Oxidative Phosphorylation and Mitochondria

A2 · 13 min

Oxidative phosphorylation is the final stage of aerobic respiration and the one that makes most of the ATP. Hydrogen delivered by reduced NAD and reduced FAD is split into protons and electrons; the electrons pass along a chain of carriers in the inner mitochondrial membrane, and the energy they release builds a proton gradient that drives ATP synthase. This is chemiosmosis, the same mechanism that makes ATP in chloroplasts. Paper 4 regularly asks for the sequence of events, the role of oxygen, the effect of inhibitors, and how the structure of a mitochondrion suits its function.

The structure of a mitochondrion

Mitochondria are rod-shaped or oval organelles, typically about $0.5$ to $1\ \mu\text{m}$ wide and $1$ to $5\ \mu\text{m}$ long, so their internal structure can only be seen with an electron microscope. Cells with high energy demands (muscle, the proximal convoluted tubule of the kidney, sperm midpieces, liver cells, neurones) contain many mitochondria with densely packed cristae.

Structure and function of the mitochondrion

Structure	How it relates to function
Double membrane (envelope): outer and inner membranes	Separates the reactions of respiration from the cytoplasm; the outer membrane contains transport proteins that let pyruvate, ADP, ATP and phosphate pass through
Inner membrane folded into cristae	Gives a large surface area for the many electron transport chains and ATP synthase molecules, so more ATP can be made
Inner membrane impermeable to protons (except through ATP synthase)	Allows a proton gradient to be built up and maintained, so protons can only return through ATP synthase
ATP synthase (stalked particles) on the inner membrane, projecting into the matrix	Site of ATP synthesis by chemiosmosis
Intermembrane space: narrow gap between the membranes	Its small volume means that pumping protons into it quickly creates a high proton concentration (steep gradient)
Matrix	Contains the enzymes of the link reaction and Krebs cycle, plus NAD, FAD, coenzyme A, oxaloacetate
Circular DNA in the matrix	Codes for some mitochondrial proteins, including some components of the electron transport chain
70S ribosomes in the matrix	Synthesise the proteins coded by mitochondrial DNA

On an electron micrograph look for: two membranes close together at the edge, the inner one folded into shelf-like cristae; a granular matrix, sometimes with small dark granules or strands of DNA. Do not confuse cristae with the stacked thylakoids of a chloroplast, which form grana and are not continuous with the envelope.

The electron transport chain

The electron transport chain (ETC) is a series of protein complexes and carrier molecules embedded in the inner mitochondrial membrane. Each carrier can accept electrons (becoming reduced) and pass them to the next (becoming oxidised again). Each carrier has a slightly greater affinity for electrons than the one before, so electrons flow "downhill" in energy along the chain, releasing energy in small steps. The syllabus does not require the names of the carriers.

The steps of oxidative phosphorylation

Oxidative phosphorylation, step by step

Delivery of hydrogen. Reduced NAD and reduced FAD from glycolysis, the link reaction and the Krebs cycle release their hydrogen atoms to the electron transport chain on the inner membrane. NAD and FAD are regenerated (oxidised) and return to accept more hydrogen.
Hydrogen atoms split. Each hydrogen atom splits into a proton ( $\ce{H+}$ ) and an energetic (high-energy) electron ( $\ce{e-}$ ).
Electrons pass along the chain. The energetic electrons pass from carrier to carrier in a series of redox reactions, and release energy as they do so.
Protons are pumped. The energy released is used to transfer protons across the inner membrane, from the matrix into the intermembrane space, by carrier proteins in the chain.
A proton gradient forms. Protons accumulate in the intermembrane space, creating a concentration gradient (and an electrical gradient, as the intermembrane space becomes positive relative to the matrix). This is a store of potential energy.
Chemiosmosis. Protons return to the matrix by facilitated diffusion through channels in ATP synthase, down their electrochemical gradient. The flow of protons provides the energy for ATP synthase to combine ADP and inorganic phosphate into ATP.
Oxygen is the final electron acceptor. At the end of the chain, electrons combine with oxygen and protons from the matrix to form water.

Key result

\ce{4H+ + 4e- + O2 -> 2H2O}

\ce{ADP + P_i -> ATP}\quad\text{(catalysed by ATP synthase, driven by proton flow)}

Chemiosmosis in the inner mitochondrial membrane. Electrons from reduced NAD pass along the carriers (boxes), releasing energy that pumps protons into the intermembrane space. Protons diffuse back through ATP synthase, which makes ATP. Oxygen accepts the electrons at the end of the chain.

Why oxygen is essential

Oxygen is the final electron acceptor. It removes electrons (and protons) from the end of the chain by forming water. If oxygen is absent:

the last carrier stays reduced, so electrons cannot leave the chain;
every carrier "backs up" and electron flow stops;
no protons are pumped, the proton gradient collapses and no ATP is made by chemiosmosis;
reduced NAD and reduced FAD cannot be reoxidised, so the link reaction and Krebs cycle stop for lack of NAD and FAD.

Oxygen is a very good final acceptor because it has a high affinity for electrons, so a large amount of energy is released along the whole chain.

Why reduced FAD yields less ATP than reduced NAD

Reduced NAD passes its hydrogen to the first carrier of the chain, so its electrons pass through all three proton-pumping complexes. Reduced FAD donates its hydrogen further along the chain, so its electrons bypass the first pump and fewer protons are moved across the membrane per molecule.

	Approximate ATP per molecule
Reduced NAD	about 2.5 (older textbooks: 3)
Reduced FAD	about 1.5 (older textbooks: 2)

The yield of ATP from aerobic respiration

The syllabus states that a detailed account of the total ATP yield is not expected, but you should know the order of magnitude and why it is not a whole number.

Source (per glucose)	Approximate ATP
Glycolysis, substrate-linked	2 (net)
Krebs cycle, substrate-linked	2
10 reduced NAD × about 2.5	about 25
2 reduced FAD × about 1.5	about 3
Total	about 30 to 32 (older theoretical value 38)

The actual yield is lower than the theoretical maximum because:

some protons leak back across the inner membrane without passing through ATP synthase;
energy (from the proton gradient) is used to transport pyruvate, ADP and phosphate into the mitochondrion and ATP out of it;
reduced NAD made in glycolysis is in the cytoplasm, and transferring its hydrogen into the mitochondrion can cost energy.

The energy not transferred to ATP is released as heat.

Watch out

It is protons that pass through ATP synthase, and electrons that pass along the electron transport chain. Swapping them, or writing "hydrogen ions pass along the chain", loses marks.
Protons move into the intermembrane space (not "out of the mitochondrion" or "into the cytoplasm").
Protons return through ATP synthase by facilitated diffusion, not active transport. The active step is the pumping out, using energy from electrons.
Oxygen accepts electrons (and protons) to form water. "Oxygen is needed to make ATP" is too vague; "oxygen combines with ATP" is wrong.

Inhibitors and uncouplers

Questions frequently give you an unfamiliar chemical and its action, and ask you to predict effects.

Cyanide binds to the last carrier of the chain (cytochrome oxidase) and prevents it passing electrons to oxygen. Electron flow stops, no proton gradient forms, and ATP production by oxidative phosphorylation stops. Oxygen uptake falls almost to zero even though oxygen is present. Cells rely on glycolysis and lactate fermentation, which cannot meet demand, so cyanide is rapidly fatal.
Uncouplers such as DNP (2,4-dinitrophenol) make the inner membrane permeable to protons. Electron transport continues (and oxygen uptake may even increase) but protons flow back into the matrix without passing through ATP synthase, so the energy is released as heat instead of making ATP. Brown adipose tissue in newborn mammals and hibernating animals uses a natural uncoupling protein (thermogenin) to generate heat.
Oligomycin blocks the proton channel of ATP synthase. The proton gradient becomes very steep, pumping slows, electron transport and oxygen uptake fall.

Worked examples

Explain chemiosmosis in mitochondria

Explain how ATP is synthesised during oxidative phosphorylation. [6]

Solution

Any six of:

Reduced NAD and reduced FAD release hydrogen to the electron transport chain on the inner mitochondrial membrane / cristae. [1]
Hydrogen atoms split into protons and electrons. [1]
Electrons pass along a chain of carriers, releasing energy. [1]
The energy is used to pump protons from the matrix into the intermembrane space. [1]
This produces a proton gradient / higher proton concentration in the intermembrane space. [1]
Protons diffuse back into the matrix through ATP synthase (facilitated diffusion). [1]
This provides energy for ATP synthase to phosphorylate ADP to ATP. [1]
Oxygen is the final electron acceptor, combining with electrons and protons to form water. [1]

Structure and function

Muscle cells from athletes trained for endurance were found to have mitochondria with more cristae per mitochondrion than those of untrained people. Explain the advantage of this. [3]

Solution

More cristae give a larger surface area of inner membrane. [1]
So there are more electron transport chains and more ATP synthase molecules. [1]
So the rate of oxidative phosphorylation / ATP production is greater, supplying more ATP for muscle contraction (myosin heads) over a long period. [1]

Measuring a proton gradient

Isolated mitochondria were suspended in a buffer of pH 7.4. The pH of the intermembrane space was measured as 7.0 and the pH of the matrix as 7.8 while the mitochondria were respiring.

(a) Calculate how many times greater the proton concentration was in the intermembrane space than in the matrix. (pH $= -\lg[\ce{H+}]$ ) [2]

(b) Explain how this difference arises and why it is important. [3]

Solution

(a) The pH difference is $7.8 - 7.0 = 0.8$ , so the ratio of proton concentrations is

10^{0.8} = 6.3 \text{ times greater}

(b)

Energy released by electrons passing along the electron transport chain is used to pump protons from the matrix into the intermembrane space. [1]
The inner membrane is impermeable to protons, and the intermembrane space has a small volume, so a high concentration builds up. [1]
The gradient provides the energy for ATP synthesis: protons diffuse back through ATP synthase, which phosphorylates ADP. [1]

Effect of cyanide on oxygen uptake and ATP

Cyanide stops electrons being passed from the last carrier of the electron transport chain to oxygen. Explain why, in a cell poisoned with cyanide, (a) oxygen uptake falls, (b) the Krebs cycle stops, and (c) lactate concentration rises. [6]

Solution

(a) Electrons cannot be transferred to oxygen, the final electron acceptor, so oxygen is not reduced to water and is not taken up. [2]

(b) Electron flow stops, so reduced NAD and reduced FAD cannot be oxidised by the chain. The cell runs out of NAD and FAD, which are needed as hydrogen acceptors for the dehydrogenation reactions of the Krebs cycle, so it stops. [2]

(c) The cell can only make ATP by glycolysis. To keep glycolysis running, NAD must be regenerated by reducing pyruvate to lactate using reduced NAD (lactate dehydrogenase), so lactate accumulates. [2]

Magnification from an electron micrograph

On an electron micrograph a mitochondrion measures $56\ \text{mm}$ long. The magnification is $\times 16\,000$ . Calculate the actual length of the mitochondrion in micrometres. [2]

Solution

\text{actual size} = \frac{\text{image size}}{\text{magnification}} = \frac{56\ \text{mm}}{16\,000} = 0.0035\ \text{mm}

0.0035\ \text{mm} \times 1000 = 3.5\ \mu\text{m}

An uncoupler (exam-hard)

In the 1930s DNP was briefly sold as a weight-loss drug. It allows protons to cross the inner mitochondrial membrane freely. Explain why people taking DNP lost body mass and had a raised body temperature, and suggest why it was dangerous. [5]

Solution

DNP lets protons diffuse back into the matrix without passing through ATP synthase, so the proton gradient is dissipated and less ATP is made per molecule of substrate respired. [1]
The energy released by electron transport is released as heat instead of being used to make ATP, raising body temperature. [1]
To meet its ATP needs, the body must respire much more substrate, so stored fat and glycogen are used up and body mass falls. [1]
Electron transport and oxygen uptake increase because NAD and FAD are rapidly reoxidised (no "back-pressure" from the proton gradient). [1]
Danger: the body temperature can rise uncontrollably (hyperthermia, denaturing enzymes) and cells may be starved of ATP, for example heart muscle, leading to death. [1]

Exam tip

"Describe the role of oxygen" (1 or 2 marks): final electron acceptor; combines with electrons and protons to form water; allows electrons to keep flowing / NAD to be regenerated.
Mark schemes credit the sequence: H atoms → protons + electrons → electrons along carriers → energy released → protons pumped into intermembrane space → gradient → protons diffuse through ATP synthase → ATP. Keep each link explicit.
"Relate structure to function" questions on mitochondria need the feature + the function: "cristae give a large surface area for electron transport chains and ATP synthase".
Use "inner mitochondrial membrane" rather than "the membrane". Examiners report that many candidates do not say which membrane or which direction protons move.

Summary

Oxidative phosphorylation takes place on the inner mitochondrial membrane (cristae).
Reduced NAD and reduced FAD deliver hydrogen, which splits into protons and energetic electrons.
Electrons pass along the electron transport chain, releasing energy that pumps protons from the matrix into the intermembrane space.
Protons diffuse back through ATP synthase, which makes ATP from ADP and $\ce{P_i}$ : this is chemiosmosis.
Oxygen is the final electron acceptor, forming water; without it the chain, the Krebs cycle and the link reaction stop.
Reduced FAD yields less ATP than reduced NAD because it enters the chain at a later point.
Mitochondrial features: double membrane, cristae (large surface area), proton-impermeable inner membrane, small intermembrane space, matrix enzymes, DNA and 70S ribosomes.
Aerobic respiration yields about 30 to 32 ATP per glucose; detailed counting is not required.

Practice

Question

State the precise location of the electron transport chain in a eukaryotic cell. [1]
State the role of oxygen in aerobic respiration. [2]
Explain why the inner mitochondrial membrane must be impermeable to protons. [2]
Describe how a proton gradient is set up across the inner mitochondrial membrane. [3]
Suggest why cells in the proximal convoluted tubule of the kidney contain many mitochondria. [2]
Explain why reduced FAD produces fewer ATP molecules than reduced NAD. [2]
On an electron micrograph at $\times 25\,000$ , the width of a mitochondrion is $20\ \text{mm}$ . Calculate its actual width in $\mu\text{m}$ . [2]
Oligomycin blocks the proton channel of ATP synthase. Predict and explain its effect on (a) ATP production, (b) the pH of the intermembrane space and (c) the rate of oxygen uptake. [5]
Compare the structure of a mitochondrion with that of a chloroplast, and explain how the two organelles both use chemiosmosis to make ATP. [6]

Answers

The inner mitochondrial membrane (on the cristae).
Oxygen is the final electron acceptor of the electron transport chain; it combines with electrons and protons to form water, allowing electron flow (and the regeneration of NAD and FAD) to continue.
So that a proton gradient can be built up and maintained; protons can then only return to the matrix through ATP synthase, and their flow is used to make ATP. If protons leaked back, the energy would be lost as heat.
Reduced NAD and reduced FAD release hydrogen, which splits into protons and electrons; electrons pass along the electron transport chain, releasing energy; this energy is used by carrier proteins to pump protons from the matrix into the intermembrane space, where they accumulate because the membrane is impermeable to them.
They carry out active transport (for example $\ce{Na+}$ / $\ce{K+}$ pumps in the basal membrane, and co-transport) to reabsorb glucose, amino acids and ions, which needs a lot of ATP, made by oxidative phosphorylation in mitochondria.
Reduced FAD gives its hydrogen to the chain at a later carrier than reduced NAD, so its electrons pass through fewer proton pumps; fewer protons are moved into the intermembrane space, so fewer pass through ATP synthase and less ATP is made.
$20\ \text{mm} \div 25\,000 = 0.0008\ \text{mm} = 0.8\ \mu\text{m}$ .
(a) ATP production by oxidative phosphorylation stops, because protons cannot flow through ATP synthase. (b) The pH of the intermembrane space falls (becomes more acidic) at first, because protons are still pumped in but cannot leave; the gradient becomes very steep. (c) Oxygen uptake falls: when the gradient is very steep, the pumps cannot move more protons against it, so electron transport slows, fewer electrons reach oxygen, and less oxygen is used.
Similarities: both have a double membrane (envelope); both contain circular DNA and 70S ribosomes; both have an internal membrane system carrying electron transport chains and ATP synthase. Differences: the mitochondrial inner membrane is folded into cristae, while the chloroplast has separate thylakoids stacked into grana; the chloroplast contains chlorophyll and other pigments and starch grains; the mitochondrion has a matrix, the chloroplast a stroma. Chemiosmosis: in both, energetic electrons pass along an electron transport chain, releasing energy that pumps protons across a membrane, creating a proton gradient; protons diffuse back through ATP synthase, which makes ATP. In mitochondria protons are pumped into the intermembrane space and return to the matrix, with electrons coming from reduced NAD/FAD and passing finally to oxygen; in chloroplasts protons are pumped into the thylakoid space and return to the stroma, with electrons coming from chlorophyll excited by light and passing finally to NADP (or back to PSI).

The structure of a mitochondrion

The electron transport chain

The steps of oxidative phosphorylation

Why oxygen is essential

Why reduced FAD yields less ATP than reduced NAD

The yield of ATP from aerobic respiration

Inhibitors and uncouplers

Worked examples

Practice

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