Biology › Respiration and cellular energy › The Krebs cycle, the chain, and where the ATP really comes from

The Krebs cycle, the chain, and where the ATP really comes from

By the end of the link reaction a cell has made two ATP from a molecule of glucose and filled four coenzymes with hydrogen. This lesson finishes the carbon in the Krebs cycle, then follows the hydrogen to the inner membrane, where a proton gradient does almost all the work.

Before this Glycolysis and the link reaction · Membrane structure and carrier proteins · Active transport and electrochemical gradients

Before you start

Oxygen burns the glucose in respiration, in the same way it burns petrol in an engine. The equation encourages it — glucose in, oxygen in, carbon dioxide and water out — and the overall bookkeeping really is the same as combustion. But no glucose molecule ever meets a molecule of oxygen inside you. The carbon has already left as carbon dioxide before oxygen appears, and the oxygen's entire job is to sit at the end of a chain of carriers accepting spent electrons and combining them with protons to make water. It is the drain the process empties into, and pulling the drain out is what stops everything upstream.

What you should be able to do

Describe one turn of the Krebs cycle and account for all of the carbon that enters it.
State the products of the cycle per turn and per molecule of glucose.
Explain how electron transfer along the chain produces a proton gradient across the inner membrane.
Explain how the gradient is used to make ATP, naming the enzyme and the direction of proton movement.
State the role of oxygen precisely, and say what happens to the chain without it.
Explain why a measured ATP yield falls short of the theoretical 38.

The cycle that finishes the carbon

Two acetyl groups arrive in the matrix per glucose, each carried by coenzyme A, each holding two carbons. The Krebs cycle takes them apart one at a time, and because it is a cycle it has to hand back whatever it started with.

The acetyl group is transferred from coenzyme A onto a four-carbon acceptor, oxaloacetate, giving the six-carbon citrate. Coenzyme A leaves at once and goes back for another acetyl group. Citrate is then decarboxylated and dehydrogenated to a five-carbon compound, and again to a four-carbon compound; a series of further changes regenerates oxaloacetate, and the cycle turns again. Six carbons in, two out as carbon dioxide, four handed back — which is exactly the four it borrowed.

The carbon count round the ring is the whole argument. The two carbon dioxide molecules leaving each turn are the two that arrived on the acetyl group, and the acceptor ends the turn exactly as it began it.

Per turn, the cycle yields 2 CO₂, 3 reduced NAD, 1 reduced FAD and 1 ATP. That single ATP is made by substrate-level phosphorylation, the same mechanism as in glycolysis, and it is the only ATP the cycle makes directly. Because one glucose gives two pyruvate and therefore two acetyl groups, the cycle turns twice per glucose, and every one of those figures doubles.

Krebs cycle: A closed series of reactions in the mitochondrial matrix in which an acetyl group is combined with a four-carbon acceptor and completely oxidised, releasing carbon dioxide and reducing coenzymes, and regenerating the acceptor.
Dehydrogenase: An enzyme that catalyses the removal of hydrogen from a substrate, passing it to a coenzyme such as NAD or FAD.
Decarboxylase: An enzyme that catalyses the removal of a carbon atom from a substrate, released as carbon dioxide.
FAD: A hydrogen-carrying coenzyme like NAD, but one that hands its electrons to the transport chain further along, so it yields less ATP per molecule.

Counting a whole glucose through the cycle

One molecule of glucose is respired aerobically. Calculate the total number of molecules of carbon dioxide and of reduced NAD produced by the link reaction and the Krebs cycle together, and show that the carbon balances.

The link reaction runs twice: 2 CO₂ and 2 reduced NAD. The cycle turns twice, each turn giving 2 CO₂ and 3 reduced NAD, so 4 CO₂ and 6 reduced NAD.

Totals: 6 CO₂ and 8 reduced NAD from those two stages. Add the two reduced NAD from glycolysis and the cell has ten in all.

The carbon balances neatly. Glucose brought in six carbons; two left in the link reaction and four in the cycle, giving six carbon dioxide molecules, which is why the overall equation has a 6 in front of the CO₂ and why none of the carbon ends up in the water.

What the first three stages were actually for

Add up the ATP made so far and it is disappointing: two from glycolysis, none from the link reaction, two from the cycle. Four ATP from a molecule carrying 2880 kJ per mole. Set beside that, the same glucose has produced ten reduced NAD and two reduced FAD.

Those coenzymes are the point. Each carries a pair of hydrogen atoms picked up from a substrate, and each hydrogen atom is a proton and an electron travelling together. On the inner membrane the two are separated: the electrons are handed to a chain of carriers, the protons are released into solution, and the difference in energy between the start and the end of that chain is used to build a gradient. The first three stages of respiration are, in effect, an elaborate system for loading hydrogen onto carriers so that the fourth stage can unload it.

The chain, the gradient, and the enzyme that turns

The inner mitochondrial membrane carries a series of electron carriers, arranged so that each one holds electrons a little less tightly than the next. Reduced NAD is oxidised at the first carrier, handing over its electrons and releasing its protons into the matrix. The electrons then pass from carrier to carrier, releasing energy at each transfer.

That energy is not used to make ATP directly. It is used to move protons out of the matrix, across the inner membrane, into the intermembrane space. The membrane is impermeable to protons, so they build up there: the space becomes more concentrated in protons and more positively charged than the matrix. The result is an electrochemical gradient, and it is a genuine store of potential energy, in the way a header tank of water is.

The only easy way back into the matrix is through ATP synthase, a large protein spanning the membrane with a channel through it. Protons flow back down their gradient through that channel, the flow turns part of the enzyme, and the movement drives the joining of ADP and inorganic phosphate into ATP. Making ATP this way, from a gradient rather than from a substrate, is chemiosmosis, and the whole stage is oxidative phosphorylation.

Watch the direction twice. Protons are pushed out of the matrix by the energy from the electrons, and they come back in through the synthase — out to build the gradient, in to spend it. Oxygen appears only at the very end.

Now oxygen. Electrons cannot simply pile up on the last carrier; something has to take them away, or the chain fills and stops. Oxygen is the final electron acceptor. At the end of the chain each oxygen atom takes up two electrons and, with two protons from the matrix, becomes water. That is oxygen's entire contribution — no glucose, no carbon, no burning.

The consequence of removing it is worth thinking through, because it explains the next lesson. With no oxygen, the last carrier stays reduced, so the carrier before it cannot pass its electrons on, and so on back up the chain. Within seconds nothing can hand its electrons anywhere, no protons are pumped, the gradient collapses and ATP synthase stops. Worse, reduced NAD has nowhere to unload, so oxidised NAD is never regenerated — and the link reaction, the Krebs cycle and eventually glycolysis all run out of the coenzyme they depend on.

Electron transport chain: A series of carrier proteins in the inner mitochondrial membrane that pass electrons from one to the next, releasing energy used to pump protons into the intermembrane space.
Chemiosmosis: The synthesis of ATP driven by the movement of protons down an electrochemical gradient through ATP synthase.
Oxidative phosphorylation: The formation of ATP from ADP and inorganic phosphate using energy from the electron transport chain, with oxygen as the final electron acceptor.
Final electron acceptor: The molecule that takes electrons from the end of the chain. In aerobic respiration it is oxygen, which combines with protons to form water.

Adding it up, honestly

The number most students carry into the exam is 38 ATP per glucose. It comes from a straightforward sum: 4 ATP made directly, plus 10 reduced NAD counted as 3 ATP each, plus 2 reduced FAD counted as 2 each. 4 + 30 + 4 = 38.

The trouble is that those two ratios are round numbers chosen for convenience rather than measured values. Careful work puts a reduced NAD nearer 2.5 ATP and a reduced FAD nearer 1.5, because the number of protons pumped per pair of electrons does not divide neatly by the number needed to turn the synthase once. Redo the sum with those and you get 32, not 38.

The bar for 38 is a ceiling: an arithmetic maximum assuming nothing leaks and nothing else is paid for. Every correction moves the real figure downwards, never up.

Three further costs push the measured figure lower still, and a question asking why the actual yield is less than the theoretical one wants at least two of them. The reduced NAD made by glycolysis is in the cytoplasm and cannot cross the inner membrane, so its hydrogen is carried in by a shuttle, which costs part of the gradient — and in some tissues delivers to FAD rather than NAD, which is worth less again. The membrane leaks: some protons find their way back without passing through the synthase. And the gradient is not reserved for ATP at all; it also drives the transport of pyruvate, phosphate and calcium ions into the matrix, and in brown adipose tissue it is deliberately short-circuited to make heat instead. A realistic figure for a working cell is around 30.

TRY IT — Explaining a poison

Cyanide binds irreversibly to the final carrier of the electron transport chain and prevents it passing electrons to oxygen. Explain why a cell treated with cyanide stops making ATP by oxidative phosphorylation within seconds, and why the pH of the intermembrane space rises.

Check your answer

Electrons can no longer leave the chain, so the final carrier stays reduced. The carrier before it has nowhere to pass its electrons, and the block works its way back along the chain until no transfers are happening anywhere.

No electron transfer means no energy released to pump protons. The existing gradient is used up as protons flow back through ATP synthase, and once it has gone the synthase stops turning and oxidative phosphorylation ends.

A higher pH means fewer protons, and that is the point: pumping has stopped while leakage and flow through the synthase continue, so the intermembrane space loses the protons it had accumulated.

Glycolysis carries on for a short while, but only until the cell's oxidised NAD is exhausted, since the chain is what normally regenerates it. That is why cyanide is fast and why tissues with high ATP demand fail first.

In the exam

Oxygen is the final electron acceptor and forms water. Any answer saying oxygen 'burns' or 'reacts with' glucose has described combustion, not respiration.
For chemiosmosis, name the direction. Protons are pumped from the matrix into the intermembrane space, and return through ATP synthase into the matrix. Marks are given for out and back, not for 'protons move'.
Two turns of the cycle per glucose. If a question says 'per molecule of glucose' and your answer contains 3 reduced NAD from the cycle, you have answered per turn.
Say where the energy for pumping comes from: the transfer of electrons between carriers, not from ATP and not from oxygen.
If asked why the actual yield is below the theoretical yield, give specifics — proton leakage, the gradient used for transporting other substances, the cost of shuttling cytoplasmic reduced NAD in. 'Not all reactions are efficient' scores nothing.
Reduced FAD is worth less ATP than reduced NAD because it delivers its electrons further along the chain, so fewer protons are pumped. That reason is examined more often than the numbers are.

Check yourself

A drug makes the inner mitochondrial membrane freely permeable to protons. Explain what happens to the rate of electron transport, to ATP production and to the temperature of the tissue. Then explain why a person taking such a drug loses mass rapidly but is in serious danger.

Answer

Protons can now return to the matrix anywhere, so no gradient can be maintained. ATP synthase has nothing driving protons through it and ATP production by oxidative phosphorylation falls to almost nothing.

Electron transport does not slow — it speeds up. Pumping protons into a space that no longer resists them is easy, so the carriers pass electrons faster than before, and oxygen is consumed faster than usual while producing no ATP.

The energy released at each transfer is no longer captured in a gradient, so it appears as heat, and the tissue warms. This is not hypothetical: brown adipose tissue does exactly this on purpose, using a channel protein to warm a newborn.

Mass is lost because respiratory substrates are being oxidised at a high rate to no useful end, so stored lipid and then protein are consumed quickly. The danger is that cells still need ATP and are now getting almost none of it from the mitochondrion, while body temperature climbs beyond what enzymes tolerate. Drugs of this kind were sold as slimming aids in the 1930s and killed people.

Questions

Written to the command words the boards use. Try them on paper before opening a scheme: the marks go to points made, not to length.

Question 15 marks

Explain how the reduced NAD produced by the earlier stages of respiration leads to the synthesis of ATP on the inner mitochondrial membrane.

Mark scheme

B1 reduced NAD is oxidised at the first carrier of the chain, handing over its electrons and releasing its protons into solution
B1 the electrons pass from carrier to carrier along the inner membrane, releasing energy at each transfer because each carrier holds them a little less tightly than the one before
B1 that energy is used to pump protons out of the matrix into the intermembrane space, and since the membrane is impermeable to protons an electrochemical gradient builds across it
B1 protons return to the matrix down that gradient through the channel in ATP synthase, and the flow drives the enzyme to join ADP and inorganic phosphate into ATP, which is chemiosmosis
B1 oxygen acts as the final electron acceptor at the end of the chain, taking up electrons and combining with protons to form water, so electrons do not pile up and the chain keeps running

Question 24 marks

Describe one turn of the Krebs cycle, and account for every carbon atom that enters it.

Mark scheme

B1 the two-carbon acetyl group is transferred from coenzyme A onto the four-carbon acceptor oxaloacetate, giving the six-carbon compound citrate, and coenzyme A leaves at once to collect another acetyl group
B1 citrate is decarboxylated and dehydrogenated to a five-carbon compound, and again to a four-carbon compound, so two molecules of carbon dioxide are released per turn
B1 a series of further changes regenerates oxaloacetate, so the acceptor ends the turn exactly as it began it and the cycle can turn again
B1 six carbons are present at the start of the turn, two leave as carbon dioxide, and the four handed back are the four the cycle borrowed, so the two carbons released are the two that arrived on the acetyl group

Question 34 marks

One molecule of glucose respired aerobically yields 10 reduced NAD, 2 reduced FAD and 4 ATP made directly. Calculate the theoretical ATP yield per glucose using the textbook ratios of 3 ATP per reduced NAD and 2 ATP per reduced FAD, and calculate it again using the better ratios of 2.5 and 1.5.

Mark scheme

M1 multiply each coenzyme by its ratio and add the ATP made directly by substrate-level phosphorylation
M1 10 × 3 = 30 from reduced NAD and 2 × 2 = 4 from reduced FAD, plus the 4 made directly
A1 a theoretical total of 30 + 4 + 4 = 38 ATP per molecule of glucose
A1 with the better ratios, 10 × 2.5 = 25 and 2 × 1.5 = 3, giving 25 + 3 + 4 = 32 ATP per molecule of glucose

Question 44 marks

A working cell is measured to yield around 30 molecules of ATP per molecule of glucose, rather than the 38 quoted in textbooks. Explain why the measured yield falls short of the theoretical one.

Mark scheme

B1 the ratios of 3 ATP per reduced NAD and 2 per reduced FAD are round numbers chosen for convenience rather than measured values, and using the nearer figures of 2.5 and 1.5 brings the theoretical total down to 32
B1 the reduced NAD made by glycolysis is in the cytoplasm and cannot cross the inner membrane, so its hydrogen is carried in by a shuttle that costs part of the gradient, and in some tissues delivers to FAD rather than to NAD
B1 the inner membrane leaks, so some protons find their way back into the matrix without passing through ATP synthase, and their energy is not captured as ATP
B1 the gradient is not reserved for ATP synthesis alone: it also drives the transport of pyruvate, phosphate and calcium ions into the matrix, and in brown adipose tissue it is deliberately short-circuited to make heat

Question 54 marks

A drug blocks the proton channel through ATP synthase in the inner mitochondrial membrane but does not affect the electron carriers themselves. Suggest what happens to the proton gradient, to ATP production, to the rate of electron transport and to the rate at which the tissue consumes oxygen.

Mark scheme

B1 protons can no longer return to the matrix through ATP synthase, so they accumulate in the intermembrane space and the gradient across the membrane becomes steeper
B1 ATP production by oxidative phosphorylation stops, because there is no flow of protons through the enzyme to drive it
B1 pumping further protons against an ever steeper gradient needs more energy than the transfers can supply, so electron transport slows and then stops
B1 with few electrons reaching the end of the chain, little oxygen is needed as the final electron acceptor, so oxygen consumption falls even though the carriers themselves are untouched

Question 62 marks

State the products of one complete turn of the Krebs cycle, and state how many turns take place per molecule of glucose.

Mark scheme

B1 one turn gives two molecules of carbon dioxide and one molecule of ATP
B1 one turn also gives three reduced NAD and one reduced FAD, and the cycle turns twice per molecule of glucose

Worth remembering

One turn of the Krebs cycle: 2 CO₂, 3 reduced NAD, 1 reduced FAD, 1 ATP. Two turns per glucose.
The acceptor is four carbons, citrate is six, and the acceptor is regenerated — otherwise the cycle could not turn again.
Electrons pass down the carriers; the energy pumps protons out of the matrix; the protons return through ATP synthase and ATP is made.
Oxygen's only role is as the final electron acceptor, combining with electrons and protons to form water.
38 ATP is a theoretical ceiling. Around 30 is realistic, and the shortfall has named causes.

← Spending ATP to make ATP: glycolysis and the link reaction · Running without oxygen, and burning something other than sugar →