Oxidative phosphorylation and chemiosmotic theory: suggested detail for students of A-level biology. (See also the accompanying Powerpoint figures.)

1. Introduction to oxidative phosphorylation for A-level students

The mitochondria of animal cells can use energy made available in metabolic reactions, such as those involved in the oxidation of sugars and fats, to make ATP. The process is known as "oxidative phosphorylation"; energy released by oxidation is used to drive the phosphorylation of ADP to ATP.

2. The location of the components of oxidative phosphorylation

The location of the proteins and cofactors used in oxidative phosphorylation is important in the process (see Figure 1).

NAD+ is reduced to NADH in the matrix of mitochondria by enzymes of the Krebs cycle.

The respiratory chain proteins and the ATP synthase enzyme are located in the inner mitochondrial membrane.

3. The chemiosmotic theory

The chemiosmotic theory was developed by the British biochemist, Peter Mitchell, to explain the mechanism of oxidative phosphorylation in mitochondria (and photophosphorylation in chloroplasts). He was awarded the Nobel Prize in 1978. The processes of oxidative phosphorylation are summarised in Figure 2.

NADH produced in the Krebs cycle passes electrons to the respiratory chain and becomes re-oxidized to NAD+. The electrons are transported down the chain to oxygen releasing energy (think of burning sugar or fat); the reduction of oxygen forms water. Thus, oxygen is the terminal electron acceptor of the mitochondrial respiratory chain. The respiratory chain is shown by the red box in Figure 2.

The oxidation/reduction reactions of a respiratory chain are shown in Figure 3. The oxidation/reduction components, A, B, C etc include haem groups (containing iron atoms), flavins, ubiquinone, copper atoms and clusters of iron and sulphur atoms; each can be reduced by accepting electrons and oxidized by donating them. The pathway of electron transport down the respiratory chain is complicated but well understood. The oxidation/reduction components are associated with large protein complexes which span (or traverse) the inner membrane, and cytochrome c, a smaller protein located at the outside surface of the inner membrane (see "stretch and challenge", Figure 4). The structures of the respiratory chain proteins and the positions of their oxidation/reduction components have been deduced using X-ray crystallography.

Electron transport through the complexes of the respiratory chain causes uptake of H+ from the matrix at the inside surface of the inner membrane, and the release of H+ at the outside surface (Figures 2 and 4). Protons are effectively moved (or "pumped") outwards across the inner membrane using energy released in the electron transport reactions. The movement of H+ makes the outer aqueous region positive and slightly acidic relative to the matrix of the mitochondrion. This difference between the two sides of the membrane (it is both an electrical and a pH difference) is called the protonmotive force, and it can drive protons back across the membrane through the ATP synthase enzyme. Proton flow through the enzyme causes a rotation of protein subunits in the ATP synthase which leads to ATP synthesis from ADP and phosphate. Another British biochemist, John Walker, and Paul Boyer from the US, shared the Nobel Prize for Chemistry in 1997 for their work on this reaction.

A key point to understand is that the protonmotive force is an intermediate in the transfer of energy from the oxidation/reduction reactions of the respiratory chain to ATP synthesis.

The chemiosmotic theory is supported by substantial experimental evidence. This includes observations that (i) the respiratory chain can pump protons across the mitochondrial inner membrane, (ii) dissipation of the protonmotive force with specific chemical reagents prevents ATP synthesis, and (iii) in mitochondria with blocked electron transport reactions the "artificial" generation of a protonmotive force can drive ATP synthesis.

It is important to note that the generation of the protonmotive force and ATP synthesis occur even when the outer mitochondrial membrane is removed in experiments; the outer membrane does not have a role in oxidative phosphorylation.

ATP produced in the mitochondrial matrix by oxidative phosphorylation is exported into the cell cytoplasm by a transporter protein located in the inner membrane. The same protein continuously returns ADP to the matrix for re-phosphorylation. The total pool of ATP + ADP in each cell is small and may turn over hundreds of times a day.

Bacteria do not have mitochondria. Many species of bacteria (including Escherichia coli) have respiratory chains and ATP synthases in their cytoplasmic membrane (which lies just inside their outer membrane/cell wall). Like mitochondria these bacteria perform oxidative phosphorylation with a protonmotive force as the energy intermediate.

This document was kindly provided by Martin D Brand (Buck Institute, California), Stuart J Ferguson (University of Oxford), J Baz Jackson (University of Birmingham), David G Nicholls (Buck Institute, California), Peter R Rich (University College, London).