Glycolysis

Glycolysis is the first stage of both aerobic and anaerobic respiration. It takes place in the cytoplasm and does not require oxygen. During glycolysis, one glucose molecule is split into two molecules of pyruvate, with a net production of ATP.

Phosphorylation and splitting of glucose

ATP supplies phosphate groups to glucose in a series of reactions. This phosphorylation uses two ATP molecules per glucose molecule. The resulting phosphorylated compound is split to form two molecules of triose phosphate. Each triose phosphate molecule contains three carbon atoms.

Oxidation of triose phosphate

Each triose phosphate molecule is oxidised through a series of reactions to form pyruvate. During these reactions, hydrogen and electrons are transferred to NAD, reducing it. ATP is also produced from ADP.

Because each glucose molecule produces two triose phosphate molecules, these reactions occur twice per glucose molecule. They produce four ATP molecules in total, as well as two molecules of reduced NAD and two molecules of pyruvate.

Net ATP yield of glycolysis

Four ATP molecules are produced, but two were used in the initial phosphorylation. Glycolysis therefore has a net yield of two ATP molecules per glucose molecule.

Anaerobic regeneration of NAD

When oxygen is unavailable, cells can keep producing ATP by glycolysis only if they have a supply of NAD. NAD accepts hydrogen during the oxidation of triose phosphate in glycolysis, becoming reduced NAD. To keep glycolysis going, reduced NAD must be converted back to NAD.

Converting pyruvate and regenerating NAD

Pyruvate, a product of glycolysis, can be converted along either of two pathways. In each pathway, reduced NAD supplies hydrogen and is oxidised back to NAD.

• Lactate pathway: pyruvate accepts hydrogen from reduced NAD and is converted to lactate.
• Ethanol pathway: pyruvate first loses carbon dioxide and forms ethanal. Ethanal then accepts hydrogen from reduced NAD and is converted to ethanol.

The regenerated NAD can accept hydrogen from more triose phosphate. This allows glycolysis to continue producing ATP without oxygen. The conversion of pyruvate itself does not produce additional ATP; its role is to regenerate NAD.

Link reaction

The link reaction prepares pyruvate, produced during glycolysis, for the Krebs cycle. Pyruvate enters the mitochondrial matrix by active transport, which requires energy. The link reaction takes place in the matrix.

Each pyruvate molecule contains three carbon atoms. During the reaction, one carbon is removed as carbon dioxide, leaving a two-carbon acetate group. Pyruvate is also oxidised: hydrogen is removed and accepted by NAD, forming reduced NAD.

The acetate group combines with coenzyme A to form acetylcoenzyme A. This carries the two-carbon group into the Krebs cycle, linking glycolysis to that cycle.

Krebs cycle

The Krebs cycle is a sequence of reactions in the mitochondrial matrix, the space inside the inner membrane of a mitochondrion. It is a cycle because it regenerates a molecule needed at the start, allowing the reactions to repeat.

An acetyl group, containing two carbon atoms, enters the cycle attached to coenzyme A. It combines with a four-carbon molecule to form a six-carbon molecule, and coenzyme A is released.

Regeneration and products of the Krebs cycle

The six-carbon molecule passes through a series of reactions that eventually regenerate the four-carbon molecule. Carbon dioxide is released during these reactions. Oxidation also removes electrons and hydrogen, which are accepted by coenzymes. The coenzymes are therefore reduced, producing reduced NAD and reduced FAD. These carry the electrons and hydrogen to later stages of respiration.

ATP is also produced during the cycle by substrate-level phosphorylation: a phosphate group is transferred directly from a substrate to ADP. Once the four-carbon molecule has been regenerated, it can combine with another acetyl group and the cycle can begin again.

Oxidative phosphorylation

Oxidative phosphorylation produces ATP using energy released as electrons pass along an electron transfer chain in the inner mitochondrial membrane. This energy is first used to build a proton gradient across the membrane. The flow of protons back across the membrane then drives ATP formation.

Electron transfer and the proton gradient

Reduced coenzymes, including reduced NAD and reduced FAD, donate electrons to the electron transfer chain. As the electrons pass along the chain, they release energy. This energy is used to pump protons from the mitochondrial matrix across the inner membrane into the intermembrane space.

Protons therefore become more concentrated in the intermembrane space than in the matrix. This proton gradient stores energy: protons tend to move back towards the matrix, down the gradient.

Chemiosmosis and ATP formation

Protons pass back into the matrix through ATP synthase, an enzyme embedded in the inner mitochondrial membrane. Their flow through ATP synthase provides the energy for the enzyme to catalyse the formation of ATP from ADP and phosphate. The coupling of a proton gradient to ATP synthesis is called chemiosmosis.

Oxygen as the final electron acceptor

At the end of the electron transfer chain, oxygen accepts the electrons and combines with them and protons to form water. Oxygen is therefore the final electron acceptor. Without it, electrons cannot continue to pass along the chain, so the proton gradient can no longer be maintained for ATP synthesis.

Alternative respiratory substrates

A respiratory substrate is a molecule whose breakdown provides material for respiration and ATP production. Glucose is one respiratory substrate, but the body can also use breakdown products of lipids and amino acids.

Fatty acids released from lipids can be broken down to form acetyl coenzyme A. This enters the Krebs cycle. Amino acids can also provide material for the cycle: after their amino group has been removed, their remaining carbon-containing skeleton can be converted into acetyl coenzyme A or into another compound that enters the cycle.

In the Krebs cycle, carbon-containing compounds are oxidised, and coenzymes are reduced. The reduced coenzymes carry electrons to later reactions that help drive ATP production. Lipids and amino acids can therefore provide material for respiration without first being converted into glucose.

Investigating respiration rate in single-celled organisms

Single-celled organisms such as yeast release carbon dioxide during respiration. Measuring how much carbon dioxide a yeast culture produces in a fixed time gives a measure of its respiration rate under the conditions used.

To investigate the effect of temperature, prepare yeast cultures with the same volume, yeast concentration and substrate concentration. Place the cultures in water baths set to different temperatures. Allow them to reach their assigned temperatures using the same procedure for each culture.

Connect each culture to a gas syringe using an airtight apparatus. Measure the volume of carbon dioxide collected over the same fixed time for each culture. The temperature is the independent variable being changed; carbon dioxide production is the measurement used to assess its effect on respiration.

Calculating and comparing rates

For each culture, divide the volume of carbon dioxide collected by the time over which it was collected:

For example, if a culture produces $$12\ \text{cm}^3$$ of carbon dioxide in $$4$$ minutes, its rate is $$3\ \text{cm}^3\text{/min}$$. Compare the rates at the different temperatures to see how temperature affects carbon dioxide production.

Repeat the investigation with separate cultures at each temperature. The results show how much the measurements vary, and a mean rate can be calculated for each temperature by adding the repeat rates and dividing by the number of repeats.

Controlling the investigation

Keep culture volume, yeast concentration, substrate concentration and measurement time constant. Use the same type of apparatus and the same measurement procedure throughout. Otherwise, a difference in carbon dioxide production might be caused by one of these factors rather than temperature.

A suitable control is a mixture containing the same substrate but no yeast, tested under the same conditions at each temperature. It helps identify carbon dioxide produced by influences other than the yeast. Consistent conditions and controls make the comparison between temperatures more reliable.