Oxidative phosphorylation constitutes the final and 3rd stage of oxidation of carbohydrates, lipids and amino acids. During this process, energy of oxidation is used to synthesise ATP. It occurs in the mitochondria and begins with the oxidation of reduced cofactors, NADH and FADH2 by the electron transport chain.

Reduced cofactors such as NADH and FADH2 are good reducing agents. Oxygen is a very good oxidizing agent – one of the strongest known to chemistry. So the oxidation of any kind of reduced carbon compound is highly favourable thermodynamically. Now we see why oxygen presented such a threat – and such an opportunity – to life on this planet, when it first began to accumulate in the atmosphere, billions of years ago, thanks to photosynthesis. Oxygen not only provided a vast new energy source, but it also presented the danger of uncontrolled oxidation processes (not only fires, but also chemical chain reactions like lipid peroxidation, which is what happens when you leave butter outside the refrigerator for too long in the summer and it gets rancid).

 

Oxidation of reduced cofactors: energetics

The calculations in Figure 23.1 show you how much energy is released by the reoxidation of NADH and FADH2. Since oxygen is such a good oxidizing agent, both reactions have very large negative Gibbs free energy changes. NADH is a little stronger, as a reducing agent, so its free energy change is a bit larger. But both numbers are much larger than any covalent bond strength. So, the reoxidation of one mole of reduced cofactor liberates enough energy to make several moles of ATP. IF the energy of the redox reaction can be harnessed to ATP production!

However, so much energy is released by the reaction between the reduced cofactors and oxygen that the direct reaction, even if it were catalyzed, would be energetically wasteful: no covalent bond could contain more than a fraction of the energy released. So, instead, nature uses electron transport chains. The reducing equivalents** from the reduced cofactors are passed to oxygen indirectly. So, instead of releasing all the available energy at once, the reoxidation of NADH and FADH2 by oxygen is broken up into several distinct processes with smaller free energy changes. This is just like lowering ourselves gently from floor to floor (in an elevator) when moving from the fifth floor of a building to the first; for, although jumping out the fifth floor window is an effective way to get to the ground, it is dangerous and the process releases so much energy.

**Three types of electron transfers occur in the electron transport chain;

1. Direct transfer as in the reduction of Fe3+ to Fe2+
2. Transfer of a hydrogen atom (H+ + e–)
3. Transfer of a hydride ion (:H–) bearing two electrons

We use the term reducing equivalent to signify electrons transferred in all these types of transfers.

In an electron transport chain, we pass reducing equivalents from molecule to molecule, always moving in the direction of higher reduction potential, until we get to oxygen, the “terminal electron acceptor” (that is, the oxidant with the most positive reduction potential). To accomplish this, we need to have a special set of electron carriers, molecules with appropriate reduction potentials, intermediate between NADH and oxygen.

There are many different electron transport chains in biology. For example, E. coli has a quite different chain from the eukaryotic mitochondrion (the chain we are going to look at in the most detail). But the general principles are always the same.

 

Mitochondrial Electron transport Chain

Figure 23.2  shows the overall plan of mitochondrial electron transport. This is a highly simplified plan; in reality, each of the complexes noted here is a very elaborate system of proteins and associated redox-active species, such as cofactors and metal ions. Reducing equivalents from NADH are passed to a called coenzyme Q; and then to the small heme-protein cytochrome c; and finally to oxygen. Each of these transfers is catalyzed by a “complex”. A complex is a big enzyme: an assembly of proteins and other molecules.

FADH2 is oxidized along a convergent pathway, but its reducing equivalents enter the chain a little lower down, since it is a weaker reducing agent than NADH. Complex II is really just succinate dehydrogenase of the citric acid cycle, which we have already seen. Complex II then passes its reducing equivalents to coenzyme Q.

The electron carriers of the Electron Transport chain: Coenzyme Q (ubiquinone)

The Q in coenzyme Q stands for quinone. A quinone is an organic functional group with two ketone oxygens in a conjugated six-membered ring. Quinones are oxidizing agents and they become reduced to hydroquinones, as shown. This is a two-electron process accompanied by two protons, and it goes via distinct one-electron steps, so there is a free radical intermediate called a “semiquinone”(·QH). The fully reduced hydroquinone form of coenzyme Q is called ubiquinol (QH2) (Figure 23.3). Coenzyme Q bears a long hydrophobic “tail” (built from “isoprene” units) as one of the substituents on the ring, and this tail keeps the molecule associated with the membrane. It can freely move in the membrane carrying electrons from one ETC complex to another.

 

Cytochrome C

Cytochromes are a large class of heme proteins participating in redox reactions. Their name is Greek for “coloured stuff in the cell”; they were first identified and classified based on their colours (optical absorption spectra). The respiratory electron transport chain involves several cytochromes, notably the small protein cytochrome c. In cytochrome c, the heme ring is covalently linked to the polypeptide via cysteine residues (Figure 23.4), but not all hemeproteins have covalently-linked heme; in some cases, such as hemoglobin, the heme is bound non-covalently. (See Stryer Fig. 18.16)

 

Heme is a prosthetic group (non-peptide component) of many proteins – hemeproteins, and it is a particular example of a larger class of molecules called porphyrins, all of which have similar ring structures. The heme ring joins four pyrrole rings (five-membered rings with one N atom) together into a tetrapyrrole ring, which forms a large conjugated aromatic system. The four N atoms are positioned to form a pocket that can bind a metal ion. In heme, that metal is iron. Heme proteins perform a multitude of biological roles, but they always involve transition metal chemistry – ligands binding to the iron atom or redox processes.

Cytochrome c is a soluble protein in the mitochondrial intermembrane space and shuttles electrons from complex III of the ETC to complex IV. The iron atom of heme acts as the redox active component and carries one electron at a time.

Cytochrome C (Fe3+)  +  e    →    Cytochrome C (Fe2+)

The complexes are enzymes that catalyze individual electron-transfer steps in the chain. We can refer to the complexes by their enzyme activities (e.g., NADH:ubiquinone oxidoreductase), but mostly we just refer to them by number: I, II, III, and IV. The complexes are integral membrane proteins; they are buried deeply in the inner mitochondrial membrane. If we gently disrupt the membrane, we can solubilize and purify the individual complexes and measure their catalytic (electron-transfer) activities in the test tube.

Complex I (NADH dehydrogenase):

Complex I catalyzes two processes which are obligately coupled to each other (Figure 23.5).

1. Transfer of a hydride ion from NADH and a H+ from the matrix to coenzyme Q (Exergonic)

NADH + H+  +  Q    →    NAD+  +  QH2

2. Transfer of four H+ from the matrix to the intermembrane space (Endergonic)

NADH  +  5H+N  +  Q    →    NAD+ + QH2 + 4HP

(N: matrix side of the inner mitochondrial membrane;
P: the intermembrane space side of the inner mitochondrial membrane)

Complex II (Succinate dehydrogenase):

Complex II is the TCA cycle enzyme succinate dehydrogenase. It transfers electrons from FADH2 generated by this reaction to coenzyme Q (Figure 23.6). It does NOT pump protons from the matrix to the intermembrane space.

Complex III (Cytochrome c reductase):

Complex II transfers electrons from QH2 to cytochrome c with the simultaneous transfer of four protons from the matrix to the intermembrane space.

Since each cytochrome c can carry only one electron, oxidation of one mole of QH2 requires two moles of oxidized cytochrome c.

The redox reaction:

QH2  +  2 Cyt C (oxidized)     →    Q  +  2 Cyt C (reduced)  +  2 H+

 

The net equation for Complex III:

QH2  +  2 Cyt C (oxidized)  +  2 H+N    →   Q  +  2 Cyt C (reduced)  +  4 H+P

The Electron Transport System also called the Electron Transport Chain, is a chain of reactions that converts redox energy available from oxidation of NADH and FADH2, into proton-motive force which is used to synthesize ATP through conformational changes in the ATP synthase complex through a process called oxidative phosphorylation.

• Oxidative phosphorylation is the last step of cellular respiration.
• This stage consists of a series of electron transfer from organic compounds to oxygen while simultaneously releasing energy during the process.
• In aerobic respiration, the final electron acceptor is the molecular oxygen while in anaerobic respiration there are other acceptors like sulfate.
• This chain of reactions is important as it involves breaking down of ATP into ADP and resynthesizing it in the process to ATP, thus utilizing the limited ATPs in the body about 300 times in a day.
• The electron flow takes place in four large protein complexes that are embedded in the inner mitochondrial membrane, together called the respiratory chain or the electron-transport chain.
• This stage is crucial in energy synthesis as all oxidative steps in the degradation of carbohydrates, fats, and amino acids converge at this final stage of cellular respiration, in which the energy of oxidation drives the synthesis of ATP.

Electron Transport Chain Location

• As the citric acid cycle takes place in the mitochondria, the high energy electrons are also present within the mitochondria. As a result, the electron transport chain in eukaryotes also takes place in the mitochondria.
• The mitochondrion is a double-membraned organelle that consists of an outer membrane and an inner membrane that is folded into a series of ridges called cristae.
• There are two compartments in the mitochondria; the matrix and the intermembrane space.
• The outer membrane is highly permeable to ions. It contains enzymes necessary for citric acid cycles while the inner membrane is impermeable to various ions and contains uncharged molecules, electron transport chain and ATP synthesizing enzymes.
• The number of electron transport chains in the mitochondria depends on the location and function of the cell. In the liver mitochondria, there are 10, 000 sets of electron transport chains while the heart mitochondria have three times the number of electron transport chain as in the liver mitochondria.
• The intermembrane space contains enzymes like adenylate kinase, and the matrix contains ATP, ADP, AMP, NAD, NADP, and various ions like Ca2+, Mg2+, etc.

Electron Transport Chain Components/ Electron carriers

• Electrons in the chain are transferred from substrate to oxygen through a series of electron carriers.
• There are about 15 different chemical groups that accept or transfer electrons through the electron chain.

a. FMN (Flavin Mononucleotide)

• At the beginning of the electron transfer chain, the electrons from NADH are transferred to the flavin Mononucleotide (FMN) reducing it to FMNH2.

NAD + H+ + FMN  →  NAD + FMNH2

• The transfer of electrons is catalyzed by the action of NADH dehydrogenase.
• The electrons are further transferred to a series of iron-sulfur complexes (Fe-S) which have a higher relative affinity towards the electrons.

b. Ubiquinone (Co-enzyme-Q)

• Between the flavoproteins and cytochromes are other electron carriers termed ubiquinone (UQ).
• Ubiquinone is the only electron carrier in the respiratory chain that is not bound attached to a protein. This allows the molecule to move between the flavoproteins and the cytochromes.
• Once the electrons are transferred from FMNH2 via the Fe-S centers to the ubiquinone, it becomes UQH2 and the oxidized form of flavoprotein (FMN) is released.

FMNH2 + UQ  →  FMN + UQH2

c. Cytochromes

• The next electron carriers are cytochromes that are red or brown colored proteins containing a heme group that carries the electrons in a sequence from ubiquinone to the molecular oxygen.
• However, each cytochrome, like Fe-S centers, only transfers a single electron whereas other electron carriers like FMN and ubiquinone transfer two electrons.
• There are five types of cytochromes between ubiquinone and the molecular oxygen, each designated as a, b, c, and so on.
• These are named on the basis of their ability to absorb light of different wavelengths (cytochrome a absorbs the longest wavelength, b absorbs the next longest wavelength and so on).

Electron Transport Chain Equation

The electron transport chain consists of a series of oxidation-reduction reactions that lead to the release of energy. A summary of the reactions in the electron transport chain is:

NADH + 1/2O2 + H+ + ADP + Pi  →  NAD+ + ATP + H2O

Electron Transport Chain Complexes

A chain of four enzyme complexes is present in the electron transport chain that catalyzes the transfer of electrons through different electron carriers to the molecular oxygen.

a. Complex I (Mitochondrial complex I)

• Complex I in the electron transport chain is formed of NADH dehydrogenases and the Fe-S centers that catalyzes the transfer of two electrons from NADH to ubiquinone (UQ).
• At the same time, the complex translocates four H+ ions through the membrane, creating a proton gradient.

NADH + H+ + CoQ  →  NAD+ + CoQH2

• NADH is first oxidized to nAD+ by reducing FMN to FMNH2 in a two-step electron transfer.
• FMNH2 is then oxidized to FMN where the two electrons are first transferred to Fe-S centers and then to ubiquinone.

b. Complex II (Mitochondrial complex II)

• Complex II consists of succinic dehydrogenase, FAD, and Fe-S centers.
• The enzyme complex catalyzes the transfer of electrons from other donors like fatty acids and glycerol-3 phosphate to ubiquinone through FAD and Fe-S centers.
• This complex runs parallel to the Complex II, but Complex II doesn’t translocate H+ across the membrane, as in Complex I.

Succinate + FADH2 + CoQ  →  Fumarate + FAD+ + CoQH2

c. Complex III (Mitochondrial complex III)

• Complex III consists of cytochrome b, c, and a specific Fe-S center.
• The enzyme complex, cytochrome reductase, catalyzes the transfer of two electrons from reduced CoQH2 to two molecules of cytochrome c.
• Meanwhile, the protons (H+) from the ubiquinone are release across the membrane aiding to the proton gradient.
• The CoQH2 is oxidized back to CoQ while the iron center (Fe3+) in the cytochrome c is reduced to Fe2+.

CoQH2 + 2 cytc c (Fe3+)  →  CoQ + 2 cytc c (Fe2+) + 4H+

d. Complex IV (Mitochondrial complex IV)

• Complex IV consists of cytochrome a and a3, which is also termed cytochrome oxidase.
• This is the last complex of the chain and is involved in the transfer of two electrons from cytochrome c to molecular oxygen (O2) forming water.
• In the meantime, four protons are translocated across the membrane aiding the proton gradient.

4 cytc c (Fe 2+) + O2   →  4cytc c (Fe3+) + H2O

Figure: Electron Transport Chain. Image created with biorender.com

Electron Transport Chain Steps

The following steps are involved in electron transfer chains which involve the movement of electrons from NADH to molecular oxygen:

1. Transfer of electrons from NADH to Ubiquinone (UQ)

• NADH is produced in different other cycles by the α-ketoglutarate dehydrogenase, isocitrate dehydrogenase, and malate dehydrogenase reactions of the TCA cycle, by the pyruvate dehydrogenase reaction that converts pyruvate to acetyl-CoA, by β-oxidation of fatty acids, and by other oxidation reactions.
• NADH produced in the mitochondrial matrix is transferred into the intermembrane space.
• The NADH then transfers the electrons to FMN present in the intermembrane space via the complex I (NADH dehydrogenase).
• FMN then passes the electrons to the Fe-S center (one electron to one Fe-S center) which then transfers the electrons, one at a time to CoQ forming semiquinone and then ubiquinol.
• The electron transfer creates energy which is used to pump two protons across the membrane creating a potential gradient.
• The protons move back to the matrix through the pore in the ATP synthase complex, forming energy in the form of ATP.

2. Transfer of electrons from FADH2 to CoQ

• The oxidation of succinate to fumarate results in the reduction of FAD to FADH2.
• The electrons from FADH2 enter the electron transport chain catalyzed by complex II, succinic dehydrogenase.
• Like in complex I, the electrons reach CoQ through a series of Fe-S centers.
• However, complex II doesn’t pump any protons across the membrane.

3. Transfer of electrons from CoQH2 to cytochrome c

• The reduced CoQH2 transfer electrons through cytochrome b and c1 which finally reaches cytochrome c.
• Complex II (cytochrome reductase) catalyzes this process where the Fe3+ present in the cytochrome is reduced to Fe2+.
• Each cytochrome transfers one electron each and thus two molecules of cytochrome are reduced for the transfer of electrons for every NADH oxidized.
• Energy is produced during the transfer of electrons which is utilized to pump protons across the membrane aiding to the potential gradient.
• The protons move back to the matrix through the pore in the ATP synthase complex, forming energy in the form of ATP like in the first step.

4. Transfer of electrons from cytochrome c to molecular oxygen

• The final step in the electron transfer chain is catalyzed by complex IV (cytochrome oxidase) where electrons are transferred from cytochrome c to molecular oxygen.
• Since two electrons are required to reduce one molecule of oxygen to water, for each NADH oxidized half of oxygen is reduced to water.
• Similarly, the Fe2+ of the cytochrome c is oxidized to Fe3+. The energy released during this process is used to pump protons across the membrane.
• The transfer of protons back to the matrix results in the formation of ATP.

Electron Transport Chain Products

The end products of the electron transport chain are:

30-32 ATPs and 44 moles of H2O

StageDirect products (net)Ultimate ATP yield (net)Glycolysis2 ATP2 ATP 2 NADH3-5 ATPPyruvate oxidation2 NADH5 ATPCitric acid cycle2 ATP/GTP2 ATP 6 NADH15 ATP 2 FADH23 ATPTotal 30-32 ATP

Table Source: Khan Academy

Note: In some cases, we can see the production of 38 ATPs also.

Frequently Asked Questions (FAQs) (Revision questions and answers)

Where is the electron transport chain located?
The electron transport chain is located in the mitochondria of a cell.

What is the purpose of the electron transport chain?
The purpose of electron transfer chains is the production of ATPs.

What does the electron transport chain do?
The electron transport chain produces ATPs from the precursors (NADH and FADH) of previous cycles.

What are the three main steps in the electron transport chain?
The three main steps of the electron transfer chain are:
a. Transfer of electrons from NADH and FADH2 to CoQ
b. Transfer of electrons from CoQ to cytochrome c
c. Transfer of electrons from cytochrome c to molecular oxygen.

Where are the proteins of the electron transport chain located?
The proteins of the electron transport chain are located in the inner mitochondrial membrane of the mitochondria.

What are the products of the electron transport chain?
The products of the electron transport chains are ATPs and water.

What is the final electron acceptor of the electron transport chain?
The final electron acceptor in aerobic respiration is molecular oxygen while in anaerobic respiration, it can be sulfate or other molecules.

How many ATPs are formed in the electron transport chain?
A total of 30-32 ATPs are formed in the electron transport chain. But it depends upon the ATP per glucose in cellular respiration. In some cases, we can see the production of 38 ATPs also.

How many ATPs are utilized in the electron transport chain?
No ATPs are utilized in the electron transport chain.

What is the main function of the electron transport chain?
The main function of the electron transport chain is the production of ATPs from NADH and FADH.

What is the role of oxygen in the electron transport chain?
Oxygen in the electron transport chain is the final electron acceptor.

How does electron transport chain work in cellular respiration?
Electron transport chain is the final stage of cellular respiration where most of the ATPs or energy is produced from glucose.

References

1. Jain JL, Jain S, and Jain N (2005). Fundamentals of Biochemistry. S. Chand and Company.
2. Nelson DL and Cox MM. Lehninger Principles of Biochemistry. Fourth Edition.
3. Berg JM et al. (2012) Biochemistry. Seventh Edition. W. H Freeman and Company.