Chemiosmotic Hypothesis:


The chemiosmotic hypothesis was developed in 1961 by Peter D. Mitchell which suggests that most ATP synthesis in respiring cells comes from the electrochemical gradient across the inner membranes of mitochondria by using the energy of NADH and FADH2 formed from the breaking down of energy-rich molecules, such as glucose.

The diagram below shows the structure of a mitochondria:

Molecules such as glucose are metabolized through glycolysis to produce acetyl CoA as an energy-rich intermediate. The oxidation of acetyl CoA in the mitochondrial matrix is coupled to the reduction of a carrier molecule such as NAD and FAD. The carriers pass electrons to the electron transport chain (ETC) in the inner mitochondrial membrane, which in turn pass them to other proteins in the ETC. The energy available in the electrons is used to pump protons from the matrix across the inner mitochondrial membrane, storing energy in the form of a trans-membrane electrochemical gradient. The protons move back across the inner membrane through the enzyme ATP synthase. The flow of protons back into the matrix of the mitochondrion via ATP synthase provides enough energy for ADP to combine with inorganic phosphate to form ATP. The electrons and protons at the last pump in the ETC are taken up by oxygen to form water.

Diagram showing the structure of mitochodrial ATP synthase (F1F0 ATPase)

atpase                          pi
In all cells, chemiosmosis involves the PROTON-MOTIVE FORCE (PMF) in some step. This can be described as the storing of energy as a combination of a proton and voltage gradient across a membrane. The chemical potential energy refers to the difference in concentration of the protons and the electrical potential energy as a consequence of the charge separation (when the protons move without a counter-ion).
In most cases the proton motive force is generated by an ETC which acts as both an electron and proton pump, pumping electrons in opposite directions, creating a separation of charge. In the mitochondria, free energy released from the electron transport chain is used to move protons from the mitochondrial matrix to the inter-membrane space of the mitochondria. Moving the protons to the outer parts of the mitochondria creates a higher concentration of positively charged particles, resulting in a slightly positive and slightly negative side. This charge difference results in an electro-chemical gradient. This gradient is composed of both the pH gradient and the electrical gradient. The pH gradient is a result of the H+ ion concentration difference. Together the electro-chemical gradient of protons is both a concentration and charge difference and is often called the proton motive force. The PMF needs to be about 50 kJ/mol for the ATP synthase to be able to make ATP.
CHEMIOSMOTIC PHOSPHORYLATION is the third pathway that produces ATP from inorganic phosphate and an ADP molecule. This process is part of OXIDATIVE PHOSPHORYLATION. The complete breakdown of glucose in the presence of oxygen is called cellular respiration. The last steps of this process occur in mitochondria. The reduced molecules NADH and FADH2 are generated by the Krebs cycle and glycolysis. These molecules pass electrons to an electron transport chain, which uses the energy released to create a proton gradient across the inner mitochondrial membrane. ATP synthase then uses the energy stored in this gradient to make ATP. This process is called oxidative phosphorylation because oxygen is the final electron acceptor and the energy released by reducing oxygen to water is used to phosphorylate ADP and generate ATP.


Questions to ask when studying cellular respiration:


1. where is glycolysis occurring?

2. what are the 10 enzyme involved?

3. what are the reactants and products formed?

4. is there any gain of loss at the end of glycolysis?

5. what does this process require in order to continue?

6. is there any cofactors necessary for the catalyzed reactions to continue?


1. what is the reactant and product involved?

2. what is the enzyme involved?

3. is there any cofactors needed for this process to continue?

4. at the end of this reaction what happens to the products formed (Acetyl-CoA)?

5. where in the cell does this process occur?


1. how many ATP, NADH and FADH2 is generated?

2. is ATP used in this process?

3. what product of respiration is formed at this stage through the metabolism of Acety-CoA?

hint- Glucose + Oxygen —-> Carbon Dioxide + Water + Energy

C6H12O6 + 6O2 —-> 6CO2 + 6H2O + Energy

4. where in the cell does this process occur?


1. what do you understand by the chemiosmosis theory?

2. where in the cell is the ETC located?

3. what is the product of respiration formed at this stage?

4. how is ATP generated by this process?

5. what do you understand by the terms ATP synthase, Complexes 1, 2, 3 and 4?

6. how do protons and electrons flow across and along the organelle’s membrane?








It is made up of four complexes which is found on the inner membrane of the mitochondria. The goal of this chain is to break down NADH and FADH2 and pump H+ into the outer compartment of the mitochondria. In this reaction the Electron Transport Chain creates a gradient which is used to produce ATP similar to chloroplast. Electrons move down an energy gradient until they meet the ultimate electron acceptor-> oxygen gas (O2).

  • Electron Transport Phosphorylation typically produces 32 ATP’s.
  • ATP is generated as H+ moves down its concentration gradient through a special enzyme called ATP syntase.