In 1961, Peter Mitchell proposed the now widely accepted chemiosmotic coupling hypothesis to explain ATP synthesis as a result of electron transport (ETS) and oxidative phosphorylation. It consists of the following principles:

1. Energy from electron transport drives an active transport system.

2. The active transport system pumps protons out of the mitochondrial matrix into the intermembrane space.

3. An electrochemical gradient of protons is created, with a lower pH value outside the inner mitochondrial membrane than inside. The protons on the outside have a thermodynamic tendency to flow back in, so as to equalize pH on both sides of the membrane.

4. When protons do flow back into the matrix, the free energy arising from the gradient (21 kJ/mol of protons) is dissipated, with some of it being used to drive the synthesis of ATP.

Evidence supporting the chemiosmotic coupling hypothesis:

1. Mitochondria do pump protons and establish a pH gradient across their inner membrane.

2. Oxidative phosphorylation requires an intact inner membrane. If the inner membrane is damaged, protons can leak back into the mitochondrial matrix and destroy the proton gradient. Thus, there would be no energy to make ATP.

3. Key electron transport proteins span the inner membrane. Thus, they are perfectly positioned to serve as pumps.

4. Agents that uncouple ETS from oxidative phosphorylation dissipate the proton gradient.

5. If one creates an artificial proton gradient across the mitochondrial inner membrane by incubating mitochondria in an acid solution, ATP is produced by the mitochondria in the absence of electron transport.