plant metabolism

image from paper: Photosynthetic metabolism of C3 plants shows highly cooperative regulation under changing environments: A systems biological analysis


Enzymes (the proteins that speed up chemical reactions in cells without being used up in the reactions) regulate just about every

metabolic activity. In biochemical reactions, one or more specific enzymes are associated with the myriad forms of energy con- version that take place within cells. In some cases, these enzymes help build molecules that store energy in chemical bonds through a process called anabolism. Use of this energy to perform work often requires chemical bonds to be broken through catabolism. Most reactions of photosynthesis are anabolic because they involve construction of molecules that are stored for energy, whereas reactions in cellular respiration (referred to as “respiration” in the text) are generally catabolic, since in per- forming work, they release energy held in chemical bonds. Photosynthesis builds organic compounds by combining carbon di- oxide and water, forming carbohydrates. Respiration, on the other hand, breaks down those carbohydrates, producing carbon dioxide and water, which may be used once again in photosynthesis. As we will see in the sections to follow, this photosynthe- sis-respiration cycle is keyed by an enzyme complex that splits water molecules and releases electrons that function, at least temporarily, in storing biochemical energy. These electrons transfer energy from one form to another through oxidation- reduction reactions.

Introduction to metabolism: anabolism and catabolism | Khan Academy


Playlist-Enzymes/ Photosynthesis/respiration

Oxidation-Reduction Reactions

The processes of both photosynthesis and respiration include many oxidation-reduction reactions. Oxidation is the loss of one or more electrons; it involves removal of electrons from a compound. Reduction is the gain of one or more electrons; it involves the addition of electrons to a compound. In most oxidation-reduction reactions, oxidation of one compound is cou- pled with reduction of another compound catalyzed by the same enzyme or enzyme complex. When an electron is removed, a proton may follow, with the result that a hydrogen atom is often removed during oxidation and added during reduction. Oxy- gen is usually the oxidizing agent (i.e., the final acceptor of the electron), but oxidations can occur without oxygen being in- volved.

Krebs Cycle & Oxidative Phosphorylation

Krebs / citric acid cycle | Cellular respiration

Oxidation and reduction | Redox reactions and electrochemistry | Chemistry

Oxidation and reduction review from biological point-of-view | Biomolecules


The Essence of Photosynthesis

The energy-storing process of photosynthesis takes place in chloroplasts and other parts of green organisms in the pres- ence of light. The light energy stored in a simple sugar molecule is produced from carbon dioxide (CO2) present in the air and water (H2O) absorbed by the plant. When the carbon dioxide and water (H2O) are combined and ultimately a sugar molecule (C6H12O6, glucose) is produced in a chloroplast, oxygen gas (O2) is released as a by-product. The oxygen diffuses out into the atmosphere. The overall process can be depicted by the equation that follows. The equation should not, however, be taken literally because there are many intermediate steps to the process, and glucose is not the immediate first product of photosyn- thesis.


6CO2 + 12H2O + light energy chlorophyll carbon     water     enzymes

C6H12O6    +   6O2    + 6H2O


glucose            oxygen          water

Photosynthesis takes place in chloroplasts (see Figs. 3.4 and 3.11) or in cells with membranes in which chlorophyll is embedded. The principals in the process are carbon dioxide, water, light, and chlorophyll; a brief examination of each fol- lows.

Carbon Dioxide


Less than 1% of all the water absorbed by plants is used in photosynthesis; most of the remainder is transpired or incorporated into cytoplasm, vacuoles, and other materials. The water used is the source of electrons involved in photosynthesis, and the oxygen released is a by-product, even though carbon dioxide also contains oxygen. This has been demonstrated by conducting photosynthetic experiments using either carbon dioxide or water containing isotopes of oxygen. When the isotope is used only in the water, it appears in the oxygen gas released. If, however, it is used only in the carbon dioxide, it is confined to the sugar and water produced and never appears in the oxygen gas, demonstrating clearly that the water is the sole source of the oxygen released.

If water is in short supply, it may indirectly become a limiting factor in photosynthesis; under such circumstances, the stomata usually close and sharply reduce the carbon dioxide supply.


Light that is too intense may change the way in which some of a cell’s metabolism takes place. For example, higher light intensities and temperatures may change the ratio of carbon dioxide to oxygen in the interiors of leaves, which, in turn, may accelerate photorespiration (discussed on p. 177). Photorespiration is typically considered to be a wasteful process that uses oxygen and releases carbon dioxide, although it may help some plants to survive under adverse conditions. It differs from common aerobic respiration in its chemical pathways.

Photooxidation, which involves the destruction (“bleaching”) of chlorophyll by light, may also occur. High light intensities may cause chlorophyll molecules to go to a different excited state. The energy released from the excited chlorophyll is passed to oxygen molecules, which become highly reactive and bleach the chlorophyll. In the fall, photooxidation plays a significant role in the breakdown of chlorophyll in leaves, resulting in the autumn colors discussed in Chapter 7. High light intensities may also cause an increase in transpiration, resulting in the closing of stomata. A sharp reduction in the available carbon dioxide supply inevitably follows.


There are several different types of chlorophyll molecules, all of which contain one atom of magnesium. They are very simi- lar in structure to the heme of hemoglobin, the iron–containing red pigment that transports oxygen in blood. Each molecule has a long lipid tail, which anchors the chlorophyll molecule in the lipid layers of the thylakoid membranes (Fig. 10.4).

Chloroplasts of most plants contain two major kinds of chlorophyll associated with the thylakoid membranes. Chloro- phyll a is blue-green in color and has the formula C55H72MgN4O5. Chlorophyll b is yellow-green in color and has the formula C55H70MgN4O6. Usually, a chloroplast has about three times more chlorophyll a than b. The more chlorophyll a there is in a cell, the brighter green the cell and the tissue of which it is a part appear to be. When a molecule of chlorophyll b absorbs light, it transfers the energy to a -molecule of chlorophyll a. Chlorophyll b, then, makes it possible for photosynthesis to take place over a broader spectrum of light than would be possible with chlorophyll a alone (see Fig. 10.7).

Other such pigments include carotenoids (yellowish to orange pigments), phycobilins (blue or red pigments found in cyanobacteria and red algae), and several other types of chlorophyll. Chlorophylls c, d, and possibly e take the place of chlo- rophyll b in certain algae, and several other photosynthetic pigments are found in bacteria. The various chlorophylls are all closely related and differ from one another only slightly in the structure of their molecules.

In chloroplasts, about 250 to 400 pigment molecules are grouped as a light-harvesting complex called a photosynthetic unit, with countless numbers of these units in each granum. Two types of these photosynthetic units function together in the chloroplasts of green plants, bringing about the first phase of photosynthesis, the light-dependent reactions, which are dis- cussed in the next section, “Introduction to the Major Steps of Photosynthesis.”


What are Electrons and Excitation?


Electrons and photons: absorption and transmission of light

Photoelectric effect | Electronic structure of atoms | Chemistry | Khan Academy

Leaf Pigments and Light



Photo-excitation of Chlorophyll


Introduction to the Major Steps of Photosynthesis

The process of photosynthesis takes place in two series of steps called the light-dependent reactions and the light- independent reactions. Although the light-independent reactions use products of the light-dependent reactions, both proc- esses occur simultaneously.

The Light-Dependent Reactions

The light-dependent reactions are the first major steps in the conversion of light energy to biochemical energy. The reactions are initiated when units of light energy (photons) strike chlorophyll molecules embedded in the thylakoid membranes of chloroplasts.

Our knowledge of the light-dependent reactions essentially began in the 1930s in England through a discovery by Robin Hill, a biochemist. He found that a solution of fragmented and whole chloroplasts, isolated from leaves that had been ground up and centrifuged, could briefly produce oxygen if an electron acceptor was present to receive electrons from water. In 1951, it was shown that NADP (nicotin-amide adenine dinucleotide phosphate), which is derived from the B vitamin niacin, was a natural electron acceptor in this reaction. In honor of its discoverer, the process became known as the Hill reaction.

During the light-dependent reactions,

  1. water molecules are split apart, producing electrons and hydrogen ions, and oxygen gas is released;
  2. the electrons from the split water molecules are passed along an electron transport system;
  3. energy-storing ATP molecules are produced;
  4. some hydrogen from the split water molecules is involved in the reduction of NADP to form NADPH (reduced nicoti- namide adenine dinucleotide phosphate), which carries hydrogen and is used in the second phase of photosynthesis, the light-independent reactions.

Conceptual overview of light dependent reactions

Light-Harvesting: The Antenna Complex

Photosynthesis Splitting H2O to O2 in Light Dependent Reactions 10 6 2018

PhotosynthesisView full playlist


plant videos 3 physiology

ps 1 – light harvesting

ps 2 – electron transport

ps 3 – reaction centers

The Light-Independent Reactions

The light-independent reactions (or carbon-fixing and reducing reactions) complete the conversion of light energy to chemi- cal energy in the form of ATP and NADPH. Some scientists refer to the light-independent reactions as the dark reactions because they don’t directly require light, but darkness has nothing to do with their functioning. In fact, even though light is not directly required in the same sense as it is for the light-dependent reactions, light is nevertheless required for the activa- tion of the enzymes involved, and the processes normally can occur only in daylight.

The light-independent reactions are a series of reactions that take place outside of the grana in the stroma of the chloro- plast (see page 40), if the products of the light-dependent reactions are available. They may initially proceed in different ways, depending on the particular kind of plant involved, but they all go through the Calvin cycle, discovered and elucidated by Dr. Melvin Calvin of the University of California. In 1961, Calvin received a Nobel Prize for unraveling how this most widespread type of light-independent reactions takes place.

In this cycle, carbon dioxide (CO2) from the air is combined with a 5-carbon sugar (RuBP, ribulose bisphosphate), and then the combined molecules are converted, through several steps, to sugars, such as glucose (C6H12O6). Energy and electrons involved in these steps are furnished by the ATP molecules and NADPH produced during the light-dependent reactions. Some of the sugars that are produced during the light-independent reactions are recycled, while others are stored as starch or

other polysaccharides (simple sugars strung together in chains). A summary of simplified photosynthetic reactions is shown in Figure 10.5. More detailed diagrams of the light-dependent and light-independent reactions are shown in Figures 10.8, 10.9, and 10.12.

Two molecules of a 3-carbon sugar compound (3PGA—an abbreviation of 3-phosphoglyceric acid) are shown as the first stable substance produced when carbon dioxide from the air and RuBP are combined and then converted during the light-independent reactions (Fig. 10.5). Some grasses and also many plants of arid regions fix carbon differently. They pro- duce a 4-carbon acid as the first product, followed by the Calvin cycle. This 4-carbon pathway is discussed, along with an- other variation found mostly in desert plants, in the next section, “A Closer Look at Photosynthesis.”

3. A Closer Look at Photosynthesis

A great deal has been learned about photosynthesis since 1772 when Joseph Priestly (1733–1804), a naturalist in England, re- ceived a medal for demonstrating that a sprig of mint “restored” oxygen so that a mouse could live in air that had been used up by a burning candle. Seven years later, Jan Ingen-Housz (1730–1799) of Holland, who visited England to treat the royal family for smallpox, carefully repeated Priestly’s demonstrations. He showed that the air was restored only when green parts of plants were receiving sunlight.

In 1782, Jean Senebier (1742–1809), a Swiss pastor, discovered that the photosynthetic process required carbon dioxide, and in 1796, Ingen-Housz showed that carbon went into the nutrition of the plant. The final component of the overall photo- synthetic reaction was explained in 1804 by another Swiss, Nicholas Theodore de Saussure (1767–1845), who showed that water was involved in the process.

A little current information about the details of photosynthesis is given in the following modest amplification of the pre- ceding section, “Introduction to the Major Steps of Photosynthesis.” Those who wish more information are referred to the reading list at the end of the chapter.

The Light-Dependent Reactions Reexamined

As noted earlier, light has characteristics of both waves and particles. Sir Isaac Newton, while experimenting with a prism over 300 years ago, produced a spectrum of colors from visible white light and postulated that light consisted of a series of discrete particles he called “corpuscles.”

Newton’s theory only partially explained light phenomena, however, and by the middle of the 19th century, James Max- well and others showed that light and all other parts of the extensive electromagnetic spectrum travel in waves.

By the late 1800s, with the discovery that a photoelectric effect can be produced in all metals, the wave theory also be- came inadequate to explain certain attributes of light. When a metal is exposed to radiation of a critical wavelength, it be- comes positively charged because the radiation forces electrons out of the metal atoms. The ability of light to force electrons from metal atoms depends on its particular wavelength—its energy content—and not on its intensity or brightness. The shorter the wavelength, the greater the energy, and vice versa.

In 1905, Albert Einstein proposed that the photoelectric effect results from discrete particles of light energy he called photons. In 1921, he received the Nobel Prize in physics for this work. Both waves and particles (photons) are today almost universally recognized as aspects of light. The energy (quantum) of a photon is not the same for all kinds of light; those of longer wavelengths have lower energy, and those of shorter wavelengths have proportionately higher energy.

Chlorophylls, the principal pigments of photosynthetic systems, absorb light primarily in the violet to blue and also in the red wavelengths; they reflect green wavelengths, which is why leaves appear green. This was first ingeniously demonstrated in 1882 by T. W. Engelmann. He focused a tiny spectrum of light on a filament (single row of cells) of Spirogyra, a freshwater alga. The alga had been mounted in a drop of water on a microscope slide containing bacteria that move toward an oxygen source. As shown in Fig. 10.6, the bacteria assembled in greatest numbers along the algal filament in the blue and red portions of the spectrum, demonstrating that oxygen production is directly related to the light the chlorophyll absorbs. An analysis using living material is called a bioassay. Information gained from bioassays often is significant.

Each pigment has its own distinctive pattern of light absorption, which is referred to as the pigment’s absorption spec- trum (Fig. 10.7). When a pigment absorbs light, the energy levels of some of the pigment’s electrons are raised. When this occurs, the energy may be emitted immediately as light (a phenomenon called fluorescence). In chlorophyll, the emitted light is characteristically in the red part of the visible light spectrum, so an extract of chlorophyll placed in light (especially ultra- violet or blue light) will appear red. The absorbed energy may also be emitted as light after a delay (a phenomenon called phosphorescence); it may otherwise be converted to heat. The most important use of the absorbed energy, however, is its storage in chemical bonds for photosynthesis.

Photosystems The two types of photosynthetic units present in most chloroplasts make up photosystems known as photo- system I and photosystem II (Fig. 10.8). Photosystem II received its “II” designation because it was discovered after photo- system I, but we know now that the events of photosynthesis that take place in photosystem II come before those of photosys- tem I.

While both photosystems produce ATP, only organisms that have photosystems I and II can produce NADPH and oxy-

gen as a consequence of electron flow. It is likely that evolutionary events led to organisms that possess both photosystems. At least 2.8 billion years ago, photosynthetic forms of bacteria (cyanobacteria) are believed to have evolved from primitive bacteria with the development of chlorophyll a and photosystem II. As a consequence of the oxygen-generating steps in pho- tosystem II that have evolved, humans and other organisms are dependent upon photosynthetic organisms that generate oxy- gen in the air we breathe today. Since photosynthetic organisms can generate oxygen from water, this very process in the future could be exploited to sustain human life during space travel and colonization of other planets.

Each photosynthetic unit of photosystem I consists of 200 or more molecules of chlorophyll a, small amounts of chloro- phyll b, carotenoid pigment with protein attached, and a special reaction-center molecule of chlorophyll called P700. Although all pigments in a photosystem can absorb photons, the reaction-center molecule is the only one that can actually use the light energy. The remaining photosystem pigments are called antenna pigments because together they function somewhat like an antenna in gathering and passing light energy to the reaction-center molecule (see Fig. 10.9). There are also iron-sulfur pro- teins that are the primary electron acceptors for photosystem I (i.e., iron-sulfur proteins first receive electrons from P700).

A photosynthetic unit of photosystem II consists of chlorophyll a, b-carotene (the precursor of vitamin A) attached to protein, a little chlorophyll b, and a reaction–center molecule of chlorophyll a; these special molecules are called P680.

The letter P stands for pigment and the numbers 700 and 680 of the reaction-center molecules of chlorophyll a refer to peaks in the absorption spectra of light with wavelengths of 700 and 680 nanometers, respectively. These peaks differ slightly from those of the otherwise identical chlorophyll a molecules of the photosynthetic units. A primary electron acceptor called pheophytin (or Pheo) is also present in photosystem II. One reaction-center molecule was found by Johann Deisenhofer and Hartmot Michel of Germany to have over 10,000 atoms. They received a Nobel Prize in 1988 for their work in determining the atomic structure.

Water-Splitting (Photolysis) When photons of light are absorbed by P680 molecules of a photosystem II reaction center (located near the inner surface of a thylakoid membrane), the light energy boosts electrons to a higher energy level. This is referred to as exciting electrons. Excited electrons are unstable and can rapidly revert back to their lower energy level, releasing absorbed en- ergy, perhaps as heat. However, during photosynthesis, the excited electrons are passed to an acceptor molecule, called pheo- phytin, at the beginning of an electron transport system.

From pheophytin, electrons are shuttled to another acceptor, PQ (plastoquinone), within the thylakoid membrane.4 PQ is mo- bile and moves through the lipid bilayer toward the inner side of the thylakoid membrane, unloading electrons to the cytochromes that are next in line. Electrons extracted from water by a manganese-containing oxygen-evolving complex (OEC) replace the elec- trons lost by the P680 molecule. It has been suggested that there is an oxidation-reduction system, usually designated Z, operating between water and P680. Transfer of electrons from Z to P680 reoxidizes Z and prepares it for accepting additional electrons from the OEC. Recent investigations indicate that for each two water molecules that are split, one molecule of oxygen is produced, along with four protons and four electrons.

This metabolic pathway eventually evolved in photosynthetic bacteria (cyanobacteria). The abundance of water, as an electron source, probably facilitated the mechanism that generated oxygen as a by-product of photosynthesis. This process increased the sup- ply of oxygen in earth’s atmosphere and made it possible for energy-efficient aerobic respiration to evolve.

Electron Flow and Photophospho-rylation The high-energy acceptor molecule PQ releases electrons that originated from photosystem II to an electron transport system, which functions something like a downhill bucket brigade. This electron transport system consists of iron-containing pigments called cytochromes and other electron transfer molecules, plus plastocyanin—a pro- tein that contains copper. When electrons pass along the electron transport system and protons subsequently move across the thy- lakoid membrane by chemiosmosis (see next section, “Chemiosmosis”), ATP molecules are formed from ADP in the process of – photophosphorylation.

A somewhat similar series of events occurs in photosystem I. When photons of light are absorbed by P700 molecules in a photo- synthetic unit, the energy excites electrons, which are transferred to an iron-sulfur acceptor molecule -designated Fe-S. This then passes electrons to another iron-sulfur acceptor molecule, Fd (ferredoxin), which, in turn, releases them to a carrier molecule desig- nated as FAD (flavin adenine dinucleotide). FAD contains flavoprotein, which assists in the reduction of NADP to NADPH. The electrons removed from the P700 molecule are replaced by electrons from photosystem II via the electron transport system outlined previously. This overall movement of electrons from water to photosystem II to photosystem I to NADP is called noncyclic electron flow, because it goes in one direction only. The production of ATP that correspondingly occurs is designated as noncyclic photophos- phorylation.

Photosystem I can also work independently of photosystem II. When it does, the electrons boosted from P700 reaction-center molecules (of photosystem I) are passed from ferredoxin to plastoquinone (instead of NADPH) and back into the reaction center of photosystem I. This process is cyclic electron flow. ATP generated by cyclic electron flow is called cyclic photophosphoryla- tion, but water molecules are not split and neither NADPH nor oxygen are produced in this process.

Chemiosmosis The oxygen-evolving complex on the inside of a thylakoid membrane catalyzes the splitting of water molecules, producing protons and electrons, as well as oxygen gas. These electrons used to replenish those excited in chlorophyll are then trans- ferred in bucket-brigade fashion through an electron transport system, ultimately reducing NADP to NADPH. As electrons travel through this transport system, additional protons move from the stroma to the inside of the thylakoid membrane in specific oxidation- reduction reactions when electrons pass from photosystem II to PQ. These protons join with the protons from the split water mole-

cules and thereby contribute to an accumulation of four protons toward the inside of the thylakoid membrane (an area also known as the thylakoid lumen).

Although some protons are used in the production of NADPH on the stroma side of the thylakoid membrane, there is still a net accumulation of protons in the thylakoid lumen from the splitting of water molecules and electron transport. This establishes a proton gradient, giving special proteins in the thylakiod membrane the potential for moving protons from the thylakoid lumen back to the stroma. Movement of protons across the membrane is thought to be a source of energy for the synthesis of ATP. The action has been described as similar to the movement of molecules during osmosis and has been called chemiosmosis, or the Mitchell theory, after its author, Peter Mitchell. In this concept, protons move across a thylakoid membrane through protein channels called ATPase. With the proton movement, ADP and phosphate (P) combine, producing ATP (Fig. 10.9). This chemiosmotic mechanism for connecting electron flow with conversion of ADP to ATP is essentially the same as that of oxida- tive phosphorylation in mitochondria (see Fig. 10.14), except that in mitochondria, oxygen (instead of NADP) is the terminal electron acceptor.

The Light-Independent Reactions Reexamined

We have seen how synthesis of ATP and NADPH is set in motion during the light-dependent reactions. Both of these substances play key roles in the synthesis of carbohydrate from carbon dioxide from the atmosphere, which during the light-independent re- actions reaches the interior of chlor-enchyma tissues via stomata. As indicated earlier, the light-independent reactions are really a whole series of reactions, each mediated by an enzyme in this major phase of photosynthesis.

The light-independent reactions take place in the stroma of the chloroplasts and, as long as the products of the light- dependent reactions are present, they do not directly need light in the same sense that the light-dependent reactions do. How- ever, they normally take place only during daylight hours because some of the enzymes involved in the light-independent reactions require light for their activation or conversion to a form in which they can actively catalyze steps of the light- independent reactions.

The Calvin Cycle The heart of the light-independent reactions is the Calvin cycle, during which carbon dioxide is fixed and converted to carbohydrate. The carbohydrate produced during these reactions facilitates growth, including the develop- ment of leaves, stems, roots, flowers, and other plant structures. From an ecological standpoint, this process is essential be- cause it is the main biosynthetic pathway through which carbon enters the web of life. As discussed next, the Calvin cycle, or photosynthetic carbon reduction (PCR) pathway (Fig. 10.10), runs in five main steps:

  1. Six molecules of carbon dioxide (CO2) from the air combine with six molecules of ribulose 1,5-bisphosphate (RuBP, the 5- carbon sugar continually being formed while photosynthesis is occurring), with the aid of the enzyme rubisco (RuBP car- boxylase/oxygenase).5
  2. The resulting six 6-carbon unstable complexes are immediately split into twelve 3-carbon molecules known as 3- phosphoglyceric acid (3PGA), the first stable compound formed in photosynthesis.
  3. The NADPH (which has been temporarily holding the hydrogen and electrons released during the light-dependent reac- tions) and ATP (also from the light-dependent reactions) supply energy and electrons that chemically reduce the 3PGA to twelve molecules of glyceraldehyde 3-phosphate (GA3P, 3-carbon sugar phosphate).
  4. Ten of the twelve glyceraldehyde 3-phosphate molecules are restructured, using another six ATPs, and become six 5- carbon molecules of RuBP, the sugar with which the Calvin cycle was initiated.
  5. This leaves a net gain of two GA3P molecules, which can contribute either to an increase in the carbohydrate content of the plant (glucose, starch, cellulose, or related substances) or can be used in pathways that lead to the net gain of lipids and amino acids.

Since rubisco catalyzes formation of the 3-carbon compound 3PGA as the first isolated product in these light– independent reactions, plants demonstrating this process are called C3 plants. However, as indicated by its name, the enzyme RuBP carboxylase/oxygenase has the potential to fix both CO2 through its carboxylase activity described by the Calvin cycle and O2 through its oxygenase activity. The oxygenase activity of rubisco makes C3 plants vulnerable to a process called photorespiration.

Photorespiration While the carboxylase activity of rubisco ultimately results in accumulation of carbohydrates, this same enzyme can exhibit an oxygenase activity that catalyzes combination of oxygen with RuBP and a subsequent pathway that releases carbon dioxide. This photorespiration may be interpreted as a wasteful process that competes with the carbon-fixing role of photosynthesis. However, the oxygenase activity of rubisco responsible for photorespiration is not necessarily an omi- nous alternative because it provides a salvage pathway to allow C3 plants to survive under hot, dry conditions. Evidence sug- gests that photorespiration helps to dissipate ATP and accumulated electrons from the light reactions, thereby preventing photooxidative damage.

Factors that determine the extent of this photorespiration in C3 plants include temperature, CO2:O2 ratio, and kinetic prop- erties of rubisco. Hot and dry climates generally promote photorespiration because stomata are closed under these conditions,

leading to a decreased CO2:O2 ratio within the leaf and near rubisco complexes. Oxygen gas can accumulate from photolysis in a leaf with closed stomata and photorespiration is more likely to occur, particularly when the carbon dioxide concentration drops roughly below 50 parts per million. When temperatures are cooler and moisture is present, the stomata are more likely to open, which leads to an increased CO2:O2 ratio. Under milder climatic conditions, C3 plants are more efficient at fixing carbon dioxide.

Photorespiration requires cooperation among chloroplasts, peroxisomes, and mitochondria to facilitate shuttling of in- termediates along the photorespiratory pathway. The products of photorespiration are the 2-carbon phosphoglycolic acid, which is processed to some extent in peroxisomes and eventually released as carbon dioxide in mitochondria, and the 3- carbon phosphoglyceric acid that can reenter the Calvin cycle. No ATP is produced by photorespiration.

The 4-Carbon Pathway Sugar cane, corn, sorghum, and at least 1,000 other species of tropical grasses or arid region plants sub- ject to high temperature stresses have a distinctive leaf -anatomy called Kranz anatomy (Fig. 10.11). Kranz anatomy leaves have two forms of chloroplasts. In the bundle sheath cells surrounding the veins, there are large chloroplasts, often with few to no grana. The large chloroplasts have numerous starch grains, and the grana, when present, are poorly developed. The chloroplasts of the mesophyll cells are much smaller, usually lack starch grains, and have well-developed grana.

Plants with Kranz anatomy produce a 4-carbon compound, oxaloacetic acid, instead of the 3-carbon 3–phosphoglyceric acid during the initial steps of the light-independent reactions. Oxaloacetic acid is produced when a 3-carbon compound, phosphoenolpyruvate (PEP), and carbon dioxide are combined in mesophyll cells with the aid of a different carbon-fixing enzyme, PEP carboxylase. Depending on the species, the oxaloacetic acid may then be converted to aspartic, malic, or other acids. PEP carboxylase is not sensitive to oxygen and, hence, has a greater enzyme affinity for carbon dioxide, so there is no photorespiratory loss of carbon dioxide captured in the organic acids. The carbon dioxide is transported to the bundle sheath cells where it is released and enters the normal Calvin cycle just as in C3 plants. The carbon dioxide concentration can be kept high in relation to the oxygen concentration in the bundle sheath cells, thereby keeping the reaction of rubisco with oxygen very low (Fig. 10.12).

Because the PEP system produces 4-carbon compounds, plants having this system are called 4-carbon, or C4, plants, to distinguish them from plants that have only the 3-carbon, or C3, system. Besides Kranz anatomy, C4 plants have other charac- teristic features:

  1. High concentrations of PEP carboxylase are found in the mesophyll cells. This is significant because PEP carboxylase allows the conversion of carbon dioxide to carbohydrate at much lower concentrations than does rubisco (found only in the bundle sheath cells), the corresponding enzyme in the Calvin cycle.
  2. The optimum temperatures for C4 photosynthesis are much higher than those for C3 photosynthesis, allowing C4 plants to thrive under conditions that would adversely affect C3 plants.

Obviously, the C4 pathway furnishes carbon dioxide to the Calvin cycle in a more roundabout way than does the C3 path- way, but the advantage of this extra pathway is a major reduction of photorespiration in C4 plants. Also, in C4 plants, the C4 pathway in the mesophyll cells results in carbon dioxide being picked up even at low concentrations (via the enzyme PEP car- boxylase) and in carbon dioxide being concentrated in the bundle sheath, where the Calvin cycle takes place almost exclusively. The Calvin cycle enzyme, rubisco, will catalyze the reaction in which RuBP will react with carbon dioxide rather than oxygen. Consequently, C4 plants, which typically photosynthesize at higher temperatures, have photosynthetic rates that are two to three times higher than those of C3 plants. However, at lower temperatures, C3 photosynthesis is more efficient than that of C4 plants because the cost of photorespiration at those temperatures is usually less than the two extra ATPs required for the C4 pathway.

CAM Photosynthesis Crassulacean acid metabolism (CAM) photosynthesis is found in plants of about 30 families, includ- ing cacti, stonecrops, orchids, bromeliads, and other succulents that are often stressed by limited availability of water. A few succulents do not have CAM photosynthesis, however, and several nonsucculent plants do. Many CAM plants are facultative C3 plants that can switch to C3 photosynthesis during the day after a good rain or when night temperatures are high. Plants with CAM photosynthesis typically do not have a well-defined palisade mesophyll in the leaves, and, in contrast to the chloroplasts of the bundle sheath cells of C4 plants, those of CAM photosynthesis plants resemble the mesophyll cell chloro- plasts of C3 plants.

CAM photosynthesis is similar to C4 photosynthesis in that 4-carbon compounds are produced during the light–independent reactions. In these plants, however, the organic acids (mainly malic acid) accumulate at night and break down during the day, re- leasing carbon dioxide. The enzyme PEP carboxylase is responsible for converting the carbon dioxide plus PEP to organic acids at night when the stomata are open. During daylight, the organic acids diffuse out of the cell vacuoles in which they are stored and are converted back to carbon dioxide for use in the Calvin cycle. A much larger amount of carbon dioxide can be converted to carbohydrate each day than would otherwise be possible, since the stomata of such plants are closed during the day to conserve water. This arrangement allows the plants to function well under conditions of both limited water supply and high light intensity (Fig. 10.13).

Other Significant Processes That Occur in Chloroplasts

In addition to photosynthesis, there are two very important sets of biochemical reactions that take place in chloroplasts (and also in the proplastids of roots).

  1. Sulfates are reduced to sulfide via several steps involving ATP and enzymes. The sulfide is rapidly converted into impor- tant sulfur-containing amino acids, such as methionine and cysteine, which are part of the building blocks for proteins, anthocyanin pigments, chlorophylls, and several other cellular components.
  2. Nitrates are reduced to organic compounds. Initially, the nitrates are reduced to nitrites in the cytoplasm. The nitrites are then transported into chloroplasts (or root proplastids) where, through several enzyme-mediated steps, they are converted to ammonia. The ammonia is then converted to amino groups that are integral parts of several important amino acids such as glutamine and aspartic acid. Glutamine is an important form of nitrogen storage in roots or specialized stems such as those of carrots, beets, and potato tubers.


The solar energy that is converted into biochemical energy by the process of photosynthesis today is stored in various organic compounds such as wood, while coal and oil contain energy originally captured by green organisms in the geological past. If the organic compounds are burned, the energy is released very rapidly in the form of heat and light, and much of the usable energy is lost. Living organisms, however, “burn” their energy-containing compounds in numerous, small, enzyme-controlled steps that release tiny amounts of immediately usable energy. The released energy is usually stored in ATP molecules, which allows the available energy to be used more efficiently and the process to be controlled more precisely.

The Essence of Respiration

Respiration is essentially the release of energy from glucose molecules that are broken down to individual carbon dioxide molecules. The process takes place in all active cells 24 hours a day, regardless of whether or not photosynthesis happens to be occurring simultaneously in the same cells. It is initiated in the cytoplasm and completed in the mitochondria. The energy, stored in chemical bonds containing high-energy electrons, is released from simple sugar molecules that are broken down during a series of steps controlled by enzymes. No oxygen is needed to initiate the process, but in aerobic respiration (the most widespread form of respiration), the process cannot be completed without oxygen gas (O2). The controlled release of energy is the significant event; carbon dioxide (CO2) and water (H2O) are the by-products. Aerobic respiration can be summed up in the following equation, but bear in mind that respiration, like photosynthesis, is a complex process that in- volves many steps not reflected in a simplified equation.

C6H12O6 + 6O2 enzymes 6CO2 + 6H2O + energy glucose                      oxygen   ¾¾®      carbon                     water


Anaerobic respiration and fermentation are two forms of respiration that were probably carried on in the geological past when there was no oxygen in the atmosphere. These forms of respiration are still carried on today by certain bacteria and other organisms in the absence of oxygen gas. Anaerobic respiration and fermentation release less than 6% of the energy re- leased from a molecule of glucose by aerobic respiration. The two forms differ from one another in the manner in which hy- drogen released from the glucose is combined with other substances (see the discussion on p. 183). Fermentation is very im- portant industrially, particularly in the brewing and baking industries. Two well-known forms of fermentation are illustrated by the following equations:

C6H12O6 enzymes 2C2H5OH + 2CO2 + energy (ATP) glucose    õõõã          ethyl                       carbon

alcohol        dioxide

C6H12O6    enzymes 2C3H6O3 + energy (ATP)

glucose õõõã lactic acid

The relatively small amount of energy released during these inefficient forms of respiration is partly stored in two ATP molecules. The actual amount of energy stored is roughly only 29% of the approximately 48 Kcals6 of energy released in anaerobic respiration.

Introduction to the Major Steps of Respiration


In most forms of carbohydrate respiration, the first major phase takes place in the cytoplasm and requires no oxygen gas (O2). This phase, called glycolysis, involves three main steps and several smaller ones, each controlled by an enzyme. During the process, a small amount of energy is released, and some hydrogen atoms are removed from compounds derived from a glu- cose molecule. The essence of this complex series of steps is as follows:

  1. In a series of reactions, the glucose molecule becomes a fructose molecule carrying two phosphates (P).
  2. This sugar (fructose) molecule is split into two 3-carbon fragments called glyceraldehyde 3-phosphate (GA3P).
  3. Some hydrogen, energy, and water are removed from these 3-carbon fragments, leaving pyruvic acid (Fig. 10.14).

Two ATP molecules supply the energy needed to start the process of glycolysis. By the time pyruvic acid has been formed, however, four ATP molecules have been produced from the energy released along the way, for a net gain of two ATP molecules. A great deal of the energy originally in the glucose molecule remains in the pyruvic acid. The hydrogen ions and high-energy electrons released during the process are picked up and temporarily held by an acceptor molecule, NAD (nicotinamide adenine dinucleotide). What happens to them next depends on the kind of respiration involved: aerobic respi- ration, true anaerobic respiration, or fermentation.

Aerobic Respiration

In aerobic respiration (the most common type of respiration), glycolysis is followed by two major stages: the citric acid cycle and electron transport. Both stages occur in the mitochondria and involve many smaller steps, each of which is controlled by enzymes (see Fig. 10.14).

The Citric Acid (Krebs) Cycle The citric acid cycle was originally named the Krebs cycle after Hans Krebs, a British biochemist who received a Nobel Prize in 1953 for his unraveling of many of the complex reactions that take place in respira- tion. The name citric acid cycle, or tricarboxylic acid (TCA) cycle, reflects the important role played by several organic acids during the process.

Before entering the citric acid cycle, which takes place in the fluid matrix located within the compartments formed by the cristae of mitochondria (see Fig. 3.13), carbon dioxide is released from pyruvic acid that was produced by glycolysis. What remains is restructured to a 2-carbon acetyl group. This acetyl group combines with an acceptor molecule called coen- zyme A (CoA). This combination (called acetyl CoA) then enters the citric acid cycle, which is a series of biochemical reac- tions that are catalyzed by enzymes. Little of the energy originally trapped in the glucose molecule is released during glyco- lysis. As the citric acid cycle proceeds, however, high-energy electrons and hydrogen are successively removed. This re- moval takes place from a series of organic acids and, after transfer, ultimately produces compounds such as NADH (re- duced nicotinamide adenine dinucleotide) and FADH2 (reduced flavin adenine dinucleotide), as well as a small amount of ATP. Carbon dioxide is produced as a by-product while the cycle is proceeding.

Electron Transport Much of the energy originally in the glucose molecule now has been transferred to the acceptors NAD and FAD, which became NADH and FADH2, respectively. NADH and FADH2 are electron donors to an electron transport system consisting of special acceptor molecules arranged in a precise sequence on the inner membrane of mitochondria. The electrons flow through a series of carrier molecules, many of which are part of protein complexes, down an energy gradient. Some of these electron carriers also accept protons and release them to the intermembrane space of the mitochondrion. Shut- tling of protons in this way causes a build-up of protons outside the mitochondrial matrix, thereby establishing an electro- chemical gradient. Through the process of chemiosmosis, additional protein complexes couple the transport of protons back into the matrix with phosphorylation of ADP to form ATP. The production of ATP stops if there are no electron donors or electron acceptor oxygen.

The acceptor molecules include iron-containing proteins called cytochromes. Energy is released in small increments at each step along the system, and ATP is produced from ADP and P. As the final step in aerobic respiration (see Fig. 10.14), oxygen acts as the ultimate electron acceptor, producing water as it combines with hydrogen.

By the time the process is complete, the recoverable energy locked in a molecule of glucose has been released and is stored in ATP molecules. This stored energy is then available for use in the synthesis of other molecules and for growth, active transport, and a host of other metabolic processes. Aerobic respiration produces a net gain of 36 ATP molecules from one glucose molecule, using up six molecules of oxygen and producing six molecules of carbon dioxide and a net total of six molecules of water. For each mole (180 grams) of glucose aerobically respired, 686 Kcal of energy is re- leased, with about 39% of it being stored in ATP molecules and the remainder being released as heat.

Anaerobic Respiration and Fermentation

In living organisms, glucose molecules often may undergo glycolysis without enough oxygen being available to complete

aerobic respiration. In such cases, the hydrogen released during glycolysis is simply transferred from the hydrogen acceptor molecules back to the pyruvic acid after it has been formed, creating ethyl alcohol in some organisms, and lactic acid or simi- lar substances in others. A little energy is released during either fermentation or true anaerobic respiration, but most of it re- mains locked up in the alcohol, lactic acid, or other compounds produced.

In true anaerobic respiration, the hydrogen removed from the glucose molecule during glycolysis is combined with an inorganic ion, as, for example, when sulfur bacteria (discussed in Chapter 17) convert sulfate (SO4) to sulfur (S) or another sulfur compound or when certain cellulose bacteria produce methane gas (CH4) by combining the hydrogen with carbon di- oxide.

Oxygen gas is not required to make these compounds, but few organisms can live long without oxygen, and many that carry on fermentation can also respire aerobically. If oxygen becomes available, the remaining energy can be released by further breakdown of these compounds. About 7% of the total energy in a glucose molecule is removed during anaerobic respiration or fermentation. So much of that energy goes into the making of the alcohol or the lactic acid or is dissipated as heat that there is a net gain of only two ATP molecules (compared with 36 ATP molecules produced in aerobic respiration). The forms of anaerobic respiration are adaptive to the organisms that have them in that they recycle NAD and allow glycoly- sis to continue.

Living cells can tolerate only certain concentrations of alcohol. In media in which yeasts are fermenting sugars, for exam- ple, once the alcohol concentration builds up beyond 12%, the cells die and fermentation ceases. This is why most wines have an alcohol concentration of about 12% (24 proof).

Many bacteria carry on both fermentation and true anaerobic respiration simultaneously, making it difficult to distinguish between the two processes. Some texts use the terms anaerobic respiration and fermentation interchangeably to designate respiration occurring in the presence of little or no oxygen gas.

Factors Affecting the Rate of Respiration


Temperature plays a major role in the rate at which the various respiratory reactions occur. For example, when air tempera- tures rise from 20°C (68°F) to 30°C (86°F), the respiration rates of plants double and sometimes even triple. The faster respi- ration occurs, the faster the energy is released from sugar molecules, with an accompanying decrease in weight. In growing plants, this weight loss is more than offset by the production of new sugar by photosynthesis. In harvested fruits, seeds, and vegetables, however, respiration continues without sugar replacement, and some water loss also occurs. Respiring cells con- vert energy stored as starch or sugar primarily to ATP, but much of the energy is lost in the form of heat, with only 39% be- ing stored as ATP. Most fresh foods are kept under refrigeration, not only to lower the respiration rate and retard water loss, but also to dissipate the heat. Keeping the temperatures down is also important to prevent the growth and reproduction of food-spoiling molds and bacteria, which may thrive at warmer temperatures.

Heat inactivates most enzymes at temperatures above 40°C (104°F), but a few organisms, such as various cyanobacteria and algae in the hot springs of Yellowstone National Park and similar places, have adapted in such a way that they are able to thrive at temperatures exceeding 60°C (140°F)—heat that would kill other organisms of comparable size almost instantly.


Water inside the cells and their organelles act as a medium in which the enzymatic reactions can take place. Living cells often have a water content of more than 90%, but the cells of mature seeds may have a water content of less than 10%. When water content becomes this low, respiration does not cease completely, but it continues at a drastically reduced rate, resulting in only very tiny amounts of heat being released and of carbon dioxide being given off. Seeds may remain viable (capable of germinat- ing) for many years if stored under dry conditions. If they come in contact with water, however, they swell by imbibition. Respi- ration rates then increase rapidly. If the wet seeds happen to be in an unrefrigerated storage bin, the temperature may increase to the point of killing the seeds. In fact, if fungi and bacteria begin to grow on the seeds, temperatures from their respiration can become so high that spontaneous combustion can sometimes occur.


If flooding sharply reduces the oxygen supply available to the roots of trees and house plants, their respiration and growth rates may be decreased. They may even die if the condition persists too long. When foods are stored, however, it helps to bring about lower rates of respiration by reducing the oxygen in the storage areas. In fact, it is a common commercial practice to reduce the oxygen present in warehouses where crops are stored. The oxygen content is reduced to as little as 1% to 3% by pumping in nitrogen gas, while maintaining low temperatures and humidity. Oxygen concentration is not reduced below 1% because that can result in an undesirable increase in fermentation.

3. A Closer Look at Respiration

Respiration, like photosynthesis, is a very complex process, and, as with photosynthesis, it is beyond the scope of this book to explore the subject in great detail. The following amplification of information already discussed is modest, and those who wish further information are referred to the reading list at the end of the chapter.

Glycolysis Reexamined

As previously discussed, this initial phase of all forms of respiration brings about the conversion of each 6-carbon glucose molecule to two 3-carbon pyruvic acid molecules via three main steps, each mediated by enzymes. The three main steps are as follows:

  1. Phosphorylation, whereby the 6-carbon sugars receive phosphates
  2. Sugar cleavage, which involves the splitting of 6–carbon fructose into two 3-carbon sugar fragments
  3. Pyruvic acid formation, which involves the oxidation of the sugar fragments

Energy needed to initiate the process is furnished by an ATP molecule, which also furnishes the phosphate group for the phos- phorylation of the sugar glucose to yield glucose 6-phosphate. Another ATP, with the aid of the enzyme fructokinase, yields fruc- tose bisphosphate (fructose 1,6-diphosphate). As a result of the cleavage of the fructose bisphosphate, two different 3-carbon sugars are produced, but ultimately, only two glyceraldehyde 3-phosphate (GA3P) molecules remain. These two 3-carbon sugars are oxi- dized to two 3-carbon acids, and, in the successive production of several of these acids, phosphate groups are removed from the acids. The phosphate groups combine with ADP, producing a net direct gain of two ATP molecules during glycolysis. In addition, hydrogen is removed as GA3P is oxidized. This hydrogen is picked up by the acceptor molecule, NAD, which becomes NADH. Glycolysis, which requires no oxygen gas, is summarized in Figure 10.14.

Transition Step to the Citric Acid (Krebs) Cycle

Before a pyruvic acid molecule enters the citric acid cycle, which takes place in the mitochondria, a molecule of carbon dioxide is removed and a molecule of NADH is produced, leaving an acetyl fragment. The 2-carbon fragment is then bonded to a large mole- cule called coenzyme A. Coenzyme A consists of a combination of the B vitamin pantothenic acid and a nucleotide. Pantothenic acid is one of several B vitamins essential to respiration in both plants and animals; others include thiamine (vitamin B1), niacin, and riboflavin. The bonded acetyl fragment and coenzyme A molecule is referred to as acetyl CoA. The following equation summarizes the fate of the two pyruvic acid molecules following glycolysis and leading to the citric acid cycle:

2 pyruvic acid + 2 CoA + 2 NAD õõõõã 2 acetyl CoA + 2 NADH + 2CO2

In addition to pyruvic acid, fats and amino acids can also be converted to acetyl CoA and enter the process at this point. The NADH molecules donate their hydrogen to an electron transport system (discussed in the section, “Electron Transport and Oxidative Phosphorylation”), and the acetyl CoA enters the citric acid cycle (see Fig. 10.14).

The Citric Acid (Krebs) Cycle Reexamined

In the citric acid cycle, acetyl CoA is first combined with oxaloacetic acid, a 4-carbon compound, producing citric acid, a 6- carbon compound. The citric acid cycle is kept going by oxaloacetic acid, which is produced in small amounts, but is an inter- mediate product rather than a starting substance or an end product of the cycle. As the cycle progresses, a carbon dioxide is removed, producing a 5-carbon compound. Then another carbon dioxide is removed, producing a 4-carbon compound. This 4- carbon compound, through additional steps, is converted back to oxaloacetic acid, the substance with which the cycle began, and the cycle is repeated.

Each full cycle uses up a 2-carbon acetyl group and releases two carbon dioxide molecules while regenerating an ox- aloacetic acid molecule for the next turn of the cycle. Some hydrogen is removed during the process and is picked up by FAD and NAD. One molecule of ATP, three molecules of NADH, and one molecule of FADH2 are produced for each turn of the cycle. The citric acid cycle may be summarized as follows:

oxaloacetic + acetyl + ADP + P + 3 NAD + FAD õõõõã acid         CoA

oxaloacetic + CoA + ATP + 3 NADH + H+ + FADH2 + 2CO2 acid

The hydrogen carried by NAD and FAD can mostly be traced to the acetyl groups and to water molecules added to some compounds in the citric acid cycle. The FAD and FADH2 are now known to be intermediate compounds. Ubiquinol, a com- ponent of the electron transport system, receives electrons from either NADH or FADH2.

Electron Transport and Oxidative Phosphorylation

After completion of the citric acid cycle, the glucose molecule has been totally dismantled, and some of its energy has been transferred to ATP molecules. A considerable portion of the energy was transferred to NAD and FAD when they were used to pick up hydrogen and electrons from the molecules derived from glucose as they were broken down during glycolysis and the citric acid cycle. This energy is released as the hydrogen and electrons are passed along an electron transport system. This sys- tem, like the electron transport system of photosynthesis, functions something like a high-speed bucket brigade in passing along electrons from their source to their destination. Several of the electron carriers in the transport system are cytochromes. They are very specific and, as electrons flow along the system, they can transfer their electrons only to other specific acceptors. When the electrons reach the end of the system, they are picked up by oxygen and combine with hydrogen ions, forming water.

Part of the energy that is released during the movement of electrons along the electron transport system can be used to make ATP in a process called oxidative phosphorylation. If hydrogen ion and electron transport begins with NADH, which was produced inside the mitochondria (i.e., during the conversion of pyruvic acid to acetyl CoA and during the citric acid cycle), enough energy is produced to yield three ATP molecules from each NADH molecule. Similarly, if hydrogen ion and electron transport begins either with FADH2 or with NADH, produced outside the mitochondria (i.e., during glycolysis), two ATP molecules are produced.

The manner in which ATP is produced during the operation of the respiratory electron transport system involves essen- tially the same chemiosmotic concept that was applied earlier to proton movement across thylakoid membranes.

The chemiosmosis theory concerning electron transport and proton movement across membranes was proposed in the 1960s by Peter Mitchell, a British biochemist, and is now widely accepted as the explanation for the movements in both pho- tosynthesis and respiration. In respiration, oxidative phosphorylation is energized by a gradient of protons (H+) that flow by chemiosmosis across the inner membrane of a mitochondrion. Mitchell, who received a Nobel Prize for his work in 1978, surmised that protons are “pumped” from the matrix of the mitochondria to the region between the two membranes (see Fig. 3.13) as electrons flow from their source in NADH molecules along the electron transport system, which is located in the inner membrane. The protons are believed to “diffuse” back into the matrix via channels provided by an enzyme complex known as the F1 particle (an ATPase), releasing energy that is used to synthesize ATP.

If we retrace our steps through the entire process of aerobic respiration, we find that glycolysis yields four -molecules of ATP and two molecules of NADH (from which more molecules of ATP are formed), for a total of eight ATP from the con- version of glucose to two pyruvic acid molecules. Two ATP are used in the process, however, leaving a net gain of six ATP.

When two pyruvic acid molecules are converted to two acetyl CoA in the mitochondria, two more NADH molecules (which will generate six molecules of ATP) are produced. The two acetyl CoA molecules metabolized in the citric acid cycle yield two molecules of ATP, two molecules of FADH2 (from which four ATP are formed), and six molecules of NADH (which cause the formation of 18 molecules of ATP), making a citric acid cycle total of 24 ATP. A grand total of 36 ATP is produced for the aerobic respiration of one glucose molecule (Table 10.1). The 36 ATP molecules represent about 39% of the energy originally present in the glucose molecule. The remaining energy is lost as heat or is unavailable. Aerobic respiration is still about 18 times more efficient than anaerobic respiration.

A condensed comparison between photosynthesis and respiration is shown in Table 10.2.


While photosynthesis and respiration are the main processes through which plants grow, develop, reproduce, and survive, there are many additional processes that contribute toward these activities. Most of these use intermediate steps, but they could not function without photosynthesis and respiration. Some of the essential compounds produced from additional path- ways include sugar phosphates and nucleotides, nucleic acids, amino acids, proteins, chlorophylls, cytochromes, carotenoids, fatty acids, oils, and waxes.

Metabolic processes not required for normal growth and development are generally referred to as secondary metabolism. Although not essential, many of the products from secondary metabolism enable plants to survive and persist under special conditions. These products provide the plant with unique colors, aromas, poisons, and other compounds that may attract or deter other organisms or give them a competitive edge in nature. Humans have exploited many secondary compounds from plants for medicinal, culinary, or other purposes. It has been estimated that 50,000 to 100,000 such compounds exist in plants with only a few thousand of these thus far having been identified. Secondary metabolic products may be derived from modi- fication of amino acids and related compounds to produce alkaloids or through specialized conversions such as the shikimic acid pathway (phenolics) and mevalonic acid pathway (terpenoids). Examples of these compounds are shown in Table 10.3. Lignin, which is a component of secondary cell walls, is, for example, synthesized through the shikimic acid pathway. Be- cause it is hard to digest and is toxic to some predators, it protects plants from herbivorous animals.


Sugars produced through photosynthesis may undergo many transformations. Some sugars are used directly in respiration, but others not needed for that purpose may be transformed into lipids, proteins, or other carbohydrates. Among the most im- portant carbohydrates produced from simple sugars are sucrose, starch, and cellulose. Much of the organic matter produced through photosynthesis is eventually used in the building of protoplasm and cell walls. This conversion process is called as- similation.

When photosynthesis is taking place, sugar may be produced faster than it can be used or transported away to other parts of the plant. When this happens, the excess sugar may be converted to large, insoluble molecules, such as starch or oils, tem- porarily stored in the chloroplasts and then later changed back to a soluble form that is transported to other cells. The conver- sion of starch and other insoluble carbohydrates to soluble forms is called digestion (Fig. 10.15). The process is nearly al- ways one of hydrolysis, in which water is taken up and, with the aid of enzymes, the links of the chains of simple sugars that comprise the molecules of starch and similar carbohydrates are broken by the addition of water. The disaccharide malt sugar (maltose), for example, is transformed to two molecules of glucose, with the aid of an enzyme (maltase), by the addition of one molecule of water, as follows:


C12H22O11 + H2O   maltase   2C6H12O6

maltose water (enzyme) glucose

Fats are broken down to their component fatty acids and glycerol, and proteins are digested to their amino acid building blocks in similar fashion. Digestion is carried on in any cell where there may be stored food, with very little energy being released in the process. In animals, special digestive organs also play a role in digestion, but plants have no such additional “help” in the process. In both plants and animals, digestion within cells is similar and is a normal part of metabolism.

Except in insect-trapping plants, digestion takes place within plant cells where the carbohydrates, fats, or proteins are stored, while in animals, digestion usually occurs outside of the cells in the digestive tract. Apart from the location, the proc- ess is essentially similar in plants and animals.


  1. Enzymes catalyze reactions of metabolism. Many of these include oxidation-reduction reactions. Oxidation is loss of electrons; reduction is gain of electrons.
  2. Photosynthesis is an anabolic process that combines carbon dioxide and water in the presence of light with the aid of chlo- rophyll; oxygen is a by-product. All life depends on photosynthesis, which takes place in chloroplasts.
  3. Carbon dioxide constitutes 0.037% of the atmosphere, but the percentage has been rising in recent years. Increased car- bon dioxide levels have potential to elevate global temperatures through the “greenhouse effect.”
  4. Chlorophyll b and carotenoids are antenna pigments that direct light energy to chlorophyll a. Photosynthetic units con- taining chlorophylls and accessory pigments absorb units of light energy, become excited, and pass this energy to accep- tors during the light-dependent reactions of photosynthesis.
  5. During the light-dependent reactions of photosynthesis, which occur in thylakoid membranes of chloroplasts, water molecules are split, and oxygen gas is released. Hydrogen ions and electrons are released from water and transferred to produce NADPH and ATP.
  6. The two types of photosynthetic units present in most chloroplasts are photosystems I and II. The events that take place in photosystem II come before those of photosystem I. Each photosystem has a reaction-center molecule of chlorophyll a that boosts electrons to a higher energy level when it is excited by light energy.
  7. Photosystem II boosts electron excitation to a level that, when it encounters photosystem I, has the potential to reduce NADP to NADPH through noncyclic electron flow. Photosystem I, by itself, can cycle electrons for generation of ATP. Electron transport while the photosystems are operating and proton movement across thylakoid membranes are both in- volved in ATP production.
  8. The light-independent reactions occur through a series of reactions known as the Calvin cycle, which takes place in the stroma of chloroplasts. In the first step, carbon dioxide is combined with RuBP through catalytic action of the enzyme rubisco to form two molecules of the 3-carbon compound, GA3P. The ATP and NADPH from the light-dependent reac- tions furnish energy to eventually convert GA3P to 6-carbon carbohydrates. This cycle also regenerates RuBP to enable continued carbon fixation.
  9. In the light-independent reactions of C4 plants, 4-carbon oxaloacetic acid is initially produced instead of 3-carbon PGA. In the leaf mesophyll of C4 plants, there are large chloroplasts, which contain rubisco in the bundle sheaths, and small

chloroplasts, which contain higher concentrations of PEP carboxylase that facilitate the conversion of carbon dioxide to carbohydrate at much lower concentrations than is possible in C3 plants.

  1. CAM photosynthesis occurs in succulent plants whose stomata are closed and admit little CO2 during the day. Regular photosynthesis occurs as the 4-carbon compounds that accumulate at night are converted back to carbon dioxide during the day.
  2. Respiration is a catabolic process that takes place in the cytoplasm and mitochondria of cells. The energy is released, with the aid of enzymes, from simple sugar and organic acid molecules.
  3. In aerobic respiration, stored energy release requires oxygen; CO2 and water are by-products of the process.
  4. Anaerobic respiration and fermentation do not require oxygen gas, and much less energy is released. The remaining energy is in the ethyl alcohol, lactic acid, or other such substances produced. Some released energy is stored in ATP molecules. Temperature, available water, and environmental oxygen affect respiration rates.
  5. Glycolysis, which occurs in the cytoplasm, requires no molecular oxygen; two phosphates are added to a 6-carbon sugar molecule, and the prepared molecule is split into two 3-carbon sugars (GA3P). Some hydrogen, energy, and water are removed from the GA3P, producing pyruvic acid. There is a net gain of two ATP molecules. Hydrogen ions and elec- trons released during glycolysis are picked up by NAD, which becomes NADH.
  6. In aerobic respiration, which occurs in the mitochondria, pyruvic acid loses some CO2, is restructured, and becomes ace- tyl CoA. Energy, CO2, and hydrogen are removed from the acetyl CoA in the citric acid cycle, which involves enzyme- catalyzed reactions of a series of organic acids.
  7. NADH passes the hydrogen gained during glycolysis and the citric acid cycle along an electron transport system; small increments of energy are released and partially stored in ATP molecules, and the hydrogen is combined with oxygen gas, forming water in the final step of aerobic respiration.
  8. Hydrogen removed from glucose during glycolysis is combined with an inorganic ion in anaerobic respiration. The hy- drogen is combined with the pyruvic acid or one of its derivatives in fermentation. Both processes occur in the absence of oxygen gas, with only about 7% of the total energy in the glucose molecule being released, for a net gain of two ATP molecules.
  9. Two molecules of NADH and two ATP molecules are gained during glycolysis when two 3-carbon pyruvic acid mole- cules are produced from a single glucose molecule. Another molecule of NADH is produced when the pyruvic acid molecule is restructured and becomes acetyl CoA prior to entry into the citric acid cycle.
  10. In the citric acid cycle, acetyl CoA combines with 4-carbon oxaloacetic acid, producing first a 6-carbon compound, next a 5-carbon compound, and then several 4-carbon compounds. The last 4-carbon compound is oxaloacetic acid. Two CO2 molecules are also released during this process.
  11. Some hydrogen removed during the citric acid cycle is picked up by FAD and NAD; one molecule of ATP, three mole- cules of NADH, and one molecule of FADH2 are produced during one complete cycle. Energy associated with electrons and/or with hydrogen picked up by NAD and FAD is gradually released as the electrons are passed along the electron transport system; some of this energy is transferred to ATP molecules during oxidative phosphorylation.
  12. Energy used in ATP synthesis during oxidative phosphor-ylation is believed to be derived from a gradient of protons formed across the inner membrane of a mitochondrion, while electrons are moving in the electron transport system by chemiosmosis.
  13. Altogether, 38 ATP molecules are produced during the complete aerobic respiration of one glucose molecule; two are used to prime the process, so there is a net gain of 36 ATP molecules.
  14. In addition to photosynthesis and respiration, other metabolic pathways are required for growth, development, reproduc- tion, and survival. Essential products of additional pathways include nucleotides, proteins, chlorophylls, and fatty acids. Secondary metabolites include alkaloids, phenolics, and terpenoids.
  15. Conversion of sugar produced by photosynthesis to fats, proteins, complex carbohydrates, and other substances is termed assimilation. Digestion takes place within plant cells with the aid of enzymes. During digestion, large insoluble mole- cules are broken down by hydrolysis to smaller soluble forms that can be transported to other parts of the plant.

ps 3 – reaction centers

Two photosynthetic reaction centers are arranged in tandem in photosynthesis of algae and plants

In green algae about eight photons are required (quantum requirement: pho- tons absorbed per molecule O2 produced) for the photosynthetic water split- ting (section 2.4). Instead of the term quantum requirement, one often uses the reciprocal term quantum yield (molecules of O2 produced per photon absorbed). According to the color of irradiated light (action spectrum) the quantum yield dropped very sharply when algae were illuminated with red light above a wavelength of 680 nm (Fig. 3.14). This effect, named “red drop,” remained unexplained since algae contain chlorophyll, which absorbs light at 700 nm. Robert Emerson and coworkers (USA) solved this problem in 1957 when they observed that the quantum yield in the spectral range above 680 nm increased dramatically when algae were illuminated with orange light (650 nm) and red light simultaneously. Then the quantum yield was higher than the sum of both yields when irradiated separately with the light of each wavelength. This Emerson effect led to the conclusion that two differ- ent reaction centers are involved in photosynthesis of green algae (and also of cyanobacteria and higher plants). In 1960 Robert Hill (Cambridge, UK) postulated a reaction scheme (Fig. 3.15) in which two reaction centers are

O2 release in green algae


Z scheme
noncyclic electron transport
photosynthetic complexes

Figure 3.17 Schematic presentation of the localization of the photosynthetic complexes and the H-ATP synthase in the thylakoid membrane. Transport of electrons between PS II and the cytochrome-b6/f complex is mediated by plastohydroquinone (PQH2), and that between the cytochrome-b6/f complex and PS I by plastocyanin (PC). Water splitting occurs on the luminal side of the membrane, and the formation of NADPH and ATP on the stromal side. The electrochemical gradient of protons pumped into the lumen drives ATP synthesis. The number of protons transported to the lumen during electron transport and the proton requirement of ATP synthesis is not known (section 4.4).

Arranged in tandem and connected by an electron transport chain containing cytochrome-b6 and cytochrome-f (cytochrome-f is a cytochrome of the c type; see section 3.7). Light energy of 700 nm was sufficient for the excitation of reaction center I, whereas excitation of the other reaction center II required light of higher energy with a wavelength of 680 nm. The electron flow accord- ing to the redox potentials of the intermediates shows a zigzag, leading to the name Z scheme. The numbering of the two photosystems corresponds to the sequence of their discovery. Photosystem II (PS II) can use light up to a wave- length of 680 nm, whereas photosystem I (PS I) can utilize light with a wave- length up to 700 nm. The sequence of the two photosystems makes it possible that at PS II a very strong oxidant is generated for the oxidation of water and at PS I a very strong reductant is produced for the reduction of NADP (see also Fig. 3.3).

Figure 3.16 gives an overview of electron transport through the photo- synthetic complexes; the carriers of electron transport are drawn according to their electric potential (see also Fig. 3.11). Figure 3.17 shows how the photosynthetic complexes are arranged in the thylakoid membrane. There is a potential difference of about 1.2 volt between the process of water oxi- dation and NADP reduction. The absorbed photons of 680 and 700 nm together correspond to a total potential difference of 3.45 volt (see section 2.2, equation 2.7). Thus, only about one-third of the energy of the pho- tons absorbed by the two photosystems is used to transfer electrons from

water to NADP. In addition to this, about one-eighth of the light energy absorbed by the two photosystems is conserved by pumping protons into the lumen of the thylakoids via PS II and the cytochrome-b6/f complex (Fig. 3.17). This proton transport leads to the formation of a proton gradi- ent between the lumen and the stroma sp ace. An H-ATP synthase, also located in the thylakoid membrane, uses the energy of the proton gradient to synthesize ATP. Thus about half the absorbed light energy of the two photosystems is not used for chemical work but is dissipated as heat. The significance of the loss of energy as heat during photosynthetic electron transport has been discussed in section 2.3.

Water is split by photosystem II

The groups of Horst Witt and Wolfgang Saenger (both in Berlin) resolved the three-dimensional structure of PS II by X-ray structure analysis of crys- tals from the PS II of the thermophilic cyanobacteria Thermosynechococcus elongatis. The subsequent X-ray structure analysis of PS I revealed that PS II and PS I are constructed after the same basic principles as the reaction

centers of purple bacteria (section 3.4). This, and sequence analyses, clearly demonstrate that all these photosystems have a common origin. Thus PS II also has a chl-a pair in the center, although the distance between the two molecules is so large that probably only one of the two chl-a molecules reacts with the exciton. Two arms, each with one chl-a and one pheophy- tin molecule, are connected with this central pair as in the purple bacteria shown in Figure 3.10. Also in the cyanobacteria, only one of these arms appears to be involved in the electron transport.

photosystem II complex

In contrast to the bacterial reaction center the excitation of the reaction center results in an electron transfer via the chl-a monomer to pheophytin (Phe), and from there to a tightly bound plastoquinone (QA), thus forming a semiquinone radical (Fig. 3.18). The electron is then further transferred to a loosely bound plastoquinone (QB). This  plastoquinone  (PQ)  (Fig. 3.19) accepts two electrons and two protons one after the other and is thus reduced to  hydroquinone  (PQH2).  The  hydroquinone  is  released  from the photosynthesis complex and may be regarded as the final product of photosystem II. This sequence, consisting of a transfer of a single electron between (chl-a)2 and QA and the transfer of two electrons between QA and QB, corresponds to the reaction sequence shown for Rb. sphaeroides (Fig. 3.11). The only difference is that the quinones are ubiquinone or menaqui- none in bacteria and plastoquinone in photosystem II.

However, the similarity between the reaction sequence in PS II and the photosystem of the purple bacteria applies only to the electron acceptor region. The electron donor function in PS II of plants is completely differ- ent from that in purple bacteria. The electron deficit in (chl-a)2 caused by non-cyclic electron transport is compensated for by electrons derived from the oxidation of water. In the transport of electrons from water to chloro- phyll manganese cations and a tyrosine residue are involved. The (chl-a)2 radical with a redox potential of about 1.1 volt is such a strong oxidant that it can withdraw an electron from a tyrosine residue in the protein of the reaction center and a tyrosine radical remains. This reactive tyrosine residue is often designated as Z. The electron deficit in the tyrosine radical is restored by oxidation of a manganese ion (Fig. 3.20). The PS II com- plex contains several manganese ions, probably four, which are close to each other. This arrangement of Mn ions is called the Mn cluster. The Mn cluster depicts a redox system that can take up and release four electrons. During this process the Mn ions probably change between the oxidation state Mn3 and Mn4. To liberate one molecule of O2 from water, the reaction center must with- draw four electrons and thus capture four excitons. The time differences between the capture of the single exciton in the reaction center depends on the intensity of illumination. If oxidation of water were to proceed stepwise, oxygen radicals could be formed as intermediary products, especially at low light intensities. Oxygen radicals have a destructive effect on biomolecules such as lipids and proteins (section 3.10). The water splitting machinery of the Mn clusters minimizes the formation of oxygen radical intermediates by  supplying the reaction center via tyrosine with four electrons one after the other (Fig. 3.20). The Mn cluster is transformed during this transfer from the ground oxidation state stepwise to four different oxidation states (these have been designated as S0 and S1–S4).

water splitting

Experiments by Pierre Joliot (France) and Bessel Kok (USA) presented evidence that the water splitting apparatus can be in five different oxida- tion states (Fig. 3.21). When chloroplasts that were kept in the dark were then illuminated by a series of light pulses, an oscillation of the oxygen release was observed. Whereas after the first two light pulses almost no O2 was released, the O2 release was maximal after three pulses and then after a further four pulses, and so on. An increasing number of light pulses, how- ever, dampened the oscillation. This can be explained by pulses that do not cause excitation of PS II and thus desynchronize the oscillation. In dark- ened chloroplasts the water splitting apparatus is in the S1 state. After the fourth oxidation state (S4) has been reached, O2 is released in one reaction and the Mn cluster returns to its ground oxidation state (S0). During this reaction, protons from water are released to the lumen of the thylakoids. The formal description of this reaction is:

equation water spolitting

Figuratively speaking, the four electrons needed in the reaction center are loaned in advance by the Mn cluster and then repaid at one stroke by oxidizing water to synthesize one oxygen molecule. In this way the Mn clus- ter minimizes the formation of oxygen radicals in photosystem II. Despite this safety device, still some oxygen radicals are formed in the PS II com- plex which damage the proteins of the complex. The consequences will be discussed in section 3.10.

Photosystem II complex is very similar to the reaction center in purple bacteria

Photosystem II is a complex consisting of at least 20 different subunits (Table 3.2), only two of which are involved in the actual reaction center. For the sake of simplicity the scheme of the PS II complex shown in Fig.

3.22 contains only some of these subunits. The PS II complex is surrounded by an antenna consisting of light harvesting complexes (Fig. 2.13).

The center of the PS II complex is a heterodimer consisting of the sub- units D1 and D2 with six chl-a, two pheophytin, two plastoquinone, and one to two carotenoid molecules bound to it. The D1 and D2 proteins are homologous to each other and also to the L proteins and M proteins from the reaction center of the purple bacteria (section 3.4). As in purple bacteria, only the pheophytin molecule bound to the D1 protein of PS II is involved in electron transport. QA is bound to the D2 protein, whereas QB is bound to the D1 protein. The Mn cluster is probably enclosed by both the D1 and D2 proteins. The tyrosine that is reactive in electron transfer is a constituent

of D1. The subunits O, P, Q stabilize the Mn cluster. The two subunits CP 43 and CP 47 (CP means chlorophyll protein) each bind about 15 chloro- phyll molecules and form the core complex of the antenna shown in Figure

2.10. CP 43 and CP 47 flank both sides of the D1-D2 complex. Cyt-b559 does not seem to be involved in the electron transport of PS II; possibly its func- tion is to protect the PS II complex from light damage. The inner and outer light harvesting complexes of LHC II are arranged at the periphery.

The D1 protein of the PS II complex has a high turnover; it is constantly being resynthesized. It seems that the D1 protein wears out during its func- tion, perhaps through damage by oxygen radicals, which still occurs despite all the protection mechanisms. It has been estimated that the D1 protein is replaced after 106 to 107 catalytic cycles of the PS II reaction center.

A number of compounds that are similar in their structure to plasto- quinone can block the plastoquinone binding site at the D1 protein, caus- ing inhibition of photosynthesis. Such compounds are used as weed killers (herbicides). Before the effect of these compounds is discussed in detail, some general aspects of the application of herbicides shall be introduced.

Table protein components

Table 3.4: Protein components of photosystem I (list not complete)

photosystem II complex

Mechanized agriculture usually necessitates the use of herbicides

About 50% of the money spent worldwide for plant protection is expended for herbicides. The high cost of labor is one of the main reasons for using herbicides in agriculture. It is cheaper and faster to keep a field free of weeds by using herbicides rather than manual labor. Weed control in agriculture is necessary not only to decrease harvest losses by weed competition, but also because weeds hinder the operation of harvesting machineries; fields free of weeds are a prerequisite for a mechanized agriculture. The herbicides usu- ally block a specific reaction of the plant metabolism and have a low toxic- ity for animals and humans. A large number of herbicides (examples will be given at the end of this section) inhibit photosystem II by being antagonists to plastoquinone. To achieve substantial inhibition the herbicide molecule has to bind to most of the many photosynthetic reaction centers. To be effective, 125 to 4,000 g of these herbicides have to be applied per hectare.

In an attempt to reduce the amount of herbicides applied to the soil, new efficient herbicides have been developed that inhibit key biosynthetic processes such as the synthesis of fatty acids, amino acids (sections 10.1 and 10.4), carotenoids, or chlorophyll. There are also herbicides that act as analogues of phytohormones or mitosis inhibitors. Some of these herbi- cides are effective with amounts as low as 5 g per hectare.

Some herbicides are taken up only by the roots and  others  by  the leaves. To keep the railway tracks free of weeds, nonselective herbicides are employed, which destroy the complete vegetation. Nonselective herbicides are also used in agriculture, e.g., to combat weeds in citrus plantations. In the latter case, herbicides are applied that are only taken up by the leaves to combat herbaceous plants at the ground level. Especially interesting are selec- tive herbicides that combat only weeds and effect cultivars as little as possible (sections 12.2 and 15.3). Selectivity can be due to different uptake efficien- cies of the herbicide in different plants, different sensitivities of the metabo- lism towards the herbicide, or different ability of the plants to detoxify the herbicide. Important mechanisms that plants utilize to detoxify herbicides and other foreign compounds (xenobiotics) are the introduction of hydroxyl groups by P-450 monooxygenases (section 18.2) and the formation of glu- tathione conjugates (section 12.2). Selective herbicides have the advantage that weeds can be destroyed at a later growth stage of the cultivars where the dead weeds form a mulch layer conserving water and preventing erosion.

In some cases, the application of herbicides has led to the evolution of herbicide-resistant plant mutants (section  10.4).  Conventional  breeding has used such mutated plants to generate herbicide-resistant cultivars. In contrast to the occurrence of herbicide resistance by accidental mutations, nowadays genetic engineering is employed on a very large scale to generate cultivars which are resistant to a certain herbicide, allowing weed control in the presence of the growing cultivar (section 22.6)

A large number of herbicides inhibit photosynthesis: the urea deriva- tive DCMU (Diuron, DuPont), the triazine Atrazine (earlier Ciba Geigy), Bentazon (BASF) (Fig. 3.23), and many similar compounds function as herbicides by binding to the plastoquinone binding site on the D1 pro- tein and thus blocking the photosynthetic electron transport. Nowadays, DCMU is not often used, as the required dosage is high and its degradation is slow. It is, however, often used in the laboratory to inhibit photosynthe- sis in an experiment (e.g., of leaves or isolated chloroplasts). Atrazine acts selectively: maize plants are relatively insensitive to this herbicide since they have a particularly efficient mechanism for its detoxification (section 12.2). Because of its relatively slow degradation in the soil, the use of Atrazine has been restricted in some countries, e.g., Germany. In areas where cer- tain herbicides have been used continuously over the years, some weeds have become resistant to these herbicides. In some cases, the resistance can be traced back to mutations resulting in a single amino acid change in the D1-proteins. These changes do not markedly affect photosynthesis of these weeds, but they do decrease binding of the herbicides to the D1-protein.

The cytochrome-b6/f complex mediates electron transport between photosystem II and  photosystem I Iron atoms in cytochromes and in iron-sulfur centers have a central function as redox carriers


Cytochromes occur in all organisms except a few obligate  anaerobes. These are proteins to which one to two tetrapyrrole rings  are  bound. These tetrapyrroles are very similar to the chromophores of chlorophylls. However, chlorophylls contain Mg as the central atom in the tetrapyr- role, whereas the cytochromes have an iron atom (Fig. 3.24). The tetrapy- rrole ring of the cytochromes with iron as the central atom is called the heme. The bound iron atom can change between the oxidation states Fe and Fe so that cytochromes function as a one-electron-carrier, in con- trast to quinones, NAD(P) and FAD, which transfer two electrons together with protons.

Cytochromes are divided into three main groups, the cytochromes-a, –b, and –c. These correspond to heme-a, –b, and –c. Heme-b may be regarded as the basic structure (Fig. 3.24). In heme-c the -SH-group of a cysteine is added to each of the two vinyl groups of heme-b. In this way heme-c is

covalently bound by a sulfur bridge to the protein of the cytochrome. Such a mode of covalent binding has already been shown for phycocyanin in Figure 2.15, and there is actually a structural relationship between the correspond- ing apoproteins. In heme-a (not shown) an isoprenoid side chain consisting of three isoprene units is attached to one of the vinyl groups of heme-b. This side chain functions as a hydrophobic membrane anchor, similar to that found in quinones (Figs. 3.5 and 3.19). Heme-a is mentioned here only for the sake of completeness. It plays no role in photosynthesis, but it does have a function in the mitochondrial electron transport chain (section 5.5).


The iron atom in the heme can form up to six coordinative bonds. Four of these bonds are formed with the nitrogen atoms of the tetrapyrrole ring. This ring has a planar structure. The two remaining bonds of the Fe atom coordinate with two histidine residues, which are positioned vertically to the tetrapyrrole plane (Fig. 3.25). Cyt-f (f  foliar, in leaves) contains, like cyt-c, one heme-c and therefore belongs to the c-type cytochromes. In cyt-f one bond of the Fe atom coordinates with the terminal amino group of the protein and the other with a histidine residue.

Iron-sulfur centers are of general importance as electron carriers in elec- tron transport chains and thus also in photosynthetic electron transport. Cysteine residues of proteins within iron-sulfur centers (Fig. 3.26) are coor- dinatively or covalently bound to Fe atoms. These iron atoms are linked to each other by S-bridges. Upon acidification of the proteins, the sulfur between the Fe atoms is released as H2S and for this reason it has been called labile sulfur. Iron-sulfur centers occur mainly as 2Fe-2S or 4Fe-4S centers. The Fe atoms in these centers are present in the oxidation states

Fe and Fe. Irrespective of the number of Fe atoms in a center, the oxidized and reduced state of the center differs only by a single charge. For this reason, iron-sulfur centers can take up and transfer only one electron. Various iron-sulfur centers have very different redox potentials, depending on the surrounding protein.

axial ligands
copper Cu anion

The electron transport by the cytochrome-b6/f complex is coupled to a proton transport

Plastohydroquinone (PQH2) formed by PS II diffuses through the lipid phase of the thylakoid membrane and transfers its electrons to the cytochrome-b6/f complex (Fig. 3.17). This complex then transfers the electrons to plastocy- anin, which is thus reduced. Therefore the cytochrome-b6/f complex has also been called plastohydroquinone-plastocyanin oxidoreductase. Plastocyanin is a protein with a molecular mass of 10.5 kDa, containing a copper atom, which is coordinatively bound to one cysteine, one methionine, and two histidine residues of the protein (Fig. 3.27). This copper atom alternates between the oxidation states Cu and Cu and thus is able to take up and transfer one electron. Plastocyanin is soluble in water and is located in the thylakoid lumen.

Electron transport through the cyt-b6/f complex proceeds along a poten- tial difference gradient of about 0.4 V (Fig. 3.16). The energy liberated by the transfer of the electron down this redox gradient is conserved by trans- porting protons to the thylakoid lumen. The cyt-b6/f complex is a mem- brane protein consisting of at least eight subunits. The main components of this complex are four subunits: cyt-b6, cyt-f, an iron-sulfur protein called Rieske protein after its discoverer, and a subunit IV. Additionally, there are some smaller peptides and a chlorophyll and a carotenoid of unknown function. The Rieske protein has a 2Fe-2S center with the very positive redox potential of 0.3 V, untypical of such iron-sulfur centers.

The cyt-b6/f complex has an asymmetric structure (Fig. 3.28). Cyt-b6 and subunit IV span the membrane. Cyt-b6 containing two heme-b molecules is almost vertically arranged to the membrane and forms a redox chain across

one iron-sulfur protein. The amino acid sequence of cyt-b in the cyt-b/c1 complex of bacteria and in mitochondria corresponds to the sum of the sequences of cyt-b6 and the subunit IV in the cyt-b6/f complex. Apparently during evolution the cyt-b gene was cleaved into two genes, for cyt-b6 and subunit IV. Whereas in plants the cyt-b6/f complex reduces plastocyanin, the cyt-b/c1 complex of bacteria and mitochondria reduces cyt-c. Cyt-c is a very small cytochrome molecule that is water-soluble and, like plasto- cyanin, transfers redox equivalents from the cyt-b6/f complex to the next complex along the aqueous phase. In cyanobacteria, which also possess a cyt-b6/f complex, the electrons are transferred from this complex to photo- system I via cyt-c instead of plastocyanin. The great similarity between the cyt-b6/f complex in plants and the cyt-b/c1 complexes in bacteria and mito- chondria suggests that these complexes have basically similar functions in photosynthesis and in mitochondrial oxidation: they are proton transloca- tors that are driven by a hydroquinone-plastocyanin (or -cyt-c) reductase.

The interplay of PS II and the cyt-b6/f complex electron transport causes the transport of protons from the stroma space to the thylakoid lumen. The principle of this transport is explained in the schematic presentations of Figures 3.28 and 3.29. A crucial point is that the reduction and oxida- tion of the quinone occur at different sides of the thylakoid membrane. The required protons for the reduction of PQ (Qb) by the PS II complex are taken up from the stroma space. Subsequently PQH2 diffuses across the lipid phase of the membrane to the binding site in the lumenal region of the cyt-b6/f complex where it is oxidized by the Rieske protein and cyt-f to yield reduced plastocyanin. The protons of this reaction are released into the thylakoid lumen. According to this scheme, the capture of four excitons by the PS II complex transfers four protons from the stroma space to the lumen. In addition four protons produced during water splitting by PS II are released into the lumen as well.


proton transport

The number of protons pumped through the cyt-b6/f complex can be doubled by a Q-cycle

Studies with mitochondria indicated that during electron transport through the cyt-b/c1 complex, the number of protons transferred per transported electron is larger than four (Fig. 3.29). Peter Mitchell (Great Britain), who established the chemiosmotic hypothesis of energy conservation (section 4.1), also postulated a so-called Q-cycle, by which the number of trans- ported protons for each electron transferred through the cyt-b/c1 complex is doubled. It later became apparent that the Q-cycle also has a role in pho- tosynthetic electron transport.

Figure 3.30 shows the principle of Q-cycle operation in the photosyn- thesis of chloroplasts. The cytb6/f complex contains two different bind- ing sites for conversion of quinones, one located at the stromal side and the other at the luminal side of the thylakoid membrane (Fig. 3.28). The plastohydroquinone (PQH2) formed in the PS II complex is oxidized by the Rieske iron-sulfur center at the binding site adjacent to the lumen. Due to its very positive redox potential, the Rieske protein tears off one electron from the plastohydroquinone. Because its redox potential is very negative, the remaining semiquinone is unstable and transfers its electron to the first heme-b of the cyt-b6 (bp) and from there to the other heme-b (bn), thus rais- ing the redox potential of heme bn to about –0.1 V. In this way a total of four protons are transported to the thylakoid lumen per two molecules of plastohydroquinone oxidized. Of the two plastoquinone molecules (PQ) formed, only one molecule returns to the PS II complex.

protons released

The other PQ dif- fuses away from the cyt-b6/f complex through the lipid phase of the mem- brane to the stromal binding site of the cyt-b6/f complex to be reduced via semiquinone to hydroquinone by the high reduction potential of heme-bn. This is accompanied by the uptake of two protons from the stromal space. The hydroquinone thus regenerated diffuses through the membrane back to the luminal binding site where it is oxidized in turn by the Rieske protein, and so on. In total, the number of transported protons is doubled by the Q-cycle (1/2  1/4  1/8  1/16  1/n  1). The fully operating Q-cycle transports four electrons through the cyt-b6/f complex which results in total to the transfer of eight protons from the stroma to the lumen. The func- tion of this Q-cycle in mitochondrial oxidation is now undisputed, while its function in photosynthetic electron transport is still a matter of contro- versy. The analogy of the cyt-b6/f complex to the cyt-b/c1 complex suggests that the Q-cycle also plays an important role in chloroplasts. So far, the operation of a Q-cycle in plants has been observed mainly under low light conditions. The Q-cycle is perhaps suppressed by a high proton gradient generated across the thylakoid membrane, for instance, by irradiation with high light intensity. In this way the flow of electrons through the Q-cycle could be adjusted to the energy demand of the plant cell.

Photosystem I reduces NADP

Plastocyanin that has been reduced by the cyt-b6/f complex diffuses through the lumen of the thylakoids, binds to a positively charged binding site of PS I, transfers its electron, and the resulting oxidized form diffuses back to the cyt-b6/f complex (Fig. 3.31).

Also the reaction center of PS I with an absorption maximum of 700 nm contains a chlorophyll pair (chl-a)2 (Fig. 3.31). As in PS II, the excitation caused by a photon reacts probably with only one of the two chlorophyll molecules. The resulting (chl-a)2 is then reduced by plastocyanin. It is assumed that (chl-a)2 transfers its electron to a chl-a monomer (A0), which then transfers the electron to a strongly bound phylloquinone (Q) (Fig. 3.32). Phylloquinone contains the same phytol side chain as chl-a and its function corresponds to QA in PS II. The electron is transferred from the semiphylloquinone to an iron-sulfur center named FX. FX is a 4Fe-4S center

reaction electron transport
photosystem I complex

with a very negative redox potential. It transfers one electron to two other 4Fe-4S centers (FA, FB), which in turn reduce ferredoxin, a protein with a molecular mass of 11 kDa with a 2Fe-2S center. Ferredoxin also takes up and transfers only one electron. The reduction occurs at the stromal side of the thylakoid membrane. For this purpose, the ferredoxin binds at a posi- tively charged binding site on subunit D of PS I (Fig. 3.33). The reduction of NADP by ferredoxin, catalyzed by ferredoxin-NADP reductase, yields NADPH as an end product of the photosynthetic electron transport.

The PS I complex consists of at least 17 different subunits, of which some are shown in Table 3.4. The center of the PS I complex is a heterodimer (as is the center of PS II) consisting of subunits A and B (Fig. 3.33). The molec- ular masses of A and B (each 82–83 kDa) correspond approximately to the sum of the molecular masses of the PS II subunits D1 and CP43, and D2 and CP47, respectively (Table 3.2). In fact, both subunits A and B have a dou- ble function. Like D1 and D2 in PS II, they bind chromophores (chl-a) and redox carriers (phylloquinone, FeX) of the reaction center and, additionally, they contain about 100 chl-a molecules as antennae pigments. Thus, the het- erodimer of A and B represents the reaction center and the core antenna as well. The three-dimensional structure of photosystem I in cyanobacteria, green algae and plants has been resolved. The principal structure of pho- tosystem I, with a central pair of chl-a molecules and two branches, each with two chlorophyll molecules, is very similar to photosystem II and to the bacterial photosystem (Fig. 3.10). It has not been definitely clarified whether both or just one of these branches are involved in the electron transport. The Fe-S-centers FA and FB are ascribed to subunit C, and subunit F is con- sidered to be the binding site for plastocyanin.

table protein
cyclic transport photosystem I

The light energy driving the cyclic electron transport of PS I is only utilized for the synthesis of ATP

Besides the noncyclic electron transport discussed so far, cyclic electron transfer can also take place in which the electrons from the excited pho- tosystem I are transferred back to the ground state of PS I, probably via the cyt-b6/f complex (Fig. 3.34). The energy thus released is used only for the synthesis of ATP, and NADPH is not formed. This electron transport is termed cyclic photophosphorylation. In intact leaves, and even in iso- lated intact chloroplasts, it is quite difficult to differentiate experimentally between cyclic and non-cyclic photophosphorylation. It has been a matter of debate as to whether and to what extent cyclic photophosphorylation occurs in a leaf under normal physiological conditions. Recent evaluations of the proton stoichiometry of photophosphorylation (see section 4.4) sug- gest that the yield of ATP in noncyclic electron transport is not sufficient for the requirements of CO2 assimilation, and therefore cyclic photophos- phorylation seems to be required to synthesize the lacking ATP. Moreover, cyclic photophosphorylation must operate at very high rates in the bundle sheath chloroplasts of certain C4 plants (section 8.4). These cells have a high demand for ATP and they contain high PS I activity but very little PSPresumably, the cyclic electron flow is governed by the redox state of theacceptor of the photosystem in such a way that by increasing the reduction of the NADP system, and consequently of ferredoxin, the diversion of the

electrons in the cycle is enhanced. The function of cyclic electron transport is probably to adjust the rates of ATP and NADPH formation according to the plant’s demand.

Despite intensive investigations, the pathway of electron flow from PS I to the cyt-b6/f complex in cyclic electron transport remains unresolved. It has been proposed that cyclic electron transport is structurally separated from the linear electron transport chain in a super complex. Most experi- ments on cyclic electron transport have been carried out with isolated thy- lakoid membranes that catalyze only cyclic electron transport when redox mediators, such as ferredoxin or flavin adenine mononucleotide (FMN, Fig. 5.16), have been added. Cyclic electron transport is inhibited by the antibiotic antimycin A. It is not clear at which site this inhibitor functions. Antimycin A does not inhibit noncyclic electron transport.

Surprisingly, proteins of the NADP dehydrogenase complex of the mitochondrial respiratory chain (section 5.5) have also been identified in the thylakoid membrane of chloroplasts. The function of these proteins in chloroplasts is still not known. The proteins of this complex occur very fre- quently in chloroplasts from bundle sheath cells of C4 plants, which have little PS II but a particularly high cyclic photophosphorylation activity (section 8.4). These observations raise the possibility that in cyclic electron transport the flow of electrons from NADPH or ferredoxin to plastoqui- none proceeds via a complex similar to the mitochondrial NADH dehydro- genase complex. As will be shown in section 5.5, the mitochondrial NADH dehydrogenase complex transfers electrons from NADH to ubiquinone. Results indicate that an additional pathway for a cyclic electron transport exists in which electrons are directly transferred via a plastoquinone reduct- ase from ferredoxin to plastoquinone.

In the absence of other acceptors electrons can be transferred from photosystem I to oxygen

When ferredoxin is very highly reduced, it is possible that electrons are

transferred from PS I to oxygen to form superoxide radicals (•O) (Fig.

3.35). This process is called the Mehler reaction. The superoxide radical reduces metal ions present in the cell such as Fe3 and Cu2 (Mn):

The hydroxyl radical (•OH) is a very aggressive substance and damages enzymes and lipids by oxidation. The plant cell has no protective enzymes against •OH. Therefore it is essential that a reduction of the metal ions be prevented by rapid elimination of •O by superoxide dismutase. But hydrogen peroxide (H2O2) also has a damaging effect on many enzymes. To prevent such damage, hydrogen peroxide is eliminated by an ascorbate

peroxidase located in the thylakoid membrane. Ascorbate, an important anti- oxidant in plant cells (Fig. 3.36), is oxidized by this enzyme and converted to the radical monodehydroascorbate, which is spontaneously reconverted by photosystem I to ascorbate via reduced ferredoxin. Monodehydroascorbate can be also reduced to ascorbate by an NAD(P)H-dependent monodehy- droascorbate reductase that is present in the chloroplast stroma and the cytosol.

As an alternative to the preceding reaction, two molecules of mono- dehydroascorbate can dismutate to ascorbate and dehydroascorbate. Dehydroascorbate is reconverted to ascorbate by reduction with glutath- ione in a reaction catalyzed by dehydroascorbate reductase present in the stroma (Fig. 3.37). Glutathione (GSH) occurs as an antioxidant in all plant

cells (section 12.2). It is a tripeptide composed of the amino acids glutamate, cysteine, and glycine (Fig. 3.38). Oxidation of GSH results in the forma- tion of a disulfide (GSSG) between the cysteine residues of two glutathione molecules. Reduction of GSSG is catalyzed by a glutathione reductase with NADPH as the reductant (Fig. 3.37).

The major function of the Mehler-ascorbate-peroxidase cycle is to dis- sipate excessive excitation energy of photosystem I as heat. The absorption of a total of eight excitons via PS I results in the formation of two super- oxide radicals and two molecules of reduced ferredoxin, the latter serving as a reductant for eliminating H2O2 (Fig. 3.35). The transfer of electrons to oxygen by the Mehler reaction is a reversal of the water splitting of PS

II. As will be discussed in the following section, the Mehler reaction occurs when ferredoxin is very highly reduced. The only gain of this reaction is the generation of a proton gradient from electron transport through PS II and the cyt-b6/f complex. This proton gradient can be used for the synthesis of ATP if ADP is present. But since there is usually a shortage in ADP under the conditions of the Mehler reaction, it mostly results in the formation of a high pH gradient. A feature common to the Mehler reaction and cyclic electron transport is that there is no net production of NADPH. For this reason, electron transport via the Mehler reaction has been termed pseudo- cyclic electron transport.

Yet another group of antioxidants was recently found in plants, the so- called peroxiredoxins. These proteins, comprising -SH groups as redox car- riers, have been known in the animal world for some time. Ten different peroxiredoxin genes have been identified in the model plant Arabidopsis. Peroxiredoxins, being present in chloroplasts as well as in other cell com- partments, differ from the aforementioned antioxidants glutathione and ascorbate in that they reduce a remarkably wide spectrum of peroxides, such as H2O2, alkylperoxides, and peroxinitrites. In chloroplasts, oxidized peroxiredoxins are reduced by photosynthetic electron transport of photo- system I with ferredoxin and thioredoxin as intermediates.

Instead of ferredoxin, PS I can also reduce methylviologen. Methylviologen, also called paraquat, is used commercially as a herbicide

(Fig. 3.39). The herbicidal effect is due to the reduction of oxygen to super- oxide radicals. Additionally, paraquat competes with dehydroascorbate for the reducing equivalents provided by photosystem I. Therefore, in the pres- ence of paraquat, ascorbate is no longer regenerated from dehydroascor- bate and the ascorbate peroxidase reaction can no longer proceed. The increased production of superoxide and decreased detoxification of hydro- gen peroxide in the presence of paraquat causes severe oxidative damage to mesophyll cells, noticeable by a bleaching of the leaves. In the past, paraquat has been used to destroy marijuana fields in South America.

Regulatory processes control the distribution of the captured photons between the two photosystems

Linear photosynthetic electron transport through the two photosystems requires the even distribution of the captured excitons between them. As discussed in section 2.4, the excitons are transferred preferentially to the chromophore which requires the least energy for excitation. Photosystem I (P700) being on a lower energetic level than PS II (Fig. 3.16) requires less energy for excitation than photosystem II (P680). In an unrestricted compe- tition between the two photosystems, excitons would primarily be directed to PS I. Due to this imbalance, the distribution of the excitons between the two photosystems must be regulated. The spatial separation of PS I and PS II and their antennae in the thylakoid membrane plays an important role in this regulation.

In chloroplasts, the thylakoid  membranes  are  present  in  two  differ- ent arrays, as stacked and unstacked membranes. The outer surface of the unstacked membranes has free access to the stromal space; these mem- branes are called stromal lamellae (Fig. 3.40). In the stacked membranes, the neighboring thylakoid membranes are in direct contact with each other. These membrane stacks can be seen as grains (grana) in light microscopy and are therefore called granal lamellae.

ATP synthase and the PS I complex (including its light harvesting com- plexes, not further discussed here) are located either in the stromal lamellae or in the outer membrane region of the granal lamellae. Therefore, these proteins have free access to ADP and NADP in the stroma. The PS II complex, on the other hand, is primarily located in the granal lamellae. Peripheral LHC II subunits attached to the PS II complex (section 2.4) contain a protein chain protruding from the membrane, which can prob- ably interact with the LHC II subunit of the adjacent membrane and thus

cause tight membrane stacking. The cyt-b6/f complexes are only present in stacked membranes. Since the proteins of PS I and F-ATP-synthase project into the stroma space, they do not fit into the space between the stacked membranes. Thus the PS II complexes in the stacked membranes are sepa- rated spatially from the PS I complexes in the unstacked membranes. It is assumed that this prevents an uncontrolled spillover of excitons from PS II to PS I.

However, the spatial separation of the two photosystems and thus the spillover of excitons from PS II to PS I can be regulated. For example, if the excitation of PS II is greater than that of PS I, plastohydroquinone accumulates, which cannot be oxidized rapidly enough via the cyt-b6/f com- plex by PS I. Under these conditions, a protein kinase is activated, which phosphorylates the hydroxyl groups of threonine residues of peripheral LHC II subunits, causing a conformational change of the LHC protein. As a result of this, the affinity to PS II is decreased and the LHC II subunits dissociate from the PS II complexes. Furthermore, due to the changed con- formation, LHC II subunits can now bind to PS I, mediated by the H sub- unit of PS II. This LHC II-PS I complex purposely increases the spillover of excitons from LHC II to PS I. In this way the accumulation of reduced plastoquinone decreases the excitation of PS II and enhances the excitation of PS I. A protein phosphatase facilitates the reversal of this regulation. This regulatory process, which has been simplified here, enables an opti- mized distribution of the captured photons between the two photosystems, independent of the spectral quality of the absorbed light.

Excess light energy is eliminated as heat

Plants face the general problem that the energy of irradiated light can be much higher than the demand of photosynthetic metabolites such as NADPH and ATP. This is the case when very high light intensities are present and the metabolism cannot keep pace. Such a situation arises at low temperatures, when the metabolism is slowed down because of decreased enzyme activities (cold stress) or at high temperatures, when stomata close to prevent loss of water. Excess excitation of the photosystems could result in an excessive reduction of the components of the photosynthetic electron transport.

Very high excitation of photosystem II, recognized by the accumulation of plastohydroquinone, results in damage to the photosynthetic appara- tus, termed photoinhibition. A major cause of this damage is an overexcita- tion of the reaction center, by which chlorophyll molecules attain a triplet state, resulting in the formation of aggressive singlet oxygen (section 2.3). The damaging effect of triplet chlorophyll can be demonstrated by placing

a small amount of chlorophyll under the human skin, which after illumina- tion causes severe tissue damage. This photodynamic principle is utilized in medicine for the selective therapy of skin cancer. Carotenoids (e.g., carotene, Fig. 2.9) are able to convert the triplet state of chlorophyll and the singlet state of oxygen to the corresponding ground states by forming a triplet carotenoid, which dissipates its energy as heat. In this way carotenoids have an important protective function. If under certain conditions this protective function of carotenoids is una- ble to cope with excessive excitation of PS II, the remaining singlet oxy- gen has a damaging effect on the PS II complex. The site of this damage could be the D1 protein of the photosynthetic reaction center in PS II, which already under normal photosynthetic conditions experiences a high turnover (see section 3.6). When the rate of D1-protein damage exceeds the rate of its resynthesis, the rate of photosynthesis is decreased, resulting in photoinhibition.

Plants have developed several mechanisms to protect the photosynthetic apparatus from light damage. One mechanism is chloroplast avoidance movement, in which chloroplasts move under high light conditions from the cell surface to the side walls of the cells. Another way is to dissipate the energy arising from an excess of excitons as heat. This process is termed nonphotochemical quenching of exciton energy. Although our knowledge of this quenching process is still incomplete, it is undisputed that zeaxan- thin plays an important role. Zeaxanthin causes the dissipation of exciton energy to heat by interacting with a chlorophyll-binding protein (CP 22) of photosystem II. Zeaxanthin is formed by the reduction of the diepoxide vio- laxanthin. The reduction proceeds with ascorbate as the reductant and the monoepoxide antheraxanthin is formed as an intermediate. Zeaxanthin can be reconverted to violaxanthin by epoxidation which requires NADPH and O2 (Fig. 3.41). Formation of zeaxanthin by diepoxidase takes place on the luminal side of the thylakoid membrane at an optimum pH of 5.0, whereas the regeneration of violaxanthin by the epoxidase proceeding at the stro- mal side of the thylakoid membrane occurs at about pH 7.6. Therefore, the formation of zeaxanthin requires a high pH gradient across the thylakoid membrane. As discussed in connection with the Mehler reaction (section 3.9), a high pH gradient can be an indicator of the high excitation state of photosystem II. When there is too much excitation energy, an increased pH gradient initiates zeaxanthin synthesis, dissipating excess energy of the PS II complex as heat. This mechanism explains how under strong sunlight most plants convert 50% to 70% of all the absorbed photons to heat. The non-photochemical quenching of excitation energy is the primary way for plants to protect themselves from too much light energy. In comparison, the Mehler reaction (section 3.9) and photorespiration (section 7.7) under

Mahler reaction

Figure 3.35 A schemefor the Mehler reaction.Upon strong reduction offerredoxin, electrons aretransferred by the Mehlerreaction to oxygen andsuperoxide is formed. Theelimination of this highlyaggressive superoxideradical involves reactionscatalyzed by superoxidedismutase and ascorbateperoxidase

release of excited electron
photosystem protein complexes

elker and Or: Soil Hydrology and Biophysics

Transport in Plants

ps 2 – electron transport

Photosynthesis is an electron transport process

The previous chapter described how photons are captured by an antenna and conducted as excitons to the reaction centers. This chapter deals with the function of these reaction centers and describes how photon energy is converted to chemical energy to be utilized by the cell. As mentioned in Chapter 2, plant photosynthesis probably evolved from bacterial pho- tosynthesis, so that the basic mechanisms of the photosynthetic reactions are alike in bacteria and plants. Bacteria have proved to be very suitable objects for studying the principles of photosynthesis since their reaction centers are more simply structured than those of plants and they are more easily isolated. For this reason, first bacterial photosynthesis and then plant photosynthesis will be described.

The photosynthetic machinery is constructed from modules

The photosynthetic machinery of bacteria is constructed from defined com- plexes, which also appear as components of the photosynthetic machinery in plants. As will be described in Chapter 5, some of these complexes are also components of mitochondrial  electron  transport.  These  complexes can be thought of as modules that developed at an early stage of evolu- tion and have been combined in various ways for different purposes. For easier understanding, the functions of these modules in photosynthesis will be treated first as black boxes and a detailed description of their structure and function will be given later.

the photosynthetic apparatus of purple bacteria

E °

Figure 3.1 Schematic presentation of the photosynthetic apparatus of purple bacteria. The energy of a captured exciton in the reaction center elevates an electron to a negative redox state. The electron is transferred to the ground state via an electron transport chain  including the cytochrome-b/c1 complex and cytochrome-c (the latter is not shown). Free energy of this process is conserved by formation of a proton potential which is used partly for synthesis of ATP and partly to enable an electron flow for the formation of NADH from electron donors such as H2S.


Oxidation state shows the total number of electrons which have been removed from an element (a positive oxidation state) or added to an element (a negative oxidation state) to get to its present state.

  • Oxidation involves an increase in oxidation state
  • Reduction involves a decrease in oxidation state

Section 6.1 – An Introduction to Oxidation Reduction Reactions (10:27)

Section 6.2 – Oxidation Numbers (22:09)

Section 6.4 – Voltaic Cells (24:17)

0.3: Electrochemical Potential

n a galvanic cell, current is produced when electrons flow externally through the circuit from the anode to the cathode because of a difference in potential energy between the two electrodes in the electrochemical cell. In the Zn/Cu system, the valence electrons in zinc have a substantially higher potential energy than the valence electrons in copper because of shielding of the s electrons of zinc by the electrons in filled d orbitals. Hence electrons flow spontaneously from zinc to copper(II) ions, forming zinc(II) ions and metallic copper. Just like water flowing spontaneously downhill, which can be made to do work by forcing a waterwheel, the flow of electrons from a higher potential energy to a lower one can also be harnessed to perform work….

Redox reactions can be balanced using the half-reaction method. The standard cell potential is a measure of the driving force for the reaction. \(E°_{cell} = E°_{cathode} − E°_{anode} \] The flow of electrons in an electrochemical cell depends on the identity of the reacting substances, the difference in the potential energy of their valence electrons, and their concentrations. The potential of the cell under standard conditions (1 M for solutions, 1 atm for gases, pure solids or liquids for other substances) and at a fixed temperature (25°C) is called the standard cell potential (E°cell). Only the difference between the potentials of two electrodes can be measured. The potential of the standard hydrogen electrode (SHE) is defined as 0 V under standard conditions. The potential of a half-reaction measured against the SHE under standard conditions is called its standard electrode potential. By convention, all tabulated values of standard electrode potentials are listed as standard reduction potentials. The overall cell potential is the reduction potential of the reductive half-reaction minus the reduction potential of the oxidative half-reaction (E°cell = E°cathode − E°anode). The standard cell potential is a measure of the driving force for a given redox reaction. If E°cell is positive, the reaction will occur spontaneously under standard conditions. If E°cell is negative, then the reaction is not spontaneous under standard conditions, although it will proceed spontaneously in the opposite direction. 

Cyclic Electron Transport in Photosynthesis

Photosynthetic reaction center

The reaction center is in the thylakoid membrane. It transfers light energy to a dimer of chlorophyll pigment molecules near the periplasmic (or thylakoid lumen) side of the membrane. This dimer is called a special pair because of its fundamental role in photosynthesis. This special pair is slightly different in PSI and PSII reaction center. In PSII, it absorbs photons with a wavelength of 680 nm, and it is therefore called P680. In PSI, it absorbs photons at 700 nm, and it is called P700. In bacteria, the special pair is called P760, P840, P870, or P960. “P” here means pigment, and the number following it is the wavelength of light absorbed.

If an electron of the special pair in the reaction center becomes excited, it cannot transfer this energy to another pigment using resonance energy transfer. In normal circumstances, the electron should return to the ground state, but, because the reaction center is arranged so that a suitable electron acceptor is nearby, the excited electron can move from the initial molecule to the acceptor. This process results in the formation of a positive charge on the special pair (due to the loss of an electron) and a negative charge on the acceptor and is, hence, referred to as photoinduced charge separation. In other words, electrons in pigment molecules can exist at specific energy levels. Under normal circumstances, they exist at the lowest possible energy level they can. However, if there is enough energy to move them into the next energy level, they can absorb that energy and occupy that higher energy level. The light they absorb contains the necessary amount of energy needed to push them into the next level. Any light that does not have enough or has too much energy cannot be absorbed and is reflected. The electron in the higher energy level, however, does not want to be there; the electron is unstable and must return to its normal lower energy level. To do this, it must release the energy that has put it into the higher energy state to begin with. This can happen various ways. The extra energy can be converted into molecular motion and lost as heat. Some of the extra energy can be lost as heat energy, while the rest is lost as light. (This re-emission of light energy is called fluorescence.) The energy, but not the e- itself, can be passed onto another molecule. (This is called resonance.) The energy and the e- can be transferred to another molecule. Plant pigments usually utilize the last two of these reactions to convert the sun’s energy into their own.

This initial charge separation occurs in less than 10 picoseconds (10−11 seconds). In their high-energy states, the special pigment and the acceptor could undergo charge recombination; that is, the electron on the acceptor could move back to neutralize the positive charge on the special pair. Its return to the special pair would waste a valuable high-energy electron and simply convert the absorbed light energy into heat. In the case of PSII, this backflow of electrons can produce reactive oxygen species leading to photoinhibition.[1][2] Three factors in the structure of the reaction center work together to suppress charge recombination nearly completely.

  • Another electron acceptor is less than 10 Å away from the first acceptor, and so the electron is rapidly transferred farther away from the special pair.
  • An electron donor is less than 10 Å away from the special pair, and so the positive charge is neutralized by the transfer of another electron
  • The electron transfer back from the electron acceptor to the positively charged special pair is especially slow. The rate of an electron transfer reaction increases with its thermodynamic favorability up to a point and then decreases. The back transfer is so favourable that it takes place in the inverted region where electron-transfer rates become slower.[1]

Thus, electron transfer proceeds efficiently from the first electron acceptor to the next, creating an electron transport chain that ends if it has reached NADPH.


Purple bacteria have only one reaction center (Fig. 3.1). In this reac- tion center the energy of the absorbed photon excites an electron, which will be elevated to a negative redox state. The excited electron is transferred back to the ground state by an electron transport chain, called the cyto- chrome-b/c1 complex, and the released energy is transformed to a chemical compound (NADH), which is then used for the synthesis of biomass (e.g., proteins and carbohydrates). Generation of energy is based on coupling the electron transport with the transport of protons across the membrane. In this way the energy of the excited electron is conserved as an electrochemical H-potential across the membrane. The photosynthetic reaction cent- ers and the main components of the electron transport chain are always located in a membrane.

Via ATP-synthase the energy of the H-potential is used to synthesize ATP from ADP and phosphate. Since the excited electrons in purple bacte- ria return to the ground state of the reaction center, this electron transport is called cyclic electron transport. In purple bacteria the proton gradient is also used to reduce NAD via an additional electron transport chain named the NADH dehydrogenase complex (Fig. 3.1). By consuming the energy of the H-potential, electrons are transferred from a reduced compound (e.g., organic acids or hydrogen sulfide) to NAD. The ATP and NADH formed by bacterial photosynthesis are used for the synthesis of organic matter; especially important is the synthesis of carbohydrates from CO2 via the Calvin cycle (see Chapter 6).

The reaction center of green sulfur bacteria (Fig. 3.2) is homologous to that of purple bacteria, indicating that they have both evolved from a com- mon ancestor. ATP is also formed in green sulfur bacteria by cyclic electron transport. The electron transport chain (cytochrome-b/c1 complex) and the ATP-synthase involved here are very similar to those in purple bacteria. However, in contrast to purple bacteria, green sulfur bacteria are able to synthesize NADH by a noncyclic electron transport process. In this case, the excited electrons are transferred to the ferredoxin-NAD-reductase complex, which reduces NAD to NADH. Since the excited electrons in this noncyc- lic pathway do not return to the ground state, an electron deficit remains in the reaction center and is replenished by electron donors such as H2S, ulti- mately being oxidized to sulfate.

Cyanobacteria and plants use water as an electron donor in photosyn thesis (Fig. 3.3). As oxygen is liberated, this process is called oxygenic pho- tosynthesis. Two photosystems designated II and I are arranged in tandem. The machinery of oxygenic photosynthesis is built by modules that have already been described in bacterial photosynthesis. The structure of the reaction center of photosystem II corresponds to that of the reaction center of purple bacteria, and that of photosystem I corresponds to the reaction center of green sulfur bacteria. The enzymes ATP synthase and ferredoxin- NADP-reductase are very similar to those of photosynthetic bacteria. The

Figure 3.2 Schematic presentation of the photosynthetic apparatus in green sulfur bacteria. In contrast to the scheme in Figure 3.1, part of the electrons elevated to a negative redox state is transferred via an electron transport chain (ferredoxin- NAD reductase) to NAD, yielding NADH. The electron deficit arising in the reaction center is compensated by electron donors such as H2S (see also Fig. 3.1).

Figure 3.3 Schematic presentation of the photosynthetic apparatus of cyanobacteria and plants. The two sequentially arranged reaction centers correspond in their function to the photosynthetic reaction centers of purple bacteria and green sulfur bacteria (shown in Figs. 3.1 and 3.2).

electron transport chain of the cytochrome-b6/f complex has the same basic structure as the cytochrome-b/c1 complex in bacteria.

Four excitons are required in oxygenic photosynthesis to split one mol- ecule of water:

H2O  NADP  4 excitons → 1/2 O2  NADPH  H

In this noncyclic electron transport, electrons are transferred to NADP and protons are transported across the membrane to generate a proton gradient that drives the synthesis of ATP. Thus, for each mol of NADPH formed by oxygenic photosynthesis, about 1.5 molecules of ATP are gener- ated simultaneously (section 4.4). Most of this ATP and NADPH are used for CO2 and nitrate assimilation to synthesize carbohydrates and amino acids. Oxygenic photosynthesis in plants takes place in the chloroplasts, a cell organelle of the plastid family (section 1.3).


Redox Reactions

o-Oxidation and reduction review from biological point-of-view | Biomolecules | MCAT | Khan Academy

Oxidation and reduction in cellular respiration | Biology | Khan Academy

A reductant and an oxidant are formed during photosynthesis

In the 1920s Otto Warburg (Berlin) postulated that the energy of light is transferred to CO2 and that the CO2, activated in this way, reacts with water to form a carbohydrate, accompanied by the release of oxygen. According to this hypothesis, the oxygen released by photosynthesis was derived from the CO2. In 1931 this hypothesis was opposed by Cornelis van Niel (USA) by postulating that during photosynthesis a reductant is formed, which then reacts with CO2. The so-called van Niel equation describes photosynthesis in the following way: He proposed that a compound H2A is split by light energy into a reduc- ing compound (2H) and an oxidizing compound (A). For oxygenic photo- synthesis of cyanobacteria or plants, it can be rewritten as:

equation redox CO2 2H2A + light
equation redox

In this equation the oxygen released during photosynthesis is derived from water. In 1937 Robert Hill (Cambridge, UK) proved that a reductant is actu- ally formed in the course of photosynthesis. He was the first to succeed in isolating chloroplasts with photosynthetic activity, which, however, had no intact envelope membranes and consisted only of thylakoid membranes. When these chloroplasts were illuminated in the presence of Fe3 com- pounds (initially ferrioxalate, later ferricyanide ([Fe  (CN)6]3)),  Robert Hill observed an evolution of oxygen accompanied by the reduction of the Fe3-compounds to the Fe2 form.

Fe equation

This “Hill reaction” proved that the photochemical splitting of water can be separated from the reduction of the CO2. Therefore the complete reac- tion of photosynthetic CO2 assimilation can be divided into two reactions:

  • The so-called light reaction, in which water is split by photon energy to yield reductive power (NADPH) and chemical energy (ATP); and
  • the so-called dark reaction (Chapter 6), in which CO2 is assimilated at the expense of the reductive power and of ATP.

In 1952 the Dutchman Louis Duysens made a very important observa- tion that helped explain the mechanism of photosynthesis. When illumi- nating isolated membranes of the purple bacterium Rhodospirillum rubrum with short light pulses, he found a decrease in light absorption at 890 nm, which was immediately reversed when the bacteria were darkened again. The same “bleaching” effect was found at 870 nm in the purple bacte- rium Rhodobacter sphaeroides. Later, Bessil Kok (USA) and Horst Witt (Germany) also found similar pigment bleaching at 700 nm and 680 nm in chloroplasts. This bleaching was attributed to the primary reaction of pho- tosynthesis, and the corresponding pigments of the reaction centers were named P870 (Rb. sphaeroides) and P680 and P700 (chloroplasts). When an oxidant (e.g., [Fe(CN)6]3) was added, this bleaching effect could also be achieved in the dark. These results indicated that these absorption changes of the pigments were due to a redox reaction. This was the first indication that chlorophyll can be oxidized. Electron spin resonance measurements revealed that radicals are formed during this “bleaching.” “Bleaching” could also be observed at the very low temperature of 1 K. This showed that in the electron transfer leading to the formation of radicals, the reac- tion partners are located so close to each other that thermal oscillation of the reaction partners (normally the precondition for a chemical reaction) is not required for this redox reaction. Spectroscopic measurements indicated that the reaction partner of this primary redox reaction are two closely adjacent chlorophyll molecules arranged as a pair, called a “special pair.”

The basic structure of a photosynthetic reaction center has been resolved by X-ray structure analysis

The reaction centers of purple bacteria proved to be especially suitable objects for explaining the structure and function of the photosynthetic machinery. It was a great step forward when in 1970 Roderick Clayton (USA) developed a method for isolating reaction centers from purple bacte- ria. Analysis of the components of the reaction centers of the different purple bacteria (shown in Table 3.1 for the reaction center of Rhodobacter sphaer- oides as an example) revealed that the reaction centers had the same basic structure in all the purple bacteria investigated. The minimum structure

consists of the three subunits L, M, and H (light, medium, and heavy). Subunits L and M are peptides with a similar amino acid sequence. They are homologous. The reaction center of Rb. sphaeroides contains four bac- teriochlorophyll-a (Bchl-a, Fig. 3.4) and two bacteriopheophytin-a (Bphe- a). Pheophytins differ from chlorophylls in that they lack magnesium as the central atom. In addition, the reaction center contains an iron atom that is not part of a heme. It is therefore called a non-heme iron. Furthermore, the reaction center is comprised of two molecules of ubiquinone (Fig. 3.5), which are designated as QA and QB. QA is tightly bound to the reaction center, whereas QB is only loosely associated with it.


Figure 3.5 Ubiquinone. The long isoprenoid side chain mediates the lipophilic character and membrane anchorage.

X-ray structure analysis of the photosynthetic reaction center

If ordered crystals can be prepared from a protein, it is possible to analyze the spherical structure of the protein molecule by X-ray structure analysis. In this method a protein crystal is exposed to X-ray irradiation. The elec- trons of the atoms in the molecule cause a scattering of X-rays. Diffraction is observed when the irradiation passes through a regular repeating struc- ture. The corresponding diffraction pattern, consisting of many single reflections, is measured by an X-ray film positioned behind the crystal or by an alternative detector. The principle is demonstrated in Figure 3.6. To obtain as many reflections as possible, the crystal, mounted in a capillary, is rotated. From a few dozen to up to several hundred exposures are required for one set of data, depending on the form of the crystal and the size of the crystal lattice. To evaluate a new protein structure, several sets of data are required in which the protein has been changed by the incorporation or binding of a heavy metal ion. With the help of elaborate computer pro- grams, it is possible to reconstruct the spherical structure of the exposed protein molecules by applying the rules for scattering X-rays by atoms of various electron densities. slowly and the diffraction pattern is monitored on an X-ray film. Nowadays much more sensitive detector systems (image platers) are used instead of films. The diffraction pattern shown was obtained by the structural analysis of the reaction center of

Rb. sphaeroides. (Courtesy of H. Michel, Frankfurt.)

X-ray structure analysis requires a highly technical expenditure and is very time-consuming, but the actual limiting factor in the elucidation of a spherical structure is usually the preparation of suitable single crystals. Until 1980 it was thought to be impossible to prepare crystals suitable for X-ray structure analysis from hydrophobic membrane proteins. The appli- cation of the detergent N,N-dimethyldodecylamine-N-oxide (Fig. 3.7) was a great step forward in helping to solve this problem. This detergent forms water-soluble protein-detergent micelles with  membrane  proteins.  With the addition of ammonium sulfate or polyethylene glycol is was then pos- sible to crystallize membrane proteins. The micelles form a regular lattice in these crystals (Fig. 3.8). The protein in the crystal remains in its native state since the hydrophobic regions of the membrane protein, which nor- mally border on the hydrophobic membrane, are covered by the hydropho- bic chains of the detergent.

Using this procedure, Hartmut Michel (Munich) succeeded in obtaining crystals from the reaction center of the purple bacterium Rhodopseudomonas viridis and, together with his colleagues Johann Deisenhofer and Robert Huber, performed an X-ray structure analysis of these crystals. The immense amount of time invested in these investigations is illustrated by the fact that the evaluation of the stored data sets alone took two and a half years (nowa- days modern computer programs would do it very much faster). The X-ray structure analysis of a photosynthetic reaction center successfully elucidated for the first time the three-dimensional structure of a membrane protein. For this work, Michel, Deisenhofer, and Huber were awarded the Nobel Prize in Chemistry in 1988. Using the same method, the reaction center of Rb. sphaeroides was analyzed and it turned out that the basic structures of the two reaction centers are astonishingly similar.

Figure 3.6 Schematic presentation of X-ray structural analysis of a protein crystal. A capillary containing the crystal rotates slowly and the diffraction pattern is monitored on an X-ray film. Nowadays muc more sensitive detector systems (image platers) are used instead of films. The diffraction pattern shown was obtained by the structural analysis of the reaction center of Rb. sphaeroides. (Courtesy of H. Michel, Frankfurt.)


The reaction center of Rhodopseudomonas viridis has a symmetric structure

Figure 3.9 shows the three-dimensional structure of the reaction center of the purple bacterium Rhodopseudomonas viridis. The molecule has a cylin- drical shape and is about 8 nm long. The homologous subunits L (red) and

A micelle is formed after solubilization of a membrane protein

Figure 3.8 A micelle is formed after solubilization of a membrane protein with detergent. The hydrophobic region of the membrane proteins, the membrane lipids, and the detergent are shown in black and the hydrophilic regions in red. Crystal structures can be formed by association of the hydrophilic regions of the detergent micelle.

M (black) are arranged symmetrically and enclose the chlorophyll and phe- ophytin molecules. The H subunit is attached like a lid to the lower part of the cylinder. In the same projection as in Figure 3.9, Figure 3.10 shows the location of the chromophores in the protein molecule. All the chromophores are positioned as pairs divided by a symmetry axis. Two Bchl-a molecules (DM, DL) can be recognized in the upper part of the structure. The two tetrapy- rrole rings are so close (0.3 nm) that their orbitals overlap in the excited state. This confirmed the actual existence of the “special pair” of chloro- phyll molecules, postulated earlier from spectroscopic investigations, as the

site of the primary redox process of photosynthesis. The chromophores are arranged underneath the chlorophyll pair in two nearly identical branches, both comprised of a Bchl-a (BA, BB) monomer and a bacteriopheophytin (A, B). Whereas the chlorophyll pair (DM, DL) is bound by both sub- units L and M, the chlorophyll BA and the pheophytin A are associated with subunit L, and BB and B with subunit M. The quinone ring of QA is bound via hydrogen bonds and hydrophobic interaction to subunit M, whereas the loosely associated QB is bound to subunit L.



Photosystem II Function: The P680 Reaction Center

How does a reaction center function?

The analysis of the structure and extensive kinetic investigations allowed a detailed description of the function of the bacterial reaction center. The kinetic investigations included measurements by absorption and fluores- cence spectroscopy after light flashes in the range of less than 1013 s, as well as measurements of nuclear spin and electron spin resonance. Although the reaction center shows a symmetry with two almost identical branches of chromophores, electron transfer proceeds only along the right branch (the L side, Fig. 3.10). The chlorophyll monomer (BB) on the M side is in close contact with a carotenoid molecule, which abolishes a harmful triplet state of chlorophylls in the reaction center (sections 2.3 and 3.7). The function of the pheophytin (B) on the M side and of the non-heme iron is not yet fully understood.

the reaction center with the reaction partners arranged according to their electrochemical potential

Figure 3.11 presents a scheme of the reaction center with the reaction partners arranged according to their electrochemical potential. The exciton of the primary reaction is provided by the antenna (section 2.4) which then excites the chlorophyll pair. This primary excitation state has only a very short half-life time, then a charge separation occurs, and, as a result of the large potential difference, an electron is removed within picoseconds to reduce bacteriopheophytin (Bphe).

equation Exciton

The electron is probably transferred first to the Bchl-monomer (BA) and then to the pheophytin molecule (A). The second electron transfer proceeds with a half-time of 0.9 picoseconds, about four times as fast as the elec- tron transfer to BA. The pheophytin radical has a tendency to return to the ground state by a return of the translocated electron to the Bchl-monomer (BA). To prevent this, within 200 picoseconds a high potential difference withdraws the electron from the pheophytin radical to a quinone (QA) (Fig. 3.11). The semiquinone radical thus formed, in response to a further poten- tial difference, transfers its electron to the loosely bound ubiquinone QB. After a second electron transfer, first ubisemiquinone and then ubihydroqui- none are formed (Fig. 3.12). In contrast to the very labile radical intermedi- ates of the pathway, ubihydroquinone is a stable reductant. However, this stability has its price. For the formation of ubihydroquinone as a first sta- ble product from the primary excitation state of the chlorophyll, more than half of the exciton energy is dissipated as heat.

Ubiquinone (Fig. 3.5) contains a hydrophobic isoprenoid side chain, by which it is soluble in the lipid phase of the photosynthetic membrane. The same function of an isoprenoid side chain has already been discussed in the case of chlorophyll (section 2.2). In contrast to chlorophyll, pheophytin, and QA, which are all tightly bound to proteins, ubihydroquinone QB is only loosely associated with the reaction center and can be exchanged by another ubiquinone. Ubihydroquinone remains in the membrane phase, is able to diffuse rapidly along the membrane, and functions as a transport metabolite for reducing equivalents in the membrane phase. It feeds the electrons into the cytochrome-b/c1 complex, also located in the membrane. The electrons are then transferred back to the reaction center through the cytochrome-b/c1

complex and via cytochrome-c. Energy is conserved during this electron transport as a proton potential (section 4.1), which is used for ATP-synthe- sis. The structure and mechanism of the cytochrome-b/c1 complex and of ATP-synthase will be described in section 3.7 and Chapter 4, respectively.

In summary, the cyclic electron transport of the purple bacteria resembles a simple electric circuit (Fig. 3.13). The two pairs of chlorophyll and pheophy- tin, between which an electron is transferred by light energy, may be regarded as the two plates of a capacitor between which a voltage is generated, driv- ing a flux of electrons, a current. Voltage drops via a resistor and a large amount of the electron energy is dissipated as heat. This resistor functions

as an electron trap, and withdraws the electrons rapidly from the capacitor. A generator utilizes the remaining voltage to produce chemical energy.

cyclic electron transport in photosynthesis

Figure 3.11 Schematic presentation of cyclic electron transport in photosynthesis of Rb. sphaeroides. The excited state symbolized by a star results in a charge separation; an electron is transferred via pheophytin, the quinones QA, QB, and the cyt-b/c complex to the positively charged chlorophyll radical. Q: quinone, Q•  : semiquinone radical, QH2: hydroquinone.

cyclic electronsport as a circuit

Figure 3.12 Reduction ofa quinone by one electronresults in a semiquinoneradical and furtherreduction to hydroquinone.Q: quinone, Q•  :semiquinone radical, QH2:hydroquinone.ExcitonGeneratorCyt-b/c1complexElectrontrap+ –Chlorophyll dimerHeatchemicalworkATP

Figure 3.13 Cyclic electrontransport of photosynthesisdrawn as an electricalcircuit.