1. Respiration

2. Cellular respiration — glucose broken down, removal of hydrogen ions and electrons by dehydrogenase enzymes releasing ATP. The role of ATP in the transfer of energy and the phosphorylation of molecules by ATP. ATP is used to transfer energy to synthetic pathways and other cellular processes where energy is required. Overview of respiration reactions During cellular respiration glucose is broken down to release energy. Some respiration reactions involve the removal of hydrogen ions and electrons – these reactions are controlled by dehydrogenase enzymes. The hydrogen ions and electrons eventually pass through the cytochrome system resulting in the production of ATP from ADP and inorganic phosphate. The role of ATP is to transfer the energy released by respiration to cell processes that require energy, e.g. synthesis reactions, like protein synthesis and DNA replication as well as other cell processes like active transport. ATP also transfers phosphate to other molecules during phosphorylation reactions. Energy for cell processes like protein synthesis Energy released from respiration ADP + Pi ATP

3. Metabolic pathways of cellular respiration. The breakdown of glucose to pyruvate in the cytoplasm in glycolysis, and the progression pathways in the presence or absence of oxygen (fermentation). Respiration reactions Glycolysis Glycolysis is a series of reactions, each catalysed by a different enzyme. Glycolysis occurs in the cytoplasm and it results in a molecule of glucose being broken down to two molecules of pyruvate. The first part of glycolysis uses 2 ATP for each glucose molecule broken down –this is the energy investment phase. Later glycolysis reactions result in the production of 4 ATP – this is the energy payoff phase. Net ATP production Since glycolysis uses 2 ATP but produces 4 ATP, there is a net gain of two ATP for each glucose molecule broken down.

4. Phosphorylation Phosphorylation reactions occur twice during glycolysis. The first phosphorylation makes a product that can follow metabolic pathways other than glycolysis, e.g. it can follow a pathway leading to the production of glycogen. The second phosphorylation is an irreversible reaction catalysed by the enzyme phosphofructokinase – the product of this reaction must continue along the glycolytic pathway. Dehydrogenation reactions During the energy payoff stage, H + ions are released in a reaction catalysed by a dehydrogenase enzyme. These are accepted by a coenzyme called NAD to produce NADH. If oxygen is present, NADH will be used to produce ATP. In the absence of oxygen, NADH transfers hydrogen to pyruvate, converting it to lactic acid and regenerating NAD to go and accept more hydrogen ions. Glycolysis does not require oxygen – this stage of respiration is anaerobic. The role of the enzyme phosphofructokinase in this pathway. The phosphorylation of intermediates in glycolysis in an energy investment phase and the direct generation of ATP in an energy pay-off stage. The first phosphorylation leads to a product that can continue to a number of pathways and the second phosphorylation, catalysed by phosphofructokinase, is an irreversible reaction leading only to the glycolytic pathway.

5. Glycolysis summary Glucose Intermediate compound able to continue to other metabolic pathways ATP ADP First phosphorylation Other metabolic pathways Intermediate compound ADP ATP Intermediate compound Second phosphorylation catalysed by the enzyme phosphofructokinase Energy investment phase NAD NADH Pyruvate 4 ADP 4 ATP Energy payoff phase

6. NADH NAD. Pyruvate progresses to the citric acid cycle if oxygen is available. Pyruvate is broken down to an acetyl group that combines with coenzyme A to be transferred to the citric acid cycle as acetyl coenzyme A. Citric acid cycle If oxygen is available to the cell: Pyruvate passes from the cytoplasm into the mitochondrion matrix. Pyruvate is converted to an acetyl group and carbon dioxide. H + ions are released and bind to NAD forming NADH. The acetyl group combines with coenzyme A to form acetyl coenzyme A. Pyruvate CO 2 + Acetyl group Coenzyme A Acetyl coenzyme A

7. ADP = Pi ATP FADH FAD 3 NAD 3 NADH Acetyl coenzyme A combines with oxaloacetate to form citrate followed by the enzyme mediated steps of the cycle. This cycle results in the generation of ATP, the release of carbon dioxide and the regeneration of oxaloacetate in the matrix of the mitochondria. Acetyl coenzyme A transfers the acetyl group to oxaloacetate forming citrate – the coenzyme A is released to join another acetyl group NADH NAD Pyruvate CO 2 + Acetyl groupCoenzyme A Acetyl coenzyme A As citrate changes back to oxaloacetate,  2 CO 2 are released  3 H + ions are transferred to NAD forming NADH  One step releases energy to form a molecule of ATP from ADP  2 H + ions join FAD to form FADH 2 oxaloacetate citrate The citric acid cycle is a series of reactions, each controlled by its own enzyme. Those reactions that involve removal of H + ions and high energy electrons are controlled by enzymes called dehydrogenases 2CO 2 Citric acid cycle reactions occur in the mitochondrion matrix (this is the space in the middle of the mitochondrion) Matrix of the mitochondrion

8. Dehydrogenase enzymes remove hydrogen ions and electrons which are passed to the coenzymes NAD or FAD to form NADH or FADH 2 in glycolysis and citric acid pathways. NADH and FADH 2 release the high-energy electrons to the electron transport chain on the mitochondrial membrane and this results in the synthesis of ATP The electron transport chain as a collection of proteins attached to a membrane. NADH and FADH2 release the high- energy electrons to the electron transport chain where they pass along the chain, releasing energy. Electron transport chain In some reactions in glycolysis and the citric acid cycle, dehydrogenase enzymes remove hydrogen ions and electrons which are then passed to the coenzymes NAD and FAD forming NADH and FADH 2. NADH and FADH 2 pass high-energy electrons to the electron transport chain and release H + ions. The electron transport chain consists of a group of protein molecules that are attached to the inner membrane of a mitochondrion. The high-energy electrons pass along the electron transport chain, as they do so, energy is released. Mitochondrion Matrix of the mitochondrion Outer membrane Cristae (folds in the inner membrane where the electron transport chain is found

9. The energy released as electrons pass through the electron transport chain is used to pump H ions across the inner mitochondrial membrane. The return flow of H ions drives ATP synthase and produces the bulk of the ATP generated by cellular respiration. ATP synthesis — high energy electrons are used to pump hydrogen ions across a membrane and flow of these ions back through the membrane synthesises ATP using the membrane protein ATP synthase. The final electron acceptor is oxygen, which combines with hydrogen ions and electrons to form water. The return flow of these ions rotates part of the membrane protein ATP synthase, catalysing the synthesis of ATP. The high-energy electrons pass along the electron transport chain. As they do so, energy is released. This energy is used to pump H + ions across the inner membrane of the mitochondrion from the matrix side to inter- membrane space H + ions pass back across the membrane by passing through ATP synthase, a protein molecule that spans the membrane. As they do so energy is released and they rotate part of the ATP synthase molecule, changing the shape of the active site allowing it to join ADP and Pi to form ATP. Most of the ATP made during cellular respiration is produced in this way. At the end of the electron transport chain, electrons and a pair of hydrogen ions combine with oxygen to produce water. Oxygen is therefore the final electron acceptor in the electron transport chain, in the absence of oxygen the electron transport chain does not operate. Oxygen combines with hydrogen ions and electrons to form water Mitochondrion matrix Matrix side of membrane Inter-membrane space

10. Regulation of the respiratory pathway Conservation of resources Phosphofructokinase catalyses an irreversible reaction during glycolysis. This step is important in regulating the rate of the pathway. If a cell produces more ATP than it needs, the high concentration of ATP inhibits phosphofructokinase so slowing the rate glycolysis. When the concentration of ATP decreases again, the enzyme is no longer inhibited and glycolysis speeds up again. By slowing down the rate of the respiratory pathway when the level of ATP is greater than required, the cell does not continue to produce ATP that is not needed and so conserves resources. Regulation of the pathways of cellular respiration by feedback inhibition — regulation of ATP production, by inhibition of phosphofructokinase by ATP and citrate, synchronisation of rates of glycolysis and citric acid cycle. The cell conserves its resources by only producing ATP when required. ATP supply increases with increasing rates of glycolysis and the citric acid cycle, and decreases when these pathways slow down. If the cell produces more ATP than it needs, the ATP inhibits the action of phosphofructokinase slowing the rate of glycolysis. The rates of glycolysis and the citric acid cycle are synchronised by the inhibition of phosphofructokinase by citrate. If citrate accumulates, glycolysis slows down and when citrate consumption increases glycolysis increases the supply of acetyl groups to the citric acid cycle.

11. Synchronising glycolysis and the citric acid cycle A high concentration of citrate also inhibits phosphofructokinase. The inhibition of phosphofructokinase by citrate synchronises the rates of glycolysis and the citric acid cycle. If citric acid accumulates, glycolysis is slowed down (due to inhibition of the enzyme) resulting in less pyruvate, and less acetyl coenzyme A being produced from pyruvate. Less acetyl coenzyme A feeding into the citric acid cycle slows that cycle too. When the rate of the citric acid cycle increases and citrate is used up more quickly. Phosphofructokinase is not inhibited leading to an increase in the rate of glycolysis as well. Feedback inhibition The process that regulates and synchronises the rates of glycolysis and the citric acid cycle is an example of feedback inhibition. If citrate and ATP are produced at too high a rate, they inhibit phosphofructokinase which has the effect of slowing their production.