• Introduction
  • The basics of chemiosmosis
  • ATP synthase
  • ATP yield
  • Final thoughts
  • Sources
  • ATP synthase: An enzyme that utilizes a hydrogen ion gradient to phosphorylate ADP, producing ATP.
  • Chemiosmosis: the movement of ions across a semipermeable membrane down their electrochemical gradient
  • Electron carrier: Small organic molecules that switch between oxidized and reduced forms and transport electrons during metabolic reactions
  • FADH₂: Also known as Flavin adenine dinucleotide. It is an electron carrier that transports electrons from glycolysis and the citric acid cycle to the electron transport chain.
  • NADH: A reduced form of Nicotinamide adenine dinucleotide that carries electrons from one reaction to another. An important cofactor in metabolism consisting of two nucleotides and their phosphate groups
  • Oxidative phosphorylation: Formation of ATP via the transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers.


ATP is an essential component in various metabolic processes that take place in living organisms. In most living organisms, this is made by phosphorylating the ADP molecules that already exist in the cells. However, this process requires energy, which is provided by either breaking down nutrients derived from food, or through photosynthesis. Both of these processes rely on chemiosmosis to synthesize ATP, which is the energy currency of the cell.

Chemiosmosis refers to the movement of ions across a semipermeable membrane down their electrochemical gradient (i.e. osmosis of the ions). The most common example of this is during the formation of adenosine triphosphate (ATP) during cellular respiration, when hydrogen ions (H+) move across the mitochondrial membrane. Collectively, the electron transport chain and the production of ATP through chemiosmosis is known as oxidative phosphorylation. This accounts for approximately 90% of all ATP generated during the breakdown of glucose in aerobic respiration. 

This post explains the basic process of chemiosmosis and what makes it so important for cellular respiration. 

The basics of chemiosmosis

In cellular respiration, glucose is first broken down via glycolysis into pyruvate, before being decarboxylated into acetyl CoA. This molecule then undergoes a process of oxidation known as the citric acid cycle, producing high-energy intermediates NADH and FADH₂. These electron carriers are then transported into a series of protein complexes known as the electron transport chain (ETC). 

During the oxidation process of NADH and FADH₂, these electron carriers donate their electrons to the ETC. As the electrons move down the electron transport chain, they release a significant amount of energy that is used to pump out hydrogen ions (H+), or protons, out of the mitochondrial matrix into the intermembrane space. This produces an outward current across the mitochondrial membrane.

The difference in charge between the two sides of the membrane creates an electrochemical gradient, owing to the higher concentration of H+ ions and their positive charge in one side of the membrane. This is also known as a proton-motive force.

Like water behind a dam, the accumulation of hydrogen ions stores a significant amount of potential energy. Through simple diffusion, these hydrogen ions travel from a higher concentration to a lower concentration back across the mitochondrial membrane, carrying potential. This flow of protons fuels an enzyme called ATP synthase, which starts rotating like a tiny waterwheel/turbine. Thus, utilizing kinetic energy to power the phosphorylation process. 

ATP synthase catalyzes a reaction which adds an inorganic phosphate (Pi) to ADP, producing ATP. Thus, without chemiosmosis, there will be no hydrogen ion gradient to power ATP synthase.

In the end, electrons are passed onto their final acceptor, molecular oxygen. This reaction forms water: 2 H+ + ½ O2 → H2O.

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The phosphorylation phase of oxidative phosphorylationTakes place in the mitochondriaUtilizes the hydrogen ion gradient created by the electron transport chainFuels ATP synthaseProduces 28 ATP

ATP Synthase

ATP Synthase, also known as complex V, is a protein complex found on the membrane of the mitochondria. It acts as a proton channel and utilizes the gradient produced by the electron transport chain. The molecule itself is divided into two separate subunits; Fo and F1. Together, these subunits work as a rotational motor system that phosphorylates ADP to ATP. 

Fo is hydrophobic (repels water) and located in the inner mitochondrial membrane. This is also where the proton channel is located. As the H+ ions flow down the gradient, the channel is protonated and deprotonated repeatedly. The alternating ionization causes Fo to rotate, which also alters the orientation of the F1 subunit. 

F1 is hydrophilic (attracted to water) and faces the mitochondrial matrix. The conformational changes of the F1 allow ATP to be bound and released once it has been synthesized. This leads to the formation of ATP from ADP and inorganic phosphate (Pi). 

As the ATP concentration rises, there is less ADP for ATP Synthase to use. Thus, limiting the production of ATP when it is available in sufficient quantities. On the other hand, a higher ADP concentration works as a natural signal to produce more ATP.

ATP yield

In aerobic conditions (with the presence of oxygen), cellular respiration produces 30-32 molecules of ATP for every molecule of glucose. Two ATP (or GTP) molecules are formed during glycolysis and two ATP is produced via the citric acid cycle. Finally, 28 molecules of ATP is produced during oxidative phosphorylation.

Studies state that to produce a single molecule of ATP, four hydrogen ions must diffuse from the intermembrane space back into the matrix. Due to the fact that a single NADH pumps out 10 hydrogen ions into the intermembrane space, each NADH generates 2.5 molecules of ATP. On the other hand, FADH₂, which enters the electron transport chain in the second complex, releases 6 hydrogen ions into the intermembrane space. Thus, producing 1.5 ATP molecules.

Cellular respiration produces a maximum of 10 NADH and 2 FADH₂ per a molecule of glucose. Thus, oxidative phosphorylation produces a total of 28 ATP. Additionally, since two molecules of ATP are generated during both glycolysis and the citric acid cycle, the theoretical maximum ATP yield from a single glucose molecule is 32. 



ATP Yield



3-5 ATP

Pyruvate Oxidation



The Citric Acid Cycle


15 ATP


30-32 ATP

One thing to keep in mind is that glycolysis takes place in the cytoplasm whereas the electron transport chain is located in the mitochondria. Because NADH is unable to move through the mitochondrial membrane by itself, it must find another way to shuttle its electrons into the mitochondria. Once there, these electrons are passed on to NAD+ or FAD, which have a different ATP yield. Thus, the amount of ATP produced ranges between 30-32.

Chemiosmosis is the phosphorylation phase of oxidative phosphorylation.

Final thoughts

The chemiosmotic theory was first introduced by Peter D. Mitchell in 1961. He suggested that metabolic cells produced most of the ATP via utilizing the energy stored in an electrochemical gradient across the mitochondrial membrane.

As with every groundbreaking theory, the chemiosmotic theory was not accepted immediately by the scientific community. In those times, it was believed that the flow of electrons was stored in some other intermediates that were used to produce ATP. However, with time Mitchell’s chemiosmotic hypothesis was accepted as it gathered more scientific support. Nowadays, Mitchell’s chemiosmotic theory remains as the most well-studied theory explaining the production ATP.

Did you learn anything new about chemiosmosis? Let us know in the comments.


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