Difficulty: Intermediate to Advanced | Prerequisites: Part 1 of these study notes (energy fundamentals, redox reactions, thermodynamics), basic enzyme function.
Part 1 covered the energy principles and redox chemistry that underpin cellular respiration. This document walks through the four stages themselves: glycolysis, pyruvate oxidation, the citric acid cycle, and oxidative phosphorylation, plus what happens when oxygen is unavailable (fermentation). You should already be comfortable with OILRIG, the roles of NADH and FADH2, and the overall ATP accounting before reading on. This is where the pathway-level detail that exam questions test lives.
Glucose is split by glycolysis (cytosol, no oxygen needed), converted to acetyl CoA, run through the citric acid cycle (mitochondrial matrix), and then the loaded electron carriers feed the electron transport chain (inner mitochondrial membrane), which drives chemiosmosis to produce the bulk of ATP. Without oxygen, cells fall back to fermentation, which yields far less ATP but keeps glycolysis running by regenerating NAD+.
Glycolysis
Literally "sugar splitting." The metabolic pathway that converts one molecule of glucose (6C) into two molecules of pyruvate (3C each), occurring in the cytosol. Think of it as the first stage of fuel processing, shared by virtually all life on Earth.
Energy investment phase
The first half of glycolysis, in which 2 ATP are consumed to phosphorylate glucose and prepare it for splitting. You have to spend money to make money.
Energy payoff phase
The second half of glycolysis, in which 4 ATP and 2 NADH are produced. Net gain: 2 ATP and 2 NADH per glucose.
Pyruvate
A three-carbon molecule produced at the end of glycolysis. The fate of pyruvate depends on whether oxygen is available: if yes, it enters the mitochondrion for further oxidation; if no, it enters fermentation.
Pyruvate dehydrogenase complex
The multi-enzyme complex that catalyses the conversion of pyruvate to acetyl CoA inside the mitochondrion, releasing CO2 and reducing NAD+ to NADH.
Oxaloacetate (OAA)
The four-carbon molecule that accepts the acetyl group from acetyl CoA to start the citric acid cycle, and is regenerated at the end of each turn of the cycle.
Citrate
The six-carbon molecule formed when acetyl CoA (2C) combines with oxaloacetate (4C). The citric acid cycle is named after this molecule.
GTP (guanosine triphosphate)
An energy carrier produced during the citric acid cycle by substrate-level phosphorylation. It is readily converted to ATP.
Cristae
The folds of the inner mitochondrial membrane. The increased surface area houses the electron transport chain complexes and ATP synthase.
ATP synthase
The enzyme complex embedded in the inner mitochondrial membrane that synthesises ATP from ADP and inorganic phosphate, driven by the flow of H+ down their concentration gradient. Its structure includes a rotor, a stator, a central stalk, and three catalytic sites in the knob.
Phosphofructokinase (PFK)
The key regulatory enzyme of glycolysis. It is inhibited by high ATP and citrate levels (feedback inhibition) and stimulated by AMP and ADP, making it the main control point for the rate of glycolysis.
Glycogen
The storage form of glucose in animals. Long chains of glucose molecules that can be broken down to release glucose when energy is needed. Also a precursor for NAD+ synthesis.
Glycolysis splits one 6-carbon glucose molecule into two 3-carbon pyruvate molecules. It occurs in the cytoplasm and does not require oxygen.
Two phases:
Energy investment phase: 2 ATP are consumed to phosphorylate glucose, first to glucose-6-phosphate, then to fructose-1,6-bisphosphate. This primes the molecule for splitting.
Energy payoff phase: The 6-carbon molecule splits into two 3-carbon molecules (glyceraldehyde-3-phosphate), which are oxidised. 4 ATP are produced by substrate-level phosphorylation and 2 NAD+ are reduced to 2 NADH.
Net yield per glucose: 2 ATP + 2 NADH + 2 pyruvate.
Evolutionary significance:
Glycolysis is one of the most ancient metabolic pathways. Early prokaryotes likely used it to produce ATP before oxygen accumulated in the atmosphere. It occurs in the cytosol and requires no organelles, which is consistent with its origin before the evolution of mitochondria. It is shared by nearly all organisms on Earth, used in both cellular respiration and fermentation.
In the presence of oxygen, pyruvate is transported into the mitochondrial matrix, where a multi-enzyme complex (pyruvate dehydrogenase) catalyses three reactions in a single step:
Pyruvate is oxidised, releasing one molecule of CO2 (the carboxyl group is removed)
NAD+ is reduced to NADH
The remaining 2-carbon fragment (an acetyl group) is attached to coenzyme A, forming acetyl CoA
Summary equation:
Pyruvate + NAD+ + CoA → Acetyl CoA + NADH + CO2
Since each glucose produces 2 pyruvates, this step runs twice per glucose, yielding 2 NADH and 2 CO2 total. No ATP is produced here. This is the bridge between glycolysis and the citric acid cycle.
The citric acid cycle completes the oxidation of organic fuel that began with glycolysis. It takes place in the mitochondrial matrix and consists of 8 enzyme-catalysed steps that recycle citrate back to oxaloacetate.
Per turn of the cycle (per acetyl CoA):
Acetyl CoA (2C) joins oxaloacetate (4C) to form citrate (6C)
Two carbons are removed as 2 CO2
3 NAD+ are reduced to 3 NADH
1 FAD is reduced to 1 FADH2
1 GTP is produced (equivalent to 1 ATP, by substrate-level phosphorylation)
Oxaloacetate is regenerated, ready for the next turn
Per glucose (2 turns): 2 ATP, 6 NADH, 2 FADH2, 4 CO2.
The NADH and FADH2 produced are loaded electron carriers. They carry the majority of the energy extracted from glucose, which will be converted to ATP in the next stage. The CO2 released is the carbon that was originally part of the glucose molecule, and it is what you exhale.
This is where the heavy lifting happens. Oxidative phosphorylation comprises two coupled processes: the electron transport chain and chemiosmosis.
The Electron Transport Chain (ETC):
The ETC is a series of protein complexes (I, II, III, IV) and mobile carriers (coenzyme Q and cytochrome c) embedded in the inner mitochondrial membrane (cristae).
NADH donates electrons to Complex I; FADH2 donates to Complex II
Electrons are passed from carrier to carrier in a series of redox reactions, each releasing a small amount of energy
That energy is used to pump H+ ions from the mitochondrial matrix into the intermembrane space
Oxygen is the final electron acceptor at Complex IV: it picks up the electrons and combines with H+ to form water
The electron carriers (cytochromes, which contain iron atoms) alternate between reduced and oxidised states as they accept and donate electrons
Critical point: The ETC generates no ATP directly. It builds the H+ gradient that chemiosmosis uses to make ATP.
Chemiosmosis:
Chemiosmosis is the energy-coupling mechanism that links the ETC to ATP production.
The ETC pumps H+ into the intermembrane space, creating a steep concentration gradient (the proton-motive force)
H+ flows back down this gradient through ATP synthase, a channel-like enzyme complex
The flow of H+ spins the rotor of ATP synthase, which activates catalytic sites that join ADP + Pi to make ATP
This is where roughly 90% of the cell's ATP comes from
ATP synthase structure:
A rotor in the membrane spins as H+ flows through
A stator anchored in the membrane holds the catalytic knob stationary
A central stalk (rod) connects the rotor to the knob, transmitting the rotation
Three catalytic sites in the stationary knob combine ADP and inorganic phosphate to produce ATP
Yield: approximately 26 to 28 ATP per glucose from oxidative phosphorylation alone.
The mitochondrion's architecture is central to oxidative phosphorylation.
Outer membrane: smooth, permeable to small molecules
Inner membrane (cristae): heavily folded to increase surface area. Houses the ETC protein complexes and ATP synthase
Intermembrane space: between the two membranes. H+ ions accumulate here, creating the proton-motive force
Matrix: the innermost compartment, enclosed by the inner membrane. Site of the citric acid cycle and pyruvate oxidation
Prokaryote vs eukaryote ETC:
Prokaryotes lack mitochondria, so their ETC is located in the plasma (cytoplasm) membrane
Eukaryotes have the ETC in the inner mitochondrial membrane (cristae)
The mechanism is the same: electron carriers pump H+ to one side of a membrane, and ATP synthase on that membrane uses the gradient to make ATP
Chloroplasts use the same principle (thylakoid membrane), with H+ accumulating in the thylakoid lumen
Why fermentation exists:
Oxygen is the final electron acceptor that pulls electrons down the ETC. Without it, the ETC stalls, NADH cannot be reoxidised, and glycolysis would stop for lack of NAD+. Fermentation solves this by regenerating NAD+ from NADH so glycolysis can keep running.
Fermentation always begins with glycolysis. The fermentation reactions themselves produce no additional ATP; their sole purpose is to recycle NAD+.
Alcohol fermentation:
Performed by yeasts and some plants
Pyruvate is converted to ethanol and CO2
NADH is oxidised back to NAD+
Net reaction: C6H12O6 + 2ADP + 2Pi → 2 ethanol + 2CO2 + 2ATP
Lactic acid fermentation:
Performed by some bacteria and by animal muscle cells during intense exercise
Pyruvate is reduced directly to lactic acid (lactate)
NADH is oxidised back to NAD+
Net reaction: C6H12O6 + 2ADP + 2Pi → 2 lactic acid + 2ATP
Both types yield only 2 ATP per glucose (from glycolysis), compared with 30 to 32 from full aerobic respiration.
Alternative electron acceptors:
Some organisms in anaerobic environments (such as the bottom of a lake) use electron acceptors other than oxygen: nitrate (denitrifiers), manganese, iron, sulphate (sulphate reducers), or CO2 (methanogens). These are forms of anaerobic respiration, distinct from fermentation because they still use an ETC.
Regulation of cellular respiration:
Glycolysis is downregulated when ATP levels are high. Phosphofructokinase (PFK), the key allosteric enzyme, is inhibited by ATP and citrate and stimulated by AMP
Control of catabolism is based on regulating enzyme activity at strategic control points in the pathway
The availability of oxygen also determines which pathways operate: with oxygen, full aerobic respiration; without it, fermentation or anaerobic respiration
Excess glucose storage:
Glucose not immediately needed is stored as glycogen in animals, a long-chain polymer that can be broken down to release glucose when energy demand rises.
Students often think glycolysis requires oxygen. It does not. Glycolysis runs in the cytosol with or without oxygen present.
Students confuse fermentation with anaerobic respiration. They are different. Fermentation does not use an electron transport chain at all. Anaerobic respiration uses an ETC but with an electron acceptor other than oxygen.
Students believe lactic acid causes muscle soreness days after exercise. The burn during intense exercise is related to lactic acid buildup, but delayed-onset muscle soreness has other causes (microtrauma to muscle fibres).
Students think each turn of the citric acid cycle produces the totals listed for one glucose. It does not. The cycle turns once per acetyl CoA. Since one glucose yields two acetyl CoA, you must double the per-turn outputs to get the per-glucose figures.
Students assume the 32 ATP figure is exact. It is approximate. The number depends on which electron shuttle (malate-aspartate or glycerol-3-phosphate) transports cytosolic NADH into the mitochondrion, which varies by cell type.
⚠️ Know the inputs, outputs, and location of each of the four stages. This is the most commonly tested concept.
⚠️ Be able to explain the energy investment and energy payoff phases of glycolysis, including why 2 ATP must be spent.
⚠️ Understand why the citric acid cycle turns twice per glucose.
⚠️ Be ready to explain how chemiosmosis works: the ETC pumps H+, the gradient stores energy, ATP synthase harvests it.
⚠️ Know the difference between alcohol and lactic acid fermentation, including the organisms that perform each and the net reaction.
⚠️ Understand feedback inhibition of glycolysis by ATP, and the role of phosphofructokinase.
⚠️ Be able to explain why oxygen is necessary for oxidative phosphorylation (it is the final electron acceptor that keeps the ETC running).
True or False: Pyruvate oxidation produces ATP. (False. It produces NADH and CO2 but no ATP.)
Fill in the blank: The citric acid cycle takes place in the mitochondrial ______. (matrix)
True or False: Fermentation produces more ATP per glucose than aerobic respiration. (False. Fermentation yields only 2 ATP per glucose; aerobic respiration yields 30 to 32.)
Fill in the blank: The purpose of fermentation is to regenerate ______ so that glycolysis can continue. (NAD+)
True or False: Glycolysis occurs inside the mitochondria. (False. Glycolysis occurs in the cytosol.)
Q: What are the net products of glycolysis per glucose molecule?
A: 2 pyruvate, 2 ATP (net), and 2 NADH.
Q: Why is the energy investment phase of glycolysis necessary?
A: The cell must spend 2 ATP to phosphorylate glucose and destabilise it enough to split. Without this initial investment, the sugar molecule would not break apart efficiently.
Q: What happens to pyruvate when oxygen is available vs when it is not?
A: With oxygen, pyruvate enters the mitochondrion and is oxidised to acetyl CoA, feeding into the citric acid cycle and eventually oxidative phosphorylation. Without oxygen, pyruvate stays in the cytosol and is converted to ethanol (yeast) or lactate (muscle cells) via fermentation, which regenerates NAD+.
Q: Explain why the electron transport chain does not directly produce ATP.
A: The ETC transfers electrons through a series of protein complexes, using the released energy to pump H+ across the inner mitochondrial membrane. This creates a concentration gradient (the proton-motive force). It is ATP synthase, not the ETC, that uses the energy stored in that gradient to join ADP and Pi into ATP.
Q: How does ATP synthase work mechanically?
A: H+ ions flow down their concentration gradient from the intermembrane space through ATP synthase. This flow spins the rotor portion of the enzyme, which is connected by a central stalk to a stationary catalytic knob in the matrix. The rotation activates three catalytic sites in the knob, each of which combines ADP with inorganic phosphate to produce ATP.
Q: Why do the citric acid cycle outputs need to be doubled to get the per-glucose totals?
A: One glucose produces 2 pyruvates, each of which is converted to one acetyl CoA. Since the citric acid cycle processes one acetyl CoA per turn, it must turn twice to process all the carbon from one glucose molecule.
This material connects to photosynthesis (Chapter 10): the light reactions use an electron transport chain and chemiosmosis in the thylakoid membrane, mirroring the mitochondrial ETC. Understanding one makes the other far easier.
The regulation of glycolysis by allosteric enzymes ties back to enzyme kinetics from Chapter 5. Phosphofructokinase is a textbook example of feedback inhibition.
Fermentation connects to microbiology and food science: brewing, baking, yoghurt, sauerkraut. It also connects to exercise physiology (lactic acid buildup during anaerobic exercise) and to anaerobic environments in ecology (methanogens, sulphate reducers in deep-sea vents and lake sediments).
glycolysis, energy investment phase, energy payoff phase, pyruvate, pyruvate oxidation, pyruvate dehydrogenase, acetyl CoA, coenzyme A, citric acid cycle, Krebs cycle, TCA cycle, oxaloacetate, citrate, GTP, oxidative phosphorylation, electron transport chain, ETC, Complex I, Complex II, Complex III, Complex IV, coenzyme Q, ubiquinone, cytochrome c, cytochromes, cristae, inner mitochondrial membrane, intermembrane space, matrix, chemiosmosis, ATP synthase, proton-motive force, proton gradient, substrate-level phosphorylation, fermentation, alcohol fermentation, lactic acid fermentation, ethanol, lactate, NAD+ regeneration, phosphofructokinase, feedback inhibition, allosteric regulation, glycogen, anaerobic respiration, alternative electron acceptors, denitrifiers, methanogens, sulphate reducers, BIO 311C, Chapter 9