Chlorophyllcarotenoidprotein complexes

Essentially all the chlorophyll and most of the carotenoid in chloroplasts occur complexed to protein. There is good reason to suppose that these

Table 8.2 Major chloroplast carotenoids in various algal classes.

Chloro- Xantho- Eustigmato- Bacillario- Chryso- Hapto- Eugleno- Phaeo- Pyrro- Crypto- Rhodo- Cyano- Prochloro-Carotenoid phyta" phyceae phyceae phyceae phyceaeft phyta phyta phyta phyta phyta phyta phyta phyta a-Carotene

Echinenone

Lutein

Zeaxanthin

Neoxanthin

Yaucheriaxanthin

Yiolaxanthin

Heteroxanthin

Fucoxanthin

Diatoxanthin

Diadinoxanthin

Peridinin

Alloxanthin

Myxoxanthophyll

" These data for the Chlorophyta apply to higher plants also. Sublittoral species of green algae usually possess siphonaxanthin or siphonein instead of, or as well as, lutein.1012,1493 Some species in the Prasinophyceae contain prasinoxanthin as their major carotenoid.406'11 b ¿-carotene frequently present; Prymnesiophyceae (Haptophyceae) have similar carotenoid composition to Chrysophyceae. c Predominant carotenoid(s) present. d Predominantly esterified.

c In some dinoflagellates, fucoxanthin (and/or fucoxanthin derivatives such as 19'-hexanoyloxyfucoxanthin) replaces peridinin as the major carotenoid.627

' Carotene predominantly a in Prochlorococcus and /? in Prochloron and Prochlorotlirix.1S7'227'467 In the chlorophyll-d containing cyanophyte, Acaryochloris marina, the major carotenoids are a-carotene and zeaxanthin, present in approximately equimolar amounts.919

Fig. 8.12 Structures of chloroplast carotenoids. (a) b-carotene. (b) Lutein. (c) Siphonaxanthin. (d) Vaucheriaxanthin. (e) Fucoxanthin. (f) Diadino-xanthin. (g) Peridinin. (h) Alloxanthin.
Prasinoxanthin Absorption
Fig. 8.13 Absorption spectrum of b-carotene in petrol at a concentration of 10 mgml_1and pathlength 1 cm.

pigments can function in photosynthesis only as components of specific complexes with protein. Many different chlorophyll/carotenoid-proteins from higher plants and algae have been described in recent years. They have in most cases been liberated from the thylakoid membranes, in which they are normally embedded, with the help of detergents. It seems likely that these are variants on a relatively small number of fundamental pigment-protein types, and we shall treat them accordingly. All such photosynthetic complexes that have been examined so far contain chlorophyll a: some also contain chlorophyll b or c. The great majority of these complexes contain one or more carotenoids as well as chlorophyll. There are usually several chlorophyll molecules per polypeptide together with one, or several, carotenoid molecules. The reviews by Larkum and Barrett (1983), Thornber (1986), Anderson and Barrett (1986), Rowan (1989), Wilhelm (1990), Hiller, Anderson and Larkum (1991), Thornber et al. (1991) and Green and Durnford (1996) may be consulted for further details.

As is discussed later (§8.5), the light reactions of photosynthesis take place within two distinct pigment-protein/electron carrier systems, referred to as photosystems I and II, which contain the components required for charge separation and electron transfer, together with chlorophyll/carotenoid pigment-proteins for collecting light. In addition, associated with each complex there are pigment-proteins that do not carry out any photoreac-tions, but which function simply to harvest photons from the prevailing field and transfer the absorbed energy to the reaction centres.

By analogy with radio or TV antennae, which collect energy from the long-wavelength radio/TV electromagnetic field, photosynthetic pigment molecules are often referred to as the 'antennae' of the photosystems. For each photosystem we distinguish between the internal antenna, made up of those pigment-protein molecules built into the core complex, and the external antenna composed of the light-harvesting pigment-proteins that are more loosely associated with the complex.

Core complex I consists of two 84 kilodalton (kDa) hydrophobic proteins, which, in addition to incorporating P700 and the other electron transfer elements of photosystem I (see below), contain, as its internal antenna, 75 to 100 molecules of chlorophyll a and 12 to 15 molecules of b-carotene, these pigment molecules being distributed between the two proteins. While the core complex I does itself absorb light (in barley chloroplasts, for example, it contains about 20% of the total chlorophyll), it nevertheless has associated with it a light-harvesting pigment-protein complex to supply it with additional energy. This is referred to as light-harvesting complex I (LHC I). In higher plants it contains chlorophylls a and b (a:b ~3.5) and xanthophylls (mainly lutein), bound to at least four protein subunits (24, 21,17 and 11 kDa): in barley chloroplasts it accounts for about 18% of the total chlorophyll. The green flagellate Chlamydomo-nas reinhardtii also has an LHC I, rather similar to the higher plant complex.1476

The siphonalean green alga, Codium, has an LHC I, containing chlorophyll a and b, in which siphonaxanthin is the main carotenoid.231 In the unicellular red alga, Porphyridium cruentum, an LHC I has been found containing chlorophyll a and zeaxanthin.1475 The LHC I from the xantho-phycean alga, Pleurochloris meiringensis, was found to contain, in addition to chlorophyll a, significant amounts of all the accessory pigments (chlorophyll c, diadinoxanthin, vaucheriaxanthin ester, heteroxanthin and diatoxanthin) that are present in this organism.178 Excitation spectra of fluorescence indicated that the accessory pigments were transferring energy to chlorophyll a in the isolated LHC I. Table 8.3 summarizes the rather limited compositional data available on the photosystem I external antenna pigment-proteins of algae.

Algae and higher plants also contain another essential chlorophyll a/b-carotene protein complex, core complex II, which functions in photosystem

Table 8.3 Algal photosystem I light-harvesting pigment-proteins (LHC Is).

Number of pigment molecules per 100 molecules of Chlorophyll"

Apoprotein Chl Chl Chl M. Wt.

Algal group a b c Carotenoid (kDa) Reference

Chlorophyta

Chlorophyceae

Chlamydomonas 100 ~20 -

Bryopsidophyceae Codium 100 ~60 -

Prasinophyceae

Mantoniella 100 113 17

squamataa

Heterokontophyta

Xanthophyceae Pleurochloris 100 - 3 meiringensis

Rhodophyta

Porphyridium 100 - -cruentum

M. Wt. = molecular weight a This pigment-protein serves as the external antenna for both photosystems in Mantoniella.1186

II, the absorbed light energy being transferred to the reaction centre known as P680, which is part of the total complex. It is estimated that there are about 40 chlorophyll a molecules and one P680 reaction centre for each photosystem II unit. Most of the chlorophyll and b-carotene is present in two non-identical but similarly sized pigment-proteins, CPa-1 and CPa-2, also known as CP47 and CP43. CPa-1 is of molecular weight ^52 kDa and contains 20 to 22 molecules of chlorophyll a and 2 to 4 of b-carotene; CPa-2 is ~48 kDa and contains 20 chlorophyll a and 5 b-carotene molecules.508 These pigment-proteins function as the internal antenna of photosystem II and together they account (in barley) for about 10% of the total thylakoid chlorophyll. They are closely associated with the reaction centre part of the complex, which consists of a pair of hydrophobic proteins, D1 and D2,

Xanthophyll + carotene, 6-8

Siphonaxanthin

20-31 (10 76 polypeptides)

Prasinoxanthin 30 20, 22 Violaxanthin 7 Neoxanthin 15

1469,1123

Diadinoxanthin 18 20.7, 16.7 Heteroxanthin 6 Diatoxanthin 3 Vaucheriaxanthin ester 9

Lutein 75

18-23.5

1475

containing four to six chlorophyll a and one to two b-carotene molecules, together with two phaeophytin a, two plastoquinone and one non-haem iron. About 1% of the total thylakoid chlorophyll is contained in these reaction centres. Cytochrome b559 is also part of the complex.

Like core complex I, core complex II, as well as absorbing light directly, is supplied with additional energy by an associated light-harvesting pigment-protein complex, which is referred to as light-harvesting complex II (LHC II). In higher plants LHC II is made up of at least four individual pigment proteins - LHC Ila, b, c and d - all containing chlorophylls a and b, and the three chloroplast xanthophylls, lutein, violaxanthin and neox-anthin. Light-harvesting complex IIb is the major component containing as it does 40 to 45% of the total chloroplast chlorophyll: the other three LHC II pigment-proteins between them account for 10 to 15% of the total chlorophyll. The aggregate LHC II has a chlorophyll a:b ratio of about 1.4; for LHC IIb the ratio is about 1.33. Light-harvesting complex IIb preparations contain three slightly different apoproteins - 28, 27 and 25 kDa - possibly resulting from post-translational modification of a single polypeptide. Each apoprotein molecule is thought to bind about 15 chlorophyll molecules (eight a, six to seven b), and about three xan-thophyll molecules.

Among the various classes of algae, a large number of different light-harvesting pigment-proteins has been found in recent years. Most of these are of the LHC II type, feeding their energy to photosystem II. They all contain chlorophyll a together with, in most cases, whatever accessory chlorophyll - b or c - is present in that particular alga. In addition they contain the major light-harvesting xanthophyll carotenoid(s) characteristic of that algal class. While they all have peaks in the red region of the spectrum, due to chlorophyll(s), their main light absorption, due to a combination of chlorophyll Soret and carotenoid bands, is in the 400 to 550 nm, blue-green waveband. Table 8.4 lists a selection of these putative LHC II algal proteins, with their pigment and polypeptide composition. In Mantoniella squamata, a member of the primitive green algal class, Prasinophyceae, it appears that a single pigment-protein serves as the external antenna for both photosystems.1186

Katoh and Ehara (1990) have presented evidence that in vivo these light-harvesting proteins are organized into supramolecular assemblies. Using the mild detergent, octyl sucrose, they were able to isolate, from chloroplasts of the brown algae Petalonia fascia and Dictyota dichotoma, pigment-protein complexes of molecular weight about 700 kDa, containing in each complex about 128 molecules of chlorophyll a, 27 of

Table 8.4 Algal light-harvesting pigment-proteins (presumptive LHC IIs).

Apoprotein

Chl

Chl

Chl

M. Wt.

Algal group

a

b

c

Carotenoid

(kDa)

Reference

Chlorophyta

Chlorophyceae

Chlamydomonas

100

106

-

Lutein 12

29, 25

673

reinhardtii

Violaxanthin 11 Neoxanthin 6 b-carotene 5

Chlorella fusca

100

88

Lutein 12 Violaxanthin 6 Neoxanthin 6 Trihydroxy-a-carotene 12

30.4, 28

1469

Bryopsidophyceae

Bryopsis

100

116

Siphonaxanthin 51 Siphonein 15 Neoxanthin 28

974

Codium

100

58

25-19

231

Prasinophyceae

Mantoniella

100

113

17

Prasinoxanthin 30

20, 22

1469, 1123

squamataa

Violaxanthin 7 Neoxanthin 15

Euglenophyta

Euglena gracilis

100

49

-

Diadinoxanthin 31

26.5, 28, 26 272

Neoxanthin 9

b-carotene 1

Heterokontophyta

Xanthophyceae

Pleurochloris

100

-

6

Diadinoxanthin 16

21.9

178

meiringensis

Heteroxanthin 9 Diatoxanthin 5 Vaucheriaxanthin ester 13

Eustigmatophyceae

Nannochloropsis

100

26-44 Vaucheriaxanthin

22-25 Neoxanthin 2 b-carotene 4

26

173, 1323

Bacillariophyceae

Phaeodactylum

100

-

40

Fucoxanthin 192

18

412

tricornutum tricornutum

Table 8.4 (cont)

Apoprotein

Chl Chl Chl

M. Wt.

Algal group

abc Carotenoid

(kDa) Reference

Haptophyta

Prymnesiophyceae Pavlova lutherii 100 -Pyrrophyta (Dinophyta)

Amphidinium 100 -

carteraeb Amphidinium 100 -

carterae

21 Fucoxanthin

Haptophyta

Prymnesiophyceae Pavlova lutherii 100 -Pyrrophyta (Dinophyta)

Amphidinium 100 -

carteraeb Amphidinium 100 -

carterae

Phaeophyta

Fucus serratus 100 -Cryptophyta

Chroomonas 100 -

Prochlorophyta

Prochloron 100 42

Prochlorothrix 100 25

21 Fucoxanthin

- Peridinin 450

57 Peridinin 171

Diadinoxanthin 29

16 Fucoxanthin 70 (predominant)

65 Unidentified

(Alloxanthin?)

32 19

30-33

206, 207 610

557 185

M. Wt. = molecular weight a This pigment-protein serves as the external antenna for both photosystems in Mantoniella.1186'1192

b This is the water-soluble peridinin-chlorophyll a complex.

chlorophyll c, 69 of fucoxanthin and 8 of violaxanthin. Electron microscopy showed that each complex was discoidal in shape, being 11.2 nm in diameter and 10.2 nm in height, with a small pit at the centre of the disc.

In photosynthetic pigment-proteins the bonding between the pigments and the protein, although highly specific, is not covalent. The molecular structure of the major chlorophyll a/b light-harvesting protein of higher plants (LHC II) has been determined to 3.4 A resolution by Kuhlbrandt, Wang and Kuniyoshi (1994) using electron crystallography, and to 2.7 A resolution by Liu et al. (2004) using X-ray crystallography. The chlorophylls (8 chlorophyll a and 6 chlorophyll b per protein monomer) are attached to the polypeptide by coordination of the central magnesium atom to polar amino acid side chains (histidine, asparagine, glutamine), or to main-chain carbonyls in the hydrophobic interior of the complex. In addition, there must be hydrogen bonding between oxygen-containing ring substituents in the chlorophyll molecules, and appropriate groups in the polypeptide chains, as well as hydrophobic associations between the phytyl chains and the non-polar regions of the protein. Of the 232 amino acid residues of the polypeptide, 36% form transmembrane helices, the remainder being exposed on the inner (luminal) and outer (stromal) membrane surfaces.

There are four carotenoid molecules per LHC II monomer. The major carotenoid is the xanthophyll, lutein, of which there are two molecules located within the monomer. There is also a neoxanthin molecule. In the case of the carotenoids there are hydrophobic associations between the central hydrocarbon chain region of the carotenoid and non-polar amino acid side chains of the polypeptide. The polar groups at each end of the carotenoid molecule presumably participate in hydrogen bonding with polar groups in the polypeptide. In the case of b-carotene, which has no polar groups, all interactions with the protein must be hydrophobic. The lutein molecules are closely associated with chlorophyll a molecules, to which they can transfer energy, and the neoxanthin is similarly associated with chlorophyll b molecules to which it can transfer its energy. The fourth carotenoid site is at the monomer-monomer interface and is believed to be where one or other of the intermediates of the xanthophyll cycle - violaxanthin, antheraxanthin, zeaxanthin (see §10.2) - is located.

A very important consequence of the interaction between the pigments and the protein is that the absorption spectra of the former are modified. The absorption peaks are, to varying degrees, shifted to longer wavelength, with, in the case of the chlorophylls, some increase in complexity as well. Chlorophyll a in living cells or chloroplasts has a rather broad absorption peak in the red at about 676 nm, a shift of 9 to 15 nm relative to its position in organic solvents (usually 661 to 667 nm). Computer deconvolution of the absorption curve indicates that the peak is made up of four major forms of chlorophyll a with peaks at 662, 670, 677 and 684 nm, together with two minor forms at 692 and 703 nm.411 Isolated chlorophyll-protein complexes show similar complexity: this may possibly be due to exciton interaction between the chlorophyll molecules within each pigment-protein complex, leading to splitting of the absorption bands and/or to the presence of different types of chlorophyllprotein binding.

In the case of some chloroplast carotenoids, such as b-carotene, the absorption peaks in vivo are shifted, relative to their positions in organic solvents, to longer wavelengths to about the same extent as the chlorophyll peak. However, certain major light-harvesting carotenoids in the algae - fucoxanthin, peridinin, siphonaxanthin - have in vivo spectra shifted by about 40 nm to longer wavelengths relative to their spectra in organic solvents. This has the effect of substantially increasing absorption in the green window between the chlorophyll blue and red peaks. The diatoms and the Phaeophyta, for example, are brown in colour rather than green precisely because of their higher absorption in the green waveband. The shift in absorption to longer wavelengths is a consequence of the specific association between the carotenoid and the protein: if this association is disrupted by, say, heat denaturation, then the carotenoid spectrum reverts to the in vitro type. When a piece of brown-algal thallus is dipped in hot water it rapidly turns green. Quite low temperatures will suffice to bring about the change. When chloroplasts, or an isolated pigment-protein (chlorophylls a and c, fucoxanthin, b-carotene) from the brown alga, Hormosira sp., were subjected to a progressive temperature rise of 1°C min-1, dissociation of the fucoxanthin-protein link (measured in terms of decrease in absorbance at 535 nm) was detectable at 35°C and complete at 60 to 65°C, 50% dissociation being achieved at 44 to 48°C.698 One possibility is that the binding between protein and carotenoid is such that the polyene chain of the latter is twisted: apparently this could account for the shift of the absorption band to longer wavelengths.179

Fucoxanthin and peridinin are the dominant carotenoids in those algae that contain them, and show up in the in vivo spectra as a major shoulder in the 500 to 560 nm region (see Fig. 9.4b). Siphonaxanthin does not to the same extent dominate the carotenoid make-up of those algae that possess it, and shows up as a subsidiary peak at about 540 nm in the in vivo spectrum.1493

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