Difficulty: Intermediate | Prerequisites: Part 1 of these notes (sections 6.1–6.5), understanding of redox reactions, NAD+/NADH.
This is the second of three sets of notes on Chapter 6. It covers the three main stages of aerobic respiration: glycolysis, the citric acid cycle and oxidative phosphorylation. Part 1 covers the overview and energy basics (6.1–6.5). Part 3 covers fermentation, evolution of metabolism and diverse fuel molecules (6.13–6.16).
Aerobic respiration has three stages, always in the same order: glycolysis (in the cytoplasm), the citric acid cycle (in the mitochondrial matrix) and oxidative phosphorylation (at the inner mitochondrial membrane). Glycolysis splits glucose into two pyruvate molecules for a small ATP payoff. The citric acid cycle finishes extracting electrons and CO2. Oxidative phosphorylation, powered by the electron transport chain and chemiosmosis, generates the vast majority of ATP, roughly 26 to 28 of the approximately 32 molecules produced per glucose.
Glycolysis
The first stage of cellular respiration. A ten-step pathway in the cytoplasm that splits one molecule of glucose (6 carbons) into two molecules of pyruvate (3 carbons each), producing a net gain of 2 ATP and 2 NADH. Does not require oxygen. Think of it as the opening act: a small energy payoff that every living cell can do, with or without oxygen.
Pyruvate
A three-carbon molecule produced at the end of glycolysis. It is the starting material for either the citric acid cycle (aerobic path) or fermentation (anaerobic path). In simple terms, pyruvate is the handoff molecule between glycolysis and everything that follows.
Acetyl CoA (acetyl coenzyme A)
The two-carbon molecule (attached to coenzyme A) that enters the citric acid cycle. Formed when pyruvate is oxidised, loses one carbon as CO2, and is joined to coenzyme A. Think of coenzyme A as a taxi that drops the acetyl group off at the citric acid cycle and then leaves.
Citric acid cycle (Krebs cycle)
The second stage of aerobic respiration, occurring in the mitochondrial matrix. Each turn of the cycle processes one acetyl CoA, releasing 2 CO2, 1 ATP, 3 NADH and 1 FADH2. Two turns per glucose (because glycolysis produces two pyruvates). In simple terms, this is where the remaining carbon atoms from glucose are fully stripped of their electrons and released as CO2.
Oxidative phosphorylation
The third and final stage, occurring at the inner mitochondrial membrane. Combines the electron transport chain with chemiosmosis to produce the bulk of ATP (about 26 to 28 per glucose). Requires oxygen. Think of it as the main power generator of the cell.
Substrate-level phosphorylation
A method of making ATP by directly transferring a phosphate group from a substrate molecule to ADP. Occurs during glycolysis and the citric acid cycle. Produces a small amount of ATP compared with oxidative phosphorylation. In simple terms, this is the "manual" way of making ATP, one molecule at a time.
Chemiosmosis
The process by which ATP is synthesised when H+ ions flow down their concentration gradient through ATP synthase, a channel protein in the inner mitochondrial membrane. The H+ gradient is built by the electron transport chain pumping H+ from the matrix into the intermembrane space. Think of it as a dam: the ETC pumps water (H+) to one side, and when it flows back through the turbine (ATP synthase), electricity (ATP) is generated.
ATP synthase
The enzyme complex embedded in the inner mitochondrial membrane through which H+ ions flow, driving the synthesis of ATP from ADP and inorganic phosphate. In simple terms, it is the molecular turbine that converts the H+ gradient into ATP.
Cristae
The folds of the inner mitochondrial membrane. They increase the surface area available for the electron transport chain and ATP synthase complexes, allowing more ATP to be produced. Think of them as extra shelving in a warehouse: more surface means more machinery can be installed.
Intermembrane space
The compartment between the inner and outer mitochondrial membranes. H+ ions are pumped here by the electron transport chain, creating a high concentration of H+ (low pH) that drives chemiosmosis.
Mitochondrial matrix
The innermost compartment of the mitochondrion, enclosed by the inner membrane. Contains the enzymes of the citric acid cycle and pyruvate oxidation.
FADH2
The reduced form of flavin adenine dinucleotide, another electron carrier (alongside NADH). Produced during the citric acid cycle. Delivers its electrons to the electron transport chain at a slightly lower energy level than NADH, so it yields slightly less ATP. In simple terms, FADH2 is NADH's less energetic sibling.
Glycolysis (cytoplasm)
The citric acid cycle (mitochondrial matrix)
Oxidative phosphorylation (inner mitochondrial membrane)
Between stages 1 and 2 there is a linking step: pyruvate oxidation (also called the "chemical grooming" of pyruvate).
Occurs in the cytoplasm (not inside any organelle), which is one reason it is considered an ancient pathway.
Splits one 6-carbon glucose into two 3-carbon pyruvate molecules.
Net gain: 2 ATP (by substrate-level phosphorylation) and 2 NADH per glucose.
No CO2 is produced during glycolysis itself.
Does not require oxygen, so it is shared by both aerobic and anaerobic metabolism.
Glucose is first phosphorylated to glucose-6-phosphate (an energy investment), then eventually split.
If bacteria are fed glucose containing radioactive carbon, the radioactivity appears first in glucose-6-phosphate, then in pyruvate.
End products: pyruvate, NADH, ATP.
Pyruvate moves from the cytoplasm into the mitochondrial matrix.
Each pyruvate (3C) is oxidised: it loses one carbon as CO2 and is converted into a 2-carbon acetyl group, which is joined to coenzyme A to form acetyl CoA.
This step also produces 1 NADH per pyruvate (so 2 NADH per glucose).
Coenzyme A acts as a shuttle: it delivers the acetyl group to the citric acid cycle, then detaches and is recycled. The analogy is a limousine driver dropping off a couple at the prom.
The false statement to watch for: "Each pyruvate has a CO2 added." That is backwards. Each pyruvate loses a CO2.
Takes place in the mitochondrial matrix (with enzymes also associated with the inner membrane).
Acetyl CoA (2C) combines with oxaloacetic acid (4C) to form citrate (6C), which is then progressively broken down.
Per turn (one acetyl CoA): 2 CO2, 3 NADH, 1 FADH2, 1 ATP (by substrate-level phosphorylation).
Per glucose (two turns): 4 CO2, 6 NADH, 2 FADH2, 2 ATP.
Pyruvate is not a product of the citric acid cycle. (This is a commonly tested false-answer trap.)
If fed radioactive carbon in glucose, during the citric acid cycle the radioactivity first appears in citrate, because that is the first molecule the acetyl group's carbons become part of.
At the end of the citric acid cycle, most of the energy from the original glucose is stored in NADH (not in ATP or CO2).
Takes place at the inner mitochondrial membrane.
Two coupled processes: the electron transport chain (ETC) and chemiosmosis.
Electron transport chain:
NADH and FADH2 deliver their electrons to the chain.
Electrons pass through a series of carrier proteins, each with a slightly greater affinity for electrons.
At each transfer, a small amount of energy is released and used to pump H+ ions from the matrix into the intermembrane space, building a concentration gradient.
Oxygen is the final electron acceptor: it picks up electrons and H+ to form water.
Chemiosmosis:
The H+ gradient (high concentration in the intermembrane space, low in the matrix) represents stored energy.
H+ ions flow back into the matrix through ATP synthase, and the energy of that flow drives the synthesis of ATP.
The inner mitochondrial membrane must be intact for this to work. If it has holes, the gradient dissipates and ATP production collapses.
Cristae:
The folds of the inner membrane increase surface area, allowing more copies of the ETC and ATP synthase to be packed in.
Key application questions:
A drug that punches holes in both mitochondrial membranes would inhibit the citric acid cycle and oxidative phosphorylation (both depend on intact mitochondrial compartments). Glycolysis, which occurs in the cytoplasm, would continue.
A mutant protist missing the inner mitochondrial membrane would lose oxidative phosphorylation entirely.
If you stopped respiration after the ETC but before chemiosmosis, the lowest pH (highest H+ concentration) would be in the intermembrane space, because the ETC has been pumping H+ there.
The final electron acceptor is an oxygen atom, not water, CO2 or ADP.
By-products of cellular respiration: CO2 and water.
Glycolysis: 2 ATP + 2 NADH
Pyruvate oxidation: 2 NADH (per glucose)
Citric acid cycle: 2 ATP + 6 NADH + 2 FADH2
Oxidative phosphorylation: roughly 26 to 28 ATP (from the NADH and FADH2)
Total: approximately 32 ATP per glucose (some textbooks say 30 to 32 because the exact yield varies)
The yield can vary depending on whether NADH passes its electrons to NAD+ or FAD in certain shuttle systems.
Most ATP comes from oxidative phosphorylation, not from substrate-level phosphorylation.
The false statement to watch: "Oxidative phosphorylation yields about 12 ATP per glucose." The correct figure is about 26 to 28. The total across all stages is about 32.
ATP yield per glucose (approximate):
Stage | Location | ATP | NADH | FADH2 | CO2 |
|---|---|---|---|---|---|
Glycolysis | Cytoplasm | 2 | 2 | 0 | 0 |
Pyruvate oxidation | Mitochondrial matrix | 0 | 2 | 0 | 2 |
Citric acid cycle | Mitochondrial matrix | 2 | 6 | 2 | 4 |
Oxidative phosphorylation | Inner membrane | ~26-28 | - | - | 0 |
Total | ~32 | 10 | 2 | 6 |
Each NADH yields roughly 2.5 ATP via oxidative phosphorylation. Each FADH2 yields roughly 1.5 ATP.
Poisons like rotenone (an insecticide) and cyanide both target the electron transport chain. Rotenone blocks electron flow through the chain, so insects (and fish) cannot produce adequate ATP and die. Cyanide does the same in humans. Dinitrophenol works differently: it makes the inner mitochondrial membrane leaky to H+ ions, collapsing the gradient so that the energy is released as heat rather than captured as ATP. This is why dinitrophenol was once (dangerously) used as a weight-loss drug.
This chemistry also explains why carbon monoxide poisoning is lethal: CO binds to the same site on cytochrome c oxidase (the final ETC complex) as oxygen does, blocking the chain.
Students often think glycolysis occurs inside the mitochondria. It does not. Glycolysis takes place in the cytoplasm, outside any organelle.
A frequent error is listing the stages in the wrong order. It is always glycolysis → citric acid cycle → oxidative phosphorylation. Never glycolysis → oxidative phosphorylation → citric acid cycle.
Students sometimes believe that most ATP is produced during glycolysis or the citric acid cycle. In fact, the vast majority comes from oxidative phosphorylation.
Some students think pyruvate directly enters the citric acid cycle. It does not. Pyruvate must first be oxidised to acetyl CoA in the linking step (pyruvate oxidation).
⚠️ Know the correct order of the three stages and their locations. This is tested repeatedly.
⚠️ Substrate-level phosphorylation occurs in glycolysis and the citric acid cycle (not oxidative phosphorylation).
⚠️ The net gain from glycolysis is 2 ATP (not 4; two are invested, four are made). Also 2 NADH.
⚠️ Glycolysis is the pathway common to both aerobic and anaerobic metabolism.
⚠️ If a question describes a drug, poison or mutation that disrupts the mitochondrial membranes, work out which stages are affected by location: glycolysis (cytoplasm) survives; the citric acid cycle and oxidative phosphorylation (both mitochondrial) do not.
⚠️ After the ETC but before chemiosmosis, the intermembrane space has the lowest pH (most H+ ions).
⚠️ The total ATP yield is about 32 per glucose. "About 12 from oxidative phosphorylation" is the false answer.
Fill in the blank: The three stages of cellular respiration in order are glycolysis, __________, and oxidative phosphorylation. (The citric acid cycle.)
True or false: Glycolysis produces a net gain of 4 ATP per glucose. (False, the net gain is 2 ATP.)
Fill in the blank: Pyruvate is converted to __________ before entering the citric acid cycle. (Acetyl CoA.)
True or false: Most ATP from aerobic respiration is produced during the citric acid cycle. (False, most comes from oxidative phosphorylation.)
Fill in the blank: The folds of the inner mitochondrial membrane are called __________. (Cristae.)
Q: Which of the following lists the stages of cellular respiration in the correct order?
A: Glycolysis, the citric acid cycle, and oxidative phosphorylation.
Q: A drug creates holes in both mitochondrial membranes. Which processes are inhibited?
A: The citric acid cycle and oxidative phosphorylation. Glycolysis (cytoplasm) is unaffected.
Q: During which phases does substrate-level phosphorylation occur?
A: Glycolysis and the citric acid cycle.
Q: How many molecules of NADH are produced during glycolysis?
A: 2.
Q: Which of the following is a result of glycolysis? (a) Production of CO2, (b) Conversion of glucose to two three-carbon compounds, (c) A net loss of two ATPs, (d) Conversion of NADH to NAD+.
A: (b). Glucose is split into two molecules of pyruvate (three-carbon compounds).
Q: Where are the enzymes of the citric acid cycle located?
A: In the mitochondrial matrix and inner mitochondrial membrane.
Q: What are the end products of the citric acid cycle? (Which of the following is NOT a product: CO2, pyruvate, ATP, FADH2?)
A: Pyruvate is not a product of the citric acid cycle. CO2, ATP, NADH and FADH2 are.
Q: At the end of the citric acid cycle, most of the energy from glucose is stored in what molecule?
A: NADH.
Q: During chemiosmosis, what drives ATP synthesis?
A: H+ ions flowing through ATP synthase, down their concentration gradient.
Q: Which statement about the inner mitochondrial membrane is false? (a) ATP synthase is found there, (b) It plays a role in the production of pyruvate, (c) Electron carriers are found there, (d) A gradient of H+ exists across it.
A: (b). The inner mitochondrial membrane does not produce pyruvate; pyruvate is produced in the cytoplasm during glycolysis.
Q: If electron transport is completed but chemiosmosis has not yet occurred, where is the pH lowest?
A: In the intermembrane space, because H+ ions have been pumped there.
Q: What is the approximate total yield of ATP per molecule of glucose in aerobic respiration?
A: About 32 ATP.
This material builds directly on the redox chemistry and electron transport concepts in Part 1 (sections 6.1 to 6.5). The NADH and FADH2 produced in glycolysis and the citric acid cycle are only valuable because oxidative phosphorylation converts their electron cargo into ATP.
It connects forward to fermentation (sections 6.13 to 6.14, covered in Part 3), which is what cells do with pyruvate when oxygen is unavailable and oxidative phosphorylation cannot run.
The concept of feedback inhibition (section 6.16) applies here: when ATP levels are high, key enzymes in glycolysis and the citric acid cycle are inhibited, slowing the whole pathway.
glycolysis, citric acid cycle, Krebs cycle, TCA cycle, oxidative phosphorylation, electron transport chain, ETC, chemiosmosis, ATP synthase, substrate-level phosphorylation, pyruvate, acetyl CoA, coenzyme A, NADH, FADH2, mitochondrial matrix, inner mitochondrial membrane, cristae, intermembrane space, H+ gradient, proton motive force, ATP yield per glucose, 32 ATP, rotenone, cyanide, dinitrophenol, Chapter 6 biology, aerobic respiration stages, glucose-6-phosphate, citrate, oxaloacetic acid