The Chemiosmotic Hypothesis

The chemiosmotic hypothesis was proposed by Peter Mitchell. This hypothesis stated that a proton-motive force was responsible for driving the synthesis of ATP. In this hypothesis, protons would be pumped across the inner mitochondrial membrane as electrons went through the electron transfer chain. This would result in a proton gradient with an lower pH in the intermembrane space and a elevated pH in the matrix of the mitochondria. An intact inner mitochondrial membrane, impermeable to protons, is a requirement of such a model. The proton gradient and membrane potential are the proton-motive force that is used to drive ATP synthesis. In effect, the pH gradient acts as a "battery" which stores energy to produce ATP. Over the past several years, Mitchell's chemiosmotic hypothesis has been widely accepted as the mechanism of coupling of electron transport and ATP synthesis. He was awarded the Nobel Prize in Chemistry in 1978. This acceptance by the scientific community is a result of accumulating experimental evidence supporting the hypothesis.

Some of the evidence supporting Mitchell's chemiosmotic hypothesis is as follows.

  1. Electron transport generates a proton gradient. The pH measured on the outside is lower than that measured inside the mitochondria.
  2. Only a proton gradient is needed to synthesize ATP. Electron transport is not required as long as there is another mechanism for generating a pH gradient.
  3. A reconstitution experiment carried out by Racker & Stoeckenius (J Biol Chem 1974 Jan 25;249(2):662-3, Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphate formation) showed that the generation of a proton gradient can result in ATP synthesis in a totally artificial system. In their experiment, a mitochondrial ATPase complex from beef heart was inserted into an artificial lipid bilayer. Also inserted in this bilayer was a membrane fragment containing the protein, bacteriorhodopsin* [pic], from the purple bacteria Halobacterium, so called because the bacteriorhodopsin gives the membrane a purple color. Bacteriorhodopsin is a light-driven proton pump. Therefore, shining light on this artificial "purple membrane" formed a proton gradient, which was used by the beef heart mitochondrial ATPase to synthesize ATP.
The electron transfer chains and the ATPases are asymmetrically oriented in the inner mitochondrial membrane.  An asymmetric orientation is a requirement to establish a pH gradient. A random arrangement would not result in a net gradient of protons and therefore, no proton-motive force for the synthesis of ATP.

Compounds called uncouplers were found to collapse the pH gradient by shuttling protons back across the membrane through the compounds. One such uncoupler, dinitophenol is shown below. In the presence of the uncoupler electron transport continues, but no ATP synthesis occurs.

Can uncoupling of electron transport and ATP synthesis ever be useful to an organism? The answer is probably "Yes." Such uncoupling can generate an energetically wasteful byproduct, heat. This occurs normally in many in hibernating animals, in newborn humans, and in mammals adapted to the cold. It occurs in a specialized tissue known as brown adipose tissue. An uncoupling protein called thermogenin can accomplish this uncoupling and thus allow heat to be generated.


Note:    a new value for ATP per NADH is 2.5 and ATP per FADH2 is 1.5.
You may see the numbers ATP per NADH = 3   and   ATP per FADH 2 = 2 in some texts. For more information on the re-evaluation of the number of ATP's/nucleotide coenzyme see Hinkle, et al.  Mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Biochemistry 30:3576-82, 1991.

The different values of 30 or 32 ATP/glucose depend on the method used to transport cytoplasmic NADH, formed by glycolysis, into the mitochondria, i.e. the shuttles.