (mv) Cofactor Em (mV)

+240/250 P865 +360/386/420 P840 (32,156,157) (pH 8.1) (158,159) (PH 6.8)

HA (BPh a) -650 A, (BChl 663) - 1000" (30) (pH f 8.1)

MQA -5O/-210 A, (quinone?) -8OO* (156,157) (pH = 8.1)

-110 F, (Fe-S center) -700* (pH = 8.1)

MQB (30)

-5401-560 F A & ( 160,161 ) (pH 9.9-10.5)

*Approximate values for the RC of photosystem I (162).

Photochemistry and Photobiology, 1998, 67(1) 63

membrane helices and similar binding sites for the various cofactors. The one exception is polypeptide B that contains a Leu residue in place of the His (M180) in Rhodopseudo- monas viridis. This difference explains why Rps. viridis binds an accessory BChl h while Cf: aurantiacus can only bind an accessory BPh a molecule. Because there is no evidence for an H subunit, the RC of green filamentous bacteria is the smallest functional RC known.

The primary donor P865 has been characterized by near- infrared Fourier transform (FT) (pre)resonance Raman spectroscopy. In the proposed model (Fig. 2) the primary donor is a BChl a dimer with one axial ligand to each BChl, and one H bond between BChl, and Tyr (Ml87). The redox potential is predicted to be ca 100 mV lower than that of P870 from Rhodobacter sphaeroides, and in P865' the charge localization is estimated to be 65% in favor of one BChl as compared to 80% for Rhodobacter sphaeroides (20).

From the redox potentials of the various RC cofactors listed in Table 1 it can be seen that the RC of green filamentous bacteria operates between substantially more posi- tive redox levels than does the RC of green sulfur bacteria.

The initial photochemical reaction is a charge separation in which an electron is transferred from P865* to BA (accessory BChl a ) or to HA (BPh a ) within 7 ps at 296 K (9 ps at 320 K) (21). Some workers believe that the electron is first localized on BA and then is transferred to HA (22,23). Others believe that the electron is transferred directly from P865* to HA (24). The next step from HA to menaquinone, (MQA) requires 300400 ps (25-29), and the final step from MQA to MQB requires about 400 k s (30) or 1.3 ms (31). The quantum yield of charge separation (P+MQ,-) was orig- inally reported to be 0.6 (32), but the most recent value is 1.0 (33).

A comprehensive chapter on the RC of Cj: auraittiacus has been written by Feick et al. (30). Recently this RC has been crystallized in a triclinic form that diffracts up to 3.2 A in two directions and 4.0 A in the third direction (34).

Sulfur bacteria

The RC of green sulfur bacteria is similar to that of heliobacteria and to the RC of photosystem I in cyanobacteria and chloroplasts. The primary electron donor is called P840 and consists of a highly symmetric dimer of BChl a molecules (35-38) in a different configuration than found in purple bacteria (39). Also, the radical cation P840' shows a symmetrical distribution of electron spin density in contrast to P870' and P960' in purple bacteria (38,4041). However, the structure of P840' in terms of the resonance interactions between the two BChl a molecules in P840' from FTIR spectroscopy is thought to be close to that of P870' and P960' in purple bacterial RC (42,43). The first intermediate electron carrier (A,) is a chlorophyll molecule (BChl 663) (44) similar to chlorophyll a (45,46). The next electron carrier (A,) may be a menaquinone-7 molecule (B. Kjm, N.- U. Frigaard, H. L. Nielsen, J. Golbeck, F. Yang and H. V. Scheller, unpublished data), but the assignment is definitely controversial (47,48). Three Fe-S centers called F,, FA and F, in analogy with photosystem I serve as terminal electron acceptors (37). In the overall reaction the RC accepts an

751

813 3 1 I I I

Wavelength ( nm)

Figure 3. Absorption spectra of the RC from C j auruntiacus at 20 K (dashed curve) and the putative RC core complex from Cb. tepidum at 6 K (solid curve). The absorbance scales for the two spectra are different. The RC from C' auruntiacus contains 6 chromophores, while the RC core complex contains 24-30 chromophores (55). The data for the RC at 20 K (57) is essentially the same as that for 4 K (16). Adapted from Parot et ul. (57) and from Francke et al. (56).

electron from a membrane-bound cytochrome on the peri- plasmic side of the membrane and transfers it to a soluble ferredoxin on the cytoplasmic side of the membrane. Redox values for some of the electron carriers are listed in Table 1.

Most of the cofactors are held in the RC by interactions with two identical 65 kDa subunits (PscA) (49,50) containing 11 putative transmembrane helices as do the large subunits (PsaA, PsaB) of photosystem I. The PscA subunit further resembles PsaA and PsaB in conserving the putative binding sites for P840P700, Ao, A, and F,. A 32 kDa subunit (PscB) binds the centers F A and F B in the RC of sulfur bacteria. The electron transfer time from P840 to A,, is less than 10 ps and from A, to A, is 0.5-0.6 ns (22,32,51). By analogy with the RC of photosystem I we might expect that the transfer time from A, to Fx would be about 200 ns (52,53).

When the RC of sulfur bacteria is isolated from the cytoplasmic membrane, there are usually a few molecules of BChl a-protein (FMO-protein) bound to the RC core complex. Very recently new procedures have been developed for the isolation of FMO RC core complexes from P. aestuarii (54). Three different preparations were obtained containing about 3, 1 and 0 FMO trimers per RC core complex, respectively. The RC core complex with no FMO trimer contained a large subunit (PscA, M, = 64 kDa) and a smaller subunit (PscB, 32 m a ) but no cytochrome. The BChl a (24- 30 molecules) (55) and BChl 663 were present in a ratio of 3.5: 1. All preparations were photochemically active both at room temperature and at cryogenic temperature. They all had a shoulder in the Qr region of the absorption spectrum that developed into a separate band at 837 nm upon cooling (compare with Fig. 3). Illumination caused an oxidation of P840. At room temperature in the RC core complex essentially all of the P840' produced in a flash was rereduced within several seconds in the dark showing that the FeS centers were fully functional.

The Fe-S-type RC of sulfur bacteria is significantly different from the quinone-type RC of filamentous bacteria both in cofactor content and subunit composition. The dif-

64 John M. Olson

ferences in pigment organization are especially clear in the comparison of absorption spectra of the two RC. As shown in Fig. 3, the reduced RC-core spectrum for a sulfur bacterium (Cb. tepidum) at low temperature shows narrow bands at 797, 808, 818 and 837 nm with a pronounced shoulder at ca 834 nm (56), whereas the reduced spectrum for a filamentous bacterium (Cf: aurantiacus) at low temperature shows a broad band at 887-890 nm, relatively narrow bands at 813 nm and 757 nm and a shoulder at 787 nm (12,16,57).

The RC of both kinds of green bacteria contains bound cytochromes that serve as electron donors to P86.5' or P840+. In filamentous bacteria the cytochrome subunit (c- 554) contains four hemes (58-61), but in sulfur bacteria it is not entirely clear whether the cytochrome subunit contains one (c-551) or four (c-553) hemes (62-66).

A comprehensive chapter on the RC of green sulfur bacteria has been written by Feiler and Hauska (47).

BACTERIOCHLOROPHYLL +PROTEIN COMPLEXES In Cf: aurantiacus the RC in the cytoplasmic membrane is surrounded by pigment-protein complexes (B806-866 antenna complexes) similar to the LH2 antenna found in purple bacteria. Each complex consists of an a - and a P-polypeptide in a ratio of 1: 1 . The a-polypeptide (44 residues, 4.9 kDa) and the @-polypeptide (51 residues) both show a three-domain structure similar to the domain structure of the antenna polypeptides of purple bacteria and sequence homologies (27-30%) to the light-harvesting a- and @-polypeptides of purple bacteria (67-70). Furthermore a His residue is found within the hydrophobic a-helical domain of each polypeptide. By analogy with the antenna complexes of purple bacteria we may suppose that each polypeptide binds one BChl a molecule to the centrally located His residue.

In marked contrast to the filamentous bacteria the sulfur bacteria contain only one intrinsic pigment-protein complex: the RC core complex with 11 membrane-spanning a-helices. In addition the sulfur bacteria contain a unique water-soluble BChl a-protein that is located between the cytoplasmic membrane and the chlorosomes. This protein has been named the FMO-protein after those who first determined its structure. It is a trimer with 46.5 kDa subunits, each of which contain seven BChl a molecules. Five of the BChl a molecules are coordinated to His residues, and the other two are coordinated to a carbonyl oxygen and a water molecule, respectively. Each subunit is surrounded by a string bag of polypeptide most of which is in the P-sheet conformation (7,71,72). The FMO proteins have been sequenced from P. aestuarii (73) and Cb. tepidum (74) and found to be 78% identical and 88% similar.

A more complete description of the FMO proteins is given by Blankenship et al. (7) and Li et al. (72).

CHLOROSOMES Structural features

Chlorosomes are mostly made up of chlorophyll, but lipid, protein. carotenoid and quinone (75) are also present. The size and especially the thickness of chlorosomes depend on the species of cells, on the stage of development and on

A I- 30 nm+ CsmA

Rod element

Envelope

BChl a baseplate protein

Membrane

RC B806-866 complex

B 1-50nrn I f m A

T I

25 nm

.

CsmB

Rod element

Envelope

BChl a baseplate protein

FMO protein

RC core complex

Figure 4. Cross-sectional views of antenna system models for (A) Cf: aurantiacus and (B) green sulfur bacteria. Relatively small chlorosomes are shown in both cases. The model for the F-chlorosome (A) is based on the electron microscopy of Staehelin et al. (76). and the model for the S-chlorosome and its attachment site (B) is based on Staehelin et al. (78). The FMO trimers are represented as oblate ellipsoids of revolution, 5.7 nm X 8.3 nm (172).

growth conditions. Typically the chlorosomes from Chlo- rojlexus (designated F-chlorosomes for filamentous) are ap- proximately 100 nm long, 20-40 nm wide and 10-20 nm high (76,77). The chlorosomes from the sulfur bacteria (designated S-chlorosomes for sulfur) can be considerably larger with lengths from 70 to 180 nm and widths from 30 to 60 nm (77,78). As shown in Fig. 4 all chlorosomes consist of a core and a 2-3 nm-thick envelope. The core consists of rod elements made up largely of aggregated chlorosome chlorophyll, while the envelope is thought to be made up of a monolayer of mostly galactolipid and some protein. Each chlorosome may contain 10-30 rod elements. The rod elements of S-chlorosomes are 10 nm in diameter (78) and may contain a central hole of about 3 nm (79), whereas the rod elements of F-chlorosomes are only 5.2-6 nm in diameter (76). It is not known what controls the diameter of rod elements, but one or more chlorosome proteins are suspected. Structures resembling rod elements have been observed in preparations from trypsinized S-chlorosomes (S. Chavoshy and J. Ormerod, unpublished data).

The core of both types of chlorosomes also contains a relatively small amount of BChl a (B790/794). The molar ratio of chlorosome chlorophyll to BChl a (B794) in S-chlorosomes is about 90/1 (80), whereas in F-chlorosomes it is typically about 25/1 (81). The CD spectrum of B794 in S- chlorosomes is conservative and indicates the possibility of a BChl a dimer (80).

Green filamentous bacteria grown anaerobically contain the carotenoids, p-carotene, y-carotene and HO-y-carotene (82). Most green sulfur bacteria with BChl c or d contain


3 nm, while the envelope layer adjacent to the cytoplasm shows no substructure (76).

Spectral properties

The spectral properties of a chlorosome are determined by the chlorophylls it contains. In Fig. 5 the absorption and fluorescence emission spectra of F-chlorosomes demonstrate the presence of a large amount of BChl c, a small amount of BChl a and energy transfer from BChl c to BChl a (88). The absorption and fluorescence peaks at 745 and 750 nm, respectively, belong to BChl c, while the peaks at 795 and 800 nm belong to BChl a. The CD spectrum (not shown) demonstrates a rather strong exciton interaction among the BChl c molecules. All these properties of BChl c in both F- and ~-ch~orosomes are comp~ete~y different from those of monomeric BChl c dissolved in polar solvents. Monomeric BChl c has absorption and fluorescence peaks at ca 660 and

Figure 5. Absorption and fluorescence emission spectra of F-chlorosomes. Fluorescence was excited at 460 nm. Adapted from Brune et al. (88).

chlorobactene and OH-chlorobactene (83,84), while bacteria with BChl e contain isorenieratene and p-isorenieratene (85).

A marked structural difference between the two types of chlorosomes is in the contact region between the chlorosome and the attachment site on the cytoplasmic membrane. In the sulfur bacteria electron microscopy indicates that the attachment site is a 5-6 mm-thick crystalline structure (78). The ridges of the lattice make an angle of between 40" and 60" with the long axis of the chlorosome and have a repeating distance of -6 nm. It is assumed that the FMO protein associated with the attachment site provides the mechanical and functional coupling between the pigments in the chlorosome and the RC within the cytoplasmic membrane. Stae- helin et al. (78) suggested that the attachment site consists of FMO proteins organized as a planar lattice, and Olson (86) further proposed a two-dimensional crystal of trigonal space group P3 ,. This arrangement of FMO trimers nicely accounts for the 6 nm repeating distance between the ridges of the lattice and is consistent with a thickness of 5-6 nm above the cytoplasmic membrane. In the proposed arrangement the 3, screw axis of the trimers makes an angle of 25" with the plane of the membrane.

From linear dichroism (LD) measurements of intact cells, membrane fragments and FMO proteins from P. aestuarii, Swarthoff et al. (87) concluded that on the average the 3, axes of FMO proteins bound to the cytoplasmic membrane make angles between 55" and 90" with the membrane. Re- cent studies of LD and CD of FMO proteins bound to cytoplasmic membrane fragments from Cb. tepidum indicate that the three-fold axis of the FMO protein is perpendicular to the membrane plane (A. N. Melkozernov, J. M. Olson and R. E. Blankenship, unpublished data). These findings do not fit in with the proposed two-dimensional crystal structure for the attachment site and the observed 6 nm spacing. In Fig. 4 the attachment site for the S-chlorosome is shown as a monolayer of FMO trimers with three-fold axes perpen-

665 nm and an absorption spectrum very much like that of monomeric Chl a. The CD spectrum shows only a very small negative peak at 660 nm (89). The spectral evidence suggests that in chlorosomes BChl c exists in some kind of aggregated state.

Krasnovsky and his coworkers were among the first to demonstrate that dried films of pure chlorosome chlorophylls formed aggregates with spectral properties similar to those of chlorosomes (90). Smith et al. (91) showed that farnesyl BChl c can be induced to form a 748 nm aggregate with an absorption spectrum very similar to the S-chlorosome spectrum just by dissolving BChl c in CH2C12 and diluting the mixture in hexane. Olson and Pedersen (89) further showed that 3-S-( l-hydroxyethyl)-8-isobutyl- 12-methyl/ethyl farnesyl BChl c in CCl, forms an aggregate whose absorption and CD spectra resemble those of chlorosomes. In water-saturated CCI, the aggregates were shown to consist of 20-40 BChl c molecules (92). Finally Hirota et al. (93) showed that BChl c-lipid aggregates were formed when a protein- free extract of S-chlorosomes was diluted in aqueous me- dium. These aggregates were ellipsoidal bodies (130 X 86 nm) that showed absorption and fluorescence spectra similar to those of intact S-chlorosomes. The LD in aggregates oriented in a stretched polyacrylamide gel was as strong as that in chlorosomes. Miller et al. (94) prepared aggregates from F-chlorosomes and showed that the aggregates had absorption and CD spectra like those of intact chlorosomes. Elec- tron microscopy showed that these aggregates formed gran- ules (40-270 nm in diameter) consisting of an amorphous core of nonpolar lipids covered by a monolayer of polar lipids, but there was no evidence of rod element formation (L. A. Staehelin, J. Meehl, M. Miller and J. M. Olson, 1993, unpublished data). The absence of proteins from these model systems supports the idea that chlorosome chlorophylls form aggregates by themselves, but protein may be required for incorporation of aggregates into rod elements.

- - dicular to the membrane. The ellipsoids of revolution rep- resenting FMO trimers give the correct thickness for the attachment site but do not account for the 6 nm spacing observed by electron microscopy.

No FMO protein is found in filamentous bacteria. The membrane-associated envelope layer of F-chlorosomes is marked by very fine striations with a repeat distance of 2.5-

Role of proteins

A most significant characteristic of both F- and S-chlorosomes is the very low protein content in relation to the amount of chlorosome chlorophyll found. In F-chlorosomes the mass ratio of protein to chlorophyll has been reported to be 2.2 (7,95), and in S-chlorosomes this ratio has been found

66 John M. Olson

Figure 6. Structural models for the 740 nm BChl c oligomer with five-coordinate Mg and involving both the 3-hydroxyethyl and 13'- keto groups. (A) Antiparallel chain model. (B) Stepped, parallel chain model. Reproduced with permission from Brune et al. ( 1 10).

to be 1.2 (79) and 1.7 (96). But in a recent study of highly purified chlorosomes from Cb. tepidum Chung et al. (97) found the ratio to be only about 0.5. This ratio varied from 0.45 to 0.55 as a function of growth under light intensities from 60 to 250 FE m-* s-I. In comparison, the protein-to- BChl a ratio for the FMO protein is 6.3 (7) and for the €3866-800 antenna complex is 5.8 or 3.9, depending on whether the complex binds two or three BChl a molecules.

F-chlorosomes (Cf: aurantiacus) contain three major proteins (CsmA, CsmM and CsmN) with relative molecular masses (M,) of 3700, 11 000 and 18 000 and a minor component with M, = 5800 (95). The true mass of CsmA was later found to be 5.7 kDa (98). S-chlorosomes also contain three major proteins with molecular masses of 6.2 kDa (CsmA), 7.5 kDa (CsmB) and 14.5 kDa (CsmC) and a total of seven minor components (97,99,100). Five of these proteins (CsmA, CsmB, CsmC, CsmD and CsmE) are known to be localized in the chlorosome envelope (99,100). The sequence identity between the 5.7 kDa protein from C' aurantiacus and the 6.2 kDa proteins from various sulfur bacteria is 30% and clearly indicates homology of structure and function. Both proteins are therefore designated CsmA.

The question of organization of the chlorosome chlorophylls has been a controversial subject for at least 10 years. In 1984 Feick and Fuller (95) proposed a model for Chlo- roflexus chlorosomes in which the BChl c molecules were organized on a framework formed by the 5.7 kDa polypeptide. Wechsler et al. (101) determined the amino acid sequence for the 5.7 kDa protein and proposed seven possible binding sites for the chlorophylls. However, none of the suggested binding sites are conserved in the homologous 6.2 kDa proteins isolated from various green sulfur bacteria (102,103). Also in spite of intense effort no one has yet isolated a complex between BChl c and the putative BChl c-binding proteins. Furthermore, Wullink et al. (104) showed that antibodies prepared against the 5.7 kDa polypeptide in Chloroflexus react with the chlorosome surface, indicating that the 5.7 kDa protein is not embedded inside the rod elements of the chlorosome as predicted by the model of Feick and Fuller. Based on the primary structures of the various CsmA proteins it appears reasonable to propose that all the proteins consist of two a-helical stretches with a f.3-bend in the middle (105). The conserved triplet GHW is responsible for the bend, and the His residue might be involved in binding a BChl c molecule at the periphery of the chlorosome core (106) even though hydropathy plots of the CsmA proteins (7) indicate that the GHW region is relatively hydrophilic. The lengths of the folded CsmA polypeptides

Figure 7. Energy-minimized aggregate of 40 methyl BChlides d arranged in five stacks each of 5, 10, 10, 10 and 5 chlorins, in partial views from the outside of the tentative tubular structure (above) and the top (below). The bottom representation indicates the sector of the tubular structure (diameter ca 5.4 nm for the Mg-Mg distance) that is obtained on extension of the 40-mer aggregate. H bonds are indicated by dashed lines. Reproduced with permission from Holz- warth and Schaffner (1 14).

are about right for spanning the chlorosome envelope, and the conserved INxNAY region is also relatively hydrophilic. This is consistent with the idea that the INxNAY region is on the outside of the envelope.

The CsmA proteins appear to be the only proteins that might be directly involved with the organization of chlorosome chlorophylls, but they possess only one possible binding site for chlorophyll. This means that only one out of every seven BChl c molecules in F-chlorosomes could have a direct interaction with CsmA protein. Little is known about the function of the other chlorosome proteins. The CsmB protein (99) is found only in S-chlorosomes, so it might have something to do with binding the chlorosome to the FMO


protein between the chlorosome and the cytoplasmic membrane (106). The minor 5.8 kDa protein in F-chlorosomes might be the BChl a-binding protein (B790) ( 9 9 , but this has been disputed by Lehmann et al. (107) and Foidl et al. (108). Other proteins may be responsible for maintaining the native shape of chlorosomes, because treatment of F-chlorosomes with 0.2% lithium dodecyl sulfate causes them to assume a more nearly spherical shape without changing their absorption spectrum (109). These proteins might also be involved in binding the chlorosome to the attachment site on the cytoplasmic membrane (D. Bryant, personal observa- tion).

Models of chlorophyll organization in chlorosomes

When researchers realized the many problems inherent in the concept of chlorophyll-proteins as applied to the chlorosome antenna, many turned to the concept of chlorophyll- chlorophyll interactions and began to build models based on the unique structure of BChl c, d and e, all of which contain a hydroxyl group in the 3' position as shown in Fig. 1.

Brune et trl. (1 10) proposed two models (Fig. 6) consistent with absorbance, FTIR and resonance Raman data. In both these models the Mg atom of BChl c is five-coordinated. In one model (Fig. 6A) the BChl molecules are arranged in antiparallel chains with the molecules in each chain linked by H bonds between the 3'-hydroxyl and the 13' keto groups. The BChl molecules in opposite chains are attached by hydroxyl ligation of the central Mg atoms. In the other model (Fig. 6B) the interacting BChl c molecules are arranged in parallel, stepped chains. Each BChl molecule is linked to the next by 3'-hydroxyl-to-Mg ligation and to the one after next by 3'-hydroxyl-to-13' keto H bonding. Most of the recent large-scale models of pigment organization are similar to one or the other of these two models.

Nozawa and coworkers (1 11,112) and Matsuura et al. (1 13) have put forth rod element models that are basically a three-dimensional extension into a tube of the antiparallel chain model. The most impressive of the large-scale structural models (Fig. 7) has been developed by Holzwarth and Schaffner (1 14). Using computer molecular modeling and energy minimization, they built up aggregates of up to 40 molecules of bacteriochlorophyllide (BChlide) d. The local geometry around each chromophore is essentially that of the parallel chain model, but the large-scale structure is a tube with a diameter of 5.4 nm. In this model the BChl macrocycles are perpendicular to the surface of the tube, and the hydrocarbon tails are oriented toward the outside of the tube, leaving a hole down the center.

In the models of Nozawa and coworkers the chlorophyll macrocyles are oriented so that the tails are arranged toward the inside of the tube (1 I 1 ) or with alternate tails toward the inside and outside (1 12). In the model of Matsuura and coworkers ( 1 13) the tails are also arranged toward the inside of the tube. There is however a big problem with the tails extending toward the inside of the tube. The tubes in F- chlorosomes are substantially smaller in diameter than those in S-chlorosomes. In addition the chlorophyll tails in F-chlorosomes are ?h larger than the tails in S-chlorosomes. So it is doubtful that there is enough space inside an F-chlorosome to accommodate the larger stearyl tails. In the model of

Holzwarth and Schaffner (1 14), there is no problem with how to pack the tails. Each rod element is covered with hydrophobic tails that can promote interactions between rod elements and between rod elements and envelope lipids.

In a recent model proposed by Mizoguchi et al. (1 15) the chlorophyll macrocycles are stacked to form a one-dimensional inclined column in which the y-axis of each macro- cycle is parallel to the long axis of the column.

Buck and Struve (1 16) showed that the tclbular excitation models mentioned above can account for the Q, absorption spectrum of a spectrally homogeneous BChl c antenna (e.g. F-chlorosomes) if the following conditions are satisfied:

1. The transition moments are oriented at <54.7" to the aggregate axis in order for the aggregate spectrum to concentrate dipole strength to the red of the monomer transition at ca 670 nm.

2. Significant symmetry breaking is introduced to yield a multiplet of dipole-allowed bandheads (strongest lines) with a realistic intensity distribution.

3. Energy spacings between major bandheads are appre- ciably less than ca 100 cm-' in order to reproduce the broad featureless nonresonant hole observed by Fetisova and Mauring (1 17).

The simulated absorption spectra (but not E,,,) are relatively insensitive to the aggregate length when the length is large, because in this limit the satellite bands merge with the bandhead.

The tubular model of Buck and Struve ( 1 16) like that of Fetisova and coworkers ( 1 18.1 19) consists of several J-aggregates on the surface of a cylinder. A J-aggregate is one in which the pigments are arranged in a straight one-dimensional array with their transition moments mutually parallel. To get a realistic BChl c oligomer spectrum strong cross interactions are needed between adjacent J-aggregates; oth- erwise one gets only a weakly perturbed J-aggregate spectrum. In the model of Buck and Struve the total BChl c spectrum is a superposition of several J-aggregate spectra with weights that depend on their number and the interactions among them. Each individual J-spectrum consists of a lowest-energy bandhead and a sequence of weaker satellite bands that merge into the bandhead in the limit of large J- aggregate size.

Although it is not yet possible to present a definitive structure for the BChl aggregates in the chlorosome antenna, there are some features that can be enumerated (7):

1. Chlorosomes contain BChl c, d or e oligomers in which the basic geometry is determined by BChl-BChl interactions. Some oligomers might possibly be associated with one or more proteins.

2. The overwhelming majority of Mg atoms in the BChl c, d or e molecules are five-coordinate in chlorosomes.

3. The 13'-carbonyl of each BChl c, d or e interacts strong- ly with a functional group on another BChl.

4. The 3'-hydroxyethyl group participates in oligomer formation and is likely to be coordinated directly to a Mg atom (second BChl c, d or e molecule) and H-bonded to the 13'-carbonyl of a third BChl c, d or e molecule.

5. The BChl c, d or e molecules are close together and oriented so that there is a strong excitonic interaction.

68 John M. Olson

6. The BChl c, d or e molecules exhibit long-range order with their Q, transitions parallel to the long axis of the chlorosome.

In both F-chlorosomes and S-chlorosomes two spectral forms of aggregated BChl c have been proposed based on LD spectra (10,113,120). The two forms have peaks at 727 and 744 nm in F-chlorosomes and at 740 and 760 nm in S- chlorosomes. However the wavelength dependence of LD can be explained with a homogeneous antenna in which different exciton components have different polarization.

Additional information about chlorosomes can be found in a chapter by Oelze and Golecki (77), a chapter by Blan- kenship et al. (7) and a review by Tamiaki (121).

ENERGY TRANSFER Overall pathway

The role of carotenoid in filamentous and sulfur bacteria is somewhat variable, but the overall pathway of excitation energy transfer upon irradiation with blue light can still be written as follows:

Carotenoid (chlorosome) + BChl c, d or e (chlorosome)

BChl a (chlorosome) 4 BChl a (membrane)

BChl a (RC).

+ +

The chlorosome light-harvesting system is an example of an energy funnel in which each successive pigment pool has progressively red-shifted absorption and fluorescence emission spectra. The descending energy levels thus provide an excitation gradient into the RC (7).

Energy transfer can be conveniently divided into two types. The first type is energy transfer between closely spaced molecules in a particular pigment pool, and the second type is energy transfer between different pigment pools that are not so closely spaced. Energy transfer between BChl c molecules in the chlorosome is an example of the first type, and energy transfer between the BChl c pool and the BChl a pool in the chlorosome is an example of the second type. The first type often takes place by a relatively fast exchange mechanism, while the second type usually takes place by a relatively slow dipole-dipole mechanism that obeys the rules of Forster energy transfer. According to these rules, the lifetime (7) of the excited state in the donor molecule is given by the following expression

T = ( k , + k2 + k3)-l

where k , is the rate constant for fluorescence emission, k2 is the rate constant for radiationless deactivation, and k, is the net rate constant for energy transfer to the acceptor molecule. Sometimes there is back transfer from acceptor to do- nor, especially when the acceptor is an RC. We shall assume that this expression is also valid for energy transfer between pools of molecules in photosynthesis. Holcomb and Knox (122) have shown that the microscopic rate constant for do- nor and acceptor molecules at the periphery of adjacent pools can be estimated from the macroscopic rate between pools (compartments) according to the following equation:

F = Ni X (SK') X f

where F is the peripheral intermolecular rate, N, is the number of molecules in the donor compartment, 5 is the dynam- ical factor, r' is the effective number of transfer pathways between compartments, and f is the macroscopic intercompartmental rate of energy transfer. The value of the dynam- ical factor depends upon geometrical factors.

Researchers studying energy transfer often measure the lifetimes of excited states in both donor and acceptor molecules by changes in absorbance or fluorescence. If the decay of the exited state in the donor matches the appearance of the excited state in the acceptor, it is assumed that k, > k , + k2, and therefore T = k3-I.

Sybesma and Olson (123) first showed that excitation energy is transferred from BChl c to BChl a in green sulfur bacteria, and the overall transfer efficiency in P. aestuarii was estimated to be 33% at 294 K. Otte (124) later estimated an efficiency of 40% for excitation transfer from the chlorosomal BChl u to the FMO protein at 6 K. This latter step of the process is different in the filamentous bacteria than in the sulfur bacteria because of the large difference between the B806-B866 antenna complex of filamentous bacteria and the FMO protein.

Vos et al. (125) found that in Cb. lirnicola the transfer of excitation energy among BChl c molecules is not restricted by energy barriers, but that the excitation can move freely through the entire chlorosome (ca 10000 BChl c molecules) at physiological temperatures. A chlorosome appears to function as a common antenna for five to seven RC from which energy transfer back to the chlorosome can take place. In F-chlorosomes on the other hand annihilation studies suggested that the excitation energy is restricted to a domain of about 30 BChl c molecules (126).

Several groups have camed out studies of energy trapping in green bacteria using time-resolved fluorescence and absorption methods. Freshly isolated whole cells, isolated complexes and pure oligomeric pigments have been investigated. The data over all are consistent with a sequential energy transfer pathway from BChl c, d or e in the core of the chlorosome to BChl a in the baseplate and then to the membrane-associated BChl a complexes (B806-B866 or FMO protein) and finally to the RC primary electron donor (P865 or P840). However, a complete picture has not yet emerged because of many apparent inconsistencies in the data (7).

Time-resolved measurements of chlorosomes with plane- polarized light generally strengthen the concepts of chlorophyll organization already presented in this article. There is little depolarization of excited states within the lifetime of BChl c, d or e (126-130), but transfer to the baseplate BChl a (B792) leads to a substantial depolarization because the two pigments differ markedly in orientation (1 13). The fluorescence decay of B792 in Cf: auruntiacus (about 40 ps at room temperature) corresponds to the rise of the fluorescence emission of the B808-866 antenna complex in the membrane (131,132). The energy transfer time from B808 to B866 is about 5-6 ps (133,134). This is similar to the transfer time for the B806850 complex in purple bacteria. The energy transfer time from B866 to reduced P865 in the RC has been measured as 43 ps by Causgrove et al. (131) and as 70-90 ps by Muller et al. (132). Comparable data for green sulfur bacteria have not yet been obtained.

Mimuro et al. (120) showed that 1-hexanol (25% satura-

Photochemistty and Photobiology, 1998, 67(1) 69

tion) inhibits energy transfer from BChl a (B793) in F-chlorosomes to the B806-866 complexes in the membrane. They also suggested a specific interaction between BChl c with an absorption maximum at 771 nm and B793. In a model for the antenna system a far-red absorber, B888, was proposed as an addition to the B806-866 complex, and both functional (A793 and F813) and nonfunctional (A813 and F826) BChl a was assigned to the baseplate.

Energy transfer within chlorosomes

In Cf: aurantiacus the BChl c excited state was first observed to decay with a lifetime of 10-15 ps (7,126,132,135,136). This excited-state decay is matched by a rise component in the baseplate BChl a (B792) in most studies (131,136). More recently Savikhin et al. (137,138) found that the BChl c + BChl a energy transfer is well simulated with biexponential kinetics with lifetimes of 2-3 and 10-11 ps. According to the energy transfer model shown below,

k , k2 (forward rate constants) BChl c ++ BChl c t) BChl a

kl k3 (reverse rate constants)

the lifetime data are best fitted by values of 0.2 ps-' for k, and k2. The values of k, and k, are linked by the Boltzmann factor, exp (-AElkt), where AE is the energy separation between the lowest BChl c exciton component (752 nm) and B795. Very recently Savikhin et al. (139) also observed downhill energy transfer within light-harvesting BChl c oligomers in intact F-chlorosomes.

It is important to note that the BChl c aggregate in cells grown under low light (6 pmol rn-, s-I) has a significantly different structure than does the aggregate in cells grown under high light (60 pmol m-2 s - ' ) and that the decay time of the BChl c excited state increases from 7 ps in low-light chlorosomes to 15 ps in high-light chlorosomes (140).

Fetisova et al. (1 19) showed by picosecond fluorescence spectroscopy that the BChl c excitation lifetimes show a quasilinear dependence on chlorosome size as predicted by their model of cylindrical exciton migration within the chlorosome. Time constant values for excitation energy transfer between BChl c aggregates as well as to baseplate BChl a were calculated to be 27 and 14 ps, respectively. The hop- ping time between the antenna aggregates (rod elements) was calculated to be 4 ps for the middle layers, 7 ps for the upper layer and 14 ps for one layer of rods.

Fetisova er al. (1 18) also developed a theory of excitation energy transfer inside the chlorosome. Six exciton-coupled BChl chains (rod elements) with interchain distances of about 2 nm were claimed to yield the key spectral features of natural chlorosomes, i.e. the exciton level structure revealed by spectral hole-burning experiments and polarization of all the levels parallel to the long axis of the chlorosome. However, the 2 nm separation between chains leads to interactions between J-aggregates that are apparently too weak to explain the chlorosome absorption spectrum. A possible mechanism of excitation energy transfer with the chlorosome is the formation of a cylindrical exciton, delocalized over a tubular aggregate of BChl c chains and Forster-type transfer of such a cylindrical exciton between nearest-neighbor rod elements as well as to baseplate BChl a.

In green sulfur bacteria the lifetimes of chlorosome chlorophyll excited states (30-100 ps) are usually longer than in Cf aurantiacus (127,130,131,141) but may be shorter when certain redox-activated quenchers are oxidized (142,143).

Control by redox potential

Energy transfer in the green sulfur bacteria is apparently regulated by the redox potential in vivo (142). This effect has been observed in whole cells, isolated membranes and purified chlorosomes. Under oxidizing conditions the overall energy transfer efficiency drops from nearly 100% to 10% or less (7). Redox titration of fluorescence in isolated chlorosomes gives a midpoint potential of -146 mV at pH 7.0 with a pH dependence of -59 mV per pH unit (143).

The excited-state lifetimes of the chlorosome chlorophylls are controlled by the redox potential. At low potential there is close to 100% energy transfer from BChl c, d or e to BChl a (B795) in the baseplate (142), and the excited-state lifetime is 50-100 ps as mentioned previously. At high redox potential energy transfer is reduced to 10-20% (142), and the lifetime is reduced to 17 ps (143). In addition, there is no characteristic rise in the B795 fluorescence (143). The excited-state lifetime in the FMO protein is similarly reduced from 2 ns to 60 ps at high redox levels (144). Van Noort ef al. (145) have recently shown that the excited-state quench- ing in the chlorosome may be due to BChl c radicals. Blan- kenship et al. (7) postulate that the formation of quenchers at high redox potentials protects the bacteria from lethal photooxidation reactions (e.g. superoxide formation) resulting from the reduction of ferredoxin in the presence of oxygen. These redox effects are largely missing from the green filamentous bacteria, whose RC do not reduce ferredoxin.

Both F-chlorosomes and S-chlorosomes contain isopren- oid quinones in an approximate molar ratio of 1 quinone for every 10 chlorosorne chlorophylls (75). The S-chlorosomes contain about 70% 1 '-oxomenaquinone (chlorobiumquinone) and lesser amounts of MQ-7. The F-chlorosomes contain mostly MQ-10 and no oxomenaquinone at all. When oxomenaquinone or MQ is added to an artificial BChl c aggregate, the fluorescence properties of the BChl c change significantly. The fluorescence yield under reducing conditions is increased, and the yield under oxidizing conditions is de- creased. Oxomenaquinone is definitely more effective than MQ. These results suggest that quinones play an important but not exclusive role in regulating fluorescence and energy transfer in chlorosomes under oxic conditions.

Energy transfer within the FMO protein and to the RC core complex

Inside the trimer from P. aestuarii the 21 BChl a molecules are so close together that they interact as a single exciton in the absorption and CD spectra (146). In the trimer from Cb. tepidurn at room temperature there is a -2 ps anisotropy decay component that has no counterpart in the isotropic absorption difference kinetics (137,147). Such a component suggests an energy transfer process between seven-pigment (or more localized) states with identical difference spectra but different orientation. From the three-fold symmetry of the trimer the -2 ps anisotropy component would correspond to a -6 ps transfer time between identical subunits.

70 John M. Olson

Gulbinas et al. (148) confirmed from a study of singlet- singlet annihilation that the transfer time between subunits in the trimer is 7 ps at room temperature. It had previously been shown that the mean time of energy migration between BChl a molecules within the subunit is on the order of 1 0 s 900 fs (137,147) and that excitation equilibration leads to a localization of the excitation on BChl a #7 (146,149). It seems reasonable that energy transfer between subunits should take place mainly between BChl a #7 molecules (150).

Quite unexpectedly, the efficiency of excitation transfer from the FMO protein to the core complex was found to be only 35% at 6 K in the EMO-RC core complex from Cb. tepidum (56). Previously, efficiencies of about 20% at 6 K and room temperature had been observed in isolated membranes of P. aestuarii (124,151). These low efficiencies of energy transfer into the RC do not fit our current concept of how the excitation pathway should work.

Summary

Excited-state lifetimes (ps) and energy transfer times (de- noted by asterisks*) in filamentous and sulfur bacteria may be summarized according to the following schemes.

Green filamentous bacteria.

10-15 5* BChl c (chlorosome) -++ BChl a (chlorosome)

5-6* 40-90* ++ B800-860 complex (membrane) ++ P865 (RC)

Green suljur bacteria.

30-100 4*, 7*, 14* BChi c (chlorosome) -+-+ BChl a (chlorosome)

7* ++ FMO protein (subunit ++ subunit) -+ P840 (RC)

EVOLUTIONARY CONSIDERATIONS The existence of chlorosomes containing BChl c, d or e is the only justification for lumping green filamentous bacteria and green sulfur bacteria together as green photosynthetic bacteria. Even the two types of chlorosomes (F and S) show important differences in size and rod element morphology. In spite of these differences the organization of the chlorosome chlorophylls appears to be basically similar, and the csmAICsmA genes and proteins appear to be homologous. This means that F- and S-chlorosomes probably are descend- ed from a common ancestor chlorosome coded for by an ancestral genome. This genome contained the csmA gene and possibly other genes necessary for the assembly of rod elements from BChl c oligomers. The enzymic machinery for esterifying alcohols to BChl c precursors seems not to be overly specific in modem green bacteria (152,153), so it seems probable that the common ancestor used whatever alcohols were available.

My guess is that the chlorosome was invented by an ancestor to Cf: aurantiacus. I make this guess on the basis of the 16.5 rRNA tree of Woese (2). It seems to me that the filamentous bacteria are relatively isolated on this tree, whereas the green sulfur bacteria are grouped together with

the heliobacteria, the proteobacteria and the cyanobacteria. I suppose that the ancestors of the green sulfur bacteria were not green at all. They got along quite well with the FMO protein serving as the complete antenna system. However this antenna system is limited to normal light intensities. There would have been an evolutionary advantage to in- creasing the size of the antenna, so that the green sulfur bacteria could photosynthesize in really dim light. So how did the genes for the chlorosome get into the green sulfur bacteria? Conventional wisdom says lateral gene transfer (154,155), but the time and circumstances remain a mystery.

Acknowledgements-I thank my colleagues, Walter S. Struve (Iowa State University), Mette Miller (Odense University) and R. Clinton Fuller (University of Massachusetts) for reading the original manu- script and making helpful suggestions. I thank Robert E. Blanken- ship (Arizona State University) for providing the structures shown in Figs. 1 and 6. I also thank the reviewers for their comments.

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