Structure, function and organization of antenna polypeptides and antenna complexes from the three families of Rhodospirillaneae

J. Photochem.

113

Photobiol. B: Biol., 15 (1992) 113-140

Structure, function and organization of antenna polypeptides and antenna complexes from the three families of Rhodospirillaneaet RenC A. Brunisholz

and Herbert

Zuber

Znstitut fir Molekularbiologie und Biophysik, ETH-Hiinggerberg CH-8093 Ziirich (Switzerland) (Received

January

7, 1992; accepted

March 20, 1992)

Abstract Comparative primary structural analysis of polypeptides from antenna complexes from species of the three families of Rhodospirillaneae indicates the structural principles responsible for the formation of spectrally distinct light-harvesting complexes. In many of the characterized antenna systems the basic structural minimal unit is an alp polypeptide pair. Specific clusters of amino acid residues, in particular aromatic residues in the Cterminal domain, identify the antenna polypeptides to specific types of antenna systems, such as B880 (strong circular dichroism (CD)), B870 (weak CD), B800-850 (high), B800-850 (low) or B800-820. The core complex B880 (B1020) of species from Ectothiorhodospiraceae and Chromatiaceae apparently consists of four (alatP,j?l) or three (2a&&) chemically dissimilar antenna polypeptides respectively. There is good evidence that the so-called variable antenna complexes, such as the B800-850 (high), B800-850 (low) or B800-820 of Rp. acidophila, Rp. palustris and Cr. vinosum, are comprised of multiple forms of peripheral light-harvesting polypeptides. Structural similarities between prokaryotic and eukaryotic antenna polypeptides are discussed in terms of similar pigment organization. The structural basis for the strict organization of pigment molecules (bacteriochlorophyll (BChl) cluster) in the antenna system of purple bacteria is the hierarchical organization of the a- and &antenna polypeptides within and between the antenna complexes. On the basis of the three-domain structure of the antenna polypeptides with the central hydrophobic domain, forming a transmembrane a helix, possible arrangements of the antenna polypeptides in the three-dimensional structure of core and peripheral antenna complexes are discussed. Important structural and functional features of these polypeptides and therefore of the BChl cluster are the a/p heterodimers, the a& basic units and cyclic arrangements of these basic units. Equally important for the formation of the antenna complexes or the entire antenna are polypeptide-polypeptide, pigment-pigment and pigment-polypeptide interactions.

Keywords: Pigment populations, antenna types, structure-spectrum relations, aromatic amino acids, primary structure antenna model, antenna polypeptide heterodimer, pigment cluster, cyclic hexamers, a& basic units. 1. General

introduction

The primary processes of photosynthesis involve light absorption, excitation energy transfer and primary charge separation across the photosynthetic membrane. Bacterial ‘Dedicated

loll-1344/92/$5.00

to Professor

K. SchatIner

on the occasion

of his 60th birthday.

0 1992 - Elsevier Sequoia. All rights reserved

114

photosynthesis, with a less complex antenna system than eukaryotic photosynthesis, has been extensively studied in recent years to obtain detailed information on the mechanisms of these fast events. A number of multidisciplinary studies (involving spectroscopy, X-ray crystallography, biochemical and genetic analyses) on structure-function relations, primarily on the components of the light reactions of purple bacteria, have provided an insight into their molecular organization [l-lo]. The light reactions are mediated by distinct pigment-protein complexes, called light-harvesting and reaction centre complexes. In purple bacteria both are synthesized as intramembranebound complexes. When dissolved in organic solvents, such as acetone-methanol, the photopigment bacteriochlorophyll (BChl) a absorbs at 772 nm. However, in viva the long-wavelength band of the same pigment may shift to lower energy and spectral forms between 800 and 900 nm are observed (e.g. 820, 850, 870 and 880 nm). This large spectral complexity of the pigment is mainly a consequence of its organization into well-defined pigment-protein complexes which are denoted B800-850 or B870 for example, because of the wavelength of their absorption maxima in the near-IR spectral region [ll]. From structural analyses of antenna complexes of purple nonsulphur bacteria it has been shown that the protein matrix consists, in most cases, of a pair of chemically non-identical small hydrophobic polypeptides ((Y/P heterodimer) to which two to three BChl and one to two carotenoid molecules are bound [6, lo]. Their chemical structure and specific polypeptide aggregation are the structural basis for the general concept of light absorption and heterogeneous energy migration to the reaction centre. Excitation energy from antenna BChl molecules, which absorb at lower wavelengths such as 800, 820 or 850 nm (peripheral antenna pigments), is transferred to pigment clusters organized in a so-called core antenna complex with pigments absorbing at higher wavelengths (B870, B880). Whereas the core complex surrounds the reaction centre in a fixed stoichiometric ratio, the formation of peripheral antenna complexes, such as B800-820 or B800-850, is strongly dependent on the purple bacterial strain and species and environmental factors (light intensity, temperature, growth medium). Over recent years major interest has been focused on the molecular origin of the spectral multiplicity of purple bacterial antenna pigments, i.e. the structural basis for the efficient energy transfer cascade [6]. Within this context the structural characterization of the bacterial antenna polypeptides from different sources plays a decisive role. This paper aims to highlight recent achievements regarding the primary structural determination of antenna polypeptides from various species of the three families of the suborder Rhodospirillaneae. From a consideration of structural homology and variability, possible structural-spectral relations are discussed. In addition, ideas are presented regarding the general structural features and possible structural organization of polypeptides and BChl molecules derived from the multiplicity of primary structural data (primary structural model).

2. Photosynthetic

membranes

of purple

bacteria

Common to most purple bacteria is the formation of extensive intracytoplasmic photosynthetic membranes, which have been referred to as “chromatophores” and which are continuous with the cytoplasmic membrane. In facultative photosynthetic bacteria such as Rb. sphaeroides, Rb. capsulatus and Rp. acidophila the extent of the chromatophores is very much dependent on factors such as oxygen tension and light intensity. Moreover, purple bacteria synthesize morphologically dissimilar types of

115

chromatophores which have been identified in ultrathin sections as either lamellar (stacked or non-stacked), vesicular or tubular configurations (Table 1). As an exception, species such as Rc. tenuis and, to some extent, Rc. gelatinosus do not show the ability to enlarge the site of the components of the light-induced energy transduction. Interestingly, the strict anaerobic bacteria from the Heliobacteriaceae also lack extensive intracytoplasmic membranes. As yet, little is known of the molecular factors required for the formation of specific intracytoplasmic membranes. As illustrated in Table 1, there is apparently no correlation between the different types of membrane structure and the formation of individual antenna complexes.

TABLE

1

Type of intracytoplasmic (IC) membrane and typical near-IR (NIR) absorption characteristics (light-harvesting (LH) BChl= bulk BChl) from a selection of purple photosynthetic bacteria Family/species

Type of IC membrane

LH NIR absorption B800

B820

B850

-

-

-

Rhodospirillaneae Rr. rubrutn Rp. marina Rp. viridi? Rs. photometncum Rb. sphaeroides Rb. capsulatus Rc. gelatinosus Rc. tenuis Rp. sulphidophila Rp. acidophila 10050 Rp. acidophilab 7050 Rp. acidophilab 7750 Rp. palustib

Vesicular Lamellar Lamellar Stacks Vesicular Vesicular No ICM No ICM Vesicular Lamellar Lamellar Lamellar Lamellar

~800 ~800 = 800 ~800 ~800 ~800 ~800 ~800 ~800 =800

Ectothiorhodospiraceae E. halochloris” E. halophila E. mobilis E. shaposhnikovii E. abdelmalekii”

Stacks Stacks Stacks Stacks Stacks

~800 ~800 ~800 ~800 ~800

= 830s -

Vesicular Vesicular

~800 =800 = 795 800 800 = 800s

= 820

Chromatiaceae Cr. vinosum Cr. tepidum Cr. minuttisimum Cr. okenii Amoebobacter roseus Amoebobacter puqwnxs Thiocapsa pfennigii”

Vesicular Vesicular Vesicular Tubular

(nm)

‘BChl b-containing photosynthetic bacteria. bSynthesis of variable peripheral antenna complexes or temperature. ICM, intracytoplasmic membrane.

= 830 ~820 = 830 =850 high

= 850 = 850 = 860 = 860 = 855 = 855 ~860 =860 ~850 low

= 850s - 875 = 855

1830s = 850 = 855 = 850

= 835 = 825 830 = 830 mainly as response

B870-890

880 880 1015 880 870 870 875 875 895 880 880 880 880 880 885s, 1015 890 900s 895s 1020 890 920 885 880 890 880 1020

to light intensity

and/

116

3. Antenna

pigment-protein

complexes

of purple

bacteria

3. I. Antenna pigment populations

Within the chromatophores, the light-harvesting pigments may account for more than 90% of the total pigment content in photosynthetically grown cells. They are housed in specific antenna pigment-protein complexes (denoted as B8XO complexes, B for bulk BChl) which allow the absorbed light to be transferred efficiently as excitation energy to the special pair of the reaction centre which is located in the bilayer half exposed to the periplasm [l, 91. From biochemical and spectroscopic investigations it seems likely that, in purple non-sulphur bacteria, two main populations of antenna BChls are present: one group with absorption bands between 820 and 860 nm (pigments of peripheral antenna complexes) or between 870 and 890 nm (pigments of the core antenna complexes) and a second group which absorbs around 800 nm. The pigments of the first group have their macrocycle oriented perpendicular with respect to the plane of the membrane [lo] and are located in the same bilayer half as the special pair of the reaction centre [4, 121. They may possibly form excitonic coupled dimers like the special pair pigments. However, in contrast with the special pair, the shape of the near-IR (NIR) circular dichroism (CD) signal of these antenna pigments is usually biphasic, centred at the absorption position, indicating that they contain two pigment centres with each pigment centre being comprised of two antenna BChl molecules (see Section 4 and ref. 13). Pigments with such characteristics are found in the core complex, e.g. B880 of Rr. rubrum, and in the peripheral antenna complexes, eg. B800-850 of Rb. sphaeroides. It is worth noting that, depending on the number of carotenoids per minimal unit, two in the core complex of Rb. sphaeroides and one in the core complex of Rs. tubrum, the BChl-BChl interactions seem to be either weak or strong as interpreted from the different NIR CD intensities [14]. In addition, strong and weak CD signals of core complexes are correlated with certain antenna polypeptide structural features (Section 3.2 and refs. 15 and 16). The second group of pigments comprises BChl molecules with an absorption band at around 800 nm. This band has been exclusively identified in the accessory antenna complexes such as B800-850 or B800-820. In the case of Chlorojkus aurantiacus, a green thermophilic bacterium, the core complex shows, in addition to an 866 nm band, an 806 nm band [17]. Here, however, the energy transfer pathway to the reaction centre involves pigment species absorbing at 740 (chlorosomal pigments), 790 (socalled baseplate), 806 and 866 nm [17]. In membranes of purple bacteria the pigment absorbing at 800 nm is situated at the photosynthetic membrane-cytosol interphase [lo]. In contrast with the first group of pigments its orientation appears to be parallel to the plane of the membrane. Earlier investigations of the B800-850 antenna complexes of Rb. sphaeroides and Rb. capsulatus have attributed the B800 band to a single BChl molecule which interacts only weakly with the pair of neighbouring pigments [lo]. Recent work on the peripheral antenna systems of Rp. acidophila and Rp. palustti has demonstrated that these species may synthesize, under specific growth conditions, accessory antenna complexes with strongly coupled B800 pigments. Interestingly, these complexes have a rather low B850 or B820 band compared with the standard Rb. sphaeroides B800-850 complex. In Rp. acidophila strain 7050 (according to Pfennig [18]) such complexes were predominantly synthesized under dim light conditions, whereas high light induced the formation of the “standard” B800-850 antenna system. Rp. palustris responds similarly to low light by replacing its “normal” B800-850 with a B800-850 (low) antenna complex. It has been postulated that the molecular or-

117

ganizations of these complexes ditfer in terms of the pigment composition, the pigment organization and orientation and the antenna polypeptide composition (Section 3.2 and refs. 19-21). In the course of structural investigations on antenna polypeptides of Rp. acidophila strain 7750 an interesting observation was made which pointed to the possibility that some of the already well-characterized purple bacterial species may well be able to synthesize additional antenna complexes as originally described. The fact that the cells of Rp. acidophila strain 7750 were usually grown under so-called standard conditions, i.e. 30 “C, exclusively promoted the synthesis of a B80&850 antenna with a B850 concentration 1.5 times larger than the B800 concentration. Surprisingly, by growing strain 7750 at 22-25 “C the preferential synthesis of a B80&820 antenna complex with a dominant B800 band was induced [22]. A comparison of the W-visible absorption spectra of the different peripheral antenna complexes from Rp. acidophila strains 7050 and 7750 is shown in fig. 7 of ref. 6. Certainly, a detailed spectroscopic and structural characterization of the variable peripheral antenna systems of Rp. acidophila and Rp. palust& should evaluate structure-function relationships (see also Section 3.2). In this context, Rp. acidophila strain 10050, with only one single peripheral antenna system (Table 1) [18, 231, appears to be the antenna preparation of choice to crystallize [24] since protein structural studies have characterized this complex to be homogeneous with respect to antenna polypeptide composition [23]. The determination of the three-dimensional structure of B800-850 of Rp. acidophila strain 10050 is currently underway [24]. Temperature-controlled synthesis of peripheral antenna complexes, as observed in our laboratory with Rp. acidophila strain 7750, have been reported for Chromatium vinosum strain D, a purple sulphur bacterium [25]. Its light-harvesting pigments are known to occur in at least five different spectral forms: B795, B805, B820, B850 (845) B890 [26]. Under low light or low temperature (below approximately 35 “C) chromatophore spectral features are generated resembling those observed in Rp. acidophila and Rp. palwtris with a fraction absorbing maximally at around 800 nm and shoulders at 820 to 850 nm. From CD spectroscopic studies it has been concluded that in low and high B850 complexes the state of BChl is dissimilar [27, 281. As in Rp. palustris, antenna complexes with low B850 (as well as those with an 820 nm band) indicate BChl species absorbing at around 800 nm with strong BChl-BChl interactions (intense double CD signal with zero-crossing at around 795 nm [27, 281). In chromatophores of Rb. sphaeroides and other non-sulphur purple bacteria an intense double CD signal corresponding to the 800 nm band is not found [29]. In addition, the spectral forms B850 and B890 of Cr. vinosum exhibit mainly single negative CD bands, not double CD signals [29, 301 as observed in the “sphaeroides’‘-like antenna B800-850 and the B880 complex from Rhodospirillaneae [31]. Furthermore, resonance Raman spectroscopy applied to the peripheral antenna complexes B800-850 (high), B800-850 (low) and B800-820 of Cr. vinosum reveal that the pigments differ regarding their local environment, compared with spectrally (absorption) analogous complexes of Rhodospirillaceae [32, 331. Thus it is worth noting that, in different photosynthetic bacteria, antenna BChl may exhibit absorption bands at almost the same wavelength although the BChl-BChl and BChl-protein interactions are different. However, according to resonance Raman studies of the B890 complex of Cr. vinosum, it seems likely that the state of the BChl molecules in terms of their local environment is quite similar to that observed in core antennae of Rhodospirillaceae [32, 331. Biochemical and spectroscopic analyses have recently been undertaken on the antenna systems of another purple sulphur bacterium, Chromatium purpuratum BN5500. Its photosynthetic unit consists of a “normal” core

118

reaction centre complex together with a spectrally non-typical B830 peripheral complex with a shoulder at 800 nm [34]. Representatives of the family Ectothiorhodospiraceae are halophiles which, in contrast with species of Chromatiaceae, deposit elemental sulphur outside their cells (for a review on halophilic phototrophic bacteria, see ref. 35). The components of the light reaction of species from Ectothiorhodospiraceae have been less well characterized compared with those from Rhodospirillaneae. The antenna system of Ectothiorhodospira halophila is characterized in the NIR region by a dominant maximum at 890 nm and minor pigment species absorbing at around 800 and 850 nm [36]. The cell spectral features do not undergo large variations when growing the cells under different conditions. Two representatives of Ectothiorhodospiraceae, Ectothiorhodospira halochloti and Ectothiorhodospira abdelmalekii, contain BChl b as antenna pigment. Their cell spectra exhibit absorption maxima at 800 and 830 nm (Table 1) together with a major farred-shifted antenna absorption band at around 1020 nm, which are typical of most of the BChl b-containing organisms (e.g. core complex of Rp. viridis). From membranes of E. halochloti an antenna fraction B800/830/1020 has been isolated which according to NIR CD studies contains at least five strongly interacting BChl b molecules [37]. The CD spectra of the B800/830/1020 antenna fractions of E. halochloris and E. abdelmalekii photosynthetic membranes are very similar [37]. They exhibit a strong biphasic signal with zero-crossing at around 800 nm which has been reported as a typical feature for the peripheral antenna complexes of Cr. vinosum [28]. Owing to the observed similarity in supramolecular structure [38, 391 of the antenna system of Rp. vikfk (see above) (with only one antenna absorbing at 1015 nm) and the spectrally very complex antenna of E. halochloris and E. abdelmalekii, the composition and structure of the antenna apoproteins of the two types of BChl b-containing bacteria have received special interest. The next section summarizes the current state of data collection from comparative primary structural analyses of antenna polypeptides from Rhodospirillaneae. These results are important due to the large spectral variety amongst species of Rhodospirillaneae.

3.2. Pwple antenna polypeptides: comparative primary structural analyses as a basis for structure-spectnun considerations In addition to the spectral characterizations of the pigments of purple bacterial antenna pigment-protein complexes, there has been a considerable increase in the amount of structural information on the light-harvesting polypeptides [6,21, 23, 40-541. Early structural characterizations of the antenna systems of Rs. rubrum [40, 411 and Rb. sphaeroides [50, 511, together with topology studies [4, 551, have pointed to some significant structural principles required for the correct pigment-polypeptide association (see also Figs. l(A) and l(B)). (1) The minimal structural unit of purple non-sulphur bacterial antenna complexes usually consists of two small (50-60 amino acid residues) apoproteins, denoted as a and /3 polypeptides, which span the membrane once in a putative (Yhelix (approximately six turns equal to 22 amino acid hydrophobic residues). The flanking polar/charged N- and C-terminal domains are located on the cytosolic and periplasmic sides respectively. (2) Within the transmembrane segment both antenna polypeptide types exhibit a conserved histidine (His) residue most probably serving as fifth ligands for the strongly coupled pair of BChl molecules (B880 and B850 pigments). Similar to the

119

special pair pigments of the reaction centre they are located in the bilayer half exposed to the periplasm. (3) A conserved alanine (Ala) (a small amino acid) is postulated to be a significant structural requirement for the B880 and B850 pigments present at a consensus distance from the His (Ala-X-X-His). (4) Further possible interaction sites are aromatic amino acid clusters in the Cterminal part and the transmembrane segment of the antenna polypeptides which may account for far-red shifts of the light-harvesting complexes [40]. A comparison with other chromophore-binding proteins (e.g. bacteriorhodopsin) indicates that aromatic amino acids in general can build up the chromophore-binding pocket [40]. With the structural information as a basis, resonance Raman spectroscopy has confirmed the postulated BChl-His ligation. Molecular genetic studies of Rb. cupsufutus have shown the importance of the conserved His and Ala (His-4) residues, i.e. substantial evidence has been obtained that these residues serve as BChl-binding and interaction sites within the hydrophobic stretch [56]. In the last few years, primary structural information of antenna apoproteins from different sources and types of antenna complex (spectral variability of the antenna complexes of purple non-sulphur and purple sulphur bacteria) has been obtained. thus a fundamental basis in terms of the specific microenvironment of the functional antenna pigments has been obtained which should be of considerable help in site-directed mutagenesis experiments. As shown in Table 2, 63 (Y or p polypeptides have been isolated and sequenced from 14 different species (total of 19 strains) [21, 23, 40-543 from the three families of Rhodospirillaneae. Most of the available protein primary sequence data are summarized in Figs. l(A) and l(B). Except for one polypeptide of Ectofhiorhodospira halochloti (asparagine (Asn) instead of His) the conserved transmembrane-located His residue is the structural key element and thus is the most

Fig. 1.

120

-

-

EVKQESLSGITEGEAKEFHKIFTSSILVFFGV 1. AEIDRPVSLSGLTEGEAREFHGVFMTSFNVFIAV 2. ADLKPSLTGLTEEEAKEFHGIFVTSTVLYLAT 3. ADKSDLGYTGLTDEQAQELHSVYMSGLWPFSAV 4. MADKNDLSFTGLTDEQAQELHAVYMSGLSAFIAV 5. AEPXGSISGLTDDEAQEFHKFWVQGFVGFTAV 6. 7. AEPXGSISGLTDDEAQEFHKFWVQ S. AEDRSSLSGVSDAEAKEFHALFVS 9. ADNMSLTGLSDEEAKEFHSIFMQ 10. ADEMRNVSDEEAKEFHANFSQ 11. ANDIRPLRDFEDEEAQEFHQMVQ 12. TDIRTGLTDEECQEIHENNML 13. ANSSMTGLTEQEAQEAHGIFVQ 14. DQKSMTGLTEEEAKEFHGIFTQ 15.MRDDDDLVPPKWRPLFNNQDWNLHDIVVK 16. TDDLNKVWPSGLTVAEAEEVHHKQLIL 17. MTDDKAGPSGLSLKEAEEIHSYLID 18. ADDANKVWPSGLTTAEAEELQKGLVD 19. ADDANKVWPSGLTTAEAEELQKGLVD 20. 21. 22. 23. AVLSPEQSEELHKYVIDGARAFLGI 24. ADKPLTADQAEELHKYVIDG-AI 25. ATLTAEQSEELHKYVIDGTRVFLGL 26. NVDDPNKVWPTGLTIAESEELHKHVIDGSRIFVAI 27. ADDPNKVWPTGLTIAESEELHKHVIDGTRIFGAI 28. DKTLTGLTVEESEELHKHVIDGTRIFGAI 29. ADMKSLSGLTEQQAKEFHEQFKVTYTAFVG 30. AELSGLTDQQAKEFHEQFKVTYTAFVG 31. ASLLLSGLTEQQAKEFHEQFKVTYTAFVG 32. MNGLTEQQAKEFHAQFKVTYTAFVG

WIPGAEGYG... AWRPWIPGDEGFG..

j

03)

Fig. 1. (A) Ammo acid sequences of the a-antenna polypeptides of species from the three families of Rhodospirillaneae: 1, Rhodospitilhon rubrum B870-q 2, Rhodopseudomonas marina B880-a; 3, Rhodopseudomonas viridis 1015-a; 4, Rhodobacter sphaeroides B870-a; 5, Rhodobacter capsulatus B870-a; 6, Rhodocyclus gelatinosus DSM 149 B870-a; 7, Rhodocyclus gelatinosus DSM 151 B870-q 8, R.p. acidophila A~7050 B880-a; 9, Ectothiorhodospira halophila B890,-a; 10, Ectothiorhodospira halophila B8902-a; 11, Ectothiorhodospira halochloris B890,-a; 12, Ectothiorhodospira halochloris B890,-a; 13, Chromatium vinosum B890-a; 14, Chlorojkws aurantiacus Jlo-fl B806-866-a; 15, Rhodobacter sphaeroides B800-850-s, 16, Rhodobacter capsulatus B800-850a; 17, Rhodocyclus gelatinosus DSM 149 B800-850-a; 18, Rhodocyclus gelatinosus DSM 151 B800-850-a, 19, Rp. acidophila A~75050 B800-850-a; 20, Rp. acidophila A~7050 B800-820-a; 21, Rp. acidophila Ac7750 B800-850-a, 22, Rp. acidophila Ac7750 B800-820-a; 23, Rp. acidophila A~10050 B800-850-a; 24, Ectothiorhodospira halophila B800-850-a; 25, Rhodopseudomonaspalustris B8/850; 27, Rhodopseudomonas “French” B8/850; 26, Rhodopseudomonas palustris “French” palustris “French” B8/850; 28, Rhodopseudomonas palustris “French” B8/850; 29, Chromatium vinosum B800-850-a,; 30, Chromatium vinosum B800-820-a; 31, Chromatium vinosum B800-850oz. (B) Amino acid sequences of the Santenna polypeptides of species from the three families of Rhodospirillaneae: 1, Rhodospirillum rubrum B890-P; 2, Rhodopseudomonas marina B880-p;

3, Rhodopseudomonas viridtk BlOl5-fi; 4, Rhodobacter sphaemides B870-P; 5, Rhodobacter capsulatus B870-P; 6, Rhodoqclus gelatinosus DSM 149 B880-f3; 7, Rhodocyclus gelatinosus DSM 151 B880/3; 8, Rhodopseudomonas acidophila A~7050 B890-j3; 9, Ectothiorhodospira halophila B890,-p; 10, Ectothiorhodospira halophila B890,-p; 11, Ectothiothodospira halochloris p-Polypeptid; 12, Ectothiorhodospira halochloti /3-Polypeptid, 13, Chromatium vinosum B890r-P; 14, Chromatium vinosum B890,+; 15, Chlorojkxus aurantiacus J-lo-fl B806-866+; 16, Rhodobacter sphaemides B800-850t3; 17, Rhodobacter capsulatus B800-850-j3; 18. Rhodocyclus gelatinosw DSM 149 B80@-850-~3; 19, Rhodocyclusgelatinosus DSM 151 B800-850-/3; 20,Rhodopseudomonas acidophila A~7050 B800-850/3; 21, Rhodopseudomonas acidophila A~7050 B800-820~/3; 22, Rhodopseudomonas acidophila Ac7750 B800-850+3; 23, Rhodopseudomonas acidophila Ac7750 B800-820-a; 24, Rhodopseudomonas acidophila Ac7750 B800-820~&; 25, Rhodopseudomonas acidophila A~10050 B800-850~/3; 26, Rhodopseudomonas palustris “French” B8/850; 27, Rhodopseudomonas palustris “French” B8/ 850; 28, Rhodopseudomonas palustris “French” B8/850; 29, Chmmatium vinosum B800-850-p; 30, Chromatium vbtosum B800-820~PI; 31, Chromatium vinosum BSocr820-a; 32, Chromatium vinosum B800820-a.

bacterium)

(?)

polypeptides

2

2

Erythrobacter sp. strain OCh 114

Chlorofltxus aurantiacus strain J-lo-fl

6 5 7

10

Chromatium vinosum strain D E. halochloris (SM 1059) E. halophila (type strain, DSM 244) E. abdelmalekii strain 9804 (J. Imhoff)

6 7 7

Rp. acidophila strain 7050 (N. Pfennig) Rp. acidophila strain 7750 (N. Pfennig) Rp. palustnk strain 2.1.6 (“strain french”)

2 2 2 3 4 4 4 4 4 4

Number”

(see also Figs. l(A)

(cm

(0)

=

=,=

;”

d

f*d

;

f

fc’f

f

23, 54

50 52, 53

57

44 41

46-48

45, 49 45 c

45

.

6, 54 6, 23, 54 21, 59

40, 40, 43 42, 51, 6, 6, 58 58 6,

Reference

or growth medium).

Comments

temperature

2x((Y/P)+y 2x((Yl@+a 6+r

4a and 6p

3X(&3) 3a and 4P 4~ and 3p

2 x (0) 2 x (dP) 2 x (a)

2X(&/P)

(alPlY) 2 x (0) 2 x (dP)

‘Qpeb

and l(B))

‘Number of isolated and sequenced antenna polypeptides. bme of antenna polypeptide. CBChl b-containing species with typical red-shifted absorption bands (approximately 1020 nm). dMore antenna polypeptides may be synthesized (dependent on environmental factors such as light intensity, “Work in progress. ‘Protein sequence data identical with DNA sequences. BDeutsche Sammlung Mikroorganismen.

Green bacteria Chloroflexaceae

Erythrobacteriaceae (strict aerobic phototrophic

complexes

complexes

Chromatiaceae with at least three antenna

Ectothiorhodospiraceae with several antenna

complexes

complexes

Rhodospirillaneae with two antenna

antenna

1zF. rubrum (wild type) R.r. rubrum G-9+ (carotenoid-free mutant) Rp. marina (type strain) Rp. viridis (type strain) Rb. sphaeroides 2.4.1. (wild type) Rb. sphaeroides 26.1. (carotenoid-free mutan It) Rb. capsulatus (type strain) Rc. gelatinosus (DSM 149) Rc. gelatinosus (DSM 151) Rp. acidophila strain 10050 (N. Pfennig)

Species/strain

data of purple bacterial

Rhodospirillaneae with at least three antenna

complex

Purple bacteria Rhodospirillaneae with one antenna

Bacterium

Overview of the amino acid sequence

TABLE 2

E

122 probable ligation site for the (B1020 for BChl b) B880, B870, B850 or B820 pigments (species and antenna complex variability). Accordingly, in Figs. l(A) and l(B) all of the amino acid sequences are aligned with respect to this His residue. The ultimate microenvironment of this particular His residue should be of special interest regarding the specific spectral properties of the associated BChl molecules. A careful inspection of all the sequences reveals the following remarkable observations. Assuming an a-helical arrangement of the transmembrane segment, the positions His + 4 and His - 4 (distance of approximately one helical turn) are possible candidates for BChl interaction sites. In the membrane-spanning regions of the cz polypeptides the His+4 and His-4 amino acid residues mark conserved residues: position His- 4 is mostly Ala, whereas position His+4 for the ry polypeptides from species of Rhodospirillaceae and Ectothiorhodospiraceae is an overall conserved leucine (Leu). Interestingly, three peripheral (Ypolypeptides of Cr. vinosum (BSOCrSSO and B800-820 apoproteins) exhibit phenylalanine (Phe) in that particular position. This structural change was demonstrated in a resonance Raman study on the B80G850 (high), B800-850 (low) and B80@-820 antenna complexes of Cr. vinosum which showed that their pigments were different in terms of their local environment compared with spectrally (absorption) analogous complexes of Rhodospirillaceae [32, 331. In addition, typical peripheral aromatic clusters found in Rhodospirillaceae are partly missing in the C-terminal domain of the three (Ypolypeptides of Cr. vinosum (see below). Similar to the (r polypeptides, in the p polypeptides the residues at a four-residue distance from the intramembrane His are conserved. His - 4 represents a small residue, mostly Ala. On the other hand, in species from Rhodospirillaceae and Ectothiorhodospiraceae, position His + 4 is mainly an aromatic residue (in the core p polypeptides, mostly tryptophan (Trp); in the peripheral p polypeptides, tyrosine (Tyr) or Phe). It is worth noting that the four isolated and sequenced peripheral p polypeptides from Cr. vinosum show significant structural changes with isoleucine (Ile) (Leu) in the His+4 position (Fig. l(B)) which is again in accordance with the reported different resonance Raman spectra [32, 331. From these structural principles it is concluded that the orientation of the pigments is firstly governed by the orientation of the putative (Yhelix traversing the membrane. Secondly the (dimeric) pigments, e.g. B850, obtain their particular orientation by interacting with bulky (aliphatic or aromatic, such as Leu=His t-4 or Trp, Tyr, Phe) and small (e.g. Ala=His4) amino acids at one particular helix surface (Fig. 3., see Section 3.3). Further aromatic amino acids are present at a consensus distance from the histidines (Figs. l(A) and l(B) and ref. 6). As depicted in Figs. l(A) and l(B) most antenna polypeptides exhibit an aromatic residue in position His + 9. The typical core structural element Phe-Asn-Trp (His+9 to His+ 11) and the peripheral apoprotein cluster Trp-Phe (His+ 9, His+ 10) indicate residues of functional significance. They are apparently part of the structural requirements to form core or peripheral antenna complexes. In a recent work probing the influence of site-directed mutagenesis in the core antenna polypeptides of Rb. cupsulutus, blue shifts of BChl a (8-11 nm) were induced by substitution of Trp at position a43 (His+ 11) by Ala, Leu or Tyr [60]. It has previously been suggested [6, 15, 16, 541 that, in Rp. acidophilu strains 7050 and 7750, modifications of the aromatic residues in equivalent positions (in strain 7050 from Tyr-Trp to Phe-Leu (His+ 13, His + 14) and in strain 7750 from Tyr-Trp to Phe-Thr (Thr, threonine); see Fig. l(A)) re p resent the structural basis for shifting the 850 nm component of the B800-850 complex to approximately 820 nm which is the second absorption band of the peripheral antenna complex B800-820 (preferential

123

synthesis under low light (strain 7050) and/or low temperature (strain 7750); see Section 3.1) [6, 15, 16, 23, 541. The finding that in two peripheral apoproteins of Rp. palusti the structural element ‘Qr-Trp (His+ 13, His+ 14) is replaced by Phe-Met (Met, methionine) [21, 591 supports the functional importance of the C-terminal domain of the antenna apoproteins. Furthermore, the same two a! polypeptides, presumably the components of the B800-850 (low 850) complexes, replace Phe (His+ 10) with valine (Val) (Fig. l(A)) [21, 591. In contrast with the observed chemical microenvironment differences between peripheral pigments of Rhodospirillaceae and Chromatiaceae (agreement between resonance Raman and sequence structural data), it has been postulated that the states of the BChl molecules of the core complex B890 of Cr. vinosum are probably similar to those observed in the core antennae of Rhodospirillaneae [32, 331. This statement agrees with the primary structural work when the individual antenna polypeptides are monitored by their specific antenna-type key elements (Figs. l(A) and l(B)) in the vicinity of the transmembrane His, e.g. in the p polypeptides the typical core stretch Trp-Leu-Trp-Arg-Pro-Trp-Leu (Arg, arginine; Pro, proline) (from His + 4 to His+ 10) is present. However, it seems likely that the core complex of Cr. vinosum is comprised of two chemically different p polypeptides (with core structural domains) in addition to an (Ypolypeptide (Figs. l(A) and l(B)) pointing to a different organization of the core complex in this bacterium. Another interpretation could be that there are two core complexes present in Chromatiaceae (see below, structural organization of Ectothiorhodospiraceae antenna complexes). It is certainly interesting to note that the two core antenna /3 polypeptides are extremely homologous in the vicinity of the transmembrane His (Fig. l(B)): within the stretch His- 8 to His+ 10 (19 residues) there is only a Val/Ile change (= 95%), whereas the overall homology is 80% (nine changes within 45 residues). Recent structural studies on antenna polypeptides of E. halochlorfs and E. halophila have revealed two sets of alp-antenna polypeptides [45], each with typical primary structural elements of core complexes from Rhodospirillaneae. In an a//3 polypeptide pair from E. halochloris and in an cx polypeptide from E. halophila an additional His was found within the hydrophobic stretch, six residues away from the borderline of the N-terminal portion (cytosolically located) and the membrane-spanning domain. Assuming an cr-helical conformation of the membrane-spanning domains, the additional histidines are located approximately 9 A beyond the membrane interface on the cytosolic side. The possible additional pigment-binding sites within the hydrophobic stretch are partly in accordance with the postulated five BChl b molecules per smallest compositional unit of the B800/830/1020 antenna complex of E. halochloris which could not be dissociated into individual antenna complexes. Thus two antenna apoproteins of E. halochlonk exhibit two individual possible BChl ligation sites at either side of the membrane interfaces. The specific location of these putative BChl-binding sites in the E. halochloris antenna indicates a similar situation as that observed in the antenna apoproteins of the more complex antenna systems of higher plants (see Section 3.3). Similar to E. halochloris the four core-like polypeptides of E. halophila appear in a ratio close to 1:l:l:l. This raises the possibility of a single core antenna in Ectothiorhodospiraceae, with two chemically different (Ypolypeptides as well as two p polypeptides ((Y& basic unit, see Section 4). In addition, the BChl b-containing antenna complex B800/830/1020 of E. ha1ochlori.s (BChl b) and E. abdelmalekii apparently consists of a small (29 residues), very hydrophobic polypeptide, denoted y polypeptide [61]. A homologous component has previously been characterized in Rp. viridk [42]. Although possibly without BChl-binding capacity it has been postulated that this y apoprotein

124

reflects a crucial component responsible for the specific structural and spectral features of the B1015 antenna [42]. As in the Rp. viridzk B1015 y polypeptide the analogous component ofE. halochloti and E. abdelmalekii shows amino acid sequence heterogeneity whose significance is yet unknown (gene family for the B1015 y polypeptides?). From protein chemical and molecular genetic work on Rp. acidophila, Rp. palUrk and Cr. vinosum (see above and Figs. l(A) and l(B)) it seems most likely that species of Rhodospirillaceae and Chromatiaceae with a pronounced tendency to change their NIR absorption spectrum (between 800 and 850 nm), depending on environmental factors, contain multiple genes (gene family, coding for at least four pairs of CY/~ polypeptide pairs). Intense CD signals in the NIR region have been reported for the core antenna complexes of the three purple non-sulphur bacteria Rs. rubrum, Rp. viridis and Rp. acidophila (biphasic signal with zero-crossing at 880 nm [14]). In contrast the CD signals of the core complexes of Rb. sphaeroides, Rb. capsulatus and Rc. gelatinosus are much weaker. From the available antenna polypeptide sequences of all six species it was postulated that weak or strong pigment coupling is dependent on specific amino acid residues or domains, such as a long C-terminal portion in the @ore polypeptide [15, 161 (Fig. l(B)) which may bend back to the membrane-periplasm interphase and thus provide interactions with the dimeric antenna pigments (see also Fig. 3, Section 3.3). These putative arrangements of the C-terminal domains are supported by the fact that some characterized purple antenna polypeptides (mostly (Ypolypeptides) have a pronounced C end, in some cases with more than 30 residues (Figs. l(A) and l(B)). In addition, the C-terminal domain of the B800-850 a-antenna polypeptide from Rc. gelatinosus (two different strains, see Table 2 and Fig. l(A)) exhibits a concentration of more than 13 Ala residues within the last 21 residues [58]. From this, a hairpinlike helical structure of such antenna polypeptides is most probable. 3.3. Structural similarities of purple antenna polypeptides with reaction centre polypeptides and eukaryotic light-harvesting polypeptides One basic and fundamental structural characteristic of many prokaryotic and eukaryotic systems involved in the primary processes of photosynthesis appears to be a pair of chemically dissimilar polypeptides (heterodimers, homologous with each other) which serve as a scaffold for the antenna and reaction centre cofactors. Some of these proteins, appearing as heterodimers, are compiled in Table 3. In some cases they contain additional polypeptide components such as the H subunit in the reaction centre of purple bacteria. Recently, in connection with the structure-spectra relations in bacterial antenna systems (see above), we focused on some structural similarities between the small bacterial antenna polypeptides and the reaction centre polypeptides from Rp. viridis [6, 15, 161. Apart from the His residues (pigment ligands), aromatic amino acids may provide common interaction sites for BChls constituting the special pair and the (core) antenna pigments. In particular, it seems probable that, in the &antenna polypeptides, His - 8 (approximately two helical turns distant from His), which is an overall conserved aromatic residue (mostly Phe in all analysed species from the three families of Rhodospirillaneae), is part of the antenna pigment-binding pocket. In the reaction centre L and M polypeptides of Rp. viridis, Phe and Tyr at similar positions were identified to be part of the special pair pigment-binding pocket [62] (here His+8, sequence comparison in reversed direction, see refs. 6, 15 and 16). Within the cd helices (interconnecting helix between helices C and D) of the L and M subunits additional aromatic residues provide interaction sites to the special pair. In the (Y-and p-antenna polypeptides there are aromatic residues in similar positions

125 TABLE

3

Polypeptide organisms

pairs involved in the primary processes of photosynthesis

of prokaryotic and eukaryotic

Organism/classification

Type

Polypeptide

Species of Rhodospirillaneae Species of Rhodospirillaneae Chloroflexus aurantiacus Cyanobacteria

Core antenna Peripheral antenna Core antenna Globular antenna (phycobilisome) PS II core antenna Reaction centre Reaction centre

e.g. B880-c~, BSSO-p e.g. B800-850-(Y, B800-850-p B806-866~a, B806-866P PEC, CPC, APC (CYand B subunits) CP4O/CP47 L and M subunit L and M subunit

PS II core antenna

CP47lCP50

Reaction

DUD2

Cyanobacteria Rhodospirillaneae Chloroflexus aurantiacus Green algae (Chlnmydomonas reinhanitii) Green algae (Chlamydomonas reinhardtii) Higher plants (e.g spinach) Higher plants Higher plants Higher plants (e.g. maize)

centre

PS II core antenna

CP43lCP47

Reaction centre Reaction centre (cytochrome b559) PS I core/reaction centre

DUD2 419 kDa

designation

Al/A2

(see Figs. i(A), l(B)) and 3 (below) and Fig. 6 in ref. 16). These are located in the C-terminal domains of the antenna polypeptides and thus support the proposed structural and functional role of the C-terminal domains of the bacterial apoproteins (especially those from the a-antenna polypeptides). Structural homologies between prokaryotic and eukaryotic pigment-protein complexes have been reported for the reaction centre of purple bacteria and the reaction centre of photosystem (PS) II of higher plants [63]. From the recently determined amino acid sequences of the antenna polypeptides of E. halochloris and E. halophila [45], striking structural similarities to the eukaryotic core antenna systems have been found as shown in Figs. 2(A) and 3: two His residues (Asn in one E. halochloris polypeptide) at a consensus distance of ten residues are of special interest regarding the similar pigment organization between prokaryotic and eukaryotic antenna systems. As a consequence, it is probable that two pigment populations, at either side of the membrane, are present. Additional structural similarities are indicated in Fig. 2(A). The structural homology of 36% (ten identical positions out of 28) between the seventh membrane-spanning domain of the spinach CF47 core polypeptide (positions 499 to 526 [64, 651) and the hydrophobic stretch of the a-core-antenna polypeptide of E. hafochloti is remarkable. Similarly, by comparing the prokaryotic antenna polypeptides with the reaction centre/antenna polypeptides of PS I (maize) indicates, as illustrated in Fig. 2(B), pronounced homology [6] between the sixth membrane-spanning domain of the Al polypeptide [66] and the single membrane-spanning domain of the B880 /3 polypeptide of Rs. rubrum [40]. Positions at the discussed (Section 3.2) consensus distances from the histidines (e.g. His + 4, His - 4) are remarkably similar (Fig. 2(B)). All of these findings raise the question of whether the arrangement of the membrane-

126

E.halochloris E.halochloris E.halophila C.

vinasum

C.

vinosum

..QLSLFTHHMWIGGFLIVGAAAHAAIFMVRDYDPTT'RY... II II III II IIII I II I II rubesao-~ . . . KEFHKIFTSSILVFFGVAAFAHLLVWIWRPWVPGPNGYS

PS

W

I

Al,“*,

(.

c-end

>

Fig. 2. (A) Comparison of the amino acid sequences of the light-harvesting polypeptides of Ectothiorhodospira halochloris (two apoproteins), E. halophih (one apoprotein) and Chromatium vinosum (two antenna polypeptides) with the core apoproteins (44 (I) and 47 kDa (II)) of higher plants (only hydrophobic segments are compared between positions 500 and 530). For clarity only homology boxes behveen the 47 kDa and one E. halochloris polypeptide are shown. His (also some Met and Asn) residues at consensus distances are marked with bold letters. Arrows mark the putative membrane-spanning segments. Primary structures of the Chl u-binding apoproteins of PS II of spinach (core antenna of PS II; the 47 and 44 kDa apoproteins, so-called CP44 and CP47) were obtained from refs. 64 and 65. Primary structures of the bacterial antenna polypeptides were obtained from this work and ref. 45. (B) A comparison of the amino acid sequence of the sixth membrane-spanning domain of the Al (reaction centre polypeptide of PS I) protein [66] with that of the membrane-spanning domain of the B880 p polypeptide of R.s. rubrum [40]. ++, indicates membrane-spanning domain; II, indicates identical residues; I, indicates structurally similar residues (such as Ile and Val).

multispanning domains of the eukaryotic antenna pigment-protein complexes is similar to the postulated aggregational properties of the prokaryotic antenna polypeptide (see Section 4). Simply, is the eukaryotic protein scaffold (e.g. the seven (Y helices of the core antenna polypeptides of PS II) a multiform of the (Y/P heterodimers of the prokaryotic antenna complexes?

4. Possible structural organization of polypeptides from primary structural data (primary structural

and BCbl model)

molecules

derived

4.1. General structural principles of antenna systems The physical principles of directed (e.g. heterogeneous) energy transfer and coupled excitons [5, 671 found in the multiplicity of antenna systems should correspond to certain general principles of structure and organization of the antenna complexes. Data available today in prokaryotic antenna systems indicate the following general structural features [6-81. (1) A strict organization of pigment molecules. (2) In the matrix of the pigment-protein complexes, pigment clusters of defined size and symmetry exist forming a specific, often cyclic, system of energy transfer.

127 N-terminalends \

transmembrane helices of the a- and p-antenna polypeptides

C-terminal domain containing specific aromatic clusters (arrangement similar to the cd-helix in RC L and M subunits ?) Fig. 3. Model showing a possible schematic arrangement of the (intramembrane-located) BChl bound to two His residues per (Y helix of the a polypeptide and one His and one Asn of the /3 polypeptide of E.haZochlori.~ (located at a consensus distance). These typical structural features

are also present in higher plant (spinach) core antenna systems (see also Sections 3.2 and 3.3 and Figs. l(A), l(B) and 2(A)). A, alanine; L, leucine; N, asparagine.

(3) Within the antenna complexes, pigment dimers (or oligomers) are formed leading to the production of excitons (distance of the pigments, 10-20 A). The dimers are further organized in a cyclic system (distance, less than 30 A). (4) The pigment clusters of antenna complexes possessing varying absorption maxima, are the basis of heterogeneous energy transfer between the antenna complexes and the reaction centre. Energy transfer takes place from pigment molecules absorbing at a short wavelength to those absorbing at a long wavelength. (5) Directed energy transfer to the reaction centre is optimized by the spatial separation of the antenna complexes (pigment clusters), minimizing random walk. Fundamental to the formation of the antenna complexes, the entire antenna and the active energy transfer system are the specific structural and/or functional interactions between pigment-pigment, pigment-polypeptide and polypeptide-polypeptide molecules. These three types of interaction should be of equal importance with respect to both the structure and function of the antenna. The role of the polypeptides is primarily structural as they are responsible for the organization, i.e. the structural arrangement, of the pigment molecules in the matrix of the entire antenna [a]. Structural data indicate the following features. (1) Pigment molecules are bound specifically to polypeptides and form defined antenna complexes.

128

(2) Polypeptides determine the position, distance, orientation and environment of the pigment molecules. On this basis they also take part in the function of the antenna system. Pigment molecules also play a structural role with respect to points (3) and (4) below. (3) Polypeptides have specific association characteristics in forming oligomeric and multimeric units. This is the basis of the three-dimensional structure of antenna complexes and of the arrangement of the pigments. (4) The antenna polypeptides associate in a hierarchical order: (a) formation of microdomains of 2-4 polypeptides (small pigment clusters, heterodimers); (b) formation of antenna complexes with larger pigment clusters (domains) through association of microdomains (cyclic arrangement); (c) formation of the entire antenna through association of antenna complexes (pigment clusters). The association of antenna complexes with different absorption maxima (e.g. core and peripheral antenna complexes) is the basis of the heterogeneous energy transfer system to the reaction centre. This hierarchical organization of polypeptides and pigments should be present in the antenna systems of all photosynthetic organisms with increasing complexity from photosynthetic bacteria to higher plants. 4.2. Structural features of the light-harvesting antenna of purple bacteria Relatively simple antenna systems, on the basis of these general structural principles, are found in the intramembrane antenna of photosynthetic bacteria and the extramembrane antennae (phycobilisomes) of cyanobacteria. For instance, the primary structure of the antenna polypeptides of purple bacteria possesses characteristic, clearly recognizable structural features, which correspond to the general structural principles of polypeptide and pigment organization in the antenna complexes described above [6-8, 15, 161. The following structural elements are of significance here. (1) Two different types of antenna polypeptides, the (Y and p polypeptides, are found in the antenna complexes. The (Yand /? polypeptides are sequence homologous and structurally related. However, they differ in the typical amino acid cluster [6, 71, which is the basis for the formation of heterodimers ((Y/P). For example, a conserved His residue within a cluster of hydrophobic amino acid residues, is probably the binding site for BChl, in both the (Yand /? polypeptides; in the p polypeptides an additional His residue (probable BChl-binding site) exists. These data indicate that, in the (Y and p polypeptides, functionally different BChl molecules are bound ((Y and /3 types) and the polypeptide and BChl pairs of the heterodimer form the structural and functional basic unit of the antenna complexes and the entire antenna. In the environment of the His residues, i.e. the main BChl-binding site, specific amino acid residues are present which interact structurally (additional binding sites) and functionally (e.g. important aromatic or charged residues, red shift in absorption, variability of CD signal) with the BChl molecules (see Section 3.2). (2) The primary structure of the (Yand p polypeptides is of a three-domain nature, which is an important feature with respect to their three-dimensional structure within and at the photosynthetic membrane. The central hydrophobic domain is most probably localized within the hydrocarbon tail region and the polar (charged) N- and C-terminal domains in the polar head region or on the membrane surface. The hydrophobic domain forms a transmembrane (Y helix (length, 31-34 A; diameter, about 10 A) as postulated on the basis of IR, UV and CD spectra [68] and labelling experiments 1691. IR dichroism studies show that the (Y helices are tilted (30-35” away from the membrane plane).

129

(3) Some purple bacteria (Rhodospirillaneae, Rs. rubrum, Rb. sphaeroides, Rb. capsulatus, Rp. viridis) have an energy transfer system with defined core (B870/890, B1015) and peripheral complexes [70] and individual (Y and p polypeptides in the ratio 1:l. The (Yand p polypeptides of both complex types differ typically in primary structure, i.e. they show complex specific amino acid clusters. We thus expect core and peripheral antenna complexes to be very similar in terms of the basic arrangement of polypeptides and BChl molecules, but to show some specific differences related to differences in the organization of these complexes. In recent years, the extension of primary structural analysis of the antenna polypeptides to include Rp. acidophila, Rp. palustris, Cr. vinosum, E. halochloks and E. halophila has revealed a structural multiplicity and heterogeneity of the (Yand /3 polypeptides particularly in the peripheral antenna complexes (see Section 3.2 and ref. 6). This structural multiplicity is most probably of functional importance, eg. in changing the spectral properties of the antenna complexes with varying environmental conditions. In spite of the multiplicity of (r and p polypeptides, they still possess in all cases typical structural characteristics. This indicates that they still form the important basic heterodimers. 4.3. Possible arrangement of antenna polypeptides in the three-dimensional structure of the antenna complexes The specific structural-functional features of the antenna polypeptides, derived from primary structural data, are ultimately the basis of the three-dimensional structure of the antenna complexes and the entire antenna of purple bacteria. Several different complexes have been crystallized and studied [24, 71-731. Most promising are the crystals of the B80&850 complex from Rp. acidophila 10050, diffracting X-rays to high resolution [24]. Apart from the crystallization problem the main difficulty in X-ray structural analysis is the isolation of well-defined antenna complexes related to those that exist in vivo. In addition, X-ray structural analysis yields no data on the arrangement of the antenna complexes within the entire antenna. It has therefore been attempted to estimate possible and probable arrangements and interactions of antenna polypeptides within and between the antenna complexes of purple bacteria on the basis of their primary structural data and general structural features [6, 7, 741. The important basis for these structural considerations is the al/3 heterodimer of polypeptides and BChls and the three-domain structure of the antenna polypeptides. In the heterodimers the BChl molecules which are bound at the conserved His residues of the hydrophobic domain (central BChl) can be located quite close together (10-20 A) in the tilted (Y helices and thus may be exciton coupled (see below). As shown in proteolytic cleavage and labelling experiments [4, 55, 75-781 the Nterminal domains of the (Yand p polypeptides lie on the cytoplasmic side and the Cterminal domains point towards the periplasmic side (vertical asymmetry, vertical topography). Therefore the central cu/p BChl pair also lies asymmetrically on the periplasmic side. This specific location of the a/p BChl pair is the basis for the twodimensional energy transfer parallel to the membrane plane within and between the antenna complexes and to the special pair of the reaction centre, which is also localized on the periplasmic side. Most important for the specific association of the antenna polypeptides is the three-dimensional structure of the transmembrane a helices. It is assumed that the specific interaction of the amino acid side-chains, distributed in four rows along the long axis of the (Yhelix, and the packing of the tilted helices in the lipid environment are the decisive factors in the formation of polypeptide aggregates. Equally decisive

130

is the arrangement and interaction of structurally and functionally different BChl molecules on the periplasmic side. In this context, in a cross-section of the cr helix (see ref. 6) it becomes apparent that, with respect to the amino acid residues and the BChls, a transverse asymmetry exists determining the method of packing of the a helices: on the N-terminal side of the (Yhelix pointing towards the cytoplasm, polar amino acid residues are frequent (hydrogen bonds in polypeptide-polypeptide interactions) and on the C-terminal side pointing to the periplasm, there are hydrophobic (aromatic) residues in the environment of the central, interacting BChl molecules. The starting point for the specific association processes, which lead in hierarchical order to the formation of the entire antenna, is the formation of the (Y/B heterodimer by means of e/3 interaction in region I (Fig. 4(A)). The other three polypeptide interaction regions II (/3-a), III ((1~01) and IV (P_p) for the specific association of the a//3 heterodimers into large cyclic arrays, e.g. cyclic hexamers, are also given (Fig. 4(A)). The tilt of the CYhelices results in a reciprocal arrangement and packing on the cytoplasmic and periplasmic sides (Figs. 4(A) and 4(B)). Thus a system of specific polypeptide interactions (polar residues: hydrogen bonds) arises on the cytoplasmic side and a system of energy transfer (BChl-BChl interactions) arises on the periplasmic side. It is interesting to note that in the two-dimensional energy transfer system on the periplasmic side two BChl-BChl interactions are possible: (1) BChl WBChl p interactions for cyclic energy transfer within the hexamer and (2) BChl P_BChl p

Fig. 4. (A) Possible helix-helix interaction sites between the tilted helices of the (z- and ##antenna polypeptides in the cyclic trimer or hexamer. I,, II,, III or Iu, II”, IV: possible interaction sites on the periplasmic or cytoplasmic side respectively (fig. l(b), ref. 74). (B) Model of the possible cyclic hexamer (a&) structure with tilted (Yhelices (fig. l(c), ref. 74). (C) Basic structural and functional units: two hexamers or three hexamers connected by two or three a& basic units (fig. 2(b), ref. 74).

131

interactions for energy transfer between cyclic hexamers (Fig. 5(I)). Both interactions may be important in the specific association processes for the formation of the cyclic antenna complexes and the entire antenna. The organization of the BChl molecules in specific clusters, and thus of the specific polypeptides and polypeptide aggregates of the antenna system, is determined by function, i.e. the ultimate purpose is to form a heterogeneous excitation energy transfer system towards the reaction centre. The smallest cyclic aggregates of the heterodimers are cyclic trimers (Y& and cyclic hexamers (Y& (Fig. 4(A)). From the “reservoir” of optimally packed and interacting antenna complexes around the reaction centres and in agreement with biochemical and biophysical data, the cyclic hexamer fulfils all criteria (Fig. 4(B), Fig. 5(I)). In this antenna system, the cyclic hexamers and the contact regions between the cyclic hexamers are optimally packed. These contact regions are characterized by LY& (polypeptide or BChl) units connecting two hexamers (Fig. 4(C)). These are repeating basic structural and functional units (microdomains) coupling the (Yhelices of two cyclic hexamers and two a/p BChl pairs via two BChl p molecules. In the “reservoir” of the entire antenna a maximum of three cyclic hexamers are in contact via three cY&units (trimer of hexamers, Figs. 4(C) and 5(I)). This arrangement may also include the reaction centre which functionally couples the BChl /3 molecule with the BChl of the special pair (see below). Through these cv& BChl units cyclic energy transfer (via the heterodimers) is converted to energy transfer between the hexamers and then to directed energy transfer to the reaction centre. 4.4. Three-dimensional Structural and functional reaction centre

structure of the core and peripheral antenna complexes. organization of the energy tran$er system directed to the

It is reasonable to assume that the structural and functional principles of the heterodimers (a/p) and C& units arranged in optimally packed cyclic systems exist in both the peripheral and core complexes, particularly since the primary structures of the antenna polypeptides of both complex types are sequence homologous and show similar structural elements. The cyclic hexamer may correspond to the peripheral antenna complexes (e.g. BSOO-850) (Figs. 4(B) and 5(I)). The cyclic peripheral complex would contain six heterodimers (sixfold symmetry, cyclic energy transfer) or three (Y& units (threefold symmetry, energy transfer between the hexamers). X-ray structural data indicate that the observed threefold axis of symmetry of the polypeptide arrangement corresponds to the threefold functional symmetry of the three (r& basic units [24]. In the antenna core complex close to the reaction centre the situation seems to be more complex and less clear. Firstly, there are two possible arrangements (orientations) of the c& basic units. (1) The (Y& units may be organized as in the hexamer of the peripheral complex in which three hexamer units (36 central BChls or polypeptides per reaction centre, Figs. S(IIA) and 6(C)) or two hexamer units (24 central BChls or polypeptides per reaction centre, Fig. S(IIB) or mixed types (Fig. S(IIC)) surround the central reaction centre (hexamer model). Interaction of the BChl p molecule is possible between the antenna complexes and with the special pair of the reaction centre. (2) Six &lZ units may surround the reaction centre and six cuZ& units may interact via BChl /3 (Fig. 6(B)). D irect interaction between BChl a of the (Y& unit and the reaction centre is not possible (tetramer model). Secondly, for the various core complexes various numbers of BChl a molecules per reaction centre (between 21:l and 41:l) have been reported [79]. From the structural point of view the following possible reasons exist for this variability: (1) 36 BChls are

B

C

_

_

._

-

-

Fig. 5. (I) “Reservoir” of cyclic hexamers with six hexamers surrounding a central hexamer optimally packed by specific helix-helix interactions between the hexamers. Basis for the directed energy transfer within and between the hexamers. Circles: n helices of the CI or p polypeptide (periplasmic side). Broken circles: (Y helices of the (Y,, a2 or a.,, (us polypeptides (cytoplasmic side). Bars: BChl molecules (fig. 2(a), ref. 74). ((I) (A) Cyclic arrangement of three hexamer units surrounding a central reaction centre. (B) Cyclic arrangements of six hexamers around a central reaction centre. (C) Mixed type of (A) and (B) (figs. 3(a)-3(c), ref. 74).

A

E

133

Fig. 6. Possible arrangement of the antenna polypeptides of the core complex B1015 (e.g. Rp. viridis) surrounding the reaction centre. The three circles for each (2 or j3 polypeptide represent three cross-sections per tilted helix. (A) Possible basis for the formation of the a& units that surround the reaction centre: substitution of two alp heterodimers by 2X2 y polypeptides. Six cr.&y2 units surrounding the reaction centre associate via BChl /3 (B) or the y polypeptides (-#$ (C) (fig. 4, ref. 74). bound to the central His residue

of 36 (a plus /3) polypeptides (hexamer model); (2) 24 BChls are bound to the central His residue and 12 BChls to the His residue of the /3 chain of 24 (LTplus p) polypeptides (tetramer model). The variability of the number of BChls is due to the variability of losses of BChl during isolation of the core complexes or the variability of the number of hexamers per reaction centre. For antenna complexes which bind BChl a the hexamer model with three hexamer units seems to best fit the size and function of the photoreceptor unit of the core and reaction centre complex: a central reaction centre is surrounded by 12 hexamers and 612 reaction centres. In this photoreceptor unit the excitation energy is trapped by four (1+6/2) reaction centres (Fig. S(IIA)). In the photoreceptor unit of the antenna and reaction centre complex there should be an optimum ratio of the number of BChls ((Y, /3) to the special pair energy trap in order to guarantee an optimum directed energy transfer to the reaction centre. On the other hand, there are structural conditions (limitations) for optimum energy transfer determined by the twofold symmetry of the special pair or the L and M subunits, compared with the sixfold symmetry of the surrounding core complex, and the special pair and the L and M subunits are arranged asymmetrically. In the hexamer model (three hexamer units) (Fig. S(IIA)), the central reaction centre possesses two “entrances” to the special pair for energy transfer from the crlp pair of the antenna, and the six peripheral reaction centres possess only one “entrance” (4 reaction centre). A total of eight “entrances” per four reaction centres exist corresponding to eight contact sites of the antenna hexamers with 8 X 2 = 16 BChls. One-third of the peripheral

134

hexamers with 24 BChls are in contact with the next photoreceptor unit. In this hypothetical photoreceptor unit the core antenna complexes combine 104 BChls (12 x 12 = 144 - 16 - 24 = 104) for directed energy transfer to the reaction centre(s). The structural and functional organization of the BChl b-containing B1015 core complex visible in the electron microscope seems to correspond to both the tetramer (Fig. 6(B)) and the hexamer (Fig. 6(C)) - model with a cyclic arrangement of six a& units or 613 a& units respectively around the reaction centre. This specific structure should be related to the presence of y polypeptides (see also Section 3.2). They may form a cyclic arrangement of six a& units in two ways. (1) Instead of the a/3 heterodimer, two y polypeptides per aI& unit are bound (Fig. 6(A)) which are responsible for the cyclic arrangement of the a& units (Fig. 6(B)). The cyclic core complexes interact via BChl /3 as in the cyclic hexamers of the peripheral complexes. (2) Alternatively, the y polypeptides (Fig. 6(A)) form hexamers ($a) (Fig. 6(C)) which determines the arrangement of the hexamers containing two a& units and two -y2 units. In this way each reaction centre is connected with six a& units (24 BChls). The two models differ particularly with respect to the position of BChl p: model (1) shows strong interaction between the az& units of the core antenna and model (2) indicates possible contacts between BChl p (a/p heterodimer) and the special pair. These differences should reflect corresponding differences in the energy of the transfer system. In photosynthetic bacteria with core and peripheral antenna complexes the entire antenna is formed by the specific association of these complexes. Two different arrangements of the peripheral antenna complex, relative to the core complexes and to the reaction centre, are possible in BChl u-binding antenna complexes: (1) the core and peripheral antenna are separated, and the peripheral antenna complexes surround the core complexes (photoreceptor unit); (2) a mixture of core and peripheral antenna complexes (hexamers) surrounds the reaction centre within the photoreceptor unit. In both cases the hexamer model of antenna complexes is most reasonable.

4.5. Data and eqeriments in agreement with the structural models The bulk of the experimental data are congruous with the models given above of the core and peripheral antenna complexes: (1) the composition and size of the isolated pigment-protein complexes, BChl content, BChl to antenna polypeptide ratio and reaction centre to BChl a (b) ratio [6-8, 70, 80-821; (2) data from cross-linking experiments (arrangements of polypeptides within and between antenna complexes) [83-86]; (3) dissociation and reassociation experiments on antenna polypeptides and antenna complexes [87-901. The model of the peripheral complex fits the X-ray diffraction data [24] and the model of the core complex agrees with the electron microscopy data [39,77,91,92]. The spectroscopic data (UV, CD and IR measurements [lo]) and studies to determine the size and organization of the antenna system, i.e. the cluster of connected BChl molecules between which excitations can be freely transferred (singlet-singlet annihilation) [93-971, are also in accord with the models described. The structural information on antenna complexes or entire antenna systems of purple bacteria is still not sufficient for a detailed theoretical analysis of exciton effects or electronic state energy transfer although the models above fit most spectroscopic data. These theoretical problems, particularly with respect to the exciton effects, are

135

review by Pearlstein [S] comparing discussed in an interesting and comprehensive models described above with the spectra-based model. structure-based

the

Acknowledgments This work was supported by grants from the ETH Unterricht the Swiss National Foundation 31-25179.88.

und Forschung

and

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