The complete aerobic catabolism of one molecule of glucose yields between 36 and 38 ATP; energy obtained mostly as the reduced coenzymes NADH and FADH2  are conveyed through the electron transport system. Three of the four respiratory complexes comprising the mitochondrial respiratory chain, as well as ATP synthase, are embedded in the inner mitochondrial membrane. Coenzyme Q and cytochrome c transfer electrons between complexes, which will ultimately meet oxygen, the terminal electron acceptor. Metabolic water is the product of oxygen reduction.
Electron Transport Chain

Image: "Electron Transport Chain" by Phil Schatz. License: CC BY 4.0

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The Structure of Mitochondrion

The inner and outer membranes surround mitochondria, and the former possesses invaginations, called cristae, which house respiratory complexes. These infoldings increase the surface area of the inner membrane and effectively increase the respiratory capability of mitochondria. Localized nearby are many of the enzymes and intermediates of the TCA Cycle and β-oxidation, which occur in the matrix of the mitochondria. Other pathways, for example, glycolysis, occur in the cytosol, and NADH produced there must be transported to the matrix in the form of reducing equivalents to participate in electron transport and ATP synthesis via oxidative phosphorylation.

The mitochondrial outer membrane contains porins that permit diffusion of ions and metabolites, but the inner membrane is only freely impermeable to O2, CO2, and H2O. Passage of metabolites, such as ATP, ADP, Ca2+, and phosphate is a process mediated by transport proteins; which permit the generation of ion gradients.

The inner membrane space (IM space) separates the inner membrane from the matrix and possesses a high concentration of protons. The passage of electrons through the chain provides the energy required to translocate protons against their concentration gradient; later as we discuss oxidative phosphorylation, the logic of this will become clear.

Diagram of a mitochondrion

Electron Transport

The inner mitochondrial membrane plays host to cofactors that were reduced throughout catabolic pathways occurring in different cellular compartments. Here, the conveying of electrons through three respiratory complexes is coupled to the outward pumping of protons into the intermembrane space.

This establishes an electrochemical proton gradient that can subsequently be used to synthesize ATP, as protons flow down their concentration gradient from the intermembrane space through the Fo subunit of ATP synthase, and into the matrix through the attached F1 subunit.

Electrons from NADH are passed to Complex I as four protons are passed into the IM space, and NADH is oxidized by coenzyme Q (also called CoQ or ubiquinone).

Complex III transfers four protons into the IM space as it accepts electrons from the lipid-soluble CoQ as it is reduced by cytochrome c.

Complex IV transfer two protons in the IM space as it catalyzes the oxidation of reduced cytochrome c by O2, the terminal electron acceptor of the electron transport chain. In the matrix, protons, electrons, and O2 combine to form metabolic water.

Complex II, also called succinate dehydrogenase (from the Citric Acid Cycle), accepts electrons from FADH2  and passes them to coenzyme Q, but does not convey protons into the IM space. These electrons are then conveyed through complex III, cytochrome c, and complex IV.

Note later while reading about oxidative phosphorylation that three protons are used to synthesize one ATP molecule. FADH2 produces less ATP than NADH because complex II does not convey protons into the IM space. Electron flow from NADH is coupled to the passage of ten protons, whereas FADH2 electron flow through ETS is coupled to the passage of six protons into the IM space. Consequently, one NADH produces three ATP while one FADH2 produces two.

The electrochemical gradient formed by proton pumping can now be used as three protons at a time are sent through ATP synthase, whose F1 subunit rotates in its catalysis to generate ATP from ADP and inorganic phosphate.

Schematic diagram of the mitochondrial electron transport chain

Oxidative Phosphorylation- Two Thought Questions

Earlier I discussed differential ATP production on electrons originating from NADH and FADH2; which now presents us with an excellent opportunity to tie together several key concepts by exercising our knowledge of metabolism.

[1] How many ATP are produced from the complete oxidation of 1mol of glucose to CO2 and H2O?

A net of two ATP is produced during glycolysis, where a six-carbon glucose molecule is anaerobically catabolized into two three-carbon pyruvate molecules. Two NAD+ are reduced to NADH as well during this process.

These two pyruvate molecules are then used to produce two Acetyl-CoA molecules by pyruvate dehydrogenase, which produces 1 NADH each.

The two Acetyl-CoA molecules enter the TCA Cycle, where they condense with oxaloacetate and generate two GTP (which are converted to two ATP), six NADH, and two FADH2.

Each NADH enters the electron transport chain at complex I, where it is re-oxidized and passes its electrons to CoQ. Electrons flow from CoQ to complex III, which relays them through cytochrome c complex IV. Here, they are accepted by O2. Both Complex I and Complex IV convey four protons each into the IM space, whereas complex III pumps two.

The six NADH produced during the TCA Cycle yield sixty protons in the IM space.

During glycolysis two NADH were reduced, and there were two more generated during the pyruvate dehydrogenase reaction; four total. When added to the sixty protons produced during TCA, we now have a total of one hundred protons in the IM space.

Two FADH2 molecules enter ETS through complex II, which does not pump protons into the IM space. Therefore, the two FADH2 produced in the TCA Cycle account for twelve protons. So far we have one hundred and twelve protons in the IM space.

ATP synthase produces one ATP per three protons pumped from the IM space into the matrix; so the protons pumped during electron transport will generate thirty-seven ATP. There were two ATP generated during glycolysis, and two produced in TCA (as GTP). Now we can say that Glucose oxidation produces 41.3 ATP.

So, why do textbooks say that there are 32 produced and not 41.3?

Pi needs to be transported into the matrix, which requires a proton to accomplish. Also, two other contributing factors are that the only point that protons can exit is the ATPase (some miss the bus because they are hanging out elsewhere), and some leak out of the F0 subunit.

If we now take the transport of Pi into consideration, it takes four protons to synthesize one ATP molecule. We divide the total number of protons from NADH (100) and FADH2 (12) (=112) to get 28 ATP produced. When we add the four ATP produced as a result of glycolysis and TCA, we get 32 ATP produced per molecule of glucose.

[2] Compare this to the amount produced during the complete oxidation of a C16 fatty acid such as a palmitic acid; or an odd-numbered fatty acid such as C17 margaric acid?

The C16 fatty acid is broken down through β-oxidation to eight acetyl-CoA molecules, which condense with oxaloacetate to form citrate.  Also produced during β-oxidation are 7 NADH, and 7 FADH2 molecules. As explained in the above example, one turn of the cycle produces 3 NADH, 1 FADH2, and 1 GTP. The activation of palmitate to palmitoyl-CoA consumes two ATP equivalents as well. In all, C16 palmitoyl-CoA will produce 31 NADH, 15 FADH2, and 8 GTP. The subsequent electron transport of the coenzymes yields a total of 400 protons in the matrix (10 for each NADH, and 6 for each FADH2. 400 total protons), yielding 100 ATP. If you add in the 8 GTP that is produced and subtract 2 ATP for the activation of Palmitate to palmitoyl-CoA, you will get 106 ATP produced for the complete oxidation of C16 palmitate.

Protons conveyed into the IM space as a result of oxidation of one molecule of palmitoyl-CoA to CO2 and H2O
Enzyme catalyzing oxidation step Number of NADH or FADH2 formed Number of protons ultimately translocated into IM Space
Acyl-CoA dehydrogenase 7 FADH2 42
β-Hydroxyacyl-CoA dehydrogenase 7 NADH 70
Isocitrate dehydrogenase 8 NADH 80
α-Ketoglutarate dehydrogenase 8 NADH 80
Succinate dehydrogenase 8 FADH2 48
Malate dehydrogenase 8 NADH 80
Total 400


A C17 fatty acid such as margaric acid will produce five fewer ATP because the product of its β-oxidation’s last round is a C3 unit of propionyl-CoA; which enters the TCA cycle as succinyl-CoA. This means that it will “miss out” on the isocitrate dehydrogenase and α-ketoglutarate dehydrogenase steps, and convey twenty fewer protons into the IM space as a result.

Control of Oxidative Phosphorylation

Glycolysis, fatty acid degradation, and the TCA Cycle provide the primary sources of electrons that enter the mitochondrial electron-transport chain. Not surprisingly, control of glycolysis and the citric acid cycle is coordinated with the demand for oxidative phosphorylation. An adequate supply of electrons to feed the electron transport chain is provided by regulation of the control points of glycolysis and the citric acid cycle (phosphofructokinase, pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and α -ketoglutarate dehydrogenase) by NADH and/or certain metabolites.

Interestingly, another important regulatory effect is the inhibition of phosphofructokinase by citrate. When the demand for ATP decreases, [ATP] increases and [ADP] decreases. Because ADP activates isocitrate dehydrogenase and ATP inhibits α -ketoglutarate dehydrogenase, the citric acid cycle slows down. This causes the citrate concentration to build up. Citrate leaves the mitochondrion via a specific transport system and, once in the cytosol, acts to restrain further carbohydrate breakdown by inhibiting PFK.

The citrate concentration also builds up when the acetyl-CoA concentration increases, which occurs during the oxidation of fatty acids. The inhibition of glycolysis by fatty acid oxidation is called the glucose–fatty acid cycle. The pathway allows fatty acids to be utilized as the major fuel for oxidative metabolism in heart muscle, while conserving glucose for organs such as the brain, which require it.

Several chemical compounds inhibit the electron transport chain at different points. Rotenone and Amytal inhibit complex I, Antimycin A at complex III, and CNblocks complex IV. 2,3-Dintrophenol doesn’t directly inhibit the chain but dissipates the proton gradient.

Practice Questions about Electron Transport & Oxidative Phosphorylation

1. Which of the following would not increase as a result of a substantial increase in work performed by the cell?

  1. The utilization of oxygen.
  2. The ratio of ATP to ADP in the cell.
  3. The rate of utilization of ADP by ATP synthase.
  4. The entrance of H+into the mitochondrial matrix through ATP synthase
  5. The pumping of H+into the IM space of the mitochondria by the electron transport chain.

2. Which of the following would not increase in rate as a consequence of subjecting the cell to Aspirin, T4 or some other chemical uncoupler of oxidative phosphorylation?

  1. ATP synthesis
  2. Proton pumping by the electron transport chain.
  3. Heat generation by the mitochondria
  4. Oxygen utilization by the cell
  5. NADH oxidation by NADH dehydrogenase

3. All of the following statements regarding electron transport in mitochondria are correct, except for:

  1. It is located in the mitochondrion inner membrane.
  2. Cytochrome c accepts electrons from complex II.
  3. Cytochrome oxidase (complex IV) accepts electrons from Cytochrome c
  4. Complex I is called NADH dehydrogenase
  5. Coenzyme Q accepts electrons from complex I and complex II
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