Energy and Respiration PDF

# Energy and respiration ## 12.1 Energy **On these pages you will learn to:** - Outline the need for energy in living organisms, as illustrated by anabolic reactions, such as DNA replication and protein synthesis, active transport, movement and the maintenance of body temperature **EXTENSION**...

# Energy and respiration ## 12.1 Energy **On these pages you will learn to:** - Outline the need for energy in living organisms, as illustrated by anabolic reactions, such as DNA replication and protein synthesis, active transport, movement and the maintenance of body temperature **EXTENSION** **Laws of thermodynamics** The first law of thermodynamics states that energy cannot be created or destroyed, but only converted from one form to another. The amount of energy in the universe is always the same, although that on Earth may fluctuate slightly. The Earth obtains the majority of its energy in the form of light from our nearest star - the Sun. Most of the energy used by mankind comes initially from the sunlight via a series of energy conversions . The second law of thermodynamics states that disorder (more technically called entropy) in the universe is continuously increasing. In other words, disorder is more likely than order. For example, a heap of bricks is more likely to fall down and become scattered than it is to arrange itself into a neat column because there is less energy in a disordered system than in an ordered one. When the universe was formed, it possessed the maximum potential energy it has ever had. Since then, it has become increasingly disordered because at each energy conversion the amount of entropy increased. All living organisms require energy in order to remain alive. This energy comes initially from the Sun (or in a few instances from chemicals). Plants use solar energy to combine water and oxygen into complex organic molecules by the process of photosynthesis. Both plants and animals then break down these organic molecules to make adenosine triphosphate (ATP) that is used as the energy source to carry out processes that are essential to life. **What is energy?** Energy is defined as 'the ability to do work'. It can be considered to exist in two states: - Kinetic energy is the energy of motion. Moving objects perform work by making other objects move. - Potential energy is stored energy. An object that is not moving may still have the capacity to do so and therefore possesses potential energy. A stone on a hillside has potential energy. If it is set in motion, gravity will cause it to roll downhill and some of its potential energy will be converted into kinetic energy. Other facts about energy include: - it takes a variety of different forms, e.g. light, heat, sound, electrical, magnetic, mechanical, chemical and atomic - it can be changed from one form to another - it cannot be created or destroyed - it is measured in joules (J). **Why do organisms need energy?** Without some input of energy, natural processes tend to break down in randomness and disorder. Living organisms are highly ordered systems that require a constant input of energy to prevent them becoming disordered - a condition that would lead to their death. More particularly energy is needed for: - Anabolism, in which smaller, more simple substances are built up into larger, more complex ones, e.g. during DNA replication, in which nucleotides are joined by condensation reactions to form polynucleotides, and protein synthesis, in which amino acids are joined together to form polypeptides. - Movement both within an organism (e.g. circulation of blood) and of the organism itself (e.g. locomotion due to muscular contraction or movement of cilia and flagella). - Active transport of ions and molecules against a concentration gradient across membranes, such as the cell surface membrane and the tonoplast, e.g. the sodium-potassium pump. - Maintenance, repair and division of cells and the organelles within them. - Maintenance of body temperature in birds and mammals. These organisms are endothermic and need energy to replace that lost as heat to the surrounding environment. ## 12.2 Adenosine triphosphate (ATP) **On these pages you will learn to** - Describe the features of ATP that make it suitable as the universal energy currency - State that ATP is produced in mitochondria and chloroplasts and outline the role of ATP in cells - Outline the roles of the coenzymes NAD, FAD and coenzyme A in respiration **Structure of adenosine triphosphate (ATP)** The ATP molecule (Figure 1) is a phosphorylated nucleotide and it has three parts: - Adenine - a nitrogen-containing organic base belonging to the group called purines. - Ribose - a sugar molecule with a 5-carbon ring structure (pentose sugar) that acts as the backbone to which the other parts are attached. - Phosphates - a chain of three phosphate groups. The universal energy currency of all cells is a molecule called adenosine triphosphate (ATP) (see Figure 1). Almost every energy-requiring process in cells uses ATP. It is a small water-soluble molecule and therefore easily transported around the cell. Some of the features that help to explain why ATP is suitable as the universal energy currency include: - a one-step reaction provides an immediate source of energy (see below) - it is easily hydrolysed to release energy - a constant supply of ATP is possible as it is recycled from ADP, which is easily phosphorylated (see Figure 2) - it is a relatively small molecule that can move around the cell with ease - it is a water-soluble molecule so it can take part in metabolic reactions - the quantity of energy released and the efficiency of recycling ATP means that the needs of the cell can be satisfied. It is the three phosphate groups that are the key to how ATP is the energy currency of the cell. Each one is very negatively charged and so they repel one another. This makes the covalent bonds that link them rather unstable. These unstable covalent bonds have a low activation energy, which means they are easily broken. When they do break they release a considerable amount of energy - 30.5 kJ mol-¹ for each of the first two phosphates removed and 14.2 kJmol-¹ for the removal of the final phosphate. The terminal phosphate is removed according to the enzyme-catalysed reversible equation: $ATP + H_2O \rightleftharpoons ADP + P + 30.5 kJ$ **Synthesis of ATP** The conversion of ATP to ADP is a reversible reaction (Figure 2) and therefore energy can be used to add an inorganic phosphate to ADP to re-form ATP. The interconversion rate of ATP and ADP is phenomenal. Although there are only around 50g of ATP in the human body at any point in time, it is thought that, even at rest, a single human uses 65 kg of ATP in a 24-hour period. This means that, on average, a single ATP molecule undergoes around 1300 cycles of synthesis and hydrolysis each day. As the synthesis of ATP from ADP involves the addition of a phosphate molecule, it is a phosphorylation reaction. This phosphorylation is catalysed by the enzyme ATP synthase (sometimes called ATP synthetase) and it occurs in three ways: - Photophosphorylation that takes place in grana of the chloroplasts during photosynthesis (Topic 13.4). - Oxidative phosphorylation that takes place on the inner mitochondrial membranes of plant and animal cells, and the cell surface membrane of bacteria, during the process of electron transport (Topic 12.5). - Substrate-level phosphorylation that takes place in plant and animal cells when phosphate groups are transferred from donor molecules to ADP to make ATP. For example, in the formation of pyruvate at the end of glycolysis (Topic 12.3). In the first two, ATP is synthesised using energy released during the transfer of electrons along a chain of electron-carrier molecules in either the chloroplasts or the mitochondria. There is a difference in hydrogen ion concentration either side of certain phospholipid membranes in chloroplasts and mitochondria and it is essentially the flow of these ions across these membranes that generates ATP. **The process is referred to as the chemiosmotic theory of ATP synthesis.** Although it takes place in a similar way in both chloroplasts and mitochondria, the summary account that follows and which is shown in Figure 3 describes the process in mitochondria. - Hydrogen atoms produced during respiration are carried to the electron transport chain where they are split into protons (hydrogen ions - H+) and electrons. - As electrons pass along the electron carriers of the electron transport chain, each one being at a lower energy level than the one before, the energy released is used to pump the protons (H+) into the space between the inner and outer mitochondrial membranes. - Protons accumulate (build up) in the inter-membranal space, leading to a concentration gradient of protons (H+) between the space and the matrix. This also means that there is an electrochemical gradient between the inter-membranal space and the matrix. - As the inner mitochondrial membrane is almost impermeable to protons, they can only diffuse back through the chemiosmotic channels in the ATP synthase complexes. - As protons flow through these channels their electrical potential energy is used to combine ADP with inorganic phosphate (P₁) to produce ATP. - The phosphorylation reaction is catalysed by ATP synthase found in the head piece (Figure 3) of the ATP synthase complexes. - Once in the matrix the protons recombine with the electrons on carriers on the inner membrane to form hydrogen atoms, which in turn combine with oxygen to form water. **Role of ATP** ATP is not a good long-term energy store. Fats and carbohydrates such as glycogen, serve this purpose far better. ATP is therefore the immediate energy source of a cell. As a result, cells do not store large quantities of ATP, but rather just maintain a few seconds' supply. This is not a problem, as ATP is rapidly re-formed from ADP and inorganic phosphate (P₁) and so a little goes a long way. ATP is the source of energy for: - Anabolic processes - It provides the energy needed to build up macromolecules from their basic units, e.g. polysaccharide synthesis from monosaccharides, polypeptide synthesis from amino acids, DNA/RNA synthesis from nucleotides. - Movement - ATP provides the energy for muscle contraction, ciliary and flagellar action and movement of vesicles along microtubules within the cell. In muscle contraction, ATP provides the energy for the filaments of striated muscle to slide past one another and therefore shorten the overall length of a muscle fibre. - Active transport - ATP provides the energy necessary to move molecules or ions against a concentration gradient. This is an essential role, as every cell must maintain a precise ionic content. - Secretion - ATP is needed to form the vesicles necessary for the secretion of cell products. - Activation of chemicals - ATP makes chemicals react more readily, e.g. the phosphorylation of glucose at the start of glycolysis (Topic 12.3), e.g. activated nucleotides in DNA and mRNA synthesis. ## 12.3 Respiration - glycolysis **On these pages you will learn to:** - List the four stages in aerobic respiration (glycolysis, link reaction, Krebs cycle and oxidative phosphorylation) - State where glycolysis occurs in eukaryotic cells - Explain that ATP is synthesised in substrate-linked reactions in glycolysis - Outline glycolysis as phosphorylation of glucose and the subsequent splitting of fructose 1,6-bisphosphate (6C) into two triose phosphate molecules, which are then further oxidised to pyruvate with a small yield of ATP and reduced NAD **REMEMBER** In eukaryotic cells glycolysis takes place in the cytoplasm while the link reaction, Krebs cycle and oxidative phosphorylation all take place in the mitochondria. Cellular respiration (also just called 'respiration') is the process by which the energy in food is converted into the energy for an organism to do biological work. Glucose is the main respiratory substrate and the overall equation for the process in aerobic conditions is: $C_6H_{12}O_6 + 6O_2 \rightarrow 6CO_2 + 6H_2O + energy$ **Overview of respiration** Respiration in aerobic conditions can be divided into four stages: - Glycolysis - an enzyme-controlled pathway in which one molecule of 6-carbon glucose is converted into two 3-carbon pyruvate molecules. - Link reaction (pyruvate oxidation) - the 3-carbon pyruvate molecule is converted into carbon dioxide and a 2-carbon molecule called acetyl coenzyme A. - Krebs cycle - the introduction of acetyl coenzyme A into a cycle of eight enzyme-catalysed reactions that yield reduced coenzymes NAD and FAD and some ATP. - Oxidative phosphorylation (electron transport system) – oxidation of reduced NAD and FAD as part of an electron transport chain and ATP synthesis by chemiosmosis. Oxygen is required as a final electron acceptor and water is produced. The main respiratory pathways are summarised in Figure 1. Respiration in anaerobic conditions involves glycolysis but not the other three stages described above. The pyruvate produced is further metabolised to lactate in mammalian tissue and to ethanol (and carbon dioxide) in yeast. **Glycolysis** Glycolysis occurs in the cytoplasm of all cells of multicellular organisms and in many unicellular organisms and is the process by which a hexose (6-carbon) sugar, usually glucose, is converted into two molecules of the 3-carbon molecule, pyruvate. Although there are 10 smaller enzyme-controlled reactions in glycolysis, these can be conveniently grouped into four stages: - Activation of glucose by phosphorylation. Before glycolysis can proceed, glucose must first be made more reactive by the addition of two phosphate molecules - phosphorylation. The phosphate molecules come from the hydrolysis of two ATP molecules to ADP. This provides the energy to activate glucose (activation energy) and also prevents glucose from being transported across the cell surface membrane and out of the cell. In the process glucose is converted to fructose 1,6-bisphosphate. - Splitting of the phosphorylated hexose sugar. Fructose 1,6-bisphosphate is then split into two 3-carbon molecules known as triose phosphate. - Oxidation of triose phosphate. Hydrogen is removed from each of the two triose phosphate molecules and transferred to a hydrogen carrier molecule called nicotinamide adenine dinucleotide (NAD+) to form reduced NAD. - The production of ATP. Four enzyme reactions convert cach triosc phosphate into another 3-carbon molecule called pyruvate. In the process, two molecules of ATP are formed from ADP and two more hydrogens are produced, which become attached to a molecule of NAD+ to give reduced NAD. It must be remembered, however, that for each molecule of glucose at the start of the process, there are two molecules of triose phosphate produced. Therefore these yields must be doubled, i.e. 4 × ATP and 2 × reduced NAD. The events of glycolysis are summarised in Figure 2. The overall yield from one glucose molecule undergoing glycolysis is therefore: - two molecules of ATP (four molecules of ATP are produced, but two were used up in the initial phosphorylation of glucose and so the net increase is two molecules) - two molecules of reduced NAD (these have the potential to produce more ATP) - two molecules of pyruvate. Glycolysis was one of the earliest biochemical processes to evolve. It occurs in the cytoplasm of cells and does not require any organelle or membrane for it to take place. As it does not require oxygen it can proceed in both aerobic and anaerobic conditions. In the absence of oxygen the pyruvate produced by glycolysis can be converted into either lactate or alcohol by a process called fermentation. This is necessary in order to re-oxidise NAD so that glycolysis can continue. This is explained, along with details of the reactions, in Topic 12.6. These reactions, however, yield only a small fraction of the potential energy stored in the pyruvate molecule. In order to release this energy, most organisms use oxygen to break down pyruvate further in a process called the Krebs cycle. ## 12.4 Link reaction and Krebs cycle **On these pages you will learn to:** - State where the link reaction and the Krebs cycle occur in eukaryotic cells - Explain that ATP is synthesised in substrate-linked reactions in the Krebs cycle - Explain that, when oxygen is available, pyruvate is converted into acetyl (2C) coenzyme A in the link reaction - Outline the Krebs cycle, explaining that oxaloacetate (a 4C compound) acts as an acceptor of the 2C fragment from acetyl coenzyme A to form citrate (a 6C compound), which is reconverted to oxaloacetate in a series of small steps - Explain that reactions in the Krebs cycle involve decarboxylation and dehydrogenation and the reduction of NAD and FAD - Outline the roles of the coenzymes NAD, FAD and coenzyme A in respiration **EXTENSION** **Enzyme complex - pyruvate dehydrogenase** The oxidation of pyruvate to acetyl coenzyme A in the link reaction involves a complex series of steps with three intermediate stages. The reaction is catalysed by a multienzyme complex called pyruvate dehydrogenase. This is one of the largest enzyme complexes in organisms, consisting of 60 sub-units. Enzyme complexes like this occur frequently in organisms and are made up of individual enzymes organised in sequence so that the product of one enzyme acts as the substrate for the next enzyme in the chain. This arrangement is more efficient because, rather than depending upon a chance meeting of enzyme and substrate as would be the case if the enzymes floated freely around, the substrate is not released but rather 'handed on' to the next enzyme in the biochemical sequence. The pyruvate molecules produced during glycolysis possess potential energy that can only be released in a process called the Krebs cycle. Before they can enter the Krebs cycle, these pyruvate molecules must first be oxidised in a procedure known as the link reaction. In eukaryotic cells both the Krebs cycle and the link reaction take place exclusively inside mitochondria and these will only occur if oxygen is available. **The link reaction** The pyruvate molecules produced in the cytoplasm during glycolysis are actively transported into the matrix of mitochondria. Here pyruvate undergoes a complex series of oxidation-reduction reactions that are catalysed by a multienzyme complex (see extension). During these reactions the following changes take place: - A carbon dioxide molecule is removed from each pyruvate (= decarboxylation) by means of the enzyme pyruvate decarboxylase. - Oxididation of pyruvate results in the reduction of NAD to form reduced NAD (later used to produce ATP). - The 2-carbon molecule that is formed is called an acetyl group and combines with a cofactor called coenzyme A (COA) to produce a 2-carbon compound called acetyl coenzyme A. The overall equation can be summarised as: $pyruvate + NAD^ + + COA \rightarrow acetyl CoA + reduced NAD + CO_2$ **EXTENSION** **The importance of acetyl coenzyme A** Coenzyme A is made up of vitamin B5, the organic base adenine and the sugar ribose. It carries the acetyl group made from pyruvate into the Krebs cycle in the form of acetyl coenzyme A. The acetyl coenzyme A molecule is important because most molecules that are used by living organisms for energy are made into acetyl coenzyme A before entering the Krebs cycle. Most carbohydrates and fatty acids can be metabolised into acetyl coenzyme A to release energy. In the case of fats, these are first hydrolysed into glycerol and fatty acids. The glycerol can then be converted into triose phosphate that can be broken down during glycolysis, while the fatty acids are progressively broken down in the matrix of the mitochondria into 2-carbon fragments that are converted into acetyl coenzyme A. The reverse is also true, namely that excess carbohydrate can be made into fats via acetyl coenzyme A, making it a pivotal molecule in the interconversion of major substances in eukaryotic cells. **Krebs cycle** The Krebs cycle was named after the biochemist, Hans Krebs, who worked out its sequence in 1937. A central element of aerobic respiration, the Krebs cycle involves a series of eight small enzyme-catalysed steps that take place in the matrix of mitochondria. Its events are shown in Figure 1 and can be summarised as: - The 2-carbon acetyl coenzyme A from the link reaction combines with a 4-carbon acceptor molecule (oxaloacetate) to produce a 6-carbon molecule (citrate). - This 6-carbon molecule (citrate) is decarboxylated (loses CO₂) and dehydrogenated (loses two hydrogens) to give a 5-carbon compound, carbon dioxide and reduced NAD. - Further decarboxylation and dehydrogenation produces a 4-carbon molecule (oxaloacetate), carbon dioxide, reduced NAD and reduced FAD and a single molecule of ATP produced as a result of substrate-level phosphorylation (Topic 12.2). - The 4-carbon molecule (oxaloacetate) can now combine with a new molecule of acetyl coenzyme A to begin the cycle again. For each molecule of pyruvate, the link reaction and Krebs cycle therefore produces: - four molecules of reduced NAD. These have the potential to produce a total of 10 ATP molecules (Topic 12.5) - one molecule of reduced FAD which has the potential to produce 1.5 ATP molecules (Topic 12.5) - one molecule of ATP - three molecules of carbon dioxide. As two pyruvate molecules are produced for each original glucose molecule, these quantities must be doubled when the yields from a single glucose molecule are being calculated. **Roles of coenzymes** There are a number of coenzymes found in cells whose role it is to carry hydrogen atoms, and hence also electrons, from one compound to another. The two important hydrogen-carrying coenzymes in respiration are: - nicotinamide adenine dinucleotide (NAD), which is important in respiration - flavine adenine dinucleotide (FAD), important in aerobic respiration. In respiration, NAD is the most important carrier. It works with dehydrogenase enzymes that catalyse the removal of hydrogen atoms from substrates like citrate and transfer them to other molecules such as the hydrogen carriers involved in oxidative phosphorylation (Topic 12.5). The process works as follows: - The two hydrogen atoms removed by the dehydrogenase enzyme dissociate into hydrogen ions (protons) and electrons: $2H \rightarrow 2H^+ + 2e^- $ - Each NAD molecule in a cell exists in a form in which it has lost an electron, i.e it is oxidised and therefore exists as NAD+. - NAD+ combines with the hydrogen ions (protons) and electrons to form NADH and a hydrogen ion (proton): $NAD^ + + 2H^+ + 2e^- \rightarrow reduced NAD (NADH + H^+)$ - When the hydrogen atom is transferred to a new molecule, the reduced NAD is re-oxidised, by the reversal of the above process, to re-form NAD+. In respiration, coenzyme A has a role as a carrier of an acetyl group in the formation of acetyl CoA from pyruvate. The 2-carbon acetyl CoA can then enter the Kreb's cycle. **The significance of the Krebs cycle** The Krebs cycle performs an important role in biochemistry for four reasons: - It breaks down macromolecules into simpler ones, pyruvate is broken down into carbon dioxide. - It produces hydrogen atoms that are carried by NAD and FAD to the electron transport chain for oxidative phosphorylation and the production of ATP by chemiosmosis, which provides metabolic energy for the cell. - It regenerates the starter material (oxaloacetate), which would otherwise be completely used up. - It is a source of intermediate compounds used by cells in the manufacture of other important substances such as fatty acids, amino acids and chlorophyll. ## 12.5 Oxidative phosphorylation **On these pages you will learn to:** - State where oxidative phosphorylation occurs in eukaryotic cells - Outline the process of oxidative phosphorylation including the role of oxygen as the final electron acceptor - Explain that during oxidative phosphorylation: -energetic electrons release energy as they pass through the electron transport system - the released energy is used to transfer protons across the inner mitochondrial membrane - protons return to the mitochondrial matrix by facilitated diffusion through ATP synthase providing energy for ATP synthesis - Describe the relationship between structure and function of the mitochondrion using diagrams So far in the process of respiration, we have seen how hexose sugars such as glucose are converted to pyruvate (glycolysis) and how the 3-carbon pyruvate is fed into the Krebs cycle to yield carbon dioxide and hydrogen atoms. The carbon dioxide is a waste product and is removed during the process of gaseous exchange (Topic 9.2). The hydrogen atoms, or more precisely the electrons they contain, are however valuable as a potential source of energy. Oxidative phosphorylation (Figure 2) is the final stage in respiration and it is during this stage that most ATP molecules are synthesised. **Krebs cycle, oxidative phosphorylation and mitochondria** Mitochondria are rod-shaped organelles, between 1 µm and 10µm in diameter, that are found in all but a few eukaryotic cells. Details of their structure are given in Topic 1.6, and in summary here. Each mitochondrion is bounded by a smooth outer membrane and an inner one that is folded into extensions called cristae (Figure 1). On the large surface area provided by the cristae are ATP synthase complexes (Figure 4). The inner space, or matrix, of the mitochondrion contains 70S ribosomes and is made up of a semi-rigid material of protein, lipids and traces of circular DNA. Mitochondria are the sites of the link reaction, Krebs cycle and oxidative phosphorylation. More specifically: - The inner folded membrane (cristae) has attached to it the proteins involved in the electron transport chain and therefore enables oxidative phosphorylation to take place. - The ATP synthase complexes located in the membrane of the cristae contain ATP synthase for the synthesis of ATP by the chemiosmosis method (Topic 12.2). **The process of oxidative phosphorylation** - The hydrogen atoms produced during glycolysis, the link reaction and Krebs cycle are combined with special molecules called carriers that are attached to the mitochondrial membranes. Most hydrogen atoms are combined with nicotinamide adenine dinucleotide (NAD), although one pair from the Krebs cycle combines with flavine adenine dinucleotide (FAD). The hydrogen atoms split into their protons (H+) and electrons (e). - The first carrier to accept electrons is a complex called NADH dehydrogenase. - The electrons then pass via a carrier to a protein-cytochrome complex and finally, via another carrier to the cytochrome oxidase complex. - The sequence of transfer of electrons from one carrier to the next is called the electron transport chain. - The enzymes that catalyse these reactions are called oxidoreductases (see extension). - Each of the three complexes in the chain acts as a proton pump using energy released from the energetic electrons to drive the protons (H+) from the mitochondrial matrix into the inter-membranal space. - The protons (H+) build up in the inter-membranal space before they return by facilitated diffusion into the mitochondrial matrix through ATP synthase. - As the protons pass through ATP synthase, ADP is combined with inorganic phosphate (P₁) to produce ATP. This ATP is formed using a diffusion force similar to osmosis: the process is called chemiosmosis (Topic 12.2). - At the end of the chain the protons and electrons recombine and the hydrogen atoms so formed link with oxygen to form water. Oxygen is the final electron acceptor. These events are summarised in Figure 2. There were thought to be 3 ATP molecules produced for each reduced NAD and 2 ATP produced for each reduced FAD molecule (fewer because reduced FAD enters further along the electron transport chain). Recent research suggests that these figures are more accurately 2.5 and 1.5 ATP molecules respectively. Not all the potential energy in the 32 ATP molecules produced for each glucose molecule (Topic 12.6) is a net yield. Around 25% of the energy produced is needed to transport ADP into the matrix of the mitochondrion so that it can combine with inorganic phosphate to form ATP. The importance of oxygen in respiration is to act as the final acceptor of the hydrogen atoms produced in glycolysis and Krebs cycle. Without its role in removing hydrogen atoms at the end of the chain, the hydrogen ions and electrons would 'back up' along the chain and the process of respiration would come to a halt. This point is shown by the effect of cyanide on respiration. Most people are aware that cyanide is a very potent poison that causes death rapidly. It is lethal because it is an inhibitor of the final dehydrogenase enzyme in the electron transport chain, cytochrome oxidase. This enzyme catalyses the addition of the hydrogens to oxygen to form water. Its inhibition causes hydrogen ions and electrons to accumulate on the carriers, bringing the Krebs cycle to a halt and leaving pyruvate from glycolysis to accumulate. This pyruvate is converted to lactate as we shall see in the next topic. ## 12.6 Anaerobic respiration and energy yields **On these pages you will learn to:** - Explain the relative energy values of carbohydrate, lipid and protein as respiratory substrates and explain why lipids are particularly energy-rich - Distinguish between respiration in aerobic and anaerobic conditions in mammalian tissue and in yeast cells, contrasting the relative energy released by each - Explain the production of a small yield of ATP from respiration in anaerobic conditions in yeast and in mammalian muscle tissue, including the concept of oxygen debt - Explain how rice is adapted to grow with its roots submerged in water in terms of tolerance to ethanol from respiration in anaerobic conditions and the presence of aerenchyma We saw in Topic 12.5 that oxygen is needed if the hydrogen atoms produced in glycolysis and Krebs cycle are to be converted to water and thereby drive the production of ATP. What happens if oxygen is temporarily or permanently unavailable to a tissue or a whole organism? In the absence of oxygen, neither the Krebs cycle nor oxidative phosphorylation can take place, leaving only the anaerobic process of glycolysis as a potential source of ATP. For glycolysis to continue, its products of pyruvate and hydrogen must be constantly removed. In particular, the hydrogen must be released from the reduced NAD in order to regenerate NAD+. Without this the already tiny supply of NAD+ in cells will be entirely converted to reduced NAD, leaving no NAD+ to take up newly produced hydrogen from glycolysis. Glycolysis will then stop. The regeneration of NAD+ is achieved by the pyruvate molecule from glycolysis accepting the hydrogen from reduced NAD in a process called fermentation. In eukaryotic cells there are two main types of fermentation: alcoholic fermentation and lactate fermentation. **Alcoholic fermentation** Alcoholic fermentation occurs in certain bacteria and fungi (e.g. yeast) as well as in some cells of higher plants, e.g. root cells under waterlogged conditions. The pyruvate molecule formed at the end of glycolysis first loses a molecule of carbon dioxide (decarboxylation) to form ethanal. $CH_3COCOOH \rightarrow CH_3CHO + CO_2 $ The ethanal accepts hydrogen from reduced NAD to produce ethanol. The summary equation for this is: $ethanal (CH_3CHO) + NADH + H^+ \rightleftharpoons ethanol (C_2H_5OH) + NAD^+$ The overall equation using glucose as the starting point is: $C_6H_{12}O_6 \rightarrow 2C_2H_5OH + 2CO_2 $ Alcoholic fermentation in yeast has been exploited by humans for thousands of years, in both the brewing and baking industries. In brewing, ethanol is the important product. Yeast is grown in anaerobic conditions in which it ferments natural carbohydrates in plant products such as grapes (wine production) or barley seeds (beer production) into ethanol. The ethanol produced kills the yeast cells that make it when its concentration reaches around 15%. **Adaptation of rice to anaerobic conditions** When soils are flooded or are waterlogged, the concentration of oxygen in the soil soon decreases and creates conditions where there is very little, or no, oxygen. If plant tissues are to survive submerged in water, there needs to be a way to receive oxygen from other areas of the plant that are not submerged. Alternatively, the submerged tissues, which now have to respire in anaerobic conditions, must either remove or show tolerance to the ethanol that is produced. This ethanol is normally toxic when it builds up. Rice has a number of adaptive features that allows it to grow with its roots submerged in water. - The cells of the embryo are tolerant to the high concentrations of ethanol that build up as a result of anaerobic respiration. - The stems and roots have many air spaces between the cells, which allow oxygen to diffuse from aerial parts not submerged and allow the cells in the root tissue to respire aerobically. This type of tissue is called aerenchyma. - If the roots remain short of oxygen, they respire anaerobically and tolerate the build-up of ethanol. They also produce an abundance of the enzyme alcohol dehydrogenase, which breaks the ethanol down. - Some varieties of rice have a higher rate of anaerobic respiration to increase the rate of ATP production. **Lactate fermentation** Lactate fermentation occurs in animals as a means of overcoming a temporary shortage of oxygen. Clearly, such a mechanism has considerable survival value, for example for a baby mammal in the period immediately after birth or an animal living in water where the concentration of oxygen fluctuates. However, lactate fermentation occurs most commonly in muscles as a result of strenuous exercise. In these conditions oxygen may be used up more rapidly than it can be supplied and therefore an oxygen deficit occurs. It may be essential, however, that the muscles continue to work despite the lack of oxygen - for example if the organism is fleeing from a predator. In the absence of oxygen, each pyruvate molecule produced takes up the two hydrogen atoms from glycolysis to form lactate as shown below: $pyruvate (CH_3COCOOH ) + NADH + H^+ \rightleftharpoons lactate (CH_3CHOHCOOH)+ NAD^+$ The lactate produced will cause cramp and muscle fatigue if it is allowed to accumulate in the muscle tissue. Although muscle has a certain tolerance to lactate, it is nevertheless important that it is removed by the blood and taken to the liver. Here it is converted to glycogen in a process called the Cori cycle. Some lactate may also be oxidised to pyruvate, in the reverse of the above equation, and then enter the Krebs cycle. The individual incurs an oxygen debt, which is later repaid when oxygen is available again. Figure 1 shows how the NAD+ needed for glycolysis to continue is regenerated in both common forms of fermentation. **Energy yields from anaerobic and aerobic respiration** Energy from respiration in aerobic conditions is obtained by substrate level phosphorylation (in glycolyis and the Krebs cycle) and by oxidative phosphorylation. The number of ATP molecules produced for each glucose molecule is 32. However, the equivalent of one ATP is used in transporting some of the chemicals involved, giving a net yield of 31 ATP. Each ATP releases 30.5 kJ mol-¹ on hydrolysis, which gives a total yield of 945.5 kJ mol-¹ . In anaerobic conditions, for each glucose molecule, only 2 ATP molecules are produced by substrate level phosphorylation in glycolysis. This is a total yield of 61.0kJ mol-¹. The theoretical maximum energy yield for the complete breakdown of a glucose molecule is 2870 kJ mol-¹. This means that respiration in aerobic conditions is 33% efficient, whereas respiration in anaerobic conditions is only approximately 2% efficient. **Energy yields from other respiratory substrates** Although we normally think of glucose as the main respiratory substrate, other carbohydrates, as well as lipids and protein, may also be used in certain circumstances, without first being converted to glucose. As lipids contain relatively more C-H bonds than an equivalent mass of carbohydrate, their breakdown produces more hydrogen atoms for the electron transport chain and hence more energy. As a result, lipids release more than twice as much energy (39.4kJg-¹) than the same mass of carbohydrate (15.8 kJg-¹). Once the immediate stores of carbohydrate, such as glycogen in the liver, have run out, organisms start to metabolise lipids for energy. Protein is normally only metabolised for energy in extreme situations such as starvation. When all carbohydrate and lipid reserves have been exhausted, organisms will, as a last resort, break down their protein into amino acids. These then have the amino groups (NH2) removed before entering the respiratory pathway at a number of different points depending on their carbon content. The four-carbon and five-carbon amino acids are converted to Krebs cycle intermediates, whereas three-carbon amino acids are converted to pyruvate. Amino acids with large numbers of carbons are first converted to three-, four and five-carbon amino acids. Although the energy yield depends on the exact composition of each protein, they normally yield around 17.0kJg-¹- slightly more energy than carbohydrates. ## 12.7 Measurement of respiration and respiratory quotients **On these pages you will learn to:** - Carry out investigations, using simple respirometers, to determine the RQ of germinating seeds or small invertebrates - Carry out investigations, using simple respirometers, to measure the effect of temperature on the respiration rate of germinating seeds or small invertebrates - Define the term 'respiratory quotient' (RQ) and determine RQs from equations for respiration The rate of respiration in an organism can be determined either by measuring the volume of oxygen taken in or the volume of carbon dioxide produced. Measurements can be taken using a respirometer. **A simple respirometer** One type of simple respirometer is shown in Figure 1. It consists of two identical chambers - an experimental one containing the respiring organisms and a control one containing an equal volume of non-respiring material such as glass beads. The two chambers are connected by a U-shaped manometer tube that contains a coloured fluid. This type of respirometer is sometimes referred to as a differential respirometer because it has a built-in control chamber that makes sure that any fluctuation in temperature or pressure affects both sides of the manometer equally and so they cancel each other out. An equal volume of some carbon dioxide-absorbing material such as soda-lime is added to each chamber. In Figure 1 the apparatus has been set up to measure the rate of respiration of small invertebrates such as woodlice at different temperatures. **A simple respirometer is used as follows:** - The apparatus is left in the water bath for about 10 minutes to allow it to reach the desired temperature (equilibrate). - Screw clips A and B are kept open during this time to allow air to escape as