The chlorophylls

The chlorophylls are cyclic tetrapyrrole compounds with a magnesium atom chelated at the centre of the ring system. Chlorophylls a and b are derivatives of dihydroporphyrin: their structures are shown in Fig. 8.7a. In the coccoid marine prochlorophyte, Prochlorococcus marinus, chlorophylls a and b are replaced by the divinyl forms, a2 and b2,225,467 in which the ethyl group on ring II is replaced by a vinyl group. Chlorophyll d, which has so far been found only in cyanobacteria in the genus Acaryochloris, has a structure similar to that of chlorophyll a, except that the vinyl group on ring I is replaced by a formyl (-CHO) group. Chlorophylls a, b and d are rendered hydrophobic by the presence of a C20 isoprenoid alcohol, phytol, esterified to the propionic acid residue on ring IV.

Chlorophylls cl and c2 are porphyrins rather than dihydroporphyrins: their structures are shown in Fig. 8.7b. In those algae that possess them, the greater part of the c chlorophyll lacks the phytol group, but in certain species a small proportion is found to be present in the phytylated form.981,439,1499 In addition to containing phytylated chlorophyll c,439 the coccolithophorid, Emiliania huxleyi, has been shown by Garrido et al. (2000) to contain another non-polar form consisting of chlorophyll c2 to which the chloroplast lipid - monogalactosyldiacylglyceride - is attached, most probably by an ester link through the carboxyl group on ring IV. In the picoplanktonic alga, Pelagococcus subviridis (Chrysophyceae), isolated from the East Australian Current, Vesk and Jeffrey (1987) found in addition to chlorophylls a and c2, a new chlorophyll, which they referred to as chlorophyll c3. Jeffrey and Wright (1987) found this pigment also to be present in Emiliania huxleyi, in a proportion approximately equal to that of c2. In chlorophyll c3 the methyl group on ring II of chlorophyll c2 (Fig. 8.7b) is replaced by a methylated carboxyl group (-COOCH3).404

In Micromonas and certain other green marine flagellates in the Prasinophyceae, Ricketts (1966) found that 2 to 9% of the total chlorophyll consisted of Mg 2,4-divinylphaeoporphyrin a5 monomethyl ester, an intermediate in the biosynthesis of chlorophyll a, with a structure similar to chlorophyll c2, differing only in that the acrylic acid group attached to ring IV is reduced to a propionic acid residue (-CH2CH2COOH). Using fluorescence excitation spectra, Brown (1985) was able to show, using one of these algal species, Mantoniella, that there is excitation energy transfer from this pigment to chlorophyll a, indicating that it is a functional part of the antenna for photosynthesis, rather than merely an accumulating biosynthetic intermediate.

All photosynthetic plants contain chlorophyll a (or a2), and most classes of plant contain, in addition, either chlorophyll b (or b2), or one or more of the chlorophyll cs, or (in one known genus so far) chlorophyll d. The distribution of the chlorophylls among the different plant groups is summarized in Table 8.1. Chlorophyll a normally constitutes most of the chlorophyll present.

Among the algae, the chlorophyll a content varies widely. The ranges of concentrations found among the three major pigment classes of the littoral multicellular marine algae off Helgoland were, as a percentage of dry mass: red, 0.09 to 0.44; brown, predominantly 0.17 to 0.55; green, 0.28 to 1.53.350 The amounts per unit area of thallus, in mg dm~2, were: red, 0.5 to 2.8; brown, predominantly 4.3 to 7.6; green, 0.5 to 1.4. So far as the phytoplankton in natural waters are concerned, the chlorophyll a content is at its highest in nutrient-rich waters favouring rapid growth. Steele and Baird (1965) found that in the northern North Sea the ratio of carbon to chlorophyll a in the mixed phytoplankton population was at its lowest value of 20:1 in the spring, after which it increased to a value of about 100:1 in late summer. Assuming the phytoplankton to contain about 37% carbon, and disregarding the silica of the frustules of the diatoms present, these ratios correspond to chlorophyll a contents of about 1.8% and 0.37% of the dry mass, respectively. In the upwelling area off Peru, the average carbon:chlorophyll a ratio of the phytoplankton throughout the euphotic zone was 40 (= chlorophyll —0.9% non-SiO2

Table 8.1 Distribution of chlorophylls among different groups of plants.

Plant group a b c1 c2 c3 d

Pteridophytes, Bryophytes Algae

Euglenophyta + + - - - -Heterokontophyta

a Some c2-containing chrysophytes also contain chlorophyll c3, where it replaces chlorophyll c1.630,1290

b All of 51 diatom species examined by Stauber and Jeffrey (1988) contained chlorophyll c2, and all except 8 also contained chlorophyll c1: where c1 was absent it was usually replaced by chlorophyll c3.

c All haptophytes so far studied contain chlorophyll c2 plus either c1 or c3; some (Prymnesium, Ochrosphaera) contain all three c chlorophylls.1500 d The majority of dinoflagellates (Pyrrophyta), those containing peridinin as the major carotenoid, with one known exception contain only chlorophyll c2: those dinoflagellates in which peridinin is replaced by fucoxanthin contain both c1 and c2.627

e In the unusual cyanophyte, Acaryochloris, discovered by Miyashita et al. (1996), not only is chlorophyll d present, but it is the major photosynthetic pigment, the cellular content being at least 25 times that of chlorophyll a. Previous reports of the presence of small amounts of chlorophyll d in some Rhodophyta are now thought to be due to the presence of colonies of Acaryochloris growing epiphytically on the red algal thalli.967

f The coccoid oceanic prochlorophytes contain the divinyl forms, a2 and b2, instead of the normal forms, of chlorophylls a and b.227,467

dry mass): this population consisted almost entirely of diatoms.828 In the eastern Pacific Ocean, off La Jolla, California, USA, the carbon:chloro-phyll a ratio of the phytoplankton average about 90 (= chlorophyll —0.4% non-SiO2 dry mass) in nutrient-depleted surface waters, and about 30 (—1.2% non-SiO2 dry mass) in deeper, nutrient-rich waters.365

When grown in culture under conditions favouring high pigment content - low light intensity, high nitrogen concentration in the medium -algae usually have chlorophyll levels higher than those observed under natural conditions. Unicellular green algae such as Chlorella commonly have chlorophyll contents in the range 2 to 5% of the dry mass and Euglena gracilis has been observed to contain 3.5% chlorophyll, when grown under these conditions.

The molar ratio of chlorophyll a to chlorophyll b is about 3 in higher plants and in freshwater green algae. Marine species of green algae, both multicellular and unicellular, are characterized by low a:b ratios, in the range 1.0 to 2.3.623,973,1494 Outside the Chloro-phyta, chlorophyll b occurs only in the Euglenophyta and in the prokaryotic Prochlorophyta. In Euglena gracilis, a:b is commonly about 6, and in the Prochlorophyta values (a2:b2) of 1 to 12 have been reported.1356,187,227

In those algae which contain chlorophyll c, this pigment constitutes (on a molar basis) about the same proportion of the total chlorophyll as chlorophyll b does in the green algae and higher plants. In surveys by Jeffrey (1972, 1976) and Jeffrey et al. (1975), the range of values of the molar ratio of a to c (where c = c1 + c2) found for different classes of marine algae were: diatoms, 1.5 to 4.0 (mean, 3.0); peridinin-containing dinoflagellates, 1.6 to 4.4 (mean, 2.3); fucoxanthin-containing dinoflag-ellates, 2.6 to 5.7 (mean, 4.2); chrysomonads, 1.7 to 3.6 (mean, 2.7); cryptophytes, 2.5; and brown algae, 2.0 to 5.5 (mean, 3.6). In the majority of those algae that contained both c1 and c2 they were present in approximately equal amounts: however, ratios (c1:c2) from 2:1 to 1:5 were found. Most of the dinoflagellates and cryptomonads were found to lack chlorophyll c1. The newly defined algal class, Synurophyceae,23 contains c1 but not c2.

The light absorption properties of chlorophyll are interpreted in terms of two excited singlet states (see §3.1) - upper and lower - of the electrons. The absorption spectra of chlorophylls a and b in organic solvent are shown in Fig. 8.8. They each have a strong absorption band (Qy) in the red (corresponding to the lower singlet state) and another stronger band (the Soret band, corresponding to the upper singlet state) in the blue region, together with a number of satellite bands. The lower the wavelength of an absorption band, the higher the energy of the photons that are absorbed, and so the higher the energy level to which the electrons in the molecule are excited. The chlorophyll Qy band corresponds to excitation from the ground state to one of the rotational

Emission Spectrum Fucoxanthin
Fig. 8.8 Absorption spectra of chlorophylls a and b in diethyl ether at a concentration of 10 mg mP1 and 1 cm pathlength. Calculated from data given by French (1960). (Chlorophyll a--; chlorophyll b )

sublevels in the lowest vibrational sublevel of the lower excited singlet state. The adjoining absorption band (Qx) is at a wavelength about 47 nm shorter than the Qy peak, corresponding to an energy difference of about 14 kJ mol-1, similar to the energy spacing between vibrational sublevels. This waveband, at — 615 nm, can thus plausibly be attributed994 to excitation to the first excited vibrational sublevel above the lowest sublevel (Fig. 8.9).

The Soret band corresponds to excitation of the chlorophyll a molecule to the upper singlet state. This is very unstable and decays to the lower excited singlet state in about 10~12 seconds by a radiationless transition (see §3.1). The excited molecule can now revert to the ground state by emission of a photon - the phenomenon of fluorescence (Fig. 8.9). Since the energy change is about the same (give or take some slight difference in rotational energy levels) as that accompanying excitation of a molecule from the ground state to the lower excited singlet state, the main chlorophyll fluorescence peak is in the red (Fig. 8.10), but at a slightly longer wavelength than the Qy absorption band - being centred on 666 nm, compared to 662 nm, in diethyl ether, and 671 nm compared to 665 nm in carbon tetrachloride.174a,1217a Figure 8.10 shows the fluorescence emission spectrum of chlorophyll a in CCl4. There is an additional smaller

Lower excited singlet state

662 nm

Ground state

Vibrational sublevels

615nm

666 nm

728 nm

Vibrational sublevels

Light absorption Fluorescence

Fig. 8.9 Energy level diagram indicating the vibrational sublevels of the ground state and the lower excited singlet state of chlorophyll a (after Nobel, P. S. (1991). Physicochemical and Environmental Plant Physiology. San Diego: Academic Press.). Solid vertical lines indicate absorption of a photon by chlorophyll a dissolved in diethyl ether: dashed lines represent the transitions corresponding to emission of a photon in fluorescence.

peak in the emission spectrum at about 720 to 730 nm, corresponding to photons emitted as the chlorophyll a molecules undergo transition from the lowest vibrational sublevel of the lower excited state to the first excited vibrational sublevel of the ground state994 (Fig. 8.9). The emitted photons are at longer wavelength because the energy change is somewhat smaller.

Absorption is very low, but not zero, in the middle, green, region of the spectrum, hence the green colour of these pigments. As chlorophylls c1 and c2 are metalloporphyrins rather than metallodihydroporphyrins, they have spectra (Fig. 8.11) in which the Soret band is more intense, and the Qy band less intense, than the corresponding bands in chlorophylls a and b. Also, the Qx band (^580 nm) in the c chlorophylls is comparable in intensity to the Qy band (^630 nm). It can be seen from Figs 8.8 and 8.11 that chlorophyll a absorbs only weakly between 450 and 650 nm, and that chlorophylls b or c, when present, have the effect of increasing absorption within this window, at both the long- and the short-wavelength ends.

Chlorophyll Fluorescence Spectrum
Fig. 8.10 Fluorescence emission spectrum of chlorophyll a, dissolved at 10 6M in carbon tetrachloride (redrawn from Fig. 5 in Broyde and Brody, 1967).
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