Oxidative Phosphorylation: Mechanism and Regulation

Oxidative phosphorylation is the energy-yielding metabolic process of aerobic organisms. All oxidative breakdowns of carbohydrates, fats, and amino acids intersect at this final stage of cellular respiration. Here, the energy of oxidation fuels the synthesis of ATP (adenosine triphosphate). It occurs in the inner mitochondrial membrane in eukaryotes and the plasma membrane in prokaryotes. 

The product of the oxidation of pyruvate, cellular respiration, Krebs cycle, and glycolysis; NADH (nicotinamide adenosine diphosphate hydrogen) and FADH2 (flavin adenosine dinucleotide hydrogen), both are electron carriers and help in the generation of ATP by oxidative phosphorylation. 

Mechanisms of Oxidative Phosphorylation

The mechanism of oxidative phosphorylation is based on chemiosmotic theory, which states that the difference in proton concentration between the membranes of mitochondria acts as the reservoir for the energy generated from biological oxidation reactions. A series of events occurs during oxidative phosphorylation, broadly classified under electron transport chain and ATP synthesis.

Electron Transport Chain (ETC)

The electron transport chain consists of a series of proteins and organic molecules found in the inner membrane of the mitochondria. The electrons are transported from one member to another membrane by redox reactions. The energy generated in these reactions is captured as a protein gradient used to make ATP by chemiosmosis (the process of movement of ions across the biological membrane). 

When electrons travel through the chain, they go from higher energy level to lower energy level; that is, it moves from less electron-hungry to more electron-hungry molecules. This downhill reaction releases energy. A protein gradient forms due to the pumping of proteins from the mitochondrial matrix to the intermembrane space. The energy released catalyzes this pumping. 

Downhill reactions: spontaneous, result in a drop in free energy (less energy in products than in reactants)

The electrons that enter the transport chain arise from NADH and FADH2 molecules. There are four complexes (I, II, III, and IV) in the electron transfer chain and two mobile electron carriers (ubiquinone and cytochrome C). 

Plants have closely related compound called plastoquinone and bacteria has menaquinone as electron carrier instead of ubiquinone

Steps in the electron transport chain

  • Firstly, NADH transfer electron to complex I and converts itself back to NAD+. NADH is a perfect electron donor in redox reactions. It indicates that the electrons are at a high energy level in NADH. As the electron moves in complex I, energy gets released by a series of redox reactions. Also, the complex uses the produced energy to pump proton(H+) from the matrix to intermembrane space.
  • The FADH2 transfers the electron to complex II. It is not a good electron donor simply because the electrons are at a lower energy level in FADH2. Complex II cannot pump protons across membranes. The FADH2 converts into FAD, i.e., FADH2 oxidizes into FAD.
  • Again, the two complexes pass their electron to a small electron carrier called ubiquinone (Q), which is reduced to form QH2
  • After that, the QH2 transfers the electron to complex III. Complex III can pump the proton across the membrane. Complex III delivers the electron to cytochrome C (Cyt C).
  • Finally, the electron carrier Cyt C transfers the electron to complex IV, which transfers the electron to O2, the ultimate electron acceptor. O2 splits into two ions of oxygen which then converts into water molecules by accepting two proton molecules (H+). A total of 4 H+ is required to reduce each molecule of O2, and two molecules of H2O are formed in this process.

The complexes I, III, and IV are responsible for the pumping proton across the membrane with the help of energy released during the downhill movement of electrons.

oxidative phosphorylation

ATP Synthesis

The pumping of the proton from the matrix to intermembrane space forms an electrochemical gradient across the inner mitochondrial membrane, also called the proton motive force (PMF). Since the protons cannot pass the phospholipid bilayer of the membrane due to its hydrophobic core, it moves down their concentration gradient with the help of channel proteins with hydrophilic tunnels across the membranes.

The only channel available is ATP synthase. ATP synthase turns on by the flow of H+ ions moving down their electrochemical gradient. After the turning on of ATP synthase, the energy from the proton gradient catalyzes the addition of a phosphate to ADP, forming a molecule of ATP. 

2.5 ATP molecule is produced when electrons enter the respiratory chain at the complex I. In contrast, a 1.5 ATP molecule is produced when electrons enter the chain at complex II. The ATP formed is released in the form of heat.

Regulation of Oxidative Phosphorylation

Every chemical reaction cell requires regulation. The oxidative phosphorylation is regulated in the following ways:

The energy demand of the cell

Oxidative phosphorylation is regulated by the consumption of O2, the intracellular concentration of ADP (adenosine diphosphate), and the availability of Pi (phosphate) in the cell. Similarly, the mass-action ratio of the ATP-ADP system is another measure regulating oxidative phosphorylation. Typically, this ratio is high, but when the energy-requiring process like protein synthesis increases, the rate of breakdown of ATP to ADP and Pi increases which lowers the ratio. The oxidative phosphorylation process increases due to the lowering of the ratio to produce ATP until the mass-action ratio returns to its normal high level. In short, the rate of ATP production depends on the energy-requiring cellular activities. 

Inhibitory proteins in ischemic cells 

In ischemic cells (cells deprived of oxygen), electron transfer to O2 stops along with the pumping of protons. The proton-motive force then collapses. In such cases, ATP synthase could work reverse: hydrolyzing ATP and pumping protons outward, causing a disastrous drop in ATP levels. To prevent that, an 84 amino acids long protein molecule (IF1) binds to ATP synthase and inhibits its activity. pH less than 6.5 favors IF1 (pyruvic acid after glycolysis lowers the pH of the cytosol and mitochondrial matrix). When aerobic condition returns, pH rises due to less production of pyruvic acid leading to destabilization of IF1 and resuming the activity of ATP synthase.  

Significance of Oxidative Phosphorylation

Regenerates electron carriers

NADH and FADH2 transform their electrons into the electron transport chain and change to NAD+ and FAD. It is essential because the oxidized forms of these carriers are used in the various metabolic pathways, and these must be available to keep the metabolic process running. 

Creates Protein Gradient

The transport chain builds a protein gradient across the inner mitochondrial membrane, with a lower concentration of H+ in the matrix and a higher concentration in the intermembrane space. This gradient represents a stored form of energy used to produce ATP.

ATP Produced

The total ATP produced by the total oxidation of a glucose molecule is as follows:

Process Direct productATP produced
Glycolysis2 NADH
5+2= 7
Pyruvate oxidation2 NADH5
Krebs Cycle6 NADH
2 GTP or ATP
Total ATP= 32 molecules


  • Nelson, D., & Cox, M. (2005). Lecture notebook for Lehninger Principles of biochemistry, fourth edition (4th ed., pp. 690-719). Freeman.

Ashma Shrestha

Hello, I am Ashma Shrestha. I had recently completed my Masters degree in Medical Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology.

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