Respiration

Respiration- the process whereby energy stored in complex organic molecules (carbohydrates, fats and proteins) is used to make ATP, occurring in living cells.

Energy is the capacity to do work, and is stored in complex molecules such as fats and carbohydrates. All living organisms need energy to drive their biological processes in order to survive. Metabolic reactions that need energy include: 

• Active Transport - moving ions and molecules across a membrane against a concentration gradient.

• Secretion- large molecules made in some cells are exported by exocytosis.

• Anabolism- synthesis of large molecules from smaller ones, e.g. proteins from amino acids, steroids into cholesterol and cellulose from β-glucose.

• Replication of DNA and synthesis of organelles before a cell divides.

• Endocytosis- bulk movement of large molecules into cells.

• Movement- movement of bacterial flagella, eukaryotic cilia and undulipodia, muscle contraction and microtubule motors that move organelles around inside cells. 

• Activation of chemicals- glucose is phosphorylated at the beginning of respiration so that it is more unstable and can be broken down to release energy.

• All chemical reactions that take place within living cells are known collectively as metabolism.

ATP

• ATP stands for adenosine triphosphate and is a phosphorylated nucleotide. Each molecule consists of adenine, ribose and three phosphates.

• ATP can be hydrolysed to ADP and Pi (inorganic phosphate), releasing 30.6 kJ energy per mol. So, energy is immediately available to cells in small, manageable amounts that will not damage the cell (enzymes and proteins can denature or membranes could become too fluid if too much energy is released), so it’s easier to harness the energy and it will not be wasted. The energy released from ATP hydrolysis is an immediate source of energy for biological processes, such as DNA replication and protein synthesis.

• ATP is the ‘universal energy carrier’. It is found in all living cells, is small and soluble sot that it can move around the cell, it has high energy bonds between phosphates which break down to release energy where required, is produced where energy is released, and is an immediate source of energy.

• ATP is a relatively stable molecule that typically only breaks down via catalysis

• At any one time you have 5g of ATP, however 36-60kg may be used each day. This is possible as a person both consumes and regenerates ATP at the rate of about 1.5kg per hour. When active this increases.

• Anabolic reactions– building larger molecules from smaller molecules (condensation)

• Catabolic reactions– breaking larger molecules to form smaller molecules (hydrolysis).

• Catabolic reactions release energy that the building of ATP uses. The hydrolysis of ATP releases energy that other anabolic reactions could use.

Respiration overview

• Glycolysis- occurs in the cytoplasm which can take place in aerobic or anaerobic conditions. Glucose is broken down to two molecules of pyruvate.

• The link reaction- occurs in the matrix of the mitochondria. Pyruvate is dehydrogenated and decarboxylated `and converted to acetate.

• Krebs cycle- occurs in the matrix of the mitochondria. Acetate is dehydrogenated and decarboxylated. 

• Oxidative phosphorylation- occurs on the folded inner membrane (cristae) of mitochondria. This is where ADP is phosphorylated to ATP.

Coenzymes

• Coenzymes are needed to help enzymes carry out oxidation reactions, where hydrogen atoms are removed from substrate molecules in respiration. The hydrogen atoms are combined with coenzymes, so that they can be carried and can later be split into hydrogen ions and electrons, to the inner mitochondrial membranes where they will be involved in oxidative phosphorylation.

• Nicotinamide adenine dinucleotide is an organic, non-protein molecule that helps dehydrogenase enzymes to carry out oxidation reactions. It's made of two linked nucleotides - nicotinamide (Vitamin B3), 5-carbon sugar ribose, adenine and 2 phosphate (phosphoryl) groups. The nicotinamide ring can accept hydrogen atoms which can later be split into a hydrogen ion and an electron. 

• When a molecule of NAD has accepted 2 hydrogen atoms with their electrons, it is reduced. When it loses the electrons it is oxidised. NAD operates in glycolysis, the link reaction, Krebs cycle and during the anaerobic ethanol and lactate pathways.

• Coenzyme A is Made from pantothenic acid (a B-group vitamin), adenosine (ribose and adenine), 3 phosphate (phosphyl) groups and cysteine (amino acid).

• Its function is to carry ethanoate (acetate) groups, made from pyruvate during the link reaction, onto Krebs cycle and can also carry acetate groups that have been made from fatty acids or from some amino acids on to the Krebs cycle. 

• Flavin Adenine Dinucleotide is similar to NAD. When FAD accepts two hydrogen atoms with their electrons so that it becomes reduced.

• FAD operates during the Krebs cycle. It then acts as a reducing agent by donating electrons at the inner mitochondrial membrane during oxidative phosphorylation

• Living organisms don’t have much NAD, CoA or FAD in their cells as they are recycled/regenerated

The mitochondria

• The mitochondria are rod-shaped and between 0.5-1.0 µm in diameter and 2-5 µm long, although some can be up to 10 µm long. The mitochondria are moved around within cells by the cytoskeleton (microtubules).

• The matrix is where the link reaction and Krebs cycle takes place. It contains enzymes that catalyse the stages of these reactions, coenzyme NAD, Oxaloacetate (a 4-carbon compound that accepts acetate from the link reaction),  mitochondrial DNA (some of which codes for mitochondrial enzymes and other proteins), and mitochondrial ribosomes where these proteins are assembled.

• The outer membrane contains proteins, some of which form channels or carriers that allow the passage of molecules such as pyruvate, or other proteins such as enzymes.

• The inner membrane has a different lipid composition from the outer membrane and is impermeable to most small ions, including hydrogen ions (protons) meaning protons accumulate in the intermembrane space, building a proton gradient - a source of potential energy. It's folded into cristae to give a large surface area and embedded in it is many electron carriers and ATP synthase. 

The electron carriers of the inner membrane are protein complexes, arranged in electron transport chains:

• Each electron carrier is an enzyme, each associated with a cofactor.The cofactors can accept and donate electrons because the iron ions can become reduced (to Fe2+) by accepting an electron and oxidised (to Fe3+) by donating an electron to the next electron carrier. They are oxidoreductase enzymes as they are involved in oxidation and reduction reactions.

• Some of the electron carriers also have a coenzyme that pumps (using the energy released from the passage of electrons) protons from the matrix to the intermembrane space. Protons flow down a proton gradient, through the ATP synthase enzymes, from the imatrix into the intermembrane space - this is called chemiosmosis. The force of this flow drives the rotation of part of the enzyme and allows ADP and Pi to be joined to make ATP.

• The ATP synthase enzymes are large and protrude from the inner membrane into the matrix, are also known as stalked particles, and allow protons to pass through them.

• More active organisms have more mitochondria, larger mitochondria, and larger cristae

Glycolysis

• Glycolysis is a very ancient biochemical pathway, occurring in the cytoplasm of all living cells (prokaryotic and eukaryotic) that respire. It is an anaerobic metabolic pathway.

In glycolysis:

1. One molecule of ATP is hydrolysed and the phosphate group produced is used to convert glucose into glucose-6-phosphate. The energy from the hydrolysed ATP molecule activates the hexose sugar and prevents it from being transported out of the cell.

2. Glucose 6-phosphate is rearranged, using the enzyme isomerase, into fructose 6-phosphate. 

3. Phosphorylation via hydrolysis of a molecule of ATP occurs again forming hexose 1,6-bisphosphate.

4. The hexose 1,6-bisphosphate splits into two molecules of triose phosphate. 

5. Each triose phosphate is dehydrogenated, removing hydrogen atoms using dehydrogenase enzymes.

6. The coenzyme NAD accepts the hydrogen atoms and becomes reduced NAD.

7. Two molecules of ATP are formed, via substrate-level phosphorylation (the formation of ATP directly during glycolysis and the Krebs cycle only). 

8. The triose phosphate molecules are converted to pyruvate, which is actively transported to the mitochondrial matrix. In the process, another two molecules of ADP are phosphorylated to make two molecules of ATP.

• During aerobic respiration in animals, the triose phosphate molecules are converted into pyruvate. Pyruvate that is produced during glycolysis is transported across the inner and outer membrane to the mitochondrial matrix where the link reaction takes place.

The Link reaction

Decarboxylation- the removal of a carboxyl group.

Dehydrogenation- the removal of hydrogen atoms

1. The pyruvate molecule is decarboxylated by the enzyme pyruvate decarboxylase, removing a carboxyl group which eventually becomes carbon dioxide.

2. The pyruvate molecule is also dehydrogenated by the enzyme pyruvate dehydrogenase, removing hydrogen atoms, forming acetate.

3. The hydrogen atoms are accepted by the coenzyme NAD, becoming reduced NAD. 

4. The acetate combines with coenzyme A forming acetyl CoA.

• 2 pyruvate + 2NAD+ + 2CoA → 2CO2 + 2NADH + 2 acetyl CoA

• Coenzyme A (CoA) accepts acetate to become acetyl coenzyme A. The function of CoA is to carry acetate to the Krebs cycle.

The Krebs cycle

The Krebs cycle takes place in the mitochondrial matrix. It produces one molecule of ATP by substrate-level phosphorylation and reduces three molecules of NAD and one molecule of FAD.

1. The acetate is offloaded from coenzyme A and joins with oxaloacetate (4C), to form citrate (6C).

2. Citrate is decarboxylated and dehydrogenated forming a 5-carbon compound. The pair of hydrogen atoms is accepted by the coenzyme NAD (hydrogen acceptor), which becomes reduced NAD.

3. The 4-carbon compound is decarboxylated and dehydrogenated forming a 4-carbon compound and another molecule of reduced NAD.

4. The 4-carbon compound is changed into another 4-carbon compound. During this reaction a molecule of ADP is phosphorylated to produce a molecule of ATP - substrate-level phosphorylation.

5. The second 4-carbon compound is changed into another 4-carbon compound. It's dehydrogenated and the coenzyme FAD (hydrogen acceptor) accepts the hydrogen atoms, and becomes reduced FAD.

6. The third 4-carbon compound is further dehydrogenated and regenerates oxaloacetate and forms another molecule of reduced NAD.

Oxidative phosphorylation

1. NADH is reoxidised to form NAD+ and 2 hydrogen atoms, aided by the enzyme NADH dehydrogenase which is attached to the first electron carrier. The hydrogen atoms split into protons and electrons.  

2. The electrons are passed along electron carriers in the electron transport chain and lose energy by doing this.

3. The energy that was lost in the electron transport chain is used to pump protons into the intermembrane space creating a proton gradient– the protons will want to move back into the matrix from a high concentration to a low concentration. 

4. The H+ ions cannot diffuse through the lipid part of the membrane so they diffuse through protein channels that are associated with ATP synthase, which is linked to the synthesis of ATP. The flow of protons is chemiosmosis.

5. The flow of protons through the protein channels creates a proton motive force which drives the rotation of the ATP synthase enzyme attached to the protein channel. The rotation causes the phosphorylation of ADP to make ATP.

6. The electrons are passed from the last electron carrier in the chain to oxygen, which is the final electron acceptor. Hydrogen ions also join forming water.

Theoretical yield

• The 10 molecules of NAD can theoretically produce 26 molecules of ATP during oxidative phosphorylation (each NAD molecule can make 2.6 molecules of ATP). Together with the 4 ATP made during glycolysis and the Krebs cycle, the total yield of ATP molecules, per molecule of glucose respired, should be 30. However this is rarely achieved for the following reasons:

1. Some protons leak across the mitochondrial membrane, reducing the number of protons to generate the proton motive force.

2. Some ATP produced is used to actively transport pyruvate into the mitochondria.

3. Some ATP is used for the shuttle to bring hydrogen from reduced NAD made during glycolysis, in the cytoplasm, into the mitochondria.

Anaerobic respiration

• Anaerobic respiration is the process where ATP is produced by substrate-level phosphorylation during glycolysis in the absence of oxygen, in the cytoplasm of eukaryotic cells.

• As anaerobic respiration occurs in the absence of oxygen, the electron transport chain cannot happen so the link reaction, Krebs cycle and oxidative phosphorylation cannot happen. Therefore only glycolysis can happen and only ATP can be produced via glycolysis. The reduced NAD has to be reoxidised so that it can keep accepting hydrogen atoms in glycolysis. There are two ways that NAD can be reoxidised – lactate fermentation and alcohol fermentation.

Comparing anaerobic respiration in mammals and yeast

• Neither ethanol fermentation nor lactate fermentation produces any ATP. However, because this allows glycolysis to continue, the net gain of two molecules of ATP per molecule of glucose is still obtained

• As glucose is only partially broken down by glycolysis, the ATP yield of anaerobic respiration is only about 1/15 of that produced from aerobic respiration

• Fast twitch muscle fibres have few if any mitochondria and use glycolysis to power their short-duration contractions. They fatigue easily and appear pale in colour due to lack of electron transport proteins. They also lack myoglobin, a protein that stores oxygen in some muscles. 

• Slow twitch muscle fibres are dark red, they contain many mitochondria and are slow to fatigue. They operate aerobically for endurance exercise.

• Rate of respiration can be measured by measuring rate of CO2 consumption. As CO2 dissolves in the medium it lowers pH, which can be measured using a pH meter

Respiratory substrates

Respiratory Substrate– an organic substance that can be used for respiration.

• The more protons, the more ATP produced as most ATP is formed from the flow of protons through channel proteins during chemiosmosis. Therefore the more hydrogen atoms there are in a molecule of respiratory substrate, the more ATP can be generated when that substrate is respired. It also follows that if there are more hydrogen atoms per mole of respiratory substrate, then more oxygen is needed to respire that substance. 

Respirometer

1. In the boiling tube the volume of oxygen decreases due to respiration. The volume of carbon dioxide also decreases as the soda lime absorbs it.

2. Overall, the volume of the boiling tube decrease, causing an air pressure decrease. This causes an air pressure gradient between the boiling tube and the capillary tube, and so the drop of liquid moves towards the boiling tube, as it has a lower air pressure.

3. Ensure that the apparatus is equilibrated – use syringe to adjust the position of the fluid in the manometer.

4. Record the position of the liquid and leave the apparatus for a certain length of time to allow time for respiration, then record the new position of the liquid. Repeat and take the mean. If the diameter of the capillary tube is known, use the formula πr2lto find the volume of oxygen uptake to give the rate. 

5. The second boiling tube is a control – air pressure stays the same so no changes occur.  There is a closed tap to ensure no air can enter or leave during the experiment.

• If measuring respiration rates in photosynthetic organisms, wrap the tubes in foil or black paper to exclude light and prevent photosynthesis