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Chapter 12 - Respiration CHAPTER 12 – RESPIRATION Respiration is a process which makes ATP using energy in organic molecules such as glucose. It has three main stages - glycolysis, the Krebs cycle and oxidative phosphorylation 12.1 An outline of respiration Glucose molecules contain energy. The energy in glucose originally came from sunlight which was captured by chlorophyll molecules and used to make glucose in photosynthesis. If glucose is placed in oxygen and set alight, it burns vigorously (Fig. 12.1). Large amounts of heat energy are released as the glucose molecules combine with oxygen to form carbon dioxide and water, and the energy from the glucose is rapidly transferred to heat energy. This is an oxidation reaction: In a living cell, a similar process takes place, but in a much more controlled way. The glucose molecule is dismantled steadily, in a series of reactions catalysed by enzymes. The energy in the glucose molecule is released in small stages, and some of this energy is used to make ATP. The whole dismantling sequence has about 25 individual steps. Such a sequence is called a metabolic pathway; you can think of it as being rather like a production line in a factory with each enzyme receiving its substrate, carrying out its own small part of the overall task by catalysing the conversion of the substrate to a product, and allowing the product to pass on to the next enzyme. The metabolic pathway of respiration is divided into three main stages (Fig.12.2). Firstly, in the cytoplasm of the cell, glucose is converted to pyruvate. This stage is called glycolysis. Next, inside mitochondria, pyruvate is fed into a cycle of reactions called the Krebs cycle. Finally, still inside mitochondria, electrons produced in the Krebs cycle are passed along an electron transport chain, producing ATP in a process called oxidative phosphorylation (Fig 12.5). Fig 12.1 – A simple calorimeter with which the amount of energy released by the oxidation of glucose can be measured. 173 Chapter 12 - Respiration Fig 12.2 – the stages of respiration 12.2 Adenosine triphosphate (ATP) Adenosine triphosphate (ATP) is the short-term energy store of all cells. It is easily transported and is therefore the universal energy carrier. 12.2.1 Structure of ATP ATP is formed from the nucleotide adenosine monophosphate by the addition of two further phosphate molecules. Its structure is shown in Fig. 12.3 12.2.2 Importance of ATP The hydrolysis of ATP to ADP is catalyzed by the enzyme ATPase, and the removal of the terminal phosphate yields 30.6 kJ molˉ¹ of free energy. Further hydrolysis of ADP to AMP yields a similar quantity of energy, but the removal of the last phosphate to give adenosine produces less than half this quantity of energy. For this reason the first two phosphate bonds are often termed high energy bonds on account of the relatively large quantity of energy they yield on hydrolysis, This is misleading in that it implies that all the energy is stored in these bonds, The energy is in fact stored in the molecule as a whole, although the breaking of the bonds initiates its release. AMP and ADP may be reconverted to ATP by the addition of phosphate molecules in a process called phosphorylation, of which there are two main forms. 1. Photosynthetic phosphorylation – occurs during photosynthesis in chlorophyllcontaining cells. 2. Oxidative phosphorylation-occurs during cellular respiration in all aerobic cells. The addition of each phosphate molecule requires 30.6kJ of energy. If the energy released from any reaction is less than this, it cannot be stored as ATP and is lost as heat. The importance of ATP is therefore as a means of transferring free energy from energy-rich compounds to cellular reactions requiring it. While not the only substance to transfer energy in this way, it is by far the most abundant and hence the most important. 174 Chapter 12 - Respiration Fig 12.3 structure of ATP 12.2.3 Uses of ATP A metabolically active cell may require up to two million ATP molecules every second. ATP is the source of energy for: 1. Anabolic processes-It provides the energy needed to build up macromolecules from their component units, e.g. -polysaccharide synthesis from monosaccharides -protein synthesis from amino acids -DNA replication 2. movement – It provides the energy for many forms of cellular movement including -muscle contraction -ciliary action -spindle action in cell division 3. Active transport – it provides the energy necessary to move materials against a Concentration gradient, e.g., ion pumps. 4. Secretion – it is needed to form the vesicles necessary in the secretion of cell products. 5. Activation of chemicals – it makes chemicals more reactive, enabling them to react more readily, e.g. the phosphorylation of glucose at the start of glycolysis. 175 Chapter 12 - Respiration THE STAGES OF AEROBIC RESPIRATION Fig 12.4 Glycolysis Glycolysis (Glyco – ‘sugar’; lyso – ‘breakdown’) is the breakdown of a hexose sugar, usually glucose, into two molecules of the three-carbon compound pyruvate (pyruvic acid). It occurs in all cells; in anaerobic organisms it is the only stage of respiration. Although they contain quite large amounts of energy, glucose molecules are relatively unreactive. They must be activated before glycolysis can proceed. This is done by the addition of a phosphate group to the glucose molecule, forming glucose phosphate (Fig. 12.4). The atoms in this molecule are then rearranged to form fructose phosphate, and another phosphate group added to form fructose bisphosphate. Each of these additions of a phosphate group is done by transferring a phosphate group to the sugar from ATP. The ATP is converted into ADP in the process. So far then, far from making any ATP, glycolysis has actually used two ATP molecules! However, as you will see, more ATP molecules are made later, resulting in a net gain at the end. Next, the six-carbon fructose bisphosphate molecule is split into two three-carbon molecules, triose phosphate. Each of these is then converted to glycerate 3phosphate (GP) and then to pyruvate in a series of small steps. These steps release enough energy from the GP molecules to make some ATP. Four molecules of ATP (two for each of the two triose phosphate molecules) are made directly, there and then in the cytoplasm, using energy released as the triose phosphate molecules are gradually changed to pyruvate. So, although two molecules of ATP were put into the process at the beginning, four have been made at the end. However, this is not all the ATP which can be made in this process. The conversion of triose phosphate into GP also releases hydrogen ions (H+) and electrons (e-) which are transferred to the coenzyme NAD (nicotinamide adenine dinucleotide) to form reduced NAD. These hydrogen ions and high energy electrons are passed into a mitochondrion where they can be used to produce up to 5 ATP molecules in oxidative phosphorylation. This, however, can only happen if oxygen is available (Fig 12.6). The overall balance sheet for one molecule of glucose undergoing glycolysis is therefore: ATP molecules 176 Chapter 12 - Respiration produced 4 ATP molecules used 2 net gain of ATP molecules 2 In addition, the reduced NAD produced in glycolysis can yield up to 5 additional ATPs when oxygen is available. Fig 12.5 - The four stages of aerobic respiration- the first stage, glycolysis, occur in the cytosol. Pyruvate, the product of glycolysis, enters a mitochondrion, where cellular respiration continues with the formation of acetyl coenzyme A, the citric acid cycle and electron transport/chemiosmosis. Most ATP is synthesized by chemiosmosis. 177 Chapter 12 - Respiration Fig 12.6 -Overview of glycolysis. The energy investment phase of glycolysis (left column) leads to the splitting od sugar; ATP and NADH are produced during the energy capture phase (right colum). During glycolysis, each glucose molecule is converted to two pyruvates, with a net yeid of two ATP molecules and two NADH molecules. Krebs (tricarboxylic acid) cycle Although glycolysis releases a little of the energy from the glucose molecule, the majority still remains ‘locked-up’ in the pyruvate. These pyruvate molecules enter the mitochondria and, in the presence of oxygen, are broken down to carbon dioxide and hydrogen atoms. The process is called the Krebs cycle, after its discoverer Hans Krebs. There are a number of alternative names, notably the tricarboxylic acid cycle (TCA cycle) and citric acid cycle (Fig 12.9). While the carbon dioxide produced is removed as a waste product, the hydrogen atoms are oxidized to water in order to yield a substantial amount of free energy. The 2-carbon acetyl coenzyme A now enters the Krebs cycle (Fig.12.5) by combing with the 4-carbon oxaloacetate (oxaloacetic acid) to give the 6-carbon citrate (citric acid). Coenzyme A is reformed and may be used to combine with a further pyruvate molecule. The citrate is degraded to a 5-carbon α-ketoglutarage (α-ketoglutaric acid) and then the 4-carbon 178 Chapter 12 - Respiration oxaloacetate by the progressive loss of two carbon dioxide molecules, thus completing the cycle. At two stages in the Krebs cycle, carbon dioxide is removed from the compounds involved. This process is called decarboxylation. This carbon dioxide, plus that which was produced when pyruvate was converted to acetylcoenzyme A, diffuses out of the mitochondrion, out of the cell, and eventually out of the organism. Other important products of the Krebs cycle are electrons and hydrogen ions which are both picked up by the oxidised form of the coenzyme NAD, and some by oxidised FAD (flavin adenine dinucleotide): NAD+ + H+ + 2e- NADH Oxidised NAD reduced NAD These coenzymes can hold electrons which will then be fed into the electron transport chain to make ATP. When one glucose molecule is respired, two pyruvates are produced and they result in the production of six reduced NAD and two reduced FAD in the Krebs cycle. Two more reduced NAD molecules are produced in the conversion of pyruvate to acetylcoenzyme A. In addition, one step in the Krebs cycle makes ATP directly. Two ATP molecules are produced in this way per original glucose molecule. Fig 12.8 formation of acetyle coenzyme 179 Chapter 12 - Respiration Fig 12.9 - the formationof aectylcoenzyme A from pyruvate, and the krebs cycle. Fig 12.10 12.3.1 Importance of Krebs cycle The Krebs cycle plays an important role in the biochemistry of a cell for three main reasons: 1. It brings about the degradation of macromolecules – The 3-carbon for pyruvate is broken down to carbon dioxide 2. It provides the reducing power for the electron (hydrogen) transport systemIt produces pairs of hydrogen atoms which are ultimately the source of metabolic energy for the cell. 3. It is an interconversion center – It is valuable source of intermediate compounds used in the manufacture of other substances, e.g. fatty acids, amino acids, chlorophyll. Oxidative phosphorylation An important result of respiration is the formation of ATP. ATP is made by the 180 Chapter 12 - Respiration addition of inorganic phosphate, Pi, to ADP, a phosphorylation reaction. In respiration, this process requires oxygen and so is known as oxidative phosphorylation (Fig. 12.14). (Compare this with the way in which ATP is made in the light-dependent stages of photosynthesis, where light is required for the process of photophosphorylation.) The Krebs cycle takes place in the matrix of a mitochondrion. The next stage, in which ATP is made, happens in the inner mitochondrial membrane. Within this membrane lie the components of the electron transport chain. Reduced NAD and FAD provide electrons for the synthesis of ATP. The electrons are used to provide energy for ATP synthesis as they are passed along the electron transport chain. The electrons pass from the reduced NAD to the first member of this chain, and then from one carrier to the next. As the electrons are passed along, ATP is made. It is not certain exactly how many ATP molecules are made using the energy of four electrons from every two reduced NAD. It was originally thought to be six, but is now thought to be five or a little less. You may have noticed that there has been no oxygen involved in any of these processes so far. It is only now, right at the end of the electron transport chain, that oxygen is needed. Its role is to combine with the electrons, as they come off the end of the chain, and with hydrogen ions to form water. If there is not enough oxygen available to do this, then the electron transport chain cannot work. This produces a complete traffic jam, stopping the Krebs cycle completely. Now that we have arrived at the end of all three stages of respiration, we can draw up a complete balance sheet to show how many ATP molecules are made from one glucose molecule when oxygen is available. Fig 12.11 Electron transport system The electron transport system is the means by which the energy from the Krebs cycle, in the form of hydrogen atoms, is converted to ATP. The hydrogen atoms attached to the hydrogen carriers NAD and FAD are transferred to a chain of other carriers at progressively lower energy levels (fig). as the hydrogens pass from one carrier to the next, the energy released is harnessed to produce ATP. The series of carriers is termed the respiratory chain. The carriers in the chain include NAD, flavoprotein, coenzyme Q and iron- containing proteins called cytochromes. Initially hydrogen atoms are passed along the chain, but these later split into their protons and electrons, and only the electrons pass from carrier to carrier. For this reason, the pathway can be called the electron, or hydrogen, transport system. At the end of the chain the protons and electrons recombine, and the hydrogen atom created link with oxygen to 181 Chapter 12 - Respiration form water. This formation of ATP through the oxidation of the hydrogen atoms is called oxidative phosphorylation. It occurs in the mitochondria. The role of oxygen is to act as the final acceptor of the hydrogen atoms. While it only performs this function at the end of the many stages in respiration, oxygen is nevertheless vital as it drives the whole process. In its absence, only the anaerobic glycolysis stage can continue. The transfer of hydrogen atoms to oxygen is catalysed by the enzyme cytochrome oxidase. This enzyme is inhibited by cyanide, so preventing the removal of hydrogen atoms at the end of the respiratory chain. In these circumstances the hydrogen atoms accumulate and aerobic respiration ceases, making cyanide a most effective respiratory inhibitor. Fig 12-17 F ig 12. 12 182 Chapter 12 - Respiration Fig 12.13 Fig 12.14 ANAEROBIC RESPIRATION Respiration without oxygen Oxygen is used as a final acceptor of electrons. As this oxygen comes from the air, the process is called aerobic respiration. 183 Chapter 12 - Respiration When oxygen is not available, however, oxidative phosphorylation and the Krebs cycle cannot take place. This is also true in organisms or cells which do not contain the machinery to carry these out; human red blood cells, for example, have no mitochondria, and so can only perform glycolysis. In these circumstances, respiration takes place without oxygen. It is anaerobic. In anaerobic respiration, glycolysis takes place as usual, producing pyruvate and a small yield of ATP. If pyruvate were allowed to build up it would inhibit glycolysis, so it is converted to something else. More importantly, the reduced NAD which is produced in glycolysis must be oxidised back to NAD again or the cell would soon run out of it, bringing ATP production to a halt. There are two different solutions to the problem, both of which get rid of the pyruvate, and regenerate NAD. These are alcoholic fermentation, which is used by fungi and plants, and lactic fermentation, which is used by animals. • Alcoholic fermentation Yeast converts pyruvate to ethanol. First, carbon dioxide is removed from pyruvate to produce ethanal (Fig. 12.15). Next, the enzyme alcohol dehydrogenase (working in the reverse direction to the one its name suggests) converts the ethanal to ethanol. This step requires hydrogen which is taken from reduced NAD. This process has been used by humans for thousands of years. If yeast is provided with a supply of carbohydrate, it will carry out glycolysis and alcoholic fermentation. In bread making we provide it with starch in flour, and make use of the carbon dioxide it releases to make the bread rise. In the making of alcoholic drinks it is the ethanol which is required. • Lactic fermentation This process contributes to the discomfort you may experience in exhausted muscles. If muscle is exercising hard, it may run out of oxygen and have to respire anaerobically for a short period. The pyruvate produced by glycolysis is converted, in a single step, to lactate. The enzyme responsible for this conversion is lactate dehydrogenase, and the process requires hydrogen from reduced NAD. It is the build-up of lactate, which forms lactic acid, in muscles which causes the pain. The lactate must be broken down, and for this to take place it is transported in the blood to the liver. Here, some is converted back into glucose. This process requires oxygen, which is why you go on breathing deeply even when your strenuous exercise is over. You are supplying extra oxygen to the liver, to help it to deal with the lactate produced because of a shortage of oxygen earlier on. You are paying off an 'oxygen debt’. Although lactic acid in muscles is unpleasant, we do enjoy lactic acid in other ways! Yoghurt, soured cream and cheese all contain lactic acid, which helps to give them their distinctive flavours. The lactic acid is produced during anaerobic respiration by bacteria such as Lactobacillus bulgaricus. The bacteria are added to milk where they use the sugar lactose as a respiratory substrate. The lactic acid moves out of the bacteria into the surrounding milk. As well as providing flavour, the lactic acid lowers the pH of the milk, causing the proteins in it to coagulate. Neither alcoholic fermentation nor lactic fermentation produce any additional ATP. So anaerobic respiration provides only two molecules of ATP for every glucose molecule. This is much less than the 31 molecules of ATP which can be produced if 184 Chapter 12 - Respiration aerobic respiration, involving the Krebs cycle and oxidative phosphorylation. Fig 12.15 – Fermentation Fig 12-16 185