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Structure and Reactions of Chlorophyll

James Steer

Chlorophyll is a green compound found in leaves and green stems of plants. Initially, it was assumed that chlorophyll was a single compound but in 1864 Stokes showed by spectroscopy that chlorophyll was a mixture. If dried leaves are powdered and digested with ethanol, after concentration of the solvent, 'crystalline' chlorophyll is obtained, but if ether or aqueous acetone is used instead of ethanol, the product is 'amorphous' chlorophyll. In 1912, Willstatter et al. (1) showed that chlorophyll was a mixture of two compounds, chlorophyll-a and chlorophyll-b:

Chlorophyll-a (C55H72MgN4O5, mol. wt.: 893.49). The methyl group marked with an asterisk is replaced by an aldehyde in chlorophyll-b (C55H70MgN4O6, mol. wt.: 906.51).

The two components were separated by shaking a light petroleum solution of chlorophyll with aqueous methanol: chlorophyll-aremains in the light petroleum but chlorophyll-b is transferred into the aqueous methanol. Cholorophyll-a is a bluish-black solid and cholorophyll-b is a dark green solid, both giving a green solution in organic solutions. In natural chlorophyll there is a ratio of 3 to 1 (of a to b) of the two components.

In this process the energy absorbed by chlorophyll transforms carbon dioxide and water into carbohydrates and oxygen: CO2 + H2O (CH2O) + O2 Note: CH2O is the empirical formula of carbohydrates. Due to the green colour of chlorophyll. shown in fig. waxes and confectionary. It is capable of channelling the energy of sunlight into chemical energy through the process of photosynthesis. (2) Because of these absorbencies the light it reflects and transmits appears green.The uv/visible adsorption spectrum for chlorophyll. is in nature. It is used in colouring soaps. . The chemical energy stored by photosynthesis in carbohydrates drives biochemical reactions in nearly all living organisms. however.The intense green colour of chlorophyll is due to its strong absorbencies in the red and blue regions of the spectrum. 1. oils. Chlorophyll's most important use. Fig. 1 . in photosynthesis. it has many uses as dyes and pigments.

Hydrolysis of this (reverse of esterification) splits off phytol and gives phaeophorbide-a. This is the end of a process which starts with the removal of an electron from water. chlorophyll is at the centre of the photosynthetic oxidation-reduction reaction between carbon dioxide and water. Thus. In this higher energy state. This starts a chain of electron-transfer steps. Chlorophyll can also be reacted with a base which yields a series of phyllins. phaeophytin-a. magnesium porphyrin compounds. Chlorophyll assists this transfer as when chlorophyll absorbs light energy. Simple reactions of chlorophyll Treatment of cholorophyll-a with acid removes the magnesium ion replacing it with two hydrogen atoms giving an olive-brown solid. Meanwhile. an electron in chlorophyll is excited from a lower energy state to a higher energy state. that is carbon dioxide is reduced by water. .In the photosynthetic reaction electrons are transferred from water to carbon dioxide. this electron is more readily transferred to another molecule. Treatment of phyllins with acid gives porphyrins. Similar compounds are obtained if chlorophyll-b is used. the chlorophyll which gave up an electron can accept an electron from another molecule. Overall reaction scheme for the hydrolysis of chlorophyll. which ends with an electron being transferred to carbon dioxide.

Photosystem I . detergents are used to break down the membrane into fragments. 2 .Fig. for example. and these fragments are further broken down by the use of different detergents. One such detergent that is very commonly used is SDS-PAGE (sodium dodecyl sulfate-polyacrylamide). chlorophyll-a binding proteins are referred to as CP I. These detergents work by replacing the membrane lipids which surround integral membrane proteins. The detergents work by substituting lipids at different spots in the membrane. This is generally used as it has several advantages over other detergents: the separation can be carried out fairly rapidly and it also gives a good overall picture of the distribution of chlorophyll. Extraction of chlorophyll from plants In plants chlorophyll is associated with specific proteins. The activity and polypeptide composition can then be assayed as the particle is purified. this is also affected by the concentration of the detergent. CP 47 and CP 43. The resulting particles are separated by polyacrylamide gel electrophoresis (a standard biochemical method) in the presence of sufficient detergent to keep them 'solubilised'.Overall scheme for the reaction of alkaline with chlorophyll. With improving biochemical techniques for use on the membrane systems there has been an ever increasing success in the isolation and characterisation of these proteins. Initially.

where 1 dalton=1 a. It has been suggested that this could be explained if there is a pair of electronically interacting chlorophyll-a molecules in the ground (reduced) state (P700).u. Photosystem II . had a high apparent molecular weight and contained only chlorophyll-a. The nature of the reaction centre of chlorophyll. 8. Initial experiments done by Ogawa et al. and that the unpaired electron of the P700+ (oxidised) state is localised on only one of the chlorophyll's (11). 9. P700. 10) and a beta-carotene to P700 ratio of ~8. 7. CP I. and are thought to be responsible for the 721nm fluorescence emission maximum (12. 13). the reaction centre P700 chlorophyll-a protein. This figure shows a schematic representation of the major subfractions that can be isolated from thylakoid membranes.Fig. called Subunits II-VII. One of these complexes. PS I-110 also contains 6 to 8 polypeptides of lower molecular weight (8 to 25kDa. CP I is the most stable of the complexes and retained the photochemical activity of P700.) that do not bind to chlorophyll. The other 40 to 50 chlorophyll-a molecules of CP I act as antennas.Fig. 2 . light-harvesting complex) (3).(4) and Thornber (5) isolated two complexes by SDS-PAGE from SDS-solubilised membranes.m. These particles contain two chlorophyll-protein complexes: the reaction centre chlorophyll-a protein (CP I) and a chlorophyll a+b complex (LHC I. 3 . the reaction centre of chlorophyll in PS I. as there is conflicting evidence.Photosystem I showing the constituents of PS I-110 particles. can be isolated from any of these mixtures by treatment with SDS (sodium dodecyl sulfate) or LiDS (lithium dodecyl sulfate) followed by electrophoresis. CP I. It has a chlorophyll to P700 ratio of ~45 (6. In PS I (photosystem I) an initial solubilisation produces large particles (called PS I-110). is still unknown.

17). but are free from chlorophyll a+b complexes. probably lipid depleted (14. These core particles contain two reaction chlorophyll-a proteins. P680 is currently considered to be a chlorophyll-a dimer. These particles are large pieces of granal membranes. 16. the reaction centre chlorophyll of PS II. 15. These complexes are now named CP 47 and CP 43. Improved extraction procedures gave oxygen evolving PS II (photosystem II) particles (BBY's). P680. where P700 is clearly localised on the green complex CP I. In 1977.charge separation decays within a nanosecond. D2). It was then discovered that CPa was really two complexes: by solubilising thylakoid membranes with octyl gluside (a detergent). 3 .Photosystem II showing the constituents of BBY particles. at least in the ground state. Core particles which retain manganese have been successfully isolated with the two chlorophyll-a proteins and a limited number of other polypeptides. CP 47 and CP 43 can be purified from the other components of PS II by the use of SDS-PAGE or HPLC (high performance liquid chromatography) but they have no photochemical activity in isolation. Other detergent treatments have been employed to isolate the core particles from PS II. . a minor chlorophyll-a complex was detected by SDS-PAGE. is difficult to determine because the P680+ Pheo. The PS II reaction centre is significantly more complex than the reaction centre of PS I. 19) demonstrated the presence of the two complexes. Camm and Green (18.Fig. CP 47 and CP 43 and several non chlorophyll binding polypeptides (D1. The complex was rather unstable a contained a much lower percentage chlorophyll than CP I and was named CPa.

In the red blood cells of vertebrates. Vitamin B12 contains a cobalt ion at the centre of the porphyrin.Porphyrin derivatives Chlorophyll is essentially two parts: a substituted porphyrin ring and phytol (the long carbon chain). There are many examples of this including heme and vitamin B12. gills. It is essential to digestion and nutritional absorption in animals. Heme consists of a porphyrin similar to that in chlorophyll but with an iron(II) ion in the centre of the porphyrin. heme is bound to proteins forming hemoglobin. myoglobin. A porphyrin ring co-ordinating a copper ion (20. . the name given to hemoglobin in muscles. stores oxygen as an electron source for energyreleasing oxidation-reduction reactions. Another example is a co-ordinated iron species in which the porphyrin ring is also substituted: Octamethyltetrabenzoporphyrineisen (II) (21. Like heme. Beilstein registry number: 1168401). Some other examples of porphyrin derivatives are shown below. Hemoglobin combines with oxygen in the lungs. or other respiratory surfaces and releases it in the tissues. vitamin B12 is bright red. Beilstein registry number: 1203779). The porphyrin ring is an excellent chelating ligand. with the four nitrogen atoms binding strongly to a co-ordinated metal atom in a square planar arrangement. In muscle cells. Heme is bright red. .uk/local/projects/steer/chloro.ic.http://www.