CHEM869P-Info009

From a biochemical perspective, equilibrium is death. Most students in chemistry courses come to understand that chemical reactions move toward the equilibrium point. Many of these students also develop an intuitive understanding of Le Chatelier’s Principle which is interpreted by biochemists to state that if a system in equilibrium is perturbed, it will move back to that equilibrium via a flux of reactants. However, few students leave undergraduate courses with the realization that biological systems only achieve equilibrium after death. Biochemists view life as a transient, semi-stable state of disequilibrium. In order to maintain a complex biological system (i.e. cell, organism or ecosystem) in a stable disequilibrium, a constant input of energy is required. For almost all of the organisms with which we are familiar, the source of that life-sustaining energy is sunlight (directly or indirectly).

Light consists of packets of energy termed photons, which stream constantly from the Sun in all directions. Each photon is a discrete entity of electromagnetic radiation with a characteristic frequency of electromagnetic field vibration and wavelength. The frequency of this vibration is on the order of 10¹⁴ times per second. The energy [local] in each photon is related to the wavelength and frequency.

For any two photons, the one with more energy is the one that has the highest frequency of field vibration. However it is usually easier to measure the wavelength. Since all photons travel at a constant velocity (the speed of light, 3 x 10⁸ m/sec), the greater the frequency of a photon, the shorter the wavelength. The shorter the wavelength, the more energy the photon contains.

For comparison, the energy contained in the gamma phosphate of ATP and in the high energy electrons of NADH are also shown. The energies that the photosynthetic machinery deals with are very large relative to that in ATP. As a consequence, ATP will be produced indirectly by chemiosmotic coupling (i.e. forming a gradient of protons across a membrane). Also notice that the energy in NADH (or NADPH) is greater than found in photons with wavelengths greater than 550 nm. As a consequence, two photosystems will be required to extract the energy from two photons to excite an electron from H₂O to the level of NADPH. Also consider that the photosynthetic process produces biological energy in the form of glucose as a stable product. The amount of energy released by the total oxidation of glucose to CO₂ and H₂O is 2870 kJ/mol.

Electromagnetic radiation is produced in a great array of wavelengths, and thus amounts of energy per packet. Visible light (the wavelengths detected by the human eye) is in the wavelength range of 400 to 700 nm. Electromagnetic radiation with shorter wavelengths than visible light includes ultraviolet light, X-rays and gamma radiation. Because they have so much energy per photon, they have the potential for damaging biological molecules and tissues and are health hazards. Electromagnetic radiation with wavelengths longer than 700 nm (and less energy) include infrared light, microwaves, radar and TV and radio transmissions.

Within the visible range of 400 to 700 nm light, the human eye is able to discern different wavelengths of light and we give them the names of various colors. An example would be a rainbow in which white sunlight is diffracted into its component wavelengths of light. The colors visible are red, orange, yellow, green, blue and violet. You can get a feeling for the colors of the spectrum by going to this link.

What causes light to interact with the molecules in cells? The most simple answer is that the electrons in conjugated double bonds are able to absorb the energy from photons. Conjugated double bonds are double bonds on alternating pairs of carbon atoms in a linear or cyclic organic molecule. An example is as follows

The pi electrons in conjugated double bonds form a resonance structure that is smeared over the double bond system. These electrons are responsible for light absorption.

Electrons occupy orbitals in molecules and are usually found in pairs. Each electron in the pair has an opposite spin so that they are considered to be balanced. Two electrons with the same spin are not permitted to occupy the same orbital. Rather than trying to describe the interaction of light with biomolecules in words, it is usually easier for students if a graphic presentation is used. These are termed Jablonski diagrams. Orbitals are indicated as horizontal lines, with higher energy orbitals placed higher on the page .

Electrons are indicated by arrows, with the direction of the arrow (up or down) showing the opposite nature of the spins. Such a Jablonski diagram is shown next.

The electrons normally occupy the lowest energy level, termed the ground state. When energy is available, one of the electrons may absorb it and move up to the excited orbital. The product of this energy absorption is shown next.

There are several characteristics of the absorption process that must be understood:

Molecules with electrons in an excited state do not generally stay that way for long. There are multiple pathways for de-excitation of excited state electrons. Two of these, heat and light, are depicted in the next Jablonski diagram.

In the process of fluorescence, the electron drops from the excited state back to the ground state by emitting a photon. This second photon is different from the absorbed photon because some energy is lost in the process, so it has a longer wavelength than the initial photon. Alternately, it is possible for an excited electron to drop back to the ground state by converting the energy to heat. This latter process is often called quenching. Another mechanism for de-excitation is energy transfer to a second pigment.

If two molecules are sufficiently close to one another and their conjugated double-bond systems are properly oriented, the energy from the excited electron in the first pigment may be transferred to an electron in the second pigment molecule. Because small amounts of energy can be lost by molecular vibration between the initial absorption and the transfer, the second pigment molecule has a slightly smaller amount of energy than the first molecule. Because of this energy loss, transfer between the two pigments is unidirectional.

The above examples, involving an excited electron with a spin opposite to the electron in the ground state are called singlet states. This nomenclature is derived from a simple summing of the spins of the electrons and then adding 1 to their absolute value. Electrons have spins of either +1 or -1. Most molecules have two electrons in their relevant orbitals with opposite spins; summing these [(+1) + (-1) = 0] and adding 1 yields a final value of 1 or a singlet state. Free radicals are molecules that have single unpaired electrons (either +1 or -1) giving an absolute value of 1. Adding 1 to this spin state yields a value of 2, or a doublet state. Sometimes the spin on an excited electron can undergo a ‘flip’. When this happens, the electron is moved to a new orbital, normally forbidden for singlet molecules, with lower energy called the triplet state. This is because the two electrons having the same spins are summed to give an absolute value of 2 and adding 1 yields 3. This system sounds more complex than it really is. Just remember that a molecule with two electrons having opposite spins is in the singlet state, whereas molecules with only one electron or two electrons with the same spin are classified as being in the doublet state or triplet state, respectively.

Once in the triplet state, the electron must remain there until there is a second flip to reverse the spin on the electron again. Because electron spin flips are rare, triplet states can last a very long time in biochemical terms (even as long as a second). Once the electron in the triplet state does undergo a flip, the energy may be lost as a photon in a manner similar to fluorescence. However this emitted photon is at a longer wavelength than photons derived from fluorescence de-excitation and are called phosphorescence.

The energy in the excited singlet state electron may be used to carry out chemical reactions. The types of reactions stimulated by the absorption of light energy are diverse and include cis-trans isomerization such as found in vision, rearrangements such as in the production of Vitamin D, and addition reactions such as in the damage of DNA by ultraviolet light. With regard to photosynthesis, the importance of light is that it enables reduction and oxidation reactions.

When a molecule absorbs the energy from a photon, the excited electron is a stronger reducing agent (potential electron donor) and an unpaired electron in the ground state is a stronger oxidizing agent (potential electron acceptor). The generation of high energy electrons is the essence of photosynthesis.

Plants contain many pigments, molecules that absorb some wavelengths of visible light and not other wavelengths. Flowers and fruits obviously contain a large number of organic molecules that absorb light. Leaves, stems and roots also contain a variety of organic molecules which absorb light and could be termed pigments. Such molecules include anthocyanins, flavanoids, flavines, quinones and cytochromes, just to name a few. However, none of these should be considered as a photosynthetic pigment. Photosynthetic pigments are those molecules that are able to absorb the energy from sunlight and make it available to the photosynthetic apparatus. For land plants, there are two classes of photosynthetic pigments, the chlorophylls and the carotenoids.

The ability of chlorophyll and carotenoid molecules to absorb the energy of light and use it effectively is related to their molecular structure and to their organization within the cell. You learned above that pigments absorb the energy from photons through systems of conjugated double bonds. Examine the molecular structure of a representative chlorophyll and carotenoid molecule.

Most land plants have two forms of chlorophyll (Chl), designated as Chl a and Chl b. They differ in that one of the Chl a methyl (-CH₃) groups on the perimeter of the large ring (called a tetrapyrrole) is oxidized to form a formyl (-CH=O) group. Because of this difference, the two chlorophyll molecules have slightly different absorption spectra, and thus are able to harvest a greater variety of wavelengths. Notice the conjugated double bond system that weaves around the tetrapyrrole; this electron cloud is the component of the molecule responsible for absorption of photons. Because the electron cloud on the tetrapyrrole ring can be polarized in two different orientations, there are actually two different excited singlet states to which the electrons can be elevated. The lowest excited singlet state requires absorption of the energy in a red photon (640 to 700 nm). The second excited singlet state is at a higher energy level from the ground state and requires the absorption of the energy in a blue photon (430 to 475 nm); remember that photons with shorter wavelengths have more energy. Thus, chlorophyll absorbs photons from both the red and blue regions of the visible spectrum because it has two excited singlet states.

The electron in the second excited state is not as stable as an excited electron in the first excited singlet state. Before this energy can be used for photochemistry, it is lost as heat by a process called internal conversion and the electron drops down to the first excited singlet state. As a consequence, chlorophyll may be able to absorb blue photons, but the amount of energy available to carry out photochemistry is only that equivalent to a weaker red photon.

Because chlorophyll, the major leaf pigment, does not absorb green photons (about 530 to 570 nm), when we see light reflected by, or transmitted through, leaves, we perceive it to be green. Also notice that the chlorophyll structure includes a long hydrophobic chain that is attached to the tetrapyrrole ring by an ester bond. This chain is a phytyl group and makes the entire chlorophyll molecule insoluble in water.

Chlorophyll [local] is synthesized in the chloroplast starting with the amino acid glutamate. Elements of this pathway (many steps are omitted) are shown.

. This is an important pathway because it produces other tetrapyrrole molecules such as heme in addition to chlorophyll. The pathway is identical up to the point of Protoporphyrin IX, which is produced by the chloroplast. The chloroplast can then insert a Mg²⁺ into the center of the tetrapyrrole and initiate biosynthesis of chlorophyll, or can insert Fe²⁺ into the tetrapyrrole and form heme. The chloroplast also exports much Protoporphyrin IX to the mitochondrion, since it is used to produce cytochromes that are in high abundance in that organelle. This pathway is also important because heme serves as a substrate in the production of phytochrome, a light sensing molecule essential for normal photomorphogenesis. Thus, products of the tetrapyrrole are involved in photosynthesis (chlorophylls and cytochromes in the chloroplast), respiration (cytochromes in the mitochondrion) and development (phytochrome). The pathway for heme biosynthesis in animals is different in that aminolevulinic acid is produced from succinyl- CoA, rather than glutamic acid.

Most land plants contain a variety of carotenoids including beta-carotene, lutein, neoxanthin and violaxanthin. Their basic structure is composed of a repeating, branched five-carbon unit. Molecules formed from these five-carbon units are often called isoprenoids or terpenes. Notice that the carotenoid structures also contain systems of conjugated double bonds that are responsible for the absorption of light. Most carotenoids absorb photons in the blue region of the spectrum (400 to 500 nm) and appear to be yellow, although the primary red pigment in tomatoes is also a carotenoid named lycopene.

The synthesis of carotenoids utilizes the isoprenoid pathway (above), which is also the basis for production of such diverse molecules as those involved in aroma molecules (e.g. menthol), vitamins (e.g. vitamin A), sterols and rubber. The biochemistry involves the addition of successive five-carbon units (or multiples of five carbons) and then rearrangements, cyclizations and additions of functional groups. The pathway is also important in that the geranylgeranyl pyrophosphate intermediate is utilized to make phytol, which becomes the phytyl group ester, in the biosynthesis of chlorophyll. In animals, the isoprenoid pathway condenses two farnesyl pyrophosphate (C₁₅) to produce squalene (C₃₀) which is cyclized to form cholesterol (C₃₀).

Chlorophyll is a dangerous molecule. This is because it is an excellent photosensitizer and will rapidly cause cell damage when exposed to light which has a very large amount of energy per photon. To prevent such damage, the organization and biosynthesis of chlorophyll is carefully controlled. All chlorophyll is located within the chloroplast organelle within the cell. Inside the chloroplast it is further confined to a system of membranes called the thylakoid membranes. Even within the thylakoid membrane system, the chlorophyll molecules do not occur in an unchaperoned state; they exist as photosynthetic pigment-protein complexes. The chlorophyll molecules are bound to specific integral membrane proteins along with carotenoids and electron transfer components necessary for photosynthesis. The proteins form a structured environment in which the chlorophylls absorb photons, but in which unwanted photodynamic reactions are relatively rare. The pigment-protein complexes are arranged into arrays of hundreds of pigment molecules called photosystems. Because of the spatial and geometric organization, most of the pigments function as light-harvesters and transfer the excitation energy to other pigments before unwanted photochemistry occurs. The carotenoids perform two functions in this environment. First, they also harvest light energy and transfer it to chlorophyll molecules. Secondly, they are highly efficient at quenching triplet chlorophylls that are formed before they have a chance to react with molecular oxygen and generate damaging molecules.

These arrays of pigment-protein complexes also contain specific electron transfer components that are important in generating energy from the process of photosynthesis [local]. The local organization within the thylakoid membrane is such that there are actually two separate photosystems. Each contains an array of antenna chlorophylls and carotenoids, representing the majority of the pigment molecules. These function solely to harvest light energy and transfer it to a small number of pigment-protein complexes called reaction centers. In the reaction center, the energy from the photon is used to excite an electron to a higher energy level (also a lower redox potential) so that it can be transferred to an acceptor molecule that has a higher energy level than the original reaction center. The acceptor molecule is then considered to be reduced. In this way, the energy from photons is used to ‘pump’ electrons to higher energy levels. Each time the reaction center loses an electron, it becomes oxidized and is able to accept electrons from an external source.

The process of photosynthesis captures the energy in photons in high energy electrons (reduction potential), and uses this energy to synthesize stable biomolecules (sugars) which can be stored and oxidized at a later time. One problem with this process is that plants evolved to use electrons from water and transfer them to a cofactor like NADPH for useful biochemistry, but a single red photon does not contain sufficient energy to pump the electron to the required energy level in one step. As a consequence, plants use two different photosystems coupled in series to excite the electrons with two consecutive photons.

Using this greatly simplified scheme, a photon is first captured by the pigments of Photosystem II and the excitation energy transferred to P-680, a pigment in the reaction center. P-680 is excited to form P680* and the high-energy electron is transferred to plastoquinone (PQ) via a protein component termed D1. A second photon is similarly captured by Photosystem I and the energy transferred to the pigment P-700 in its reaction center. The electron excited in P-700 is transferred to NADP⁺ through the protein ferredoxin (Fd) to produce NADPH. When P-680 and P-700 have donated their high-energy electrons, they became oxidized (lacking an electron). To bring the system back to neutrality so that it can cycle through the process again, electrons are channeled from water to P-680, thereby forming O₂, and electrons from PQ (originally donated by P-680) are transferred to P-700 via cytochrome (Cyt) and plastocyanin (PC) intermediates. The net effect is that the energy in photons is captured in pigments and used to take the electrons from water and pump them to an energy level high enough to form NADPH. The reduced cofactor NADPH can then be used by metabolism to reduce CO₂ to the level of carbohydrates (CH₂O)_n.

The process of photosynthetic electron transfer also generates ATP. When electrons are transferred between PS-II and PS-I, part of the energy lost is conserved in the form of a proton gradient across the thylakoid membrane. In effect, the energy from light excites electrons to higher levels, which then lose the energy by transfer to molecules with lower energy, but the free energy released is used to pump protons across the membrane. The transmembrane gradient of protons represents potential chemical energy. The chloroplast thylakoid membrane contains an ATP synthase complex similar to that found in the mitochondrion that is able to transduce the trans-membrane proton gradient into the production of ATP. This entire process, including the formation of ATP is termed photophosphorylation [local].

In summary, the process of photophosphorylation is used to capture the energy in photons and convert it to high-energy electrons in the form of NADPH and biochemical energy in the form of ATP. With these two molecules, there is sufficient energy to carry out the fixation of CO₂ into sugars via the Calvin cycle and to drive the other anabolic reactions of the plant cell. Excess energy formed by photosynthesis is conserved as starch. In the dark, the plant is able to mobilize these carbohydrate reserves and obtain energy by respiration in a fashion similar to the normal lifestyle of microbes and animals.

Photosynthesis
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