CHEMICAL BIOLOGY

Chlorophylls and Carotenoids, Chemistry of

 

Hugo Scheer, Department Biologie I, Botanik, Universitat Munchen, Munchen, Germany

doi: 10.1002/9780470048672.wecb083

 

Chlorophylls and carotenoids are essential pigments of photosynthesis, in which they have complementary functions for capturing light and transducing it to biochemical energy and for protecting against its deleterious effects. Carotenoids are also present in most other organisms where they have, besides photoprotection, a variety of other functions. Typical structures of both types of pigments are reviewed in this article as well as their functions, chemical properties, spectroscopy, biosynthesis, and applications.

 

Photons contain, by biologic standards, relatively high energies (120-300 kJ/mol). In photosynthesis, this energy is captured collectively by hundreds of pigment molecules (chlorophyll, carotenoids, and biliproteins) that are organized in lightharvesting complexes (LHCs). The excitation energy is then funneled with high quantum efficiencies to reaction centers (RCs), where few specialized chlorophylls initiate the charge separation across the photosynthetic membrane. The resulting electrochemical potential is used by a vectorial electron/proton transport chain to generate energy-rich compounds (adenosine triphosphate, reduced nicotinamide adenine dinucleotide) that are used eventually to reduce atmospheric carbon dioxide to carbohydrates (see the section entitled “Photosynthesis”).

Chlorophylls (Chls) and carotenoids (Cars) are the two indispensable pigments of photosynthesis, where they have complementary functions: Chls are key pigments for the productive functions of photosynthesis, which includes light harvesting and primary energy transduction of light into an electrochemical gradient. The combination of intense light absorption and long excited state lifetimes renders them, however, highly phototoxic and potentially damaging. This finding may be the reason that only few examples exist where Chl (derivatives) have been found in nonphotosynthetic tissue and organisms and, generally, in low concentrations. Chls and their precursors are also involved in intracellular communication and regulation. Cars have essential light-protective functions in all photosynthetic organisms and in many nonphotosynthetic organisms. Cars are functionally more diverse, however. In photosynthesis, they can function in a productive way by light harvesting. They contribute to the coloration of flowers, their derivatives are photosensory pigments (rhodopsin), they are involved in the alternative photosynthesis of halobacteria that is based on H+-pumping, they act as hormones in plants (abscisic acid) and animal (retinoic acid), and they act as volatile defensive or attractive principles in plants. This short review is focused on the biochemistry and biophysics of Chls and Cars in photosynthesis, and it will provide a short outline of applications.

In the dynamic natural light environment, the combination of Chls and Cars allows photosynthetic organisms to maintain the balance between efficiently competing for light and damage by an overdose of light. Photosynthesis based on these two pigments has conquered nearly all habitats on earth where light is available, even at very low levels. It has produced the atmospheric oxygen we breathe, and it fixes -5 x 1011 tons of carbon annually. The greening and de-greening of the vegetation in temperate zones, because of the biosynthesis of Chls in spring and their degradation in fall, is probably the most obvious life process on earth, which is visible clearly from outer space (1).

Chls and Cars both absorb light very strongly. In the LHC, these absorptions cover, in combination, most of the visible and the near-infrared spectral range (350-1050 nm). The two groups of pigments differ, however, by more than three orders of magnitudes in their excited state lifetimes: Those of the Chls live for several nanoseconds (10-9 s), and those of most Cars live for only few picoseconds (10-12 s). This finding has major consequences for their potential functions. The short lifetimes of the Cars suffice only for an efficient transfer in the LHC over very short distances, whereas the much longer lifetimes of Chls allow for transfer over tens of nanometers. Only the latter are also involved in charge separation. However, the short lifetimes of Cars with relatively excited state energies are favorable for light protection, where Cars are indispensable. Whenever, under varying light (cloud cover, leaf movements) and physiologic conditions (water, temperature), the energy cannot be transformed rapidly in a productive way; high-energy side-products can be formed that can lead to severe damage sunburn. Cars are involved in minimizing this damage at several levels. Excess energy is drained from the Chls and converted into heat, triplet states of Chls are quenched, and reactive oxygen species (ROS) are quenched as well, in particular singlet oxygen. The widespread occurrence of photosynthesis in many habitats and its evolution over more than 3 x 109 years led to considerable variations of the photosynthetic apparatus, which includes the pigments. The variety within the two pigment classes and the principally similar properties of the different pigments within each of the two groups will be reviewed.

 

Chlorophylls

Structures

The basic structure of Chls is the tetrapyrrolic porphyrin macrocycle with an attached isocyclic ring (Fig. 1). This structure has been termed “phytoporphyrin” by the IUPAC commission (2). The four nitrogens bind a magnesium ion (Mg2+) as central metal. In rare cases, it is replaced by Zn2+, and it is missing in the pheophytins that are present in type II reaction centers. Three spectrally distinct types of Chls can be distinguished by the degree of unsaturation of the macrocycle; they are shown in Fig. 1. It is fully unsaturated in the phytoporphyrin type Chls, hydrogenated in ring D in the phytochlorins, and hydrogenated in ring B in the phytobacteriochlorins. The red-most absorption band (Qx) becomes increasingly intense and red-shifted with increasing saturation (Fig. 2). Green plants, green algae, cyanobacteria, and green photosynthetic bacteria contain mainly chlorin-type Chls. Bacteriochlorin-type Chls abound in the purple photosynthetic bacteria and heliobacteria, and the porphyrin-type Chls is found in brown algae and dinoflagellates (Table 1). The Chls vary even more in their peripheral substituents (Fig. 1). In most Chls, the C-17 propionic acid side chain is esterified by a long-chain alcohol, which is mainly the C20 terpenoid alcohol, phytol. Notable exceptions are the c-type Chls that are abundant (e.g., in brown algae) because they mostly carry a free acrylic side chain. Commonly varied substituents are those at C-3 and C-7, and particularly large structural variations are found among the BChls c, d, and e in green bacteria.

 

 

Figure 1. Chlorophyll structures: The three basic types are shown on the top, with the IUPAC numbering of the atoms and the ligands to the central metal given for the central phytochlorin system. (a) Only sometimes esterified and (b) acrylic side chain at C-17 (171 -172 double bond). See the internet version for color coding.

 

 

Figure 2. Type spectra of phytoporphyrin, phytochlorin, and phytobacteriochlorin-type Chls. See the internet version for color coding.

 

Occurrence and Functions

The occurrence and function(s) of the different Chls are summarized in Table 1. In combination, Chls absorb light across most of the spectral range from 350 to 1020 nm, with a gap only in the region from 500 to 600 nm where Cars and biliproteins add to light harvesting. However, no single organism contains all types of Chls; therefore, the pigmentation is an important ecologic and taxonomic criterion. Depending on their pigmentation, different organisms can occupy different ecologic niches as defined by the light quality, quantity, and its variations in time and space. The anoxygenic photosynthetic bacteria have specialized for near-infrared (NIR) light. Green bacteria that contain bacteriochlorophylls (BChls) c, d, and e harvest light in the 700-800 nm range, bacteria that contain BChls a, b, or g harvest light mainly in the range from 800-900 nm, with extremes such as Blastochloris viridis that reach 1020 nm. Oxygenic phototrophs, viz. organisms that generate oxygen from water, can only use light <730 nm. Probably because of the more demanding energetics of water splitting, their reaction centers contain phytochlorin-type Chls that absorb at wavelengths <710 nm. Therefore, their light-harvesting systems also need to absorb at higher energies. Plants, red and green algae, and cyanobacteria contain Chls a and b, few cyanobacteria contain Chl d. Brown algae and dinoflagellates contain, in addition, Chls of the c-type. They absorb in the “green gap” where all other Chls absorb only weakly (Fig. 2) and they have particularly intense absorption bands in the blue spectral region where light in clear waters prevails.

RCs contain few, more specialized Chls (Fig. 1 and Table 1, see References 3 and 4). They form a chain of pigments across the membrane, over which in a stepwise fashion electrons are transferred from the primary donor, which is a pair of Chls located on the periplasmic side of the membrane, to the acceptors on the cytoplasmic side. Two types of reaction centers can be distinguished. In type I, the primary donor is a heterodimer composed of one molecule of Chls a or d (oxygenic photosynthesis) or BChl g (anoxygenic photosynthesis), and one molecule of the respective C-132-epimer (Chla’, d’, BChl g’). The electron acceptors of the type I-RC are Chls a (or d) in oxygenic photosynthesis and Chls like 82-hydroxy-Chl a in anoxygenic photosynthesis. In type II reaction centers, the primary donor is Chl a or d (oxygenic photosynthesis), or BChl a or b (anoxygenic photosynthesis): The same pigments also act as primary acceptors, whereas the Mg-free pheophytin a and bacteriopheophytin a (or b), respectively, act as secondary acceptors.

Because of their long excited-state lifetimes, chlorophyll derivatives are rare outside phototrophic organisms. However, some chlorophyll-like pigments perform other functions in nature: Certain deep-sea fish use Chls as visual pigments; in the marine worm, Bonella viridis, a chlorophyll-derivative acts as a sex determinant; and in certain tunicates, the tunichlorins may be involved in nonphotosynthetic electron transport (5).

 

Spectroscopy

The spectral properties of Chls are described by the four-orbital model (6-8). It predicts four major absorptions termed Qy, Qx, By, and Bx, in the near ultraviolet (NUV), visible (Vis), and NIR spectral regions that are often accompanied by vibrational side bands at higher energies of the 0-0 transition. The band intensities vary among the Chls, however, and they partly overlap so that the four bands are observed only in the bacteriochlorin structures, viz. BChls a, b, g. The type of spectrum is determined mainly by the degree of unsaturation of the tetrapyrrole macrocycle (Fig. 2): In the fully unsaturated Chl c that contains a phytoporphyrin macrocyclic system, the B-bands around 400 nm are very intense and overlap, the Qy-band around 620 nm is weak, and Qx-band is even weaker and discernible only with special techniques. In phytochlorin-type Chls (Chl a, b, d, BChl c, d, e), the B-bands are reduced in intensity and still overlap, the Qy -band is increased to nearly equal intensity and red-shifted to ~660nm, and the Qx-band is only weak. In the phytobacteriochlorin-type Chls (BChl a, b, g), the B-bands are blue-shifted to <400 nm and are well separated, the Qy-band is even more increased in intensity and red-shifted to >750 nm, and the Qx-band has gained intensity and becomes clearly visible around 570 nm. By these spectral characteristics, the chlorophyll type of any given pigment can be determined readily from the spectra and other details can be detected, such as substitution pattern, ligation to the central metal (e.g., by the protein), or aggregation (see below).

Chls dissolved in organic solvents have long-lived excited states (10-8 s). Therefore, they are highly fluorescent and have significant intersystem crossing to the triplet state, which generates phosphorescence and, under aerobic conditions can generate highly toxic ROS like singlet oxygen. Chlorophyll solutions bleach rapidly by attack of the pigment by these ROS, and photooxidize cosolutes. Excess Chls or precursor porphyrin are phototoxic, their biosynthesis is controlled tightly; and deregulation is a way of herbicidal action (9-11). Chlorophylls injected into animals lead to severe “sunburn” and destruction of tissue. This effect is used in photodynamic therapy of cancer and other diseases (PDT) (12, 13).

 

Table 1. Occurence and functions of chlorophylls

 

A/a, antenna pigment; R/r, reaction center pigment. Major pigments bold caps, minor pigments in lowercase, and pigments present in few species in italics.

 

Chls aggregate readily both in nonpolar solvents and in water. Aggregates have generally red-shifted absorption of the Qy-band, and the excited state lifetimes (and thereby fluorescence and phosphorescence) are reduced drastically because of rapid conversion of the excitation energy into heat by internal conversion (IC). Formation of such aggregates is probably responsible for “concentration quenching.” In both cases, one mechanism of quenching seems to be the presence of “traps,” such as radicals. In large aggregates in which the excitation is highly delocalized over many pigment molecules, this mechanism leads generally to deexcitation that becomes more efficient with aggregate size. Exceptions are the aggregates of BChls c, d, and e present in chlorosomes of green and brown bacteria, which are fluorescent.

Aggregation is also observed in photosynthetic pigment-protein complexes, where it may be considered a major organizing force. In addition to the red-shifted absorption, an additional red shift exists of all bands and is brought about by the protein environment. Both bands are most pronounced in the phytobacteriochlorin-type BChls. In photosynthetic systems, these aggregates show much less IC. In the antenna systems, an important function of the protein is to block (and control) deexcitation channels of Chl-aggregates in a manner still unresolved mechanistically. Isolated LHCs, which contain most (≥99%) Chls of the photosynthetic apparatus therefore show high fluorescence. Light-harvesting Chls are coupled to those in the RC, such that now the excitation energy of the former is transferred efficiently to the latter. The RCs show only little fluorescence at moderate light intensities, here “photochemical quenching” by charge transfer across the membrane is the major process that is initiated by electron ejection from a Chl dimer with quantum efficiencies near 100%. Additional transfers lead to a charge separation across the photosynthetic membrane, which is otherwise an insulator, and creates a long-lived membrane potential that drives all subsequent dark reactions. Therefore, the amount of protein per pigment and the energy required for synthesis is much less in LHC than in RC. For a certain number of photosynthetic pigments, the coupling of LHC to RC in the photosynthetic apparatus reduces the biosynthetic expense considerably. Moreover, different numbers and types of LHC allow for specializations among the organisms to specific light conditions and for spatial and temporal adaptations in a highly variable light environment. High-resolution crystal structures are available of both RC and LHC from nonoxygenic and from oxygenic organisms. In combination with ultrafast time-resolved spectroscopy, site directed mutagenesis, and selective pigment modifications, these structures have greatly advanced our understanding on how this process works.

 

Chemical Properties

Chlorophylls are a subgroup of the cyclic tetrapyrroles. They are large, relatively rigid aromatic molecules. They are planar, but deviations from planarity are observed for most Chls in photosynthetic proteins. The four central nitrogens can bind a variety of central metals. The stabilities of these metal complexes vary widely. The Mg++ characteristic of Chls is lost rapidly when they are treated with mild acids, and is readily replaced by other, more stable metals like Zn, Ni, Ag, and Cu. In contrast, complexes with Ni++ are so stable that they can be demetalated only by destroying the macrocycle.

Several functional groups of Chls are reactive. The allylic long-chain esterifying alcohol, phytol, is readily hydrolyzed. Under alkaline conditions, or with bases, the isocyclic ring is modified extensively, and it is often opened. The macrocycle can be opened (photo)chemically at the methine bridges, and it is cleaved enzymatically at C-5 (14,15) during Chl degradation.

Many metals, including Mg++, have still free valences after binding to the tetrapyrrole. This finding is important for the aggregation of Chls in nonpolar solvents, in which other Chls are bound via peripheral C=O or OH groups. A particular type of aggregation occurs in the BChls c, d, and e. In the LHC of green bacteria, the chlorosomes, coordinating properties of the central Mg++, and the ligation of special peripheral substituents combine to form large aggregates of BChls c, d, and e that are nearly devoid of protein. Free valences of the central Mg++ are also critical for interactions with the proteins in photosynthetic complexes. By coordinating to suitable amino acid side chains (e.g., histidine, methionine, and glutamate) or backbone C=O-groups, they are positioned optimally for efficient energy or electron transfer. Most (B)Chls have a single extra ligand; in this case, two isomers can be formed in which binding occurs to the (inequivalent) upper or lower face of the molecule (see Fig. 1).

The esterifying alcohol, in most cases phytol (Fig. 1), comprises about 1/3 of the mass of Chls, yet its influence on the chemistry (and function) is still poorly understood. With the exception of the nonesterified Chls c, it renders Chls amphotoeric, and is important both in aggregation in polar environments, and in the positioning of Chls in photosynthetic complexes. Variations of the alcohol are frequent in phototrophic bacteria, in particular in the chlorosomes, where they contribute to formation of the fluorescent BChl c, d, and e aggregates that are unique for these pigments (16).

 

Metabolism

The first dedicated intermediate in Chl biosynthesis (17) is 5-aminolevulinic acid (ALA), which is the common precursor of all tetrapyrroles. It can be formed either in a single step from succinyl-CoA and glycine (C4-pathway), or from glutamic acid (C5-pathway) via an intermediate (Glu-tRNAglu) that is generally involved in protein synthesis. Some photosynthetic bacteria (purple bacteria) use the C4-pathway for BChl formation; most other organisms use the C5-pathway. In the second stage, which is ubiquitous for all natural tetrapyrroles, two ALA react to yield a pyrrole (porphobilinogen). Four of those then condense to a linear tetrapyrrole and then cyclize in a remarkable reaction in which one of the pyrroles is flipped, to the cyclic tetrapyrrole skeleton (uroporphyrinogen III). The next stage involves a series of decarboxylations and oxidations to yield protoporphyrin. The latter is the first of a series of increasingly colored and phototoxic products; therefore, the organisms regulate the levels of ALA tightly to avoid any over production of protoporphyrin. Any deregulation of this process, as well as mutations of the enzymes, may result in severe damage and often death (9-11). The next stage, which is dedicated to Chls, is the insertion of Mg, the esterification at the C-13 propionic acid side chain, and its cyclization to yield protochlorophyllide. The biosynthesis of the phytoporphyrin-type Chls c is practically complete at this stage and requires only peripheral modifications. For the phytochlorin-type Chls, ring D is reduced either by the light-independent protochlorophyllide-reductase (DPOR), or by the light-dependent LPOR. In angiosperms, only the latter is present; therefore, they require light for Chl biosynthesis. Subsequent reactions at the periphery, including esterification with an activated long-chain alcohol derived from the isoprenoid pathway, complete the reaction sequence to the phytochlorin-type Chls. In the phytobacteriochlorin-type c BChls, ring B is reduced, too, by an enzyme that is homologous to DPOR, which is followed again by peripheral modifications.

Because of the high phototoxicity of Chls, their degradation is tightly controlled. It has been studied in detail only for Chls a and b, where it involves early on the (light independent) opening of the macrocycle to yield the much less phototoxic open chain tetrapyrroles (bilins), which are modified over several stages and eventually are degraded to monocyclic compounds (14,15). Very little is known on the degradation of the other Chls.

 

Applications

The best source for Chl a is the cyanobacterium Spirulina platensis, which is available commercially. Chl a/b mixtures can be obtained from all green plants. All other Chls are less readily accessible, which limits their applications. Several Chl derivatives are used as dyes for food colorants (Cu-chlorophyllin) and cosmetics. The “chlorophyll” used for the latter is a complex mixture of degradation products. More recently, (B)Chl derivatives have gained increasing interest as photosensitizers in photodynamic therapy of cancer, these compounds include pigments in which the isocyclic ring is opened and/or the central metal has been removed or replaced (e.g., by Pd++) to increase phototoxcicity (12, 13).

 

Carotenoids

Structures

Most Cars are tetraterpenes that contain 6-15 conjugated double bonds. Two C20-units (originally geranyl-geraniol) are joined tail-to-tail to a chain of 32 carbon atoms bearing 8 methyl side-chains (see Reference 18 for nomemclature, and lycopene structure in Fig. 3 for numbering). Sometimes, this basic carbon C40-skeleton is either retained or only modified slightly. However, much more extensive modifications are possible (Fig. 3), including isomerization and rearrangement of the double bonds, cyclization at one or both ends, the introduction of oxygen-containing functional groups and their glycosylation or acylation, and shortening or extension of the carbon skeleton. In addition, a family of triterpenic Cars exists that are generated in a similar fashion but starting from two C15-units (farnesol). In combination, these modifications account for the more than 800 Cars known currently, each of which can form several cis-trans isomers. The number and the structural variations of Cars reflect a variety of functions. The modifications seem to be particularly far-reaching in Cars dedicated to light harvesting, as exemplified by peridinin; a highly modified C37 pigment from algae (Fig. 3).

Furthermore, many carotenoid metabolites exist that have distinct functions. One such example shown in Fig. 3 is retinal, the chromophore of visual pigments (rhodopsins) and the light-driven proton pump, bacteriorhodopsin. Other examples are the plant hormone, abscisic acid, or volatile compounds that contribute to the fragrance of roses, for example.

 

 

 

Figure 3. Selected carotenoid structures from bacteria, algae, plants, and animals, and of precursors and metabolic products with biologic function. The IUPAC numbering is given for lycopene (top right).

 

Occurrence and Functions

Car functions are as diverse as their structures. In contrast to the Chls, they are not defined to photosynthetic organisms but are rather ubiquitous in living organisms. However, animals (including humans) cannot synthesize them but rely on dietary supplies, for example of vitamin A.

The essential and indispensable function of Cars in photosynthesis is the protection of the photosynthetic apparatus. Variations of light quality (color) and intensity in the natural environment often lead to overload of the photosynthetic machinery. Whenever energy transfer in the light-harvesting systems is disturbed, or the RC cannot cope with the input from the LHC, the Cars are the prime protectants from toxic long-lived excited states of the Chls and the subsequently formed ROS (see above). This task is achieved by three major reaction mechanisms. The first is direct photoprotection: Cars have several “forbidden” states that can accept excess singlet excitations from Chls; this energy is subsequently converted rapidly into heat by IC (19). The second mechanism is quenching of Chl triplets by triplet energy transfer to the low-lying Car triplets that can no longer generate ROS. Even if ROS are formed by Chl photosensitization, Cars can detoxify them for example by energy transfer from singlet oxygen or by addition of ROS to the double bond system. All these processes require very close distances between the donor (excited Chls, ROS) and the energy-accepting Car. Therefore, all photosynthetic Chl proteins contain Cars, and they are always in contact with the Chls, as is revealed by several X-ray structures of photosynthetic complexes (20). Specialized Cars are found in the RC and in all Chl-containing LHC. In the latter, the effective Cars in protection seem to be positioned strategically at critical sites where the energy is funneled to the RC.

Because ROS attack is an important defense mechanism against infections in animals, Cars have functions in bacteria that protect them against the immune system. Many bacteria synthesize Cars for their protection, and many virulent forms of Cars are colored deeply by Cars.

Carotenoids also protect simply by their function as nonphototoxic light filters. They remove, by virtue of their high absorption, near ultraviolet and blue light region, and they degrade their excitation energy rapidly to heat by IC. Certain algae tolerant to extreme light stress contain droplets of pure Cars. Many nonphotosynthetic organisms synthesize Cars when subjected to increasing light intensities. As nonphototoxic pigments, Cars often function as “safe” colorants in nature. Examples are colors of flowers used for attracting and for communicating with pollinating animals, mainly insects, or examples of crustaceans that blend into the marine background.

Another function in photosynthetic organisms is light harvesting in the “green gap” (470-600 nm) where Chls absorb only poorly. Because of rapid IC, most Cars transfer energy only with low efficiency to Chls. However, particularly in microalgae and phototrophic bacteria they can contribute prominently to photosynthesis despite their general protective function. Two factors are important to this function. One is the evolution of Cars in which the excited state lifetime is somewhat increased, and therefore IC is reduced. The two most abundant Cars, fucox- anthin and peridinin, have lifetimes that reach 100 ps (21). The second factor is, again, a location of such Cars close enough to Chls that energy transfer becomes sufficiently effective within the short excited state lifetimes (electron exchange mechanism). Plants and some algae use (e.g., in the so-called violaxanthin cycle) subtle structural modifications to manipulate Cars such that light-harvesting Cars are converted into protecting pigments, and vice versa, in response to the light supply and the status of the photosynthetic apparatus (22). The mechanism of this switch is still unclear.

Last are Car precursors for important metabolites. Only three examples shall be given. The first example is retinal (Fig. 3), which is the chromophore of the visual pigment rhodopsin (23) and is derived from β, β’-carotene. Because the latter cannot be synthesized by mammals, they need it to be supplied as provitamin A. Retinal derivatives are also required for other regulatory functions. The second example is abscisic acid (Fig. 3), which is the plant hormone involved in the shedding of leaves in fall and in fruit ripening; it is derived from violaxanthin. Finally, certain fragrances of roses are not synthesized directly, but they are breakdown products of the flowers’ Cars.

 

Spectroscopy

Carotenoids are conjugated linear polyenes with 6-15 double bonds. The optical spectra of such pigments are characterized by some unusual features [Fig. 4, (19)]. First, the lowest energy S0 > Sltransition located in the red to NIR spectral range is “optically forbidden” for conventional one-photon excitation or emission; therefore, the related absorption and fluorescence are extremely weak. Theoretically, this is true only for C2-symmetric polyenes; but in practice, even highly asymmetric Cars like peridinin have negligible Sl absorption. Second, the most intense absorption, which is responsible for the yellow-orange color of most Cars, is a series of closely spaced, sometimes overlapping bands in the 400-550 nm range. They belong to vibrational sub-bands of an S0 > Sn absorption that are generally well resolved, but they can be broadened to a degree that they appear only as a single, broad band. With an increasing number of conjugated double bonds, these absorptions are shifted, in an asymptotic fashion, to the red. Third, at least two other transitions of intermediate energy exist, which are again forbidden and therefore are very weak. Fourth, cis -carotenoids show an additional band that is typically located 100-150 nm to the blue of the main absorption. Last, the triplet states have comparably low energy; in Cars with >7 double bonds it lies below 1250 nm, which is the energy required to generate singlet oxygen. The “forbidden” bands and the triplet state(s) are fundamentally important for the biologic functions of Cars (see above). Car triplets can quench singlet oxygen efficiently by a spin-allowed singlet-triplet exchange reaction. The “forbidden” states are important in singlet energy transfer with other Cars and, in particular, Chls. They can be reached only indirectly (e.g., after absorption into the energy-rich major absorption band and subsequent IC) or by energy transfer from neighboring pigments like Chls, but thereby contribute to energy transfer and dissipation. The fluorescence of Cars is generally negligible even from the optically allowed Sn state because of rapid IC to the lower lying forbidden states. Generally, lifetimes are a few picoseconds, and even in extreme cases they reach only 100 ps, which corresponds to fluorescence yields of ~1%.

When incorporated into proteins, the spectral properties of Cars can be modified considerably. Generally, the bands are sharpened and red-shifted, and they can become strongly optically highly even for achiral Cars because of twisting of the long-chain molecule. The red shifts are in the range of ~10nm, but they can be much larger. Spectacular cases are the color change of astaxanthin from orange to green when it is bound in the crustacean protein, α-crustaxanthin, and its reversion when the protein is denatured by boiling, or the red shift of retinals in rhodopsins.

 

 

Figure 4. Type spectra of carotenoids with well and poorly resolved bands, and of a cis-carotenoid.

 

Chemical Properties

The basic hydrocarbon skeleton of Cars is very hydrophobic, and almost all Cars are insoluble in water and soluble in nonpolar solvents. Solubility is increased by polar functional groups and conjugation to sugars (glycosylation) (Fig. 3), but truly water-soluble Cars are very rare. The conjugated double-bond system is moderately stable chemically. It is subject to rearrangements, in particular in the light, and to additions (e.g., of oxygen). The chemical properties of the highly modified Cars are determined by their particular functional groups, which in some Cars are labile. A frequent example is the epoxy-group present e.g., in violaxanthin, diadinoxanthin, fucoxanthin or peridinin. With the latter two representing >75% of the total Cars. Because of the diversity of structures and substituents, the reader is referred to specialized treatments of carotenoid chemistry (see “Further Reading” section).

 

Metabolism

Carotenoids are terpenoids, which are derived from oligomerization of activated isoprene. It can be synthesized either by the mevalonate pathway from acetyl-coenzyme A, or via the recently discovered deoxyxylulose pathway (24). The latter seems to be the pathway that leads to Cars in most photosynthetic organisms. Both pathways result in two isomers: isopentenyl pyrophosphate and dimethylallyl pyrophosphate. One molecule of the latter is condensed by prenyl transferases in a head-to-tail fashion sequentially with three molecules of isopentenyl pyrophosphate. The resulting geranylgeranyl-pyrophosphate (GGPP) constitutes an important branching point in terpenoid metabolism that leads to Chls via esterification with the tetrapyrrolic chlorophyllides (see above), to longer isoprenoids via additional condensation with isopentenyl pyrophosphate, and in particular to Cars (25), beginning with a tail-to-tail condensation of two molecules of GGPP by the first dedicated enzyme, phytoene synthase. The resulting phytoene is still uncolored because it has only three conjugated double bonds (Fig. 3). Dehydrogenation by phytoene-desaturase via phytofluene to lycopene is common to most known Cars. C30 Cars are derived, in an analogous fashion, from tail-to-tail condensation of two C15-units (farnesol-pyrophosphate) and subsequent dehydrogenations. A variety of enzymes is responsible for additional structural modifications, which includes cyclases to form the end-rings characteristic of many Cars, oxygenases to introduce OH groups which can be additionally modified, isomerases and (de)hydrogenases to modify the double-bond system, and many others, which provide the structural diversity of Cars.

 

Applications

The use of Cars as food colorants is of considerable economic importance. This task can be done directly, but is generally done indirectly by supplying Cars or carotenoid-rich algae as food additives to fish (salmon) or poultry (chicken eggs). In cosmetics, Cars are used as sunscreens. Provitamin A (P-carotene) is a dietary supplement that is given directly but more frequently is given indirectly, for example as milk supplement. Natural sources for Cars are algae like Hematococcus, plants like carrots, or genetically manipulated bacteria. Industrial scale synthetic methods have been developed.

 

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Further Reading

Britton G, Liaaen-Jensen S, Pfander H. Carotenoids. 1995. Birkhauser Verlag, Basel.

Deisenhofer J, Norris Jr. The Photosynthetic Reaction Center. 1993. Academic Press, New York.

Frank HA, Young AJ, Britton G and Cogdell RJ. The Photochemistry of Carotenoids. 1999. Kluwer, Dordrecht.

Green B, Parson W. Light-Harvesting Antennas in Photosynthesis. 2003. Kluwer, Dordrecht.

Grimm B, Porra R, Rudiger W and Scheer H. Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications. 2006. Springer, Dordrecht.

International Carotenoid Society. http://www.carotenoidsociety.org/. Scheer H. Chlorophylls. In: Chlorophylls. 1991. CRC-Press, Boca Raton, FL.

 

See Also

Cancer, Topics in Chemical Biology

Photosynthesis, Electron Transfer Chemistry in

Porphyrin and Corrin Biosynthesis

Spectroscopic Techniques: Overview of Applications in Chemical Biology

Terpenoid Biosynthesis

Vision, Topics in Chemical Biology