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Photosynthetic Pigments

There are various pigments which take part in the photosynthetic reactions. These pigments are found in the leaves and in other parts of the photosynthetic plants and other organisms. Of these the most important are the chlorophylls. These pigments occur universally in the plantkingdom, in photosynthetic protests, and in virtually all photosynthetic bacteria, except in holobacteria. In higher plants, there are two major forms of chlorophylls, chlorophyll a and chlorophyll b, which differ slightly in structure. Both contain a complex ring system called porphyrin, a lengthy hydrocarbon 'tail' and a central magnesium atom. (Haeme which is found in haemoglobin is also a porphyrin.)

Chlorophyll also occurs in photosynthetic bacteria. The only one type of chlorophyll which these organisms possess is called bacteriochlorophyll. The photosynthetic activity of these organisms, however, does not result in the evolution of oxygen.

Structure of Chlorophyll

Chlorophyll absorbs light from the blue and red regions of the visible spectrum. In case the chlorophylls were the only pigments of the chloroplast involved in photosynthesis, much of the visible spectrum would go unused. However there are other pigments called accessory pigments in photosynthetic organisms which absorb light between the red and blue regions of the visible spectrum. Carotenoids are one such group of pigments which absorb light in the blue and blue-green regions and appear rich yellow in colour. The phycobilins (phycocyanin and phycoerythrin) are found in red and blue-green algae. These pigments absorb variously in the yellow-green, yellow and orange regions. Such accessory pigments along with chlorophyll constitute the energy absorbing devices for photosynthesis. Although accessory pigments absorb light energy and supplement the function of chlorophyll, the light energy absorbed by accessory pigments must, first, be passed to chlorophyll before it can be used to do photochemical work. Carotenoids also protect chlorophylls from undergoing degradation through oxidation, particularly at high light intensities.

The Spectrum of Electromagnetic Radiation

Absorption and Action Spectra

When the amount of light absorbed by a pigment is plotted as a function of wavelength, we obtain what is called the absorption spectrum. The absorption spectrum of chlorophyll is shown in the figure below. The actual rate of photosynthesis is measured in terms of oxygen evolution or carbon dioxide utilisation. If these data are plotted as a function of wavelength, we get what is called the efficiency spectrum or action spectrum. These two spectra show close correspondence indicating that chlorophyll is the main light-capturing molecule in most of the higher plants.

Absorption Spectrum and Efficiency Spectrum of a Chlorophyll

Mechanism of Photosynthesis

Photosynthesis, as mentioned earlier, is a complex process involving many steps. By this process the chlorophyll molecules trap the energy of the sunlight and this energy is utilised for the synthesis of organic compounds (sugars) using carbon dioxide and water as inorganic ingredients. Oxygen gas is evolved as a byproduct of the process. The photosynthetic reactions can be grouped broadly into two categories: (1) Light reactions which are chemical processes occur only in the presence of light. In these reactions the light energy is absorbed and used by chlorophyll. (2) In dark reactions carbon dioxide is fixed and reduced by thermochemical mechanisms, resulting in the formation of carbohydrates. These reactions are not directly dependent on light.

Both the light and dark reactions occur within the chloroplast, but in different parts of the organelle. The rate of each set of reactions is dependent on the other, and in the dark, neither process can occur, although only light reactions contain steps directly requiring light.

Light Reactions (Photochemical Reactions)

Robert Hill, in 1983, showed that leaves found in the water to which hydrogen acceptors were added (e.g. quinone), give off oxygen when exposed to light. These reactions did not result in the synthesis of carbohydrates. The main steps in the initial reactions of photosynthesis, which are generally called Hill reactions, involve
  1. Absorption of light quantum by the green pigment chlorophyll,
  2. Transfer of this energy by the transfer of an electron which reduces a cofactor (NADP+), and
  3. Utilisation of this energy in an electron transfer chain to form ATP from ADP (photophosphorylation).
Essentially these reactions involve the utilisation of light energy and water by green plants resulting in the production of ATP, reduced coenzyme (NADPH) and oxygen. The ATP and NADPH are utilised in the dark reactions for the synthesis of sugar. The various steps in the light reactions of photosynthesis can be detailed as shown below:

The Excitation of Chlorophyll and Photoreduction

In order to understand how light energy is absorbed by chlorophyll and is converted to chemical energy we have to see the behaviour of molecules under illumination. Pure chlorophyll isolated from leaves goes into an excited state when it is illuminated. The excited chlorophyll molecules in a test tube simply return to their ground state and lose the absorbed light energy as fluorescence and heat. In intact leaf-cells, however, chlorophyll molecules behave quite differently on illumination. They do not lose their energy of excitation by simple fluorescence, but they behave much like a photoelectric cell. When intact plant cells are illuminated, high energy electrons leave the excited chlorophyll molecule and are led away from it by a chain of electron carrier enzymes very similar to those which participate in electron transport in the mitochondria during cellular respiration. The electrons are carriers of energy and when they flow downhill (from high energy level to a low energy level) from an electron carrier to another, they release their energy. The electron acceptor gets reduced in the process.

Excitation of a Molecule and the Loss of Energy on its Return to Ground State

As mentioned earlier, in the late 1930's, Robert Hill made the discovery that isolated chloroplasts could, when illuminated, reduce various artificial electron acceptors such as ferricyanide, or certain dyes with simultaneous production of oxygen. This discovery led to the identification of a number of electron carriers that function to transport electrons away from excited chlorophyll. It was found the oxidised forms of NAD and a closely related electron acceptor, namely nicotinamide adenine dinucleotide phosphate (NADP) accept electrons when chloroplasts are illuminated.

Nicotinamide Adenine Dinucleotide Phosphate

NAD is the specific electron for those dehydrogenase enzymes that are normally concerned with passing electrons to molecular oxygen, as occurs in cellular respiration. NADP on the other hand is specific for dehydrogenases that function to provide electrons for the reduction of organic substances. As we shall see later, the reduced NADP (NADP red) is the most important and most immediate reducing agent required for reduction of carbon dioxide to sugar, in the dark reactions of photosynthesis.

Non-cyclic Electron Flow

The question that naturally arises is - how is NADP reduced by excitation of chlorophyll? There is a chain of electron carriers leading from chlorophyll molecules to NADP in chloroplasts. The chain of these electron carriers is shown in the figure. When chlorophyll molecules are excited by light, electrons are 'boosted' to a high energy level and they leave the chlorophyll molecules and pass to another pigment called P 700 (P 700 itself is a specialized chlorophyll molecule which acts as a 'tap' from the complex of light absorbing molecules consisting of chlorophyll a and carotenoids). These electrons are then passed on to a specialised electron carrying protein called ferredoxin. Ferredoxin transfers electrons to NADP through a flavo-protein electron carrier, ferredoxin-NADP oxidoreductase. The light induced flow of electrons from chlorophyll to NADP is called non-cyclic electron flow which will continue until all available NADP is reduced. Noncyclic electron flow thus proceeds with the accumulation of reduced product namely NADPH or NADP red.

Cyclic Electron Flow

There is another kind of light induced electron transport from chlorophyll molecules which is called cyclic electron flow. This type of electron flow takes place when chloroplasts are illuminated in the absence of NADP as the electron acceptor. In cyclic electron flow electrons leave excited chlorophyll molecules, on illumination, pass along a circular chain of election carriers and then return to the chlorophyll molecule once again. There is no accumulation of reduced products during cyclic electron flow. This circular chain of electron carriers includes some of the electron carriers used in non-cyclic electron flow. In addition it also contains two cytochromes: cytochrome b and cytochrome f.


Cyclic Electron Flow and Photo Phosphorylation


The cyclic light-induced flow of electrons from the chlorophyll molecule results in the formation of ATP from ADP. The ATP formation occurs in the complete absence of organic substrates and also in the absence of oxygen. It clearly shows that the mechanism is quite different from oxidative phosphorylation that occurs in mitochondria during cellular respiration. However, the phosphorylation of ADP by illuminated chloroplasts is dependent on the intensity of light and the duration of illumination; the longer the chloroplasts are illuminated, the greater the amount of ATP formed. This type of light-induced phosphorylation of ADP to form ATP is called photophosphorylation or photosynthetic phosphorylation.

In the system of photophosphorylation, the only source of energy required to make ATP from ADP and inorganic phosphate, is the radiant energy supplied to the chloroplasts. The excitation of chlorophyll molecules leads to the generation of electrons with very high energy levels. As these electrons return to the chlorophyll molecules once again through the chain of electron carriers, they lose their energy. At one or more points along this chain, there are enzymatic mechanisms, similar to those in mitochondria, which convert the oxidation-reduction energy of electron flow into the phosphate bond energy of ATP. Thus, one of the important purposes of photo-induced cyclic electron flow is to transform the light energy absorbed by the chlorophyll molecules in the chloroplast, into phosphate bond energy. For this reason such phosphorylation is called specifically cyclic photophosphorylation.

Photosystem I and Photosystem II

The illumination of chloroplasts, thus leads to the formation of the two chemical agents that are necessary to carry out the biosynthesis of glucose from carbon dioxide. These chemical agents are reduced NADP red (or NADPH) and ATP.

As we have seen, there are two types of chlorophyll molecules in higher plants that evolve oxygen (chlorophyll a and chlorophyll b). The photosynthetic bacteria which do not evolve oxygen, on the other hand, have only one type of chlorophyll. Chlorophyll a is the characteristic pigment of what is called photosystem I of higher plants. It plays a primary role in photo-phosphorylation and in the reduction of NADP. Chlorophyll b is the characteristic light-absorbing pigment of photosystem II of higher plants. Photosystem II is found only in oxygen evolving photosynthetic cells and it is believed to represent the system that generates oxygen from water. Both these photosystems of higher plants can be excited independently by different wavelengths of light. This is because of the difference in the absorption spectra of chlorophyll a and chlorophyll b.

The popular hypothesis on the interrlationships between photosystem I and II and how they bring about photosphosphorylation, photoreduction and evolution of oxygen (in summary, the overall light reactions) are diagrammatically shown in the figure below. The photosystems I and II are believed to be connected to each other by a chain of electron carriers. This connecting link coordinates photophosphorylation, photoreduction of NADP and evolution of oxygen in chloroplasts.

When chlorophyll a of photosystem I is illuminated, one or more electrons are boosted to a high energy level. These are transferred to the first member of a chain of electron carriers. These electrons then pass via ferredoxin to NADP and get reduced in the process. However, this process of photoreduction of NADP cannot go on for ever, because each chlorophyll a molecule gives up only very few electrons. The release of electrons creates electron 'holes' in the chlorophyll molecules and until these are filled with electrons, the chlorophyll molecule cannot return to its original ground state.

The electrons required to return chlorophyll a of photosystem I to the ground state come from another electron transport chain connecting the photosystem I which is photosystem II. When photosystem II is illuminated chlorophyll b molecules are excited and electrons are boosted to a high energy level. These electrons pass down the connecting chain of carriers losing energy in the process. They finally enter chlorophyll a and bring it back to the ground state. As the electrons flow downhill from photosystem II to the electron holes in chlorophyll a some of their energy is trapped in high energy phosphate bonds of ATP (photophosphorylation). It is believed that there are two energy-conserving sites in the connecting chain.

The Connection between Photosystem I and Photosystem II in Oxygen evolving
Photosynthesis in Plants


The electron holes in chlorophyll b are believed to be filled with electrons arising from water, specifically, from hydroxyl ions. Removal of electrons from hydroxyl ions causes the formation of oxygen:
H2O  H+ + OH-
OH-  O2 +  H+  +  2e+

The reaction by which water undergoes light dependent cleavage is called photolysis. The complete pathway taken by electrons arising from the water molecule is as follows: They pass from water to chlorophyll b; following excitation of chlorophyll b they travel the electron transferring chain connecting photosystem II and photosystem I, then they enter the empty holes in chlorophyll a; from chlorophyll a they are again boosted to high energy levels on illumination and pass to NADP.

Mechanism of Photophosphorylation

The mechanism of ATP formation in the chloroplasts appears to be quite similar by which ATP is generated in the mitochondria during oxidative phosphorylation. In this model (chemosmotic model) there is a pumping of protons (H+ ions) across a membrane resulting in a difference in PH and in electrical charge across the membrane. The electron carriers for photophosphorylation are located in the thylakoid membranes of the chloroplast. These electron carriers are placed in such a way so as to produce a movement of protons into the interior of the thylakoid so that the inside becomes acidic with respect to the outside. This then leads to the passive movement of protons back, out of the thylakoid through protein channels in the membrane. The proteins are ATP synthetases, which are enzymes that catalyse the formation of ATP. These enzymes are activated by the lowered pH produced by the movement of (H+) ions through the channels.

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