The Stages of Respiration (OCR A-Level Biology A): Revision Notes

The Stages of Respiration

Overview of the four stages

Respiration comprises four interconnected stages, each occurring in specific cellular locations:

1. Glycolysis – occurs in the cytosol; does not require oxygen; functions in both aerobic and anaerobic respiration; converts glucose into pyruvate
2. The link reaction – occurs in the mitochondrial matrix; converts pyruvate into an acetyl group attached to coenzyme A
3. The Krebs cycle – occurs in the mitochondrial matrix; processes the acetyl group from acetyl coenzyme A
4. Oxidative phosphorylation – occurs on the cristae (inner mitochondrial membranes); electrons flow along the electron transport chain, driving proton pumping and ATP synthesis

Glycolysis

Glycolysis represents an ancient metabolic pathway that likely evolved in primitive life forms before atmospheric oxygen appeared. This anaerobic process occurs in the cytosol of all living cells and releases relatively small amounts of energy.

Ten enzyme-catalysed reactions comprise glycolysis, converting one glucose molecule into two pyruvate molecules. The pathway requires NAD coenzymes and phosphate groups from two ATP molecules.

Phosphorylation

Glucose must first be activated by phosphorylation, which raises its energy level:

• A phosphate group transfers from ATP to glucose at the carbon-6 position, forming glucose 6-phosphate
• Glucose 6-phosphate converts into its isomer, fructose 6-phosphate
• A second ATP donates another phosphate to the carbon-1 position, creating fructose 1,6-bisphosphate (the 'bis' prefix indicates phosphates attached to two different carbon atoms)
• Two ATP molecules have been consumed; the released ADP molecules will be regenerated into ATP in later stages

Splitting fructose 1,6-bisphosphate

The six-carbon fructose 1,6-bisphosphate now splits into two three-carbon molecules, each retaining one phosphate group. These molecules are called triose phosphate (triose = three-carbon sugar).

Oxidation of triose phosphate

Dehydrogenase enzymes remove hydrogen atoms from triose phosphate molecules, with NAD acting as the hydrogen acceptor. This oxidation reaction produces:

• Two molecules of reduced NAD per glucose molecule
• Sufficient energy to add phosphate ions (from the cytosol) to both triose phosphate molecules, forming two molecules of triose bisphosphate
• Energy conservation – without this phosphorylation step, energy would dissipate as heat

Conversion to pyruvate

Multiple enzyme-controlled reactions convert each triose phosphate into a three-carbon pyruvate molecule. This conversion releases enough energy to phosphorylate ADP molecules directly, producing two ATP molecules through substrate-level phosphorylation – the direct formation of ATP when converting one molecule to another, contrasting with ATP formed via oxidative phosphorylation.

Products of glycolysis

Each glucose molecule entering glycolysis yields:

• Two molecules of reduced NAD – coenzymes reduced by accepting hydrogen atoms; these diffuse into mitochondria via a shunt mechanism
• Net gain of two ATP molecules – four ATP produced minus two ATP consumed in initial phosphorylation steps
• Two molecules of pyruvate – actively transported into mitochondria for the link reaction during aerobic respiration; converted to lactate (animals) or ethanol (yeast and some bacteria) during anaerobic respiration

The link reaction

Pyruvate molecules from glycolysis cross the mitochondrial membranes into the matrix, where enzymes for the link reaction are located. Two distinct processes occur:

1. Decarboxylation – the enzyme complex pyruvate dehydrogenase removes a carboxyl group from pyruvate, releasing it as carbon dioxide ($\text{CO}_2$)
2. Dehydrogenation – the same enzyme complex removes hydrogen atoms, which NAD accepts to form reduced NAD

The resulting two-carbon acetyl group attaches to coenzyme A, forming acetyl coenzyme A (acetyl CoA), which carries the acetyl group into the Krebs cycle.

Processing two pyruvate molecules (from one glucose) through the link reaction produces:

• Two molecules of reduced NAD
• Two carbon dioxide molecules
• Two acetyl CoA molecules

The Krebs cycle

The Krebs cycle (also called the tricarboxylic acid cycle or citric acid cycle) operates in the mitochondrial matrix as a series of enzyme-controlled reactions organised cyclically. Acetyl groups from the link reaction drive this cycle.

The two-carbon acetyl group transfers from acetyl CoA to the four-carbon molecule oxaloacetate, forming the six-carbon molecule citrate. Through subsequent steps, citrate undergoes decarboxylation (releasing two $\text{CO}_2$ molecules) and dehydrogenation (releasing eight hydrogen atoms), regenerating oxaloacetate. Six hydrogen atoms reduce NAD (forming three reduced NAD molecules), while two hydrogen atoms reduce FAD (forming one reduced FAD molecule). The energy released during these transformations directly phosphorylates one ADP to ATP through substrate-level phosphorylation.

Steps in the Krebs cycle

For each turn of the cycle:

1. The two-carbon acetyl group from acetyl CoA combines with four-carbon oxaloacetate, forming six-carbon citrate; coenzyme A releases for reuse
2. Citrate undergoes dehydrogenation and decarboxylation, forming a five-carbon molecule; this releases hydrogen atoms (accepted by NAD, forming reduced NAD) and one $\text{CO}_2$ molecule
3. The five-carbon molecule undergoes further dehydrogenation and decarboxylation, forming a four-carbon compound plus reduced NAD and $\text{CO}_2$
4. Reorganisation of the four-carbon molecule provides energy for direct ATP synthesis from ADP and inorganic phosphate ($\text{P}_i$) in the matrix – another example of substrate-level phosphorylation
5. The next four-carbon compound undergoes dehydrogenation; FAD (not NAD) accepts the hydrogen atoms, becoming reduced FAD
6. In the final reaction, dehydrogenation of a four-carbon molecule regenerates oxaloacetate, completing the cycle; NAD accepts the hydrogen atoms, forming reduced NAD

Since one glucose produces two pyruvate molecules, two acetyl groups enter the Krebs cycle, creating two complete turns. The products from two turns are:

• Six molecules of reduced NAD
• Two molecules of reduced FAD
• Four carbon dioxide molecules
• Two ATP molecules (produced directly)

Oxidative phosphorylation

Oxidative phosphorylation uses energy from electron transfer to add inorganic phosphate to ADP, forming ATP. This process occurs on the cristae – the highly folded inner mitochondrial membranes where electron carriers embed alongside ATP synthase enzymes. The folded structure maximises surface area for carriers and ATP synthase.

Reduced NAD and FAD from the Krebs cycle (plus reduced NAD from the link reaction and possibly glycolysis) donate their hydrogen atoms to the electron transport chain. The hydrogen splits into protons ($\text{H}^+$) and electrons ($\text{e}^-$). Electrons transfer to NADH-coenzyme Q reductase (Complex I), while protons enter the matrix.

Electrons pass sequentially from one carrier to another, with each carrier becoming reduced (accepting electrons) then oxidised (donating electrons to the next carrier). Energy released at certain steps phosphorylates ADP to ATP. Each pair of electrons passing through the chain produces between $1.5$ and $2.5$ ATP molecules, depending on which hydrogen carrier initiated the process and whether hydrogen atoms needed transport from the cytosol.

Oxygen serves as the final electron acceptor. It combines with electrons and protons (which rejoin the electrons), reducing to form water ($\text{H}_2\text{O}$).

Chemiosmosis

Chemiosmosis describes ATP formation from energy released during electron transport along the electron transport chain. It occurs on the inner mitochondrial membranes as part of oxidative phosphorylation.

As electrons move along carrier molecules, they release energy. Reduced coenzymes become oxidised, and some released energy actively pumps protons into the intermembrane space. This creates a proton concentration gradient and electrochemical gradient. The pump uses energy from electron flow (electromotive force), not ATP.

The inner membrane remains impermeable to ions, including protons. Protons cannot cross the membrane directly but flow through ion channels associated with ATP synthase, moving down their concentration gradient. As protons flow through ATP synthase channels, the enzyme's rotating component turns, releasing energy that phosphorylates ADP with $\text{P}_i$ to form ATP.

The electrochemical gradient from chemiosmosis creates potential energy – stored energy from the protons' position in the intermembrane space. This potential energy drives ATP synthase and builds ATP molecules.

ATP yield summary

The complete oxidation of one glucose molecule through aerobic respiration produces:

Stage	Location	Substrate	Products	ATP yield	Respiration type
Glycolysis	Cytosol	Glucose (6C)	$2$ pyruvate (3C), $4$ ATP, $2$ reduced NAD	$2$ (net production)	Aerobic and anaerobic
Link reaction	Mitochondrial matrix	Pyruvate	Acetyl coenzyme A, reduced NAD, $\text{CO}_2$	—	Aerobic
Krebs cycle (per turn)	Mitochondrial matrix	$2$ acetyl groups from CoA	Oxaloacetate recycled, $2\ \text{CO}_2$, $1$ ATP, $6$ reduced NAD, $2$ reduced FAD	$2$ (two turns)	Aerobic
Oxidative phosphorylation	Cristae	$10$ reduced NAD, $2$ reduced FAD, ADP	$\text{H}_2\text{O}$, $26$ ATP	$26$–$28$	Aerobic
Total				$30$–$32$

Glycolysis produces four ATP molecules directly through substrate-level phosphorylation, but consumes two ATP, yielding a net gain of two ATP. The Krebs cycle produces two ATP directly (one per turn, two turns total) through substrate-level phosphorylation. Oxidative phosphorylation generates 26 ATP molecules from $10$ reduced NAD molecules.

Both reduced NAD and reduced FAD donate electrons to the electron transport chain, but only reduced NAD donates the hydrogen ions forming the proton gradient for chemiosmosis. Hydrogen ions from reduced FAD remain in the matrix, eventually recombining with electrons and oxygen to form water.

However, some ATP actively transports pyruvate into the mitochondrial matrix, and the mitochondrial shunt mechanism transports reduced NAD from glycolysis into the matrix. Some $\text{H}^+$ may leak out, reducing potential energy and limiting ATP production. In reality, $30$ ATP molecules may not always be achieved, with yields typically ranging from $30$–$32$ ATP per glucose.