Oxidative phosphorylation & chemiosmosis

Where it happens

Oxidative phosphorylation takes place in the inner mitochondrial membrane. This membrane is folded into ridges called cristae, which pack more membrane, and so more machinery, into each mitochondrion. The space inside it is the matrix. The narrow gap between the inner and outer membranes is the intermembrane space.

One detail matters a lot: protons (H⁺ ions) can't simply leak through the inner membrane. That's what lets the membrane hold a gradient, much as a dam holds back water.

Step 1: the electron transport chain

The electron transport chain is a series of four protein complexes (I–IV) sitting in the inner membrane, plus two small carriers that shuttle electrons between them. Electrons move "downhill" along the chain: each carrier pulls on electrons a little more strongly than the one before, and oxygen pulls hardest of all. At each step a small amount of energy is released.

1. 1NADH drops off two electrons at Complex I and becomes NAD⁺, which goes back to glycolysis and the Krebs cycle to be loaded again.
2. 2FADH₂ hands its electrons to Complex II instead, so they join the chain one step later.
3. 3A small fat-soluble carrier, ubiquinone (Q), moves through the membrane carrying electrons from Complex I or II to Complex III.
4. 4A small protein, cytochrome c, carries the electrons from Complex III to Complex IV.
5. 5At Complex IV the electrons are passed to oxygen, which also picks up H⁺ and becomes water. Oxygen is the final electron acceptor: without it, the whole chain stops.

Complexes I, III and IV use the energy released by the electrons to pump H⁺ out of the matrix and into the intermembrane space. Complex II doesn't pump any. That's why electrons from FADH₂ end up making less ATP than electrons from NADH.

Step 2: building a proton gradient

As the pumping goes on, H⁺ ions pile up in the intermembrane space. That stores energy in two ways at once: there are more protons outside than inside (a concentration difference), and the outside becomes more positive than the inside (a charge difference). Together, these are called the proton-motive force.

Because the membrane won't let H⁺ leak back, the energy stays stored, like water behind a dam waiting to flow.

Step 3: ATP synthase, a spinning motor

Protons have one main route back into the matrix: through an enzyme called ATP synthase. As H⁺ flows through it, down its gradient, part of the enzyme spins like a tiny turbine. Each turn changes the shape of the enzyme's head, and those shape changes press ADP and phosphate together into ATP.

Using a gradient of ions across a membrane to make ATP is called chemiosmosis. Peter Mitchell proposed the idea in 1961. At the time most scientists doubted it, but it turned out to be right, and it won him the Nobel Prize in 1978.

How much ATP?

Each NADH powers enough pumping for about 2.5 ATP. Each FADH₂, which skips the pumping at Complex I, gives about 1.5. These aren't whole numbers because ATP synthase needs several protons to make each ATP, and the count doesn't divide evenly.

Add everything up for one glucose and you get about 30–32 ATP. The exact number varies, for example with how the NADH made in glycolysis gets into the mitochondrion. Older textbooks give 36–38, because they rounded to 3 ATP per NADH and 2 per FADH₂. Measurements show the real values are lower.

What happens when it breaks

Blocking different parts of the system has very different effects. Working through them is one of the best ways to check that you really understand it. You can try each one in the pathway lab.

• Cyanide blocks Complex IV. Electrons can't reach oxygen, so the chain backs up like a traffic jam. Pumping stops, the gradient fades, and ATP synthase grinds to a halt. NADH can't be turned back into NAD⁺, so the Krebs cycle stalls too. Cells fall back on glycolysis and fermentation, about 2 ATP per glucose, which isn't enough for the heart and brain.
• Uncouplers such as DNP punch holes in the membrane for H⁺. Electrons keep flowing and oxygen use even goes up, but the protons leak back without passing through ATP synthase. The energy is released as heat instead of being captured as ATP. Brown fat does this on purpose, with a protein called UCP1, to keep newborns and hibernating animals warm.
• Oligomycin blocks ATP synthase itself. Protons pile up until the pumps can't push any more against the gradient, so electron flow and oxygen use slow down. This shows that the chain only runs as fast as ATP is being made and used.

Key terms

Electron transport chain

Complexes I–IV plus ubiquinone and cytochrome c, which pass electrons from NADH and FADH₂ to oxygen.

Final electron acceptor

Oxygen, which takes the electrons at the end of the chain and becomes water.

Proton-motive force

The energy stored in the H⁺ gradient: a concentration difference plus a charge difference across the inner membrane.

ATP synthase

The enzyme that uses H⁺ flowing back into the matrix to make ATP from ADP and phosphate.

Chemiosmosis

Making ATP using the energy of an ion gradient across a membrane.

Uncoupler

A substance that lets H⁺ leak across the membrane, so electron transport continues but ATP isn't made.

Oxidative phosphorylation

Electron transport plus chemiosmosis: making ATP using energy from electrons passed to oxygen.

Check yourself

Try answering in your head before you open each answer.

• 1.Where does the oxygen you breathe in end up?Show answerHide

  In water. O₂ accepts electrons at Complex IV and becomes H₂O. The CO₂ you breathe out comes from the carbon atoms of glucose, released during pyruvate oxidation and the Krebs cycle.

• 2.A drug makes the inner membrane leaky to H⁺. Predict what happens to oxygen use, ATP production and body temperature.Show answerHide

  Oxygen use goes up, because nothing holds the chain back. ATP production falls, because protons skip ATP synthase. Body temperature rises, because the energy is released as heat. This is exactly what the uncoupler DNP does.

• 3.Why does FADH₂ produce less ATP than NADH?Show answerHide

  FADH₂ gives its electrons to Complex II, which doesn't pump protons, so those electrons skip Complex I's pumping. Fewer H⁺ are pumped per pair of electrons, so less ATP is made.