In this article we will discuss about the photochemical and biosynthetic phase of photosynthesis.
Photochemical Phase of Photosynthesis:
The pigment system of the chloroplast first absorbs light energy and then passes it on via intermediates to the products of photosynthesis.
When a chlorophyll molecule absorbs a light quantum (i.e., photon) the molecule is excited; it means that it has been brought from its normal ground state to an excited state (i.e., higher energy level). All quanta are not able to lift chlorophyll to a higher state of energy.
Firstly the light is being absorbed and, therefore, the quantum absorbed possesses a sufficient amount of energy to facilitate this job. The quantum energy may be determined from the wavelength of radiation; the shorter the wavelength greater the energy.
White light, as it comes from the sun is composed of different wavelengths, ranging from the relatively long waves of red light, through successively shorter waves to violet light. Wave length (1) denotes the linear length by which a wave travels forward in one complete vibration.
This is generally shown by units such as millimicron (mµ) or Angstrom (Å)—(1 cm = 10−8 Å). When white light is passed through a glass prism, it is resolved into these colours. The band of colours is the visible spectrum.
The complete visible spectrum is composed of red, orange, yellow, green, blue, indigo and violet colours. Our eyes can perceive only the visible rays whose wave lengths are between 390 nm to 780 nm.
The wavelengths exist that we are unable to perceive with our eyes. Beyond the red are still longer, invisible rays, the infrared; and beyond the violet are shorter, invisible rays, the ultraviolet. This way, the visible rays represent only a part of the radiant energy that comes to the earth from the sun. Here only a part of the visible spectrum is effective in photosynthesis.
Chlorophyll, however, is a pigment that gives green colour to the leaves. It absorbs light in the violet and blue wavelengths, and also in the red region of the visible spectrum of the light. This portion of the spectrum between 400 nm and 700 nm is called photosynthetically active radiation (PAR). As chlorophylls reflect the green light, thus they impart green colour to the leaves.
Transfer of Energy:
Not all of the pigment molecules absorb light or are activated at once. Light energy absorbed by one pigment molecule is thought to be transferred through many other pigment molecules before reaching its site of action.
All the pigments except chlorophyll a are known as accessory pigments. Here the transfer of light energy may be from one chlorophyll a molecule to another, from chlorophyll b to chlorophyll a, from carotenoids to chlorophyll a, or from phycobilins to chlorophyll a.
The energy can be transferred from molecule to molecule as follows—the normal state of molecules is known as ground state or singlet state. When an electron of a molecule or atom absorbs a quantum of light, (light rays consist of tiny particles known as photons. The energy carried by a photon is known as quantum which is represented by hv.)
It is raised to a higher energy level which is called excited second singlet state. This is unstable and has a half-life to 10−12 seconds.
The electron comes to the next higher energy level by the loss of some of its extra energy in the form of heat. This is called as excited first singlet state and is also unstable with a half-life of 10−9 seconds.
From the first singlet state the excited electron may return to the ground state in two ways:
(i) Either losing its remaining extra energy in the form of heat, or
(ii) By losing extra energy in the form of radiant energy.
The latter process is known as fluorescence. The substances which show this property of fluorescence emit fluorescent light only during the period they are exposed to incident light. Secondly, the fluorescent light is of higher wavelength than the incident light. It is because some energy lost during the change of excited second singlet state to excited first singlet state.
The excited molecule or the atom may also lose its electronic excitation energy by ‘internal conversion’ and comes to another excited state known as triplet state which is metastable with a half-life of 10−3 seconds. In this excited state the electron carrying extra energy can take part in further reaction.
From the triplet state the excited molecule or the atom may return to the ground state in three ways:
(i) By losing its remaining extra energy in the form of heat;
(ii) By losing the extra energy in the form of radiant energy. The latter process is termed phosphorescence. The substances which show this property of phosphorescence emit phosphorescent light even after the incident radiant light is cut off. Secondly, the phosphorescent light is of higher wave length than the incident light and also fluorescent light;
(iii) The electron carrying the extra energy may be expelled from the molecule and is consumed in some further chemical reaction and a fresh normal electron returns to the molecule and is consumed in some further chemical reactions and a fresh normal electron which has now become unexcited returns to the molecule.
This is what exactly happens with excited triplet state of chlorophyll-a molecule which takes part in primary photochemical reaction in photosynthesis.
Two Pigment Systems (Photosystems I and II):
ChlorophyII is present in different forms, which have maximum absorption at different wavelengths of visible sight. Here, one of the forms shows an absorption peak at a wavelength of 673 nanometers (nm) and is called Chl a 673.
In the similar way two other forms of chlorophyll a are chl a 683 (P680) and chl a 703 (P700), with peak absorption at 683 and 703 nm, respectively. These pigments are anchored in thylakoids in separate units of organisation called photosystems. A single photosystem consists of about 250 to 400 pigment molecules.
According to Butler (1966), chlorophyll-a exists in two forms, one form with an absorption maximum at 673 nm and the other with an absorption maximum at 683 nm.
In addition to these two pigment systems or photosystems a long wave absorbing chlorophyll is also present in much smaller amounts. This is known as P 700 with an absorption maximum at 700 nm. According to Clayton (1966), this is another form of chlorophyll. Here, P stands for pigments.
The photosynthesis takes place by two photochemical processes, each process being associated with a specific group of pigments. These light absorbing group of pigments are called pigment system I or photosystem I and pigment system II or photosystem II.
Light energy for pigment system (photosystem I) is collected by chl a 683, P 700 and the carotenoids, whereas the light energy for pigment system II (photosystem II) is collected by chl a 673, chlorophyll b, phycobilins.
The primary function of the two photosystems (PS I and II), which interact with each other is to trap light energy and convert it to the chemical energy (ATP). This chemical energy (ATP) is utilised by living cells.
Now this has been proved that the chloroplasts are composed of numerous minute photosynthetic units. A photosynthetic unit is the smallest group of collaborating pigment molecules necessary to effect a photochemical act. Park and Biggins (1946) isolated the photosynthetic unit from chloroplast lamellae. They called the photosynthetic units as quantasomes.
There are two principal forms of hydrogen acceptor which can receive hydrogen from a substrate under the action of the appropriate dehydrogenase.
According to the latest nomenclature, recommended by the international commission they are:
Coenzyme I-nicotinamide adenine dinucleotide (NAD)
Coenzyme Il-nicotinamide adenine dinucleotide phosphate (NADP)
Adenosine Triphosphate (ATP):
This compound occurs in all living cells and consists of the nucleotide adenosine (A) and three phosphate radicals (—P).
For the most part it is only ATP which is able to carry out the actual transference of the high energy phosphate. (~ P) so that the ~ P formed from low energy sources is used to regenerate ATP from adenosine diphosphate (ADP) rather than be used directly.
“ATP is the main fuel of life produced in photosynthesis and oxidative phosphorylation. In both cases it is produced by an electric current, that is, energy released by a ‘dropping’ electron”.
Biosynthetic Phase of Photosynthesis:
As already has been discussed, how ATP and NADPH2 are formed during photochemical reactions in chloroplasts.
Both ATP and NADPH2 are essentially needed for assimilation of CO2 to carbohydrates. The reactions that catalyse assimilation of CO2 to carbohydrates take place in the stroma of chloroplast where all necessary enzymes are present.
These reactions are called carbon reactions (dark reactions) that lead to the photosynthetic reduction of carbon to carbohydrates.
Carbon Reactions (Dark Reactions):
This part of photosynthesis is called synthesis stage and is the anabolic counterpart of respiration, i.e., the synthesis of carbohydrate from carbon dioxide. This is called photosyntlietic assimilation as it occurs in green plants.
Whereas carbon can be assimilated by non-green plants and even by animal non-photosynthetically, there it is called dark carbon assimilation. Both these processes (dark C-assimilation and photosynthesis C-assimilation) differ in nature of end product.
Carbohydrates are formed as a result of photosynthesis C-assimilation but organic acids are formed as end products in dark C-assimilation. During this process carbon dioxide and water build up carbohydrate, oxygen being released. Energy is absorbed in form of light energy and converted into chemical energy of carbohydrate molecule.
In first phase of carbon reactions (dark reactions), CO2 is accepted by a 5-carbon molecule, ribulose-1, 5-bisphosphate (RuBP) and two molecules of 3-carbon compound, i.e., 3-phosphogIycerate (PGA) are formed.
The 3-carbon molecule, i.e., 3-phosphoglycerate, is first stable product, and therefore, this is called C3 pathway.
Formation of 3-phosphoglycerate (PGA) is known as carboxylation. This reaction is catalysed by an enzyme called ribulose bisphosphate carboxylase (i.e., Rubisco).
In addition to carboxylase activity this enzyme (Rubisco) also possesses oxygenase activity, and therefore abbreviated as Rubisco (i.e., ribulose bisphosphate carboxylase oxygenase).
The oxygenase activity of the enzyme allows O2 to complete with CO2 for combining with RuBP (i.e., ribulose bisphosphate) in photorespiration.
After carboxylation reaction, reduction of PGA takes place, where ATP and NADPH2 formed during photochemical reactions are utilised.
With the reduction of PGA (i.e., 3-phosphoglycerate), a carbohydrate, glyceraldehyde- 3-phosphate, is formed.
These 3-carbon molecules (carbohydrates) are also called triose phosphates. They are diverted from Calvin cycle and act as precursors for synthesis of sucrose and starch.
For the completion of Calvin cycle, and for its process of continuation, on its own, regeneration of the initial 5-carbon acceptor molecule, i.e., ribulose-1, 5-bisphosphate (RuBP), takes place.
The regeneration of RuBP from glyceraldehyde-3-phosphate requires another ATP molecule formed with the result of photophosphorylation during light reactions.
Path of Carbon in Photosynthesis:
Our knowledge of the path of carbon in photosynthesis comes mainly from a series of brilliant researches initiated by Melvin Calvin (1946) and his coworkers. They made use of the radioactive isotope of carbon, C14, from which they prepared radioactive carbon dioxide (C14 O2) and then fed this to unicellular algae—Chlorella and Scenedesmus—for periods of time ranging from five seconds to several minutes.
After these short periods of photosynthesis in C14O2 the cells were plunged into boiling ethyl alcohol in order to kill them. The cells were instantaneously killed and their enzymes denatured. The compounds into which C14 had been incorporated by photosynthesis were then separated from the alcoholic extract.
Among the many compounds found are:
Tetroses, pentoses, hexoses and heptoses after 90 seconds exposure.
After an exposure of only 5 seconds to C14O2 most of the radioactive carbon is found in 3-phosphoglyceric acid (3-PGA), a three carbon compound normally considered a constituent of glycolysis. Increase in the time of exposure to C14O2 to 30-90 seconds found most of the isotopic carbon in phosphoglyceraldehyde dihydroxyacetone phosphate and hexose phosphates as well as 3-PGA.
The C3 type of carbon reactions occur in the stroma of chloroplast. This C3 pathway is also called Calvin cycle after its discoverer, Melvin Calvin, who was awarded Nobel Prize for the discovery of this pathway.
Initial Acceptor of Carbon-Dioxide:
The question arises as to what compound or compounds give rise to 3-PGA. In other words, what compound is the initial acceptor of the carbon dioxide molecule? It might be anticipated that the fed C14O2 would couple with some 2-carbon compound to produce the 3-carbon compound (3-PGA), but this seems not to be true. Calvin obtained evidence that a 5-carbon compound ribulose-1, 5, bisphosphate (RuBP), is formed from ribulose-5- phosphate.
The C14O2 appears to attach to a 5-carbon compound ribulose-1, 5 bisphosphate (RuBP) to form an unstable 6-carbon compound, which then spontaneously decomposes to two PGA molecules. RuBP is now accepted to be the compound with which carbon dioxide first combines in photosynthesis under the influence of the enzyme, carboxydismutase.
This has been confirmed that the 6-carbon compound formed by combination of CO2 with RuBP is quite unstable and immediately splits to give rise to two molecules of PGA. The study of the radioactive carbon labelling of the 7-carbon (C7) and 5-carbon (C5) sugar phosphates has shown that PGA was starting material for their synthesis and therefore the precursor, not only of hexoses, but of RuBP.