Respiration

Respiration is the process that occurs in living cells and releases the energy stored in organic molecules such as glucose. The energy is immediately used to synthesis molecules of ATP from ADP and inorganic phosphate (Pi).

ATP in cells can be hydrolysed to release energy needed to drive biological processes. Microorganisms (both eukaryotic microbes such as yeast, and prokaryotes such as bacteria), plants, animals, fungi and protoctists all respire to obtain energy.

The energy that is stored in complex organic- e.g. fats, carbohydrates and proteins- is potential energy. It is also chemical energy, converted from light energy during the process of photosynthesis. When this energy is released from organic molecules, via respiration, it can be used to make ATP to drive biological processes, such as:

• active transport, endo/exocytosis, synthesis, DNA replication, cell division, movement of cilia/flagella, activation of chemicals

All the chemical reactions that take place within living cells are known collectively as metabolism or metabolic reactions. Anabolic (small molecules built up to a large molecule) and catabolic (hydrolysis of large molecules into small molecules) reactions.

ATP is the standard intermediary between energy-releasing and energy-consuming metabolic reactions in both eukaryotic and prokaryotic cells.

Each molecule of ATP consists of adenosine, which is the nitrogenous base adenine plus the 5 carbon sugar ribose, and 3 phosphate (phosphoryl) groups. ATP is relatively stable (it does not break down to ADP and Pi) when in solution in cells, but is readily hydrolysed by enzyme catalysis. However, whilst in solution, it can easily be moved from place to place within a cell.

The energy releasing hydrolysis of ATP is coupled with an energy consuming metabolic reaction. ATP is the immediate energy source for this metabolic reaction. When ATP is hydrolysed to ADP and Pi, a small quantity of energy is released for use in the cells. Cells can therefore obtain the energy they need for a process in small manageable amounts that will not cause damage or be wasteful.

ATP is referred to as the universal energy currency, as it occurs in all living cells and is a source of energy that can be used by cells in small amounts. Some energy is released from the hydrolysis of ATP as heat. The release of heat, both in respiration and during ATP hydrolysis, may appear to be inefficient and wasteful. Heat, however, helps to keep living organisms 'warm' and enables their enzyme-catalysed reactions to proceed at or near their optimum rate.

It is a biochemical pathway that occurs in the cytoplasm of all living organisms that respire, including many prokaryotes. The pathway involves a sequence of 10 reactions, each catalysed by a different enzyme, with the help of the coenzyme, NAD.

The 3 main stages are:

1. Phosphorylation of glucose to hexose bisphosphate.

2. Splitting each hexose bisphosphate molecule into two triose phosphate molecules.

3. Oxidation of triose phosphate to pyruvate.

Enzymes that catalyse oxidation and reduction reactions need the help of coenzymes that accept the hydrogen atoms removed during oxidation.

NAD (nicotinamide adenine dinucleotide) is a non-protein molecule that helps dehydrogenase enzymes to carry out oxidation reactions. NAD oxidises substrate molecules during glycolysis, the link reaction and the Krebs cycle.

NAD is synthesised in living cells from nicotinamide (vitamin B3), the 5 carbon sugar ribose, the nucleotide base adenine and 2 phosphoryl groups. Reduced NAD carries the protons and electrons to the cristae of mitochondria and delivers them to be used in oxidative phosphorylation for the generation of ATP from ADP and Pi.

When reduced NAD gives up the protons and electrons that it accepted during one of the first 3 stages of respiration, it becomes oxidised and can be reused to oxidise more substrate, in the process becoming reduced again.

Glucose is a hexose sugar, which means it contains 6 carbon atoms. Its molecules are stable and need to be activated before they can be split into 2 three-carbon compounds.

1. One molecule of ATP is hydrolysed and the released phosphoryl group is added to glucose to make hexose monophosphate.

2. Another molecule of ATP is hydrolysed and the phosphoryl group added to the hexose phosphate to form a molecule of hexose bisphosphate. This sugar has one phosphate group at carbon 1 and another at carbon 6.

The energy from the hydrolysed ATP molecules activates the hexose sugar and prevents it from being transported out of the cell.

Each molecule of hexose bisphosphate is split into 2 three-carbon molecules, triose phosphate, each with a phosphate group attached.

Although this process is anaerobic, it involves oxidation because it involves the removal of hydrogen atoms from substrate molecules.

1. Dehydrogenase enzymes, aided by the coenzyme NAD, remove hydrogens from triose phosphate.

2. The 2 molecules of NAD accept the hydrogen atoms and become reduced.

3. At this stage of glycolysis, 2 molecules of NAD are reduced for every molecule of glucose undergoing this process. Also at this stage, 4 molecules of ATP are made for every 2 triose phosphate molecules undergoing oxidation.

For each molecule of glucose:

•2 molecules of ATP; four have been made but two were used to 'kick start' the process, so the net gain is 2.

•2 molecules of reduced NAD

•2 molecules of pyruvate

1. Glycolysis

2. The Link Reaction

3. The Krebs Cycle

4. Oxidative Phosphorylation

The last 3 stages only take place under aerobic conditions. Under aerobic conditions, the pyruvate molecules from glycolysis are actively transported into the mitochondria for the link reaction.

In anaerobic conditions pyruvate is converted in the cytoplasm to lactate or ethanol. In the process, the reduced NAD molecules are reoxidised so that glycolysis can continue to run, generating two molecules of ATP for every glucose molecule metabolised.

They may be rod shaped, thread like or spherical which diameters of 0.5-1.0um and lengths of 2-5 um, but occasionally up to 10um.

All mitochondria have an inner and outer phospholipid membrane making up the envelope. The outer membrane is smooth and the inner membrane is folded into cristae, giving it a large surface area. Embedded in the inner membrane are proteins that transport electrons, and protein channels associated with ATP synthase enzymes that allow protons to diffuse through them.

Between the inner and outer membranes of the envelope there is a inter-membrane space.

The mitochondrial matrix, enclosed by the inner membrane, is semi rigid and gel like; it contains mitochondrial ribosomes, looped mitochondrial DNA and enzymes for the Link Reaction and Krebs Cycle. 

This is where the link reaction and the Krebs cycle takes place. It contains:

•enzymes that catalyse that stages of these reactions

•molecules of the coenzymes NAD and FAD (flavine adenine dinucleotide)

•oxaloacetate- the 4 carbon compound that accepts the acetyl group from the link reaction.

•mitochondrial DNA- some of which codes for mitochondrial enzymes and other proteins.

•mitochondrial ribosomes, structurally similar to prokaryotic ribosomes, where these proteins are assembled.

The phospholipid composition of the outer membrane is similar to that of membranes around the other organelles in eukaryotic cells.

It contains proteins, some of which form channels or carriers that allow the passage of molecules, such as pyruvate, into the mitochondrion.

The lipid composition of the inner membrane differs from that of the outer membrane.

This lipid bilayer is less permeable to small ions such as hydrogen ions that is the outer membrane.

The folds in the inner membrane give a large SA for the electron carriers and ATP synthase enzymes embedded in them.

The electron carriers are protein complexes arranged in electron transport chains.

Electron transport chains are involved in the final stage of aerobic respiration, oxidative phosphorylation.

The IMS between the outer and inner layers of the mitochondrial envelope is also involved in oxidative phosphorylation.

The inner membrane is in close contact with the mitochondrial matrix, so the molecules of reduced NAD and FAD can easily deliver hydrogens to the electron transport chain.

Each electron carrier protein contains a cofactor- a non protein haem group that contains an iron ion.

The iron ion can accept and donate electrons, because it can become reduced (Fe2+) by gaining an electron and then become oxidised (Fe3+) when donating the electron to the next electron carrier. Electron carrier proteins are oxido-reductase enzymes.

The electron carriers also have a coenzyme that, using energy released from the electrons, pumps protons from the matrix to the IMS.

Protons accumulate in the IMS and a proton gradient forms across the membrane. The proton gradient can produce a flow of protons through the channels in the ATP synthase enzymes to make ATP.

ATP synthase enzymes are large and protrude from the inner membrane into the matrix. Protons can pass through them.

Pyruvate produced in glycolysis is transported across the outer and inner mitochondrial membranes via a specific pyruvate-H+ symport, a transport protein that moves 2 ions or molecules in the same direction, and into the matrix. Then:

•Pyruvate is converted into a 2 carbon acetyl group during the link reaction

•the acetyl groups is oxidised during the Krebs cycle.

It occurs in the matrix. Pyruvate is decarboxylated and dehydrogenated, catalysed by a large multi enzyme complex, pyruvate dehydrogenase, which catalyses the sequence of reactions that occur during the link reaction. No ATP is produced in this reaction.

1. The carboxyl group is removed and is the origin of some of the CO2 produced in respiration. This decarboxylation of pyruvate together with the dehydrogenation produces an acetyl group.

2. The acetyl group combines with coenzyme A (CoA) to become acetyl CoA.

3. The coenzyme NAD becomes reduced.

The equation summaries the link reaction for 2 molecules of pyruvate derived from 1 molecule of glucose:

2 Pyruvate + 2NAD + 2Coa ----> 2CO2 + 2 reduced NAD + 2 acetyl CoA

Coenzyme A accepts the acetyl group and, in the form of acetyl CoA, carries the acetyl group on to the Krebs cycle.

It takes place in the mitochondrial matrix.

It is a series of enzyme catalysed reactions that oxidises the acetate from the link reaction to 2 molecules of CO2, while conserving energy by reducing the coenzymes NAD and FAD.

These reduced coenzymes then carry the H atoms to the electron transport chain in the cristae, where they will be involved in the production of more ATP molecules.

For every molecule of glucose, there are 2 turns in the Krebs cycle.

1. The acetyl group released from acetyl CoA combines with a 4 carbon compound, oxaloacetate, to form a 6 carbon compound- citrate.

2. Citrate is decarboxylated and dehydrogenated producing a 5 carbon compound, one molecule of CO2 and one molecule of reduced NAD.

3. The 5 carbon compound is further decarboxylated and dehydrogenated, forming a 4 carbon compound, and is then released from, coenzyme A.

4. This 4 carbon compound combines temporarily with and is then released from, coenzyme A. At this stage, substrate-level phosphorylation takes place, producing one molecule of ATP.

5. The 4 carbon compound is dehydrogenated, producing a different four carbon compound and a molecule of reduced ATP.

6. Rearrangement of the atoms in the 4 carbon molecule, catalysed by an isomerase enzyme, followed by a further dehydrogenation, regenerates a molecule of oxaloacetate, so the cycle can continue.

Although O2 is not directly used in the 2 systems, these stages will not occur in the absence of oxygen, so they are aerobic. By the end of the Krebs cycle, the production of CO2 from glucose is complete. Other substrates beside glucose can be respired aerobically:

• Fatty acids are broken down to many molecules of acetate that enter the Krebs cycle via
• acetyl CoA.
• Glycerol may be converted to pyruvate and enter the Krebs cycle via the link reaction. 
• Amino acids may be deaminated and the rest of the molecules can enter the Krebs cycle directly or be changed to pyruvate or acetyl CoA.

The last stage is oxidative phosphorylation. It takes place in the mitochondria, involving electron carrier proteins which are arranged in chains called electron transport chains, embedded in the inner mitochondrial membrane, and a process called chemiosmosis. The cristae give a large surface area for the electron carrier proteins and the ATP synthase enzymes.

1. Reduced NAD and reduced FAD are reoxidised when they deliver their hydrogen atoms to the electron transport chain.

2. The H atoms released from the reduced coenzymes split into protons and electrons.

3. The protons go into solution in the mitochondrial matrix.

The electrons from the hydrogen atoms pass along the chain of electron carriers. Each electron carrier protein has an iron ion at its core.

The iron ions can gain an electron, becoming reduced (Fe2+). The reduced iron ion can then donate the electron to the iron ion in the next electron carrier in the chain, becoming reoxidised to Fe3+.

As electrons pass along the chain, some of their energy is used to pump protons across the inner mitochondrial membrane, into the IMS.

As protons accumulate in the IMS, a proton gradient forms across the membrane. This generates a chemiosmotic potential that is also known as a proton motive force, pmf. They are a source of potential energy. ATP is made using the energy of the proton motive force.

Protons cannot easily diffuse through the lipid bilayer of the mitochondrial membranes, as the outer membrane has a low degree of permeability to protons and the inner membrane is impermeable to protons.

Protons can diffuse through protein channels associated with ATP synthase enzymes that are in the inner membrane. As protons diffuse down their concentration gradient through these channels, the flow of protons causes conformational change in the ATP synthase enzyme that allows ADP and Pi to combine, forming ATP. This flow of protons is known as chemiosmosis. It is coupled to the formation of ATP. This way of forming ATP, in the presence of oxygen is oxidative phosphorylation.

Oxygen is the final electron acceptor. It combines with electrons coming off the ETC and with protons, diffusing down the ATP synthase channel, forming water:

4H+ + 4 e- + O2 ---> 2H2O

The reduced coenzymes provide both protons and electrons to the electron transport chain.

The protons and electrons from the 10 molecules of reduced NAD can theoretically produce 25 molecules of ATP.

The protons and electrons from the 2 molecules of reduced FAD can theoretically produce 3 molecules of ATP.

Oxidative phosphorylation may therefore produce 28 molecules of ATP per molecule of glucose.

Glycolysis produces a net gain of 2 molecules of ATP, as does the Krebs Cycle, the link reaction doesn't produced any and oxidative phosphorylation produces 28. Theoretically the total amount of ATP that can be produced from 1 molecule of glucose is 32 molecules. This rarely happens because:

• some ATP is used to actively transport pyruvate into the mitochondria
• some ATP is used in a shuttle system that transports reduced NAD, made during glycolysis, into mitochondria.
• some protons may leak out through the outer mitochondrial membrane.

1. Oxygen cannot act as the final electron acceptor at the end of oxidative phosphorylation. Protons diffusing through channels associated with ATP synthase are not able to combine with electrons and oxygen to form water.

2. The concentration of protons increases in the matrix and reduces the proton gradient across the inner mitochondrial membrane.

3. Oxidative phosphorylation ceases.

4. Reduced NAD and FAD are not able to unload their hydrogen atoms and cannot be oxidised.

5. The link reaction and Krebs cycle stop.

For the organism to survive these adverse conditions, glycolysis can take place, but the reduced NAD generated during the oxidation of triose phosphate to pyruvate has to be reoxidised so that glycolysis can continue. These reduced coenzyme molecules cannot be reoxidised at the ETC, so another metabolic pathway must operate to reoxidise them.

Eukaryotic cells have 2 metabolic pathways to reoxidise the reduced NAD:

•Fungi, such as yeast, and plants use the ethanol fermentation pathway.

•Mammals use the lactate fermentation pathway.

Both occur in the cytoplasm of cells.

1. Each molecule of pyruvate produced in glycolysis is decarboxylated and converted into ethanal. This stage is catalysed by pyruvate decarboxylase, which has a coenzyme, thiamine diphosphate, bound to it. A molecule of CO2 is released.

2. The ethanal accepts hydrogen atoms from reduced NAD, becoming reduced to ethanol. The enzyme ethanol dehydrogenase catalyses the reaction.

3. In the process, reduced NAD is reoxidised and made available to accept more H atoms from triose phosphate, thus allowing glycolysis to continue.

1. Pyruvate, produced during glycolysis, accepts hydrogen atoms from the reduced NAD, also made during glycolysis. The enzyme lactate dehydrogenase catalyses the reaction. There are 2 outcomes:

•Pyruvate is reduced to lactate.

•The reduced NAD becomes oxidised.

2. The reoxidised NAD can accept more H atoms from triose phosphate during glycolysis, and glycolysis can continue to produced enough ATP to sustain muscle contraction for a short period.

The lactate produced in the muscle tissue is carried away from the muscles, in the blood, to the liver. When more oxygen is available, the lactate may be either:

•Converted to pyruvate, which may enter the Krebs Cycle via the Link Reaction.

•Recycled to glucose and glycogen.

If lactate wasn't removed from the muscle tissue, the pH would be lowered and this would inhibit the action of many of the enzymes involved in glycolysis and muscle contraction. 

Neither ethanol or lactate fermentation produces any ATP. However, because this allows glycolysis to continue, the net gain of 2 molecules of ATP per molecule of glucose is still obtained.

Because the glucose molecule is only partly broken down, many more molecules can undergo glycolysis per minute, and therefore the overall yield of ATP is quite large.

However, for each molecule of glucose, the yield of ATP via anaerobic respiration is about 1/15 of that produced by aerobic respiration.

1. Pour 50cm3 of cider into each of the conical flasks.

2. Using a pipette, add one drop of yeast suspension to each conical flask. Make sure that the flask is swirled to mix its contents before taking the yeast suspension out of its flask.

3. Place 4 layers of muslin over the mouth of each conical flask and secure with an elastic band- allows oxygen in but stops dust etc from getting in. Leave the flask in a warm place for a week.

4. Mix the contents of each flask and using a pipette take some out and place a drop of a haemocytometer slide with its coverslip in place.

5. Count the number of yeast cells in the centre and corner squares. Each of the 5 squares where you counted contains 16 smaller squares, so you counted cells in every 80 squares, having a total volume of 0.02mm3, multiply your cell count by 50,000 to get the number of yeast cells per cm3.

6. Repeat for 3 counts of each flask size (50, 100, 150,150, 500cm3). Tabulate and graph the data.

1. Breathe onto the underside of the cover slip to moisten it.

2. Slide the coverslip horizontally onto the slide and carefully press down with your index fingers whilst pushing with the thumbs.

3. When the coverslip is correctly positioned, you will see a 6 rainbow pattern (Newton's Rings). The depth of the central chamber is 0.1mm.

4. Place the pipette tip at the entrance to the groove and allow liquid to fill the chamber. Leave for 5 minutes before counting.

5. Place the haemocytometer slide on the microscope stage with 1 of the grids over the stage aperture. Focus, using total x40 magnification.

6. Now focus using total x100 magnification. The central portion of the grid will now fill the field of view.

7. Count the cells in the central and 4 corner cells.

The rate of respiration can be measured by measuring the rate of evolution of CO2.

A carbon dioxide dissolves in the culture medium, it lowers the pH, and this can be measured uing a pH meter.

The monosaccharide glucose is the chief respiratory substrate.

Some mammalian cells, for example brain cells and RBCs, can use only glucose for respiration.

Animals and some bacteria store carbohydrate as glycogen, which can be hydrolysed to glucose for respiration.

Plant calls store carbohydrate as starch, and this can also be hydrolysed to glucose for respiration:

•Disaccharides can be digested to monosaccharides for respiration.

•Monosaccharides such as fructose and galactose can be changed, by isomerase enzymes, to glucose for respiration.

Fatty acids are long-chain hydrocarbons with a carboxylic acid group. Hence in each molecule there are many carbon atoms, many hydrogen atoms and very few oxygen atoms. These molecules are a source of many protons for oxidative phosphorylation, and so fats produce much more ATP than an equivalent mass of carbohydrate.

1. With the aid of some energy from the hydrolysis of one ATP molecule to AMP, each fatty acid is combined with coenzyme A.

2. The fatty acid-coA complex is transported into the mitochondrial matrix, where it is broken down into 2-carbon acetyl groups, each attached to CoA.

3. This beta-oxidation pathway generates reduced NAD and FAD.

4. The acetyl groups are released from CoA and enter the Krebs Cycle by combining with the 4-carbon oxaloacetate.

For every acetyl group oxidised in the Krebs Cycle, 3 molecules of reduced NAD, one molecule of reduced FAD and one molecule of ATP, by substrate level phosphorylation, are made.

Excess amino acids, released after the digestion of proteins, are deaminated in the liver.

Deamination of an amino acid involves removal of amino group and its subsequent conversion to urea that is removed via the kidney.

The rest of the amino acid molecules, a keto acid, enters the respiratory pathway as pyruvate, acetyl CoA or a Krebs cycle acid such as oxaloacetic acid.

During fasting, starvation or prolonged exercise, when insufficient glucose or lipids are available for respiration, protein from muscle can be hydrolysed to amino acids which are then respired.

These amino acids may be converted to pyruvate or acetate and enter the Krebs cycle.

Carbohydrates    15.8kJg-1

Lipid                    39.4kJg-1

Protein                 17.0kJ-1

The greater the availability of protons for chemiosmosis, the more ATP can be produced.

Therefore the more hydrogen atoms there are in a molecule of respiratory substrate, the more ATP can be generated per molecule of substrate. 

RQ= CO2 produced/ O2 consumed. No units.

Glucose 1

Fatty acids 0.7

Amino acids 0.8-0.9 

If the RQ value is greater than 1, this indicates that some anaerobic respiration is taking place, because it shows that more CO2 is being produced than oxygen is being consumed.

Organisms that are respiring aerobically absorb oxygen and give out CO2.

If the carbon dioxide produced is absorbed by the NaOH solution (soda lime), then the only volume change within the respirometer is due to the volume of O2 absorbed by the organisms.

If oxygen is absorbed from the tube containing the organisms, then that tube has a reduced volume of air in it, exerting less pressure that the greater volume of air in the other tube.

As a result, the coloured liquid in the manometer tube rises up towards the respirometer tube.

If the original level of liquid in the mano meter is marked and the radius of the bore in the capillary tube is known, the volume of oxygen absorbed during a specific period can be calculated.

To reset the apparatus, the syringe is depressed to inject air into the system and reset the liquid in the manometer tube back to its original position. This also allows a reading of the volume of oxygen absorbed by noting the change in level of the syringe plunger, as measured from the graduated scale on the syringe barrel.

1. After placing the coloured liquid (e.g. methylene blue solution with a drop of detergent) into the manometer tube, the apparatus is connected with the taps open. This enables the air in the apparatus to connect with the atmosphere. The mass of living organisms (e.g. woodlice) to be used should be found.

2. With the taps still open the whole set up, with the living organism in place, is placed in a water bath for at least 10 minutes until it reaches the temperature of the water bath. The syringe plunger should be near the top of the scale of the syringe barrel and its level noted.

3. The levels of coloured liquid in the manometer tubes can be marked with a felt tip pen or chinagraph pencil. The taps are closed and the apparatus left in the water bath for a specific period. 

4. The change in level of the manometer liquid can be measured and the syringe barrel depressed to reset the apparatus. This also enables you to measure the volume of O2 absorbed.

5. You can then calculate the volume of O2 absorbed per minute per gram of living organism.

The effect of temperature on the rate of respiration can be investigated using the respirometer.

3 readings should be taken at each temperature.

In between each temperature reading, the apparatus and organisms should be allowed to adjust to the new temperature.

Suitable living organisms can be blowfly maggots, woodlice, yeast in glucose suspension or soaked pea seeds that are beginning to germinate.

Animal specimens should only be used over a narrow range of temperatures, such as from 10 to 40 degrees Celsius. For more extreme temperatures, fungal material should be used. 

The respirometer may be used so that a suspension of yeast, with differing concentrations of glucose solution, is placed in one of the tubes.

If the sodium hydroxide solution is omitted, the evolution of carbon dioxide during a specific time frame can be measured.