Unit II: Metabolism

Photosynthesis

Photosynthesis is defined as the process, utilized by green plants and photosynthetic bacteria, where electromagnetic radiation is converted into chemical energy and uses light energy to convert carbon dioxide and water into carbohydrates and oxygen.

• The carbohydrates formed from photosynthesis provide not only the necessary energy form the energy transfer within ecosystems, but also the carbon molecules to make a wide array of biomolecules.

• Photosynthesis is a light-driven oxidation-reduction reaction where the energy from the light is used to oxidize water, releasing oxygen gas and hydrogen ions, followed by the transfer of electrons to carbon dioxide, reducing it to organic molecules.

• Photosynthetic organisms are called autotrophs because they can synthesize chemical fuels such as glucose from carbon dioxide and water by utilizing sunlight as an energy source.

• Other organisms that obtain energy from other organisms also ultimately depend on autotrophs for energy.

• One of the essential requirements for photosynthesis is the green pigment ‘chlorophyll’ which is present in the chloroplasts of green plants and some bacteria.

• The pigment is essential for ‘capturing’ sunlight which then drives the overall process of photosynthesis.

Process/ Steps of Photosynthesis

The overall process of photosynthesis can be objectively divided into four steps/ process:

1. Absorption of light

• The first step in photosynthesis is the absorption of light by chlorophylls that are attached to the proteins in the thylakoids of chloroplasts.

• The light energy absorbed is then used to remove electrons from an electron donor like water, forming oxygen.

• The electrons are further transferred to a primary electron acceptor, quinine (Q) which is similar to CoQ in the electron transfer chain.

2. Electron Transfer

• The electrons are now further transferred from the primary electron acceptor through a chain of electron transfer molecules present in the thylakoid membrane to the final electron acceptor, which is usually NADP⁺.

• As the electrons are transferred through the membrane, protons are pumped out of the membrane, resulting in the proton gradient across the membrane.

3. Generation of ATP

• The movement of protons from the thylakoid lumen to the stroma through the F₀F₁ complex results in the generation of ATP from ADP and Pi.

• This step is identical to the step of the generation of ATP in the electron transport chain.

4. Carbon Fixation

• The NADP and ATP generated in steps 2 and 3 provide energy, and the electrons drive the process of reducing carbon into six-carbon sugar molecules.

• The first three steps of photosynthesis are directly dependent on light energy and are thus, called light reactions, whereas the reactions in this step are independent of light and thus are termed dark reactions.

Sunlight is Essential for Plants

Sunlight has a very important job in photosynthesis. Plants can't survive without it.

In the leaves of a plant are very important organelles called chloroplasts. Within the chloroplasts are specialized pigment molecules called chlorophylls. Chlorophyll can absorb light and turn it into chemical energy that the plant can use.

Since the sun gives off a mix of mostly red and blue light, these are the colors that chlorophyll absorbs best. On the other hand, green light is reflected by chlorophyll, which is why most plants have green leaves.

The energy captured by chlorophyll can be used in photosynthesis to make sugar. When a plant gets limited sunlight, photosynthesis slows down. This also means that the plant might not be getting enough sugar—its energy source. We can see that the role of sunlight is extremely important.

Sunlight and Photons

The sun is the source of almost all energy on Earth. It enables plants and other organisms to turn water and carbon dioxide into sugars through a process called photosynthesis. The sun releases light that travels many millions of miles through space. A unit of that light is known as a photon. Photons have properties of both particles and waves.

The energy of a photon determines the color of light it emits. The higher the energy, the smaller the wavelength. Wavelengths are measured in nm, or nanometers. One nanometer is one-billionth of a meter.

Sunlight absorption and Chloroplasts

Inside of the plant's leaves are organelles known as chloroplasts. Chloroplasts have parts called thylakoids. The thylakoids stack up inside chloroplasts like how you would stack plates. Chloroplasts also contain chlorophyll. The two main types of chlorophyll are chlorophyll A and B. They are pigments that absorb specific wavelengths of light. Chlorophyll A can absorb blue, violet, red, and orange light. Chlorophyll B mostly absorbs blue light.

Photosynthetic Apparatus

Chlorophyll is a dangerous molecule. This is because it is an excellent photosensitizer and will rapidly cause cell damage when exposed to light. To prevent such damage, the organization and biosynthesis of chlorophyll is carefully controlled. All chlorophyll is located within the chloroplast organelle within the cell. The chloroplast is surrounded by a pair of membranes called the 'envelope'. The non-membrane, water-soluble material within the chloroplast is called the 'stroma'. Inside the chloroplast, chlorophyll is further confined to a system of membranes called the 'thylakoid membranes'.

Even within the thylakoid membrane system, the chlorophyll molecules do not occur in an unchaperoned state; instead they exist as photosynthetic pigment-protein complexes. The chlorophyll molecules are bound to specific integral membrane proteins along with carotenoids and other components necessary for photosynthesis. The proteins form a structured environment in which the potentially photosensitizing chlorophylls absorb photons, but in which unwanted photodynamic reactions are relatively rare. The pigment-protein complexes are arranged into arrays of hundreds of pigment molecules called photosystems. Because of the spatial and geometric organization, most of the pigments function as light-harvesters and transfer the excitation energy to other pigments before unwanted photochemistry occurs. The carotenoids perform two functions in this environment. First, they harvest light energy and transfer it to chlorophyll molecules. Secondly, they are highly efficient at quenching triplet chlorophylls that are formed before they have a chance to react with molecular oxygen and generate damaging molecules.

1. Cyclic Photophosphorylation:

It is a process of photophosphorylation in which an electron expelled by the excited photo-centre is returned to it after passing through a series of electron carriers. It occurs under conditions of low light intensity, wave­length longer than 680 nm and when CO₂ fixation is inhibited.

Absence of CO₂ fixation results in non-requirement of electrons for for­mation of NADPH. Cyclic photophosphoryla­tion is performed by photosystem I only. Its photo-centre P₇₀₀ extrudes an electron with a gain of 23 kcal/mole of energy after absorb­ing a photon of light (hv). After losing the electron the photo-centre becomes oxidized.

The expelled electron passes through a series of carriers including X or A₀ (a special P₇₀₀ chlorophyll molecule), A, (a quinone), FeS complexes (FeS_X, FeS_A, FeS_B), ferredoxin, (Fd), plastoquinone (PQ), cytochrome b – f complex and plastocyanin before returning to photo Centre. While over the cytochrome com­plex, the electron energises passage of pro­tons to create a proton gradient for synthesis of ATP from ADP and inorganic phosphate.

Halo bacteria or halophile bacteria also perform photophosphorylation but ATP thus produced is not used in synthesis of food. These bacteria possess purple pigment bacteriorhodopsin attached to plasma membrane. As light falls on the pigment, it creates a proton pump which is used in ATP synthesis.

2. Non-Cyclic Photophosphorylation:

It is the normal process of photophosphorylation in which the electron expelled by the excited photo-centre does not return to it. Non-cyclic photophosphorylation is carried out in collaboration of both photosystems I and II. Electron released during photolysis of water is picked up by photo-centre of PS II called P₆₈₀. The same is extruded out when the photo Centre absorbs light energy (hv).

The extruded electron has an energy equivalent to 23 kcal/mole. It passes through a series of electron carriers— phaeophytin, PQ, cytochrome b – f complex and plastocyanin. While passing over cytochrome complex, the electron loses sufficient energy for the syn­thesis of ATP. The electron is handed over to photo Centre P₇₀₀ of PS I by plastocyanin. P₇₀₀ extrudes the electron after absorbing light energy. The extruded electron passes through special chlorophyll X, Fe-S, ferredoxin, to finally reach NADP⁺. The latter then combines with H⁺ (released during photolysis) with the help of NADP-reductase to form NADPH. This is called Z scheme due to its characteristic zig-zag shape based on redox potential of different electron carriers.

Phases of Calvin Cycle:

Photosynthetic Carbon Reduction (PCR) Cycle or Calvin cycle occurs in all photo­synthetic plants whether they have C₃ or C₄ pathways. It is divided into the following three phases— carboxylation, glycolytic reversal and regeneration of RuBP.

1. Carboxylation:

Carboxylation is the addition of carbon dioxide to another substance called acceptor. Photosynthetic carboxylation requires ribulose-1, 5-bi-phosphate or RuBP as acceptor of carbon dioxide and RuBP carboxylase-oxygenase or RuBisCo as enzyme. The enzyme was previously called carboxydismutase.

Rubisco is the most abundant protein of the biological world. It constitutes 16% of chloropiast proteins (40% of soluble leaf proteins). However, it is a slowest enzyme with a turnover of 3 carbon dioxide molecules per second. Rubisco is located in the stroma on the outer surface of thylakoid membranes.

Carbon dioxide combines with ribulose-1, 5-bio-phosphate to produce a transient inter­mediate compound called 2-carboxy 3-keto 1, 5-bi-phosphoribotol. The intermediate splits up immediately in the presence of water to form the two molecules of 3-phosphoglyceric acid or PGA. It is the first stable product of photosynthesis.

2. Glycolytic Reversal:

The processes involved in this step or phase are reversal of the processes found during glycolysis part of respiration. Phosphoglyceric acid or PGA is further phosphorylated by ATP with the help of enzyme triose phosphate kinase (phosphoglycerate kinase). It gives rise to 1, 3-biphosphoglyeerie acid.

Biphosphoglyceric acid is reduced by NADPH through the agency of enzyme glyceraldehyde 3-phosphate dehydrogenase (triose phosphate dehydrogenase). It produces glyceraldehyde 3-phosphate or 3-phosphoglyceraldehyde.

Glyceraldehyde-3-phosphate is a key product which is used in synthesis of both carbo­hydrates and fats. For forming carbohydrates, say glucose, a part of it is changed into its isomer called dihydroxyacetone-3-phosphate. The enzyme that catalyses the reaction is phosphotriose isomerase.

The two isomers condense in the presence of enzyme aldolase forming fructose 1,6- bi-phosphate.

Fructose 1,6-bi-phosphate (FBP) loses one phosphate group, forms fructose 6-phosphate (F 6-P) which is then changed to glucose-6- phosphate (G 6-P). The latter can produce glucose or become part of sucrose and polysaccharide.

As glucose is a six carbon compound, six turns of Calvin cycle are required to synthesise its one molecule.

3. Regeneration of RuBP:

Fructose 6-phosphate (F 6-P) and glyceraldehyde 3-phos­phate (GAP) react to form erythrose 4-phosphate (E 4-P) and xylulose 5-phosphate (X 5-P). Erythrose 4-phosphate combines with dihydroxy acetone 3-phosphate to produce sedoheptulose 1: 7 diphosphate (SDP)which loses a molecule of phosphate and gives rise to sedoheptulose 7-phosphate (S 7-P).

Sedoheptulose 7-phosphate reacts with glyceraldehyde 3- phosphate to produce xylulose 5-phosphate (X 5-P) and ribose 5-phosphate. (R 5-P) Both of these are changed to their isomer ribulose 5-phosphate (Ru 5-P). Ribulose 5-phosphate picks up a second phosphate from ATP to become changed into ribulose 1, 5 bi-phosphate (RuBP).

Photorespiration or Glycolate Pathway:

It is interesting to know that in the plants possessing Calvin cycle, the enzyme RuBP carboxylase can initiate the reversal of photosynthetic reactions. This process occurs when there is low CO₂, concentration but high O₂, concentration.

At mid-day, when temperature and CO₂ content are high, the affinity of RuBP carboxylase increases for O₂ but decreases for CO₂. Thus, it converts RuBP to 3-carbon compound (PGA) and a 2-carbon compound (phosphoglycolate). The phosphoglycolate is converted rapidly to glycolate in the peroxisomes.

Glycolate is further converted to glycine, serine, CO₂ and NH₃ without the generation of ATP or NADPH. Thus net result is oxidation of organic food synthesized during photosynthesis. This process is called photorespiration or glycolate pathway as it occurs at high rate in the presence of light. As already mentioned that photorespiration is a loss to the net productivity of green plants having Calvin cycle.

The green plants having Calvin cycle are C₃ plants. Overcoming photo-respiratory loss poses a challenge to plants growing in the tropics. Photorespiration occurs due to fact that the active site of enzyme Rubisco (ribulose bisphosphate carboxylase oxygenase) is same for both carboxylation and oxygenation.

The oxygenation of RuBP (ribulose bisphosphate) in the presence of O₂ is first reaction of photorespiration that leads to the formation of one molecule of phosphoglycolate, a two-carbon compound and one molecule of PGA.

Where PGA is used in Calvin cycle, and phosphoglycolate is dephosphorylated to form glycolate in the chloroplast.

From chloroplast, glycolate is diffused to peroxisome where it is oxidised to in glyoxylate. Here glyoxylate is used to form amino acid, glycine. Now, glycine enters mitochondria where two glycine molecules (4 carbons) give rise to one molecule of serine (3 carbons) and one molecule of CO₂ (one carbon).

Now, serene is taken up by peroxisome, and through a series of reactions is being converted into glycerate.

This glycerate leaves the peroxisome and enters the chloroplast, where it is phosphorylated to form PGA.

Now PGA molecule enters the Calvin cycle to make carbohydrates, but one CO₂ molecule released in mitochondria during photorespiration has to be re-fixed. This means, 75 per cent of the carbon lost by the oxygenation of RuBP is recovered and 25 per cent is lost as release of one molecule of CO₂.

Photorespiration is also known as photosynthetic carbon oxidation cycle.

This process involves an interaction of three organelles, i.e.,

(i) Chloroplast

(ii) Peroxisome, and

(iii) Mitochondria.

Under conditions of high light and limited CO₂ supply, photorespiration plays an important role for protection of plants from photo-oxidative damage. This shows that if enough CO₂ is not available to utilise light energy for carboxylation, and excess energy inflicts damage to plants.

However, photorespiration being oxygenation of RuBP, utilises part of light energy and saves the plant from photo-oxidative damage.

The relative levels of O₂ and CO₂ are responsible for determination of the occurrence of photorespiration as both of these gases (O₂ and CO₂) compete for the same active site of enzyme Rubisco.

Increased O₂ level increases photorespiration; while increased CO₂ level decreases photorespiration, and increases C₃ photosynthesis.

Hatch-Slack (C4) pathway of CO₂ fixation.

The discovery of C₄ cycle in monocots such as sugarcane, maize and sorghum has indicated that these plants have solved the problem of photorespiration. The carbon dioxide is fixed in the mesophyll cells. The initial product being a-4 carbon compound, the process is called C₄ pathway of carbon dioxide fixation.

Hatch-Slack Pathway:

Two Australian botanists Hatch and Slack (1966) discovered that there are two types of chloroplasts in sugarcane. One type restricted to bundle sheath cells have the normal grana. These chloroplasts carry on Hatch-Slack or C₄ cycle. Hence, Hatch-Slack cycle or C₄ cycle has been found in most monocots and some dicots. The plants having C₄ cycle are known as C₄ plants, and the plants C₃ (Calvin cycle) are C₃ plants.

Photorespiration occurs in C₃ plants (Calvin cycle), which leads to a 25 percent loss of the fixed CO₂. Photorespiration occurs in C₃ plants only, as the enzyme Rubisco catalysis both carboxylation and oxygenation reactions of the initial acceptor molecule that is RuBP.

In C₃ plants, photosynthesis occurs only in mesophyll cells. Photosynthesis has two types of reactions, i.e., light reactions and carbon or dark reactions.

In light reactions, ATP and NADPH₂ are produced, and as a result of photolysis of water O₂ is released.

During carbon or dark reactions, CO₂ is assimilated and carbohydrates are produced.

As both light reactions and carbon (dark) reactions occur in mesophyll cells in C₃ plants, it becomes essential for enzyme Rubisco to catalyse both oxygenation and carboxylation reactions of RuBP, simultaneously.

However, in category of C₄ plants, nature has evolved a mechanism to avoid occurrence of photorespiration, which is thought to be a harmful process.

C₄ pathway requires the presence of two types of photosynthetic cells, i.e., mesophyll cells and bundle sheath cells. The bundle sheath cells are arranged in a wreath like manner. This kind of arrangement of cells is called Kranz anatomy (Kranz: wreath). In Kranz anatomy, the mesophyll and bundle sheath cells are connected by plasmodesmata or cytoplasmic bridges.

The C₄ plants contain dimorphic chloroplasts. The chloroplasts in mesophyll cells are granal, whereas in bundle sheath cells they are agranal.

The granal chloroplasts contain thylakoids which are stacked to form grana, as formed in C₃ plants. However, in agranal chloroplasts of bundle sheath cells grana are absent and thylakoids are present only as stroma lamellae.

The presence of two types of cells (granal and agranal) allows occurrence of light and carbon (dark) reactions separately in each type.

Here, release of O₂ takes place in one type, while fixation of CO₂ catalysed by Rubisco enzyme occurs in another type of cells.

In C₄ plants (maize, sugarcane, etc.), light reactions occur in mesophyll cells, whereas CO₂ assimilation takes place in bundle sheath cells. Such arrangement of cells does not allow O₂ released in mesophyll cells to enter in bundle-sheath cells.

Hence, Rubisco enzyme, which is present only in bundle-sheath cells, does not come into contact with O₂, and thus, oxygenation of RuBP is completely avoided.

In C₄ plants, a CO₂ concentrating mechanism is present which helps in reducing the occurrence of photorespiration (i.e., oxygenation of initial acceptor RuBP). This type of CO₂ concentrating mechanism is called C₄ pathway.

For operation of C₄ pathway, both mesophyll and bundle-sheath cells are required. The main objective of C₄ pathway is to build up high concentration of CO₂ near Rubisco enzyme in bundle- sheath cells. High concentration of CO₂ near Rubisco enhances carboxylation and reduces photorespiration.

C₄ photosynthetic Carbon Cycle:

In C₄ pathway, CO₂ from the atmosphere enters through stomata into the mesophyll cells and combines with phosphoenol pyruvate (3-carbon compound). This reaction is catalysed by an enzyme known as phosphoenol pyruvate carboxylase, i.e., PEPCase. With the result, a C₄ acid, oxaloacetic acid (OAA) is formed.

The above-mentioned reaction occurs in cytosol of the mesophyll cells and is called fixation of CO₂ or carboxylation.

Since this gives rise to the first stable product C₄ acid, and therefore, known as C₄ pathway.

The next step of reaction is transport of oxalo acetic acid (OAA – 4 C compounds) from cytosol of mesophyll cells to chloroplasts of bundle-sheath cells, where it is decarboxylated to release fixed CO₂ and high concentration of CO₂ is generated near Rubisco.

The other product of decarboxylation reaction is a 3-carbon compound called pyruvic acid. Now, this is transported back to mesophyll cells, where if regenerates phosphoenol pyruvate to its own for continuation of C₄ pathway.

However, the C₄ pathway is more efficient than C₃ pathway due to absence of photorespiration in C₄ plants.

Crassulacean Acid Metabolism (CAM)

This type of metabolism, refers to a mechanism of photosynthesis, that is, different from C₃ and C₄ pathways. Crassulacean acid metabolism (CAM) is found only in succulents and other xerophytes or plants that grow in dry conditions.

In this type of metabolism, CO₂ is taken up by the leaves on green stems through stomata which remain open during night. However, during day time, stomata in such plants remain closed to conserve moisture.

The CO₂ taken up by succulent plants in night is fixed in the similar way as it takes place in C₄ plants to form malic acid, which is being stored in vacuole.

Hence, malic acid formed during night is used during day time as a source of CO₂ for photosynthesis to proceed through C₃ pathway.

Crassulacean metabolism is a kind of adaptation found in certain succulent plants such as pineapple to proceed photosynthesis without much loss of water, which generally occurs in plants with C₃ and C₄ pathways.

CAM plants are the plants, which fix carbon dioxide by CAM pathway or Crassulacean acid metabolism. It was first discovered in the plants of the Crassulaceae family. They are present in dry and arid environments. The CAM pathway is adapted to minimise water loss and photorespiration. Examples of CAM plants include cactus, pineapple, etc.

CAM Photosynthesis

CAM pathway is adapted in plants to perform photosynthesis under stress. The CAM pathway reduces photorespiration.

In CAM plants stomata are open at night and they absorb carbon dioxide at night to reduce water loss during the daytime. The process has the following steps:

1. The first step in carbon dioxide fixation is the combination of CO₂ with PEP (phosphoenolpyruvate) to form 4 carbon oxaloacetate (same as C₄ plants) in the chloroplast of mesophyll cells. The reaction is catalysed by PEPcarboxylase. This occurs at night.

2. Oxaloacetate is converted to malate and other C₄ acids. Malate is stored in vacuoles at night.

3. During the daytime, stomata remain closed, so there is no gas exchange. Malate is transported out of the vacuole and CO₂ is released by the process of decarboxylation.

4. This CO₂ finally enters the Calvin cycle and carbon fixation completes. The CO₂ which gets accumulated around RuBisCO increases the efficiency of the photosynthesis process and minimizes photorespiration.

Respiration:

We know that during photosynthesis, light energy is converted into chemical energy, and is stored in carbohydrate molecules, such as glucose and starch. Organisms make use of such energy for their activities by oxidising these high energy food molecules into simple low energy molecules, i.e., carbon dioxide and water.

The reactions involved in process of oxidation are known as respiration. The compounds that are oxidised during process of respiration are called respiratory substrates.

Technically, Respiration is defined as follows:

This is a process by which living cells break down complex high energy food molecules into simple low energy molecules, i.e., CO₂ and H₂O, releasing the energy trapped within the chemical bonds.

The energy released during oxidation of energy rich compounds is made available for activities of cells through an intermediate compound called adenosine triphosphate (ATP).

During process of respiration, the whole of energy contained in respiratory substrates is not released all at a time. It is released slowly in several steps of reactions controlled by different enzymes.

Respiration takes place in all types of living cells, and generally called cellular respiration. During the process of respiration oxygen is utilised, and CO₂ water and energy are released as products. The released energy is utilised in various energy-requiring activities of the organisms, and the carbon dioxide released during respiration is used for biosynthesis of other molecules in the cell.

As we know, important life processes, such as synthesis of proteins, fats and carbohydrates, require a certain expenditure of energy. Where does this energy come from, how is it stored, and how is it made available to the living cell, are some of the questions, which are to be answered by process of respiration.

The main facts associated with respiration are:

a. Consumption of atmospheric oxygen.

b. Oxidation and decomposition of a portion of the stored food resulting in a loss of dry weight as seen in the seeds germinating in dark.

c. Liberation of carbon dioxide and a small quantity of water (the volume of CO₂ liberated is equal to volume of O₂ consumed).

d. Release of energy by breakdown of organic food, (such as carbohydrates).

Glycolysis

Glycolysis is a series of reactions that extract energy from glucose by splitting it into two three-carbon molecules called pyruvates. Glycolysis is an ancient metabolic pathway, meaning that it evolved long ago, and it is found in the great majority of organisms alive today.

In organisms that perform cellular respiration, glycolysis is the first stage of this process. However, glycolysis doesn’t require oxygen, and many anaerobic organisms—organisms that do not use oxygen—also have this pathway.

Highlights of Glycolysis

Glycolysis has ten steps, and depending on your interests—and the classes you’re taking—you may want to know the details of all of them. However, you may also be looking for a greatest hits version of glycolysis, something that highlights the key steps and principles without tracing the fate of every single atom. Let’s start with a simplified version of the pathway that does just that.

Glycolysis takes place in the cytosol of a cell, and it can be broken down into two main phases: the energy-requiring phase, above the dotted line in the image below, and the energy-releasing phase, below the dotted line.

• Energy-requiring phase. In this phase, the starting molecule of glucose gets rearranged, and two phosphate groups are attached to it. The phosphate groups make the modified sugar—now called fructose-1,6-bisphosphate—unstable, allowing it to split in half and form two phosphate-bearing three-carbon sugars. Because the phosphates used in these steps come from ATP, two ATP molecules get used up.

The three-carbon sugars formed when the unstable sugar breaks down are different from each other. Only one—glyceraldehyde-3-phosphate—can enter the following step. However, the unfavorable sugar, DHAP, can be easily converted into the favorable one, so both finish the pathway in the end.

• Energy-releasing phase. In this phase, each three-carbon sugar is converted into another three-carbon molecule, pyruvate, through a series of reactions. In these reactions, two ATP molecules and one NADH molecule are made. Because this phase takes place twice, once for each of the two three-carbon sugars, it makes four ATP and two NADH overall.

Each reaction in glycolysis is catalyzed by its own enzyme. The most important enzyme for regulation of glycolysis is phosphofructokinase, which catalyzes formation of the unstable, two-phosphate sugar molecule, fructose-1,6-bisphosphate. Phosphofructokinase speeds up or slows down glycolysis in response to the energy needs of the cell.

Overall, glycolysis converts one six-carbon molecule of glucose into two three-carbon molecules of pyruvate. The net products of this process are two molecules of ATP (4 ATP produced − 2 ATP used up) and two molecules of NADH.

Detailed steps: Energy-requiring phase

We’ve already seen what happens on a broad level during the energy-requiring phase of glycolysis. Two ATPs are spent to form an unstable sugar with two phosphate groups, which then splits to form two three-carbon molecules that are isomers of each other.

Next, we’ll look at the individual steps in greater detail. Each step is catalyzed by its own specific enzyme, whose name is indicated below the reaction arrow in the diagram below.

Step 1. A phosphate group is transferred from ATP to glucose, making glucose-6-phosphate. Glucose-6-phosphate is more reactive than glucose, and the addition of the phosphate also traps glucose inside the cell since glucose with a phosphate can’t readily cross the membrane.

Step 2. Glucose-6-phosphate is converted into its isomer, fructose-6-phosphate.

Step 3. A phosphate group is transferred from ATP to fructose-6-phosphate, producing fructose-1,6-bisphosphate. This step is catalyzed by the enzyme phosphofructokinase, which can be regulated to speed up or slow down the glycolysis pathway.

Step 4. Fructose-1,6-bisphosphate splits to form two three-carbon sugars: dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate. They are isomers of each other, but only one—glyceraldehyde-3-phosphate—can directly continue through the next steps of glycolysis.

Step 5. DHAP is converted into glyceraldehyde-3-phosphate. The two molecules exist in equilibrium, but the equilibrium is “pulled” strongly downward, as glyceraldehyde-3-phosphate is used up. Thus, all of the DHAP is eventually converted.

Detailed steps: Energy-releasing phase

In the second half of glycolysis, the three-carbon sugars formed in the first half of the process go through a series of additional transformations, ultimately turning into pyruvate. In the process, four ATP molecules are produced, along with two molecules of NADH.

Here, we’ll look in more detail at the reactions that lead to these products. The reactions shown below happen twice for each glucose molecule since a glucose splits into two three-carbon molecules, both of which will eventually proceed through the pathway.

Step 6. Two half reactions occur simultaneously: 1) Glyceraldehyde-3-phosphate (one of the three-carbon sugars formed in the initial phase) is oxidized, and 2) NAD+ is reduced to NADH and H+ . The overall reaction is exergonic, releasing energy that is then used to phosphorylate the molecule, forming 1,3-bisphosphoglycerate.

Step 7. 1,3-bisphosphoglycerate donates one of its phosphate groups to ADP, making a molecule of ATP and turning into 3-phosphoglycerate in the process.

Step 8. 3-phosphoglycerate is converted into its isomer, 2-phosphoglycerate.

Step 9. 2-phosphoglycerate loses a molecule of water, becoming phosphoenolpyruvate (PEP). PEP is an unstable molecule, poised to lose its phosphate group in the final step of glycolysis.

Step 10. PEP readily donates its phosphate group to ADP, making a second molecule of ATP. As it loses its phosphate, PEP is converted to pyruvate, the end product of glycolysis.

The Krebs’ Cycle

Tricarboxylic acid cycle (TCA cycle) is also called citric acid cycle or Krebs cycle (after its discoverer, Sir Hans Krebs). TCA cycle or citric acid cycle is the central metabolic hub of the cell and is the gateway to the aerobic metabolism of any molecule that can be transformed into an acetyl group or dicarboxylic acid.

In glycolysis the glucose molecule is broken down in pyruvate. Although the pyruvate is converted to various fermentation products as a result of fermentation, it is oxidized fully to CO₂ in respiration.

Outline of TCA Cycle:

Though the breakdown of glucose to pyruvate by earlier described pathways (glycolysis, HMP pathway, ED pathway) yields some energy, it is the degradation of pyruvate aerobically to CO₂ via TCA cycle (an eight step process) that generates much more of the energy because the TCA cycle is one major pathway by which pyruvate is completely oxidized to carbon dioxide (CO₂).

Pyruvate is first decarboxylated and converted into acetyl-CoA, which the connecting link between glycolysis and TCA cycle and acts as the fuel for TCA cycle. Acetyl-CoA is a two carbon energy-rich molecule, which initiates TCA cycle (Fig. 24.4) and is condensed with a four-carbon intermediate, oxaloacetate, to form citrate and to begin the six-carbon stage.

The citrate is isomerized to give isocitrate, which is subsequently oxidized and decarboxylated twice to produce α-ketoglutarate, then succinyl-CoA. During it, two NADH molecules are generated and two carbons are released from the cycle as CO₂ and, as a result, four-carbon stage initiates. Succinyl-CoA is finally converted into oxaloacetate via the formation of succinate, fumarate and L-malate.

Four-carbon stage yields one FADH₂ (FAD = Flavin adenine dinucleotide) and one NADH (NAD = Nicotinamide dinucleotide) during two oxidation steps: succinate to fumarate and L-malate to oxaloacetate. GTP (a high energy molecule equivalent to ATP) is also produced during the conversion of succinyl-CoA to succinate. Finally, the oxaloacetate is reformed and becomes ready to join acetyl-CoA to proceed further.

In this way, Kreb’s cycle (TCA-cycle) generates two CO₂ molecules, three NADH molecules, one FADH₂ molecule and one GTP molecule for each acetyl-CoA molecule oxidized in the cycle.

The generation of NADH and FADH₂ molecules is associated with electron transport chain and oxidative phosphorylation. The generation of GTP molecule takes place via substrate-level phosphorylation.

The various reactions of TCA cycle and enzymes involved are given in Table 24.1.

However, the overall reaction of the TCA cycle is:

Acetyl – CoA + 3 NAD + FAD + GDP + Pi + 2H₂O → 2CO₂ + 3 NADH + FADH₂ + GTP + 2H⁺ + CoA

Significance of TCA Cycle:

TCA cycle, as stated, is a central hub in cellular metabolism. It is the gateway to the aerobic metabolism of any molecule that can be transformed into an acetyl group or dicarboxylic acid.

Although the cycle generates considerable amount of energy, its major function is to provide precursors, not only for the strong forms of fuel, but also for the building blocks of many other molecules such as amino acids, nucleotide bases, cholesterol and porphyrin.

For example, most of the carbon atoms in oorphyrins (the organic components of heme) come from succinyl-CoA. Many of the amino acids are derived from α-ketoglutarate and oxaloacetate as shown in the following. Thus the TCA cycle is an amphibolic cycle, which means that it functions not only in catabolism (breakdown) but also in anabolic (synthesis) reactions in the cell.

Electron Transport System and Oxidative Phosphorylation in Mitochondria

Ever since the wide recognition of the fluid mosaic model of cell membranes, Mitchell’ chemiosomotic theory and more recently obtained knowledge about detailed structure and function of phosphorylating complexes (ATP synthase/ATPase) in mitochondria, our concept about elec­tron transport chain and oxidative phosphorylation has also changed and become more clear.

It is now well established that the electron transport chain or system in mitochondria con­sists of four multi-protein complexes (called by Roman numerals I through IV) which are localised in the inner mitochondrial membrane and also ubiquinone (UQ or coenzyme Q) and cytochrome-c which are not tightly bound to membrane protein but act as mobile carriers between the complexes. (Fig. 16.6, 7, 9)

The composition of mitochondrial electron transport system is basically similar in most living organisms although there may be some minor variations in the nature of some of the components among groups of organisms. The electron transport system of animals and plants mitochondria is also similar; the main differences between the two are mentioned in the text that follows and are also apparent from the figures.

Complex-I:

Consists of NADH-dehydrogenase (or NADH: Ubiquinone oxidoreductase) which contains a flavoprotein (FPint) FMN (Flavin Mono Nucleotide) and is associated with non-heme iron-sulphur (Fe-S) proteins.

This complex is responsible for passing electrons (also protons) from mitochondrial NADH/NADPH to Ubiquinone (UQ):

In plants (not animals), an additional external dehydrogenase complex is present which can oxidize cytosolic NADH (Fig. 16.7)

Complex-II:

Consists of succinate dehydrogenase which contains a flavoprotein (FPs) called FAD (Flavin Adenine Dinucleotide) in its prosthetic group and is associated with nonheme iron- sulphur (Fe-S) proteins. This complex receives electrons (also protons) from succinic acid (which is oxidised in Krebs’ cycle to form fumaric acid vide reaction no. 17) and passes them to Ubiquinone (UQ).

Succinate + UQ → Fumarate + UQH₂

Complex-III:

Consists of Dihydroubiquinone (UQH₂): cytochrome-C Oxido-reductase, two forms of cytochrome b (i.e., Cyt. b 562 and Cyt. b 566 in animal mitochondria and Cyt. b 566 and Cyt. b 560 or Cyt. b 557 and Cyt. b 560 in plant mitochondria), non-heme iron sulphur (Fe-S) proteins and cytochrome C₁ (with E₀‘ = + 0.22 V.).

In addition to these, this complex in plant mitochondria is also associated with a flavoprotein (FPha) which has a high (i.e. positive) E₀‘ (+ 0.11 V) and a large absorbance change on redox change (Fig. 16.7). This complex receives electrons from UQH₂ and passes them to cytochrome-C. The protons received from UQH₂ are released out.

Complex-lV:

Consists of Cytochrome-C: Oxygen Oxidoreductase (Cytochrome Oxidase), Cytochrome-a and Cytochrome-a₃. The enzyme of this complex contains copper (Cu) in the form of two copper centres CuA & CuB. This complex receives electrons from cytochrome-c and passes them to ½ O₂. Two protons are needed and H₂O molecule is formed (terminal oxidation).

(The organisation of these four complexes is quite specific in the inner mitochondrial membrane. NADH and FADH₂ in complex I and II respectively are oxidised on the matrix side of the membrane. Ubiquinone can freely diffuse within the inner membrane. Cytochrome-c is loosely bound to the outer surface of the inner mitochondrial membrane i.e., towards outside of inner membrane and cytochrome b₅₆₆ appears to face towards outside of inner membrane and cytochrome b₅₆₀ is localised more towards inner side of the inner membrane.

In complex IV, one of the two copper centres of this complex CuA along with cytochrome a are localised towards outside of the inner membrane while CuB and cytochrome a₃ are localised in the complex towards inner side (matrix side). In plants, the additional external NADH dehydrogenase complex is localised on the outer side (towards inter membrane space) of the inner mitochondrial membrane).

The transfer of electrons from reduced coenzyme NADH to oxygen via complexes I through IV is coupled to the synthesis of ATP from ADP and inorganic phosphate (Pi) which is called as oxidative phosphorylation. Experimental findings have confirmed that there are 3 sites of phosphorylation during mitochondrial electron transport, (i) during the transport of electrons from FP_i of NADH dehydrogenise to UQ through Fe-S in complex I, (ii) from within the com­plex III to cytochrome-c and (iii) from cytochrome a to cytochrome a_} in complex IV. Thus cytochrome a, in complex IV. Thus terminal oxidation of NADH results in the formation of 3ATP molecules while oxidation of FADH., results in production of only 2ATP molecules as it bypasses the first phosphorylation site (Fig. 16.6-7).

The widely accepted mechanism of mitochondrial synthesis of ATP is based on the chemiosmotic hypothesis first proposed by Mitchell in 1961 according to which asymmetric orienta­tion of electron carriers within the inner mitochondrial membrane allows for the transfer of protons (H⁺) across the inner membrane during electron transport. It is now confirmed that mitochondrial electron transport is associated with translocation of protons from matrix to inter membrane space.

When electrons flow through complexes I, III or IV, these complexes act as proton pumps. They pump out protons across the inner membrane from matrix to inter membrane space. Be­cause inner mitochondrial membrane is impermeable to protons (H⁺), a proton electrochemi­cal gradient (∆~µ_H+) or a proton motive force is build up (H⁺ accumulate on the outside of the inner membrane in inter-membrane space which becomes acidic and positively charged while the inner side of the inner membrane i.e., matrix becomes alkaline and negatively charged). The free energy released during the electron transport is in-fact used to generate this proton motive force (proton electrochemical gradient).

(According to an estimate about 10 protons are pumped out across the inner mitochondrial membrane during electron transport by these complexes (4 protons by complex I, 4 protons by complex III and 2 protons by complex IV) for each pair of electrons that travels from NADH to ½O₂.

Although the pumping of protons across the inner membrane can be explained by “redox loop” found in complex III, but the coupling of electron transport to proton translocation at complexes I & IV is not clearly understood. Oligomycin specifically blocks conduction of H⁺ through F₀)

The free energy now stored in proton electro-chemical gradient or proton motive force can be used to carry chemical work i.e., synthesis of ATP from ADP + Pi. This is accomplished through phosphorylating complexes which are knob like structures situated on cristae in mito­chondria (Fig. 16.8A) The phosphorylating complex or F₀F₁-ATP synthase (or F₀F₁-ATPase) which is sometimes called as complex V consists of two major components, a head piece or F₁ and a basal part called F₀.

The Respiratory Quotient

The ratio of the volume of CO₂ released to the volume of O₂ taken in respiration is called as respiratory quotient and is denoted as R.Q.

R.Q. = Vol. of CO₂ / Vol. of O₂

Value of Respiratory Quotient:

The value of R.Q. depends upon the nature of the respiratory substrate (the organic food matter oxidised in respiration) and its oxidation.

(1) When carbohydrates such as hexose sugars are oxidised in respiration, the value of R.Q. is 1 or unity because vol. of CO₂ evolved equals to the vol. of O₂ absorbed as is shown by the following equation:

C₆H₁₂O₆ + 6O₂ (Glucose) → 6CO₂ + 6H₂O

R.Q. = Vol. of CO₂/ Vol. of O₂ = 6/6 = 1 or unity

(2) When fats are the respiratory substrates, the value of R.Q. becomes less than one because the fats are poorer in oxygen in comparison to carbon and they require more O₂ for their oxidation which is obvious from the following equation:

2C₅₁H₉₈O₆ (Tripalmitin) + 145O₂ → 102CO₂ + 98H₂O

R.Q. = Vol. of CO₂/ Vol. of O₂ = 102/145 = 0.7 (less than one)

(Fats are oxidised in respiration usually during the germination of fatty seeds).

The value of R.Q. is also less than one when proteins are the respiratory substrates

(3) When organic acids are oxidised in respiration the value or R.Q. becomes more than one. It is because organic acids are rich in O₂ and require less O₂ for their oxidation e.g.,

C₄H₆O₅ (Malic acid) + 3O₂ → 4CO₂ + 3H₂O

R.Q. = Vol. of CO₂/ Vol. of O₂ = 4/3 = 1.3 (more than one)

(4) Partial oxidation of carbohydrates:

In some succulent plants like Opuntia, Bryophyllum etc carbohydrates are incompletely oxidised to organic acids in dark without the evolu­tion of CO₂, hence the value of R.Q. remains O.

2C₆H₁₂O₆ (Glucose) + 3O₂ → 3C₄H₆O₅ (Malic acid) + 3H₂O

R.Q. = Vol.of CO₂/ Vol. of O₂ = 0/3 = 0

(5) During anaerobic respiration, due to the absence of O₂ the value of R.Q. is always very high rather infinite.

C₆H₁₂O₆ (Glucose) → 2CO₂ + 2C₂H₅OH (Alcohol)

R.Q. = Vol. of CO₂/ Vol. of O₂ = 2/0 = (infinite)

Significance of Respiratory Quotient:

By determining the value of R.Q. the nature of respiratory substrate can be known. For example, if the value of R.Q. is one, it indicates that carbohydrates are being oxidised during respiration. Similarly if the value is less than one it will be concluded that organic matter like fats constitute the respiratory substrates.