Overview of oxidative phosphorylation. The electron transport chain forms a proton gradient across the inner mitochondrial membrane, which drives the synthesis of ATP via chemiosmosis.

Why do we need oxygen?

You, like many other organisms, need oxygen to live. As you know if you’ve ever tried to hold your breath for too long, lack of oxygen can make you feel dizzy or even black out, and prolonged lack of oxygen can even cause death. But have you ever wondered why that’s the case, or what exactly your body does with all that oxygen?
As it turns out, the reason you need oxygen is so your cells can use this molecule during oxidative phosphorylation, the final stage of cellular respiration. Oxidative phosphorylation is made up of two closely connected components: the electron transport chain and chemiosmosis. In the electron transport chain, electrons are passed from one molecule to another, and energy released in these electron transfers is used to form an electrochemical gradient. In chemiosmosis, the energy stored in the gradient is used to make ATP.
So, where does oxygen fit into this picture? Oxygen sits at the end of the electron transport chain, where it accepts electrons and picks up protons to form water. If oxygen isn’t there to accept electrons (for instance, because a person is not breathing in enough oxygen), the electron transport chain will stop running, and ATP will no longer be produced by chemiosmosis. Without enough ATP, cells can’t carry out the reactions they need to function, and, after a long enough period of time, may even die.
In this article, we'll examine oxidative phosphorylation in depth, seeing how it provides most of the ready chemical energy (ATP) used by the cells in your body.

Overview: oxidative phosphorylation

Simple diagram of the electron transport chain. The electron transport chain is a series of proteins embedded in the inner mitochondrial membrane.
In the matrix, NADH and FADH2 deposit their electrons in the chain (at the first and second complexes of the chain, respectively).
The energetically "downhill" movement of electrons through the chain causes pumping of protons into the intermembrane space by the first, third, and fourth complexes.
Finally, the electrons are passed to oxygen, which accepts them along with protons to form water.
The proton gradient produced by proton pumping during the electron transport chain is used to synthesize ATP. Protons flow down their concentration gradient into the matrix through the membrane protein ATP synthase, causing it to spin (like a water wheel) and catalyze conversion of ADP to ATP.
The electron transport chain is a series of proteins and organic molecules found in the inner membrane of the mitochondria. Electrons are passed from one member of the transport chain to another in a series of redox reactions. Energy released in these reactions is captured as a proton gradient, which is then used to make ATP in a process called chemiosmosis. Together, the electron transport chain and chemiosmosis make up oxidative phosphorylation. The key steps of this process, shown in simplified form in the diagram above, include:
  • Delivery of electrons by NADH and FADH2_2. Reduced electron carriers (NADH and FADH2_2) from other steps of cellular respiration transfer their electrons to molecules near the beginning of the transport chain. In the process, they turn back into NAD+^+ and FAD, which can be reused in other steps of cellular respiration.
  • Electron transfer and proton pumping. As electrons are passed down the chain, they move from a higher to a lower energy level, releasing energy. Some of the energy is used to pump H+^+ ions, moving them out of the matrix and into the intermembrane space. This pumping establishes an electrochemical gradient.
  • Splitting of oxygen to form water. At the end of the electron transport chain, electrons are transferred to molecular oxygen, which splits in half and takes up H+^+ to form water.
  • Gradient-driven synthesis of ATP. As H+^+ ions flow down their gradient and back into the matrix, they pass through an enzyme called ATP synthase, which harnesses the flow of protons to synthesize ATP.
We'll look more closely at both the electron transport chain and chemiosmosis in the sections below.

The electron transport chain

The electron transport chain is a collection of membrane-embedded proteins and organic molecules, most of them organized into four large complexes labeled I to IV. In eukaryotes, many copies of these molecules are found in the inner mitochondrial membrane. In prokaryotes, the electron transport chain components are found in the plasma membrane.
As the electrons travel through the chain, they go from a higher to a lower energy level, moving from less electron-hungry to more electron-hungry molecules. Energy is released in these “downhill” electron transfers, and several of the protein complexes use the released energy to pump protons from the mitochondrial matrix to the intermembrane space, forming a proton gradient.
Image based on (and partially traced from) original diagram by Ryan Gutierrez.
Image of the electron transport chain. All the components of the chain are embedded in or attached to the inner mitochondrial membrane. In the matrix, NADH deposits electrons at Complex I, turning into NAD+ and releasing a proton into the matrix. FADH2 in the matrix deposits electrons at Complex II, turning into FAD and releasing 2 H+. The electrons from Complexes I and II are passed to the small mobile carrier Q. Q transports the electrons to Complex III, which then passes them to Cytochrome C. Cytochrome C passes the electrons to Complex IV, which then passes them to oxygen in the matrix, forming water. It takes two electrons, 1/2 O2, and and 2 H+ to form one water molecule. Complexes I, III, and IV use energy released as electrons move from a higher to a lower energy level to pump protons out of the matrix and into the intermembrane space, generating a proton gradient.
Image modified from "Oxidative phosphorylation: Figure 1", by OpenStax College, Biology (CC BY 3.0).
All of the electrons that enter the transport chain come from NADH and FADH2_2 molecules produced during earlier stages of cellular respiration: glycolysis, pyruvate oxidation, and the citric acid cycle.
  • NADH is very good at donating electrons in redox reactions (that is, its electrons are at a high energy level), so it can transfer its electrons directly to complex I, turning back into NAD+^+. As electrons move through complex I in a series of redox reactions, energy is released, and the complex uses this energy to pump protons from the matrix into the intermembrane space.
  • FADH2_2 is not as good at donating electrons as NADH (that is, its electrons are at a lower energy level), so it cannot transfer its electrons to complex I. Instead, it feeds them into the transport chain through complex II, which does not pump protons across the membrane.
Because of this "bypass," each FADH2_2 molecule causes fewer protons to be pumped (and contributes less to the proton gradient) than an NADH.
Complex I. NADH transfers its electrons to complex I. Complex I is quite large, and the part of it that receives the electrons is a flavoprotein, meaning a protein with an attached organic molecule called flavin mononucleotide (FMN). FMN is a prosthetic group, a non-protein molecule tightly bound to a protein and required for its activity, and it’s FMN that actually accepts electrons from NADH. FMN passes the electrons to another protein inside complex I, one that has iron and sulfur bound to it (called an Fe-S protein), which in turns transfers the electrons to a small, mobile carrier called ubiquinone (Q in the diagram above).
Complex II. Like NADH, FADH2_2 deposits its electrons in the electron transport chain, but it does so via complex II, bypassing complex I entirely. As a matter of fact, FADH2_2 is a part of complex II, as is the enzyme that reduces it during the citric acid cycle; unlike the other enzymes of the cycle, it’s embedded in the inner mitochondrial membrane. FADH2_2 transfers its electrons to iron-sulfur proteins within complex II, which then pass the electrons to ubiquinone (Q), the same mobile carrier that collects electrons from complex I.
Beyond the first two complexes, electrons from NADH and FADH2_2 travel exactly the same route. Both complex I and complex II pass their electrons to a small, mobile electron carrier called ubiquinone (Q), which is reduced to form QH2_2 and travels through the membrane, delivering the electrons to complex III. As electrons move through complex III, more H+^+ ions are pumped across the membrane, and the electrons are ultimately delivered to another mobile carrier called cytochrome C (cyt C). Cyt C carries the electrons to complex IV, where a final batch of H+^+ ions is pumped across the membrane. Complex IV passes the electrons to O2_2, which splits into two oxygen atoms and accepts protons from the matrix to form water. Four electrons are required to reduce each molecule of O2_2, and two water molecules are formed in the process.
Complex III. Like complex I, complex III includes an iron-sulfur (Fe-S) protein, but it also contains two proteins of another type, known as cytochromes. Cytochromes are a family of related proteins that have heme prosthetic groups containing iron ions. (Have you heard of the protein hemoglobin, which transports oxygen in the blood? Hemoglobin also has heme groups, but they bind oxygen rather than electrons.) In complex III, electrons are passed from one cytochrome to an iron-sulfur protein to a second cytochrome, then finally transferred out of the complex to a mobile electron carrier (cytochrome C). Like complex I, complex III pumps protons from the matrix into the intermembrane space, contributing to the H+^+ concentration gradient.
Complex IV. From complex III, cytochrome C delivers electrons to the last complex of the electron transport chain, complex IV. There, the electrons are passed through two more cytochromes, the second of which has a very interesting job: with the help of a nearby copper ion, it transfers electrons to O2_2, splitting oxygen to form two molecules of water. The mechanism of the transfer is pretty cool and worth looking up1^1, but basically, the heme group and the copper ion bind tightly to the oxygen molecule and hold it in place until it is fully reduced (has gained electrons and protons to form water). The protons used to form water come from the matrix, contributing to the H+^+ gradient, and complex IV also pumps protons from the matrix to the intermembrane space.
Overall, what does the electron transport chain do for the cell? It has two important functions:
  • Regenerates electron carriers. NADH and FADH2_2 pass their electrons to the electron transport chain, turning back into NAD+^+ and FAD. This is important because the oxidized forms of these electron carriers are used in glycolysis and the citric acid cycle and must be available to keep these processes running.
  • Makes a proton gradient. The transport chain builds a proton gradient across the inner mitochondrial membrane, with a higher concentration of H+^+ in the intermembrane space and a lower concentration in the matrix. This gradient represents a stored form of energy, and, as we’ll see, it can be used to make ATP.

Chemiosmosis

Complexes I, III, and IV of the electron transport chain are proton pumps. As electrons move energetically downhill, the complexes capture the released energy and use it to pump H+^+ ions from the matrix to the intermembrane space. This pumping forms an electrochemical gradient across the inner mitochondrial membrane. The gradient is sometimes called the proton-motive force, and you can think of it as a form of stored energy, kind of like a battery.
Like many other ions, protons can't pass directly through the phospholipid bilayer of the membrane because its core is too hydrophobic. Instead, H+^+ ions can move down their concentration gradient only with the help of channel proteins that form hydrophilic tunnels across the membrane.
In the inner mitochondrial membrane, H+^+ ions have just one channel available: a membrane-spanning protein known as ATP synthase. Conceptually, ATP synthase is a lot like a turbine in a hydroelectric power plant. Instead of being turned by water, it’s turned by the flow of H+^+ ions moving down their electrochemical gradient. As ATP synthase turns, it catalyzes the addition of a phosphate to ADP, capturing energy from the proton gradient as ATP.
Overview diagram of oxidative phosphorylation. The electron transport chain and ATP synthase are embedded in the inner mitochondrial membrane. NADH and FADH2 made in the citric acid cycle (in the mitochondrial matrix) deposit their electrons into the electron transport chain at complexes I and II, respectively. This step regenerates NAD+ and FAD (the oxidized carriers) for use in the citric acid cycle. The electrons flow through the electron transport chain, causing protons to be pumped from the matrix to the intermembrane space. Eventually, the electrons are passed to oxygen, which combines with protons to form water. The proton gradient generated by proton pumping during the electron transport chain is a stored form of energy. When protons flow back down their concentration gradient (from the intermembrane space to the matrix), their only route is through ATP synthase, an enzyme embedded in the inner mitochondrial membrane. When protons flow through ATP synthase, they cause it to turn (much as water turns a water wheel), and its motion catalyzes the conversion of ADP and Pi to ATP.
Image modified from "Oxidative phosphorylation: Figure 3," by Openstax College, Biology (CC BY 3.0).
This process, in which energy from a proton gradient is used to make ATP, is called chemiosmosis. More broadly, chemiosmosis can refer to any process in which energy stored in a proton gradient is used to do work. Although chemiosmosis accounts for over 80% of ATP made during glucose breakdown in cellular respiration, it’s not unique to cellular respiration. For instance, chemiosmosis is also involved in the light reactions of photosynthesis.
What would happen to the energy stored in the proton gradient if it weren't used to synthesize ATP or do other cellular work? It would be released as heat, and interestingly enough, some types of cells deliberately use the proton gradient for heat generation rather than ATP synthesis. This might seem wasteful, but it's an important strategy for animals that need to keep warm. For instance, hibernating mammals (such as bears) have specialized cells known as brown fat cells. In the brown fat cells, uncoupling proteins are produced and inserted into the inner mitochondrial membrane. These proteins are simply channels that allow protons to pass from the intermembrane space to the matrix without traveling through ATP synthase. By providing an alternate route for protons to flow back into the matrix, the uncoupling proteins allow the energy of the gradient to be dissipated as heat.

ATP yield

How many ATP do we get per glucose in cellular respiration? If you look in different books, or ask different professors, you'll probably get slightly different answers. However, most current sources estimate that the maximum ATP yield for a molecule of glucose is around 30-32 ATP2,3,4^{2,3,4}. This range is lower than previous estimates because it accounts for the necessary transport of ADP into, and ATP out of, the mitochondrion.
When authors and teachers quote different numbers of ATP molecules per glucose, it's often because they make different choices about what processes to account for in the total ATP yield. Neither the 38 ATP figure nor the 30-32 ATP figure is incorrect. They are just measuring slightly different things.
  • The 38 ATP figure assumes that every single proton pumped in the electron transport chain as a result of electrons harvested from glucose goes towards synthesizing ATP. In other words, none of the energy of the proton gradient is used to power other transport processes. (The 38 ATP figure also assumes that an efficient shuttle mechanism is used to carry electrons from NADH made in glycolysis to the electron transport chain. With an inefficient shuttle, the maximum figure in this scenario would be 36 ATP.)
  • The 30-32 ATP figure accounts for the fact that, in a real cell, not all of the energy of the proton gradient can go towards making ATP. Instead, some of it must be used to transport molecules into and out of the mitochondrial matrix. For instance, ADP must be transported into the matrix so it can be made into ATP, and ATP must be transported out so it can be used by the cell. The 30-32 ATP figure accounts for transport of ATP and ADP, which uses up energy from the proton gradient (meaning that there is less energy left to drive ATP synthesis, yielding fewer ATP)5,6^{5,6}.
In real cells, the yield of ATP per glucose molecule would likely be even lower than 30-32 ATP. Cells often use the proton gradient to transport other molecules (in addition to ATP and ADP), and some of the proton gradient's energy may also be lost if the inner mitochondrial membrane is "leaky" to protons.
Where does the figure of 30-32 ATP come from? Two net ATP are made in glycolysis, and another two ATP (or energetically equivalent GTP) are made in the citric acid cycle. Beyond those four, the remaining ATP all come from oxidative phosphorylation. Based on a lot of experimental work, it appears that four H+^+ ions must flow back into the matrix through ATP synthase to power the synthesis of one ATP molecule. When electrons from NADH move through the transport chain, about 10 H+^+ ions are pumped from the matrix to the intermembrane space, so each NADH yields about 2.5 ATP. Electrons from FADH2_2, which enter the chain at a later stage, drive pumping of only 6 H+^+, leading to production of about 1.5 ATP.
With this information, we can do a little inventory for the breakdown of one molecule of glucose:
StageDirect products (net)Ultimate ATP yield (net)
Glycolysis2 ATP2 ATP
2 NADH3-5 ATP
Pyruvate oxidation2 NADH5 ATP
Citric acid cycle2 ATP/GTP2 ATP
6 NADH15 ATP
2 FADH2_23 ATP
Total30-32 ATP
One number in this table is still not precise: the ATP yield from NADH made in glycolysis. This is because glycolysis happens in the cytosol, and NADH can't cross the inner mitochondrial membrane to deliver its electrons to complex I. Instead, it must hand its electrons off to a molecular “shuttle system” that delivers them, through a series of steps, to the electron transport chain.
  • Some cells of your body have a shuttle system that delivers electrons to the transport chain via FADH2_2. In this case, only 3 ATP are produced for the two NADH of glycolysis.
  • Other cells of your body have a shuttle system that delivers the electrons via NADH, resulting in the production of 5 ATP.
In bacteria, both glycolysis and the citric acid cycle happen in the cytosol, so no shuttle is needed and 5 ATP are produced.
30-32 ATP from the breakdown of one glucose molecule is a high-end estimate, and the real yield may be lower. For instance, some intermediates from cellular respiration may be siphoned off by the cell and used in other biosynthetic pathways, reducing the number of ATP produced. Cellular respiration is a nexus for many different metabolic pathways in the cell, forming a network that’s larger than the glucose breakdown pathways alone.

Self-check questions

  1. Cyanide acts as a poison because it inhibits complex IV, making it unable to transport electrons. How would cyanide poisoning affect 1) the electron transport chain and 2) the proton gradient across the inner mitochondrial membrane?
    Choose 1 answer:
    Choose 1 answer:
    If complex IV can no longer transfer electrons (is blocked), the rest of the electron transport chain will quickly back up and come to a halt. If electrons are no longer moving through the transport chain, protons will no longer be pumped out of the matrix and into the intermembrane space. As the protons already in the intermembrane space flow down their gradient and into the matrix, they won't be replaced, causing the gradient to weaken (and eventually disappear).
    Question adapted from OpenStax Biology.
  2. Dinitrophenol (DNP) is a chemical that acts as an uncoupling agent, making the inner mitochondrial membrane leaky to protons. It was used until 1938 as a weight-loss drug. How would DNP affect the amount of ATP produced in cellular respiration? Why do you think it is now off the market?*
    Choose 1 answer:
    Choose 1 answer:
    DNP allows protons to travel down their gradient and back into the mitochondrial matrix without passing through ATP synthase. Thus, it reduces ATP production in cellular respiration. The energy of the gradient is dissipated in the form of heat, raising body temperature. DNP was removed from the market because it caused deaths due to hyperthermia (severely elevated body temperature), and also because it was linked to cataracts and vision loss.
    Question adapted from OpenStax Biology.

Attribution

This article is adapted from “Oxidative phosphorylation,” by OpenStax Biology (CC BY 3.0). Download the original article for free at http://cnx.org/contents/185cbf87-c72e-48f5-b51e-f14f21b5eabd@9.85:37/Oxidative-Phosphorylation.
The modified article is licensed under a CC BY-NC-SA 4.0 license.

Works cited:

  1. Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). The respiratory chain consists of four complexes. In Biochemistry (5th ed., section 18.3). New York, NY: W. H. Freeman. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK22505/.
  2. Efficiency of ATP production. (2016, May 18). In Cellular respiration. Retrieved May 22, 2016 from Wikipedia: https://en.wikipedia.org/wiki/Cellular_respiration#Efficiency_of_ATP_production.
  3. Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). An accounting of ATP production by cellular respiration. In Campbell biology (10th ed., p. 176). San Francisco, CA: Pearson.
  4. Raven, P. H., Johnson, G. B., Mason, K. A., Losos, J. B., and Singer, S. R. (2014). Energy yield of aerobic respiration. In Biology (10th ed., AP ed., p. 137). New York, NY: McGraw-Hill.
  5. Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). The regulation of cellular respiration is governed primarily by the need for ATP. In Biochemistry (5th ed., section 18.6). New York, NY: W. H. Freeman. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK22448/.
  6. Cellular respiration. (2015, 13 September). Retrieved September 16, 2015 from Wikipedia: https://en.wikipedia.org/wiki/Cellular_respiration.

Additional references:

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002). Electron-transport chains and their proton pumps. In Molecular biology of the cell (4th ed.). New York, NY: Garland Science. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK26904/.
ATP-ADP translocase. (2015, January 28). Retrieved September 16, 2015 from Wikipedia: https://en.wikipedia.org/wiki/ATP%E2%80%93ADP_translocase.
Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002). Many shuttles allow movement across the mitochondrial membranes. In Biochemistry (5th ed., section 18.5). New York, NY: W. H. Freeman. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK22470/.
Brown adipose tissue. (2015, August 25). Retrieved September 16, 2015 from Wikipedia: https://en.wikipedia.org/wiki/Brown_adipose_tissue.
Caprette, D. R. (2005, 31 May). The electron transport system of mitochondria. In Experimental biosciences. Retrieved from http://www.ruf.rice.edu/~bioslabs/studies/mitochondria/mitets.html.
Chemiosmosis. (2015, August 25). Retrieved September 16, 2015 from Wikipedia: https://en.wikipedia.org/wiki/Chemiosmosis.
Gutierrez, R. (2011). Bio41 Week 6 — Biochemistry Lectures 7-9. In Biosci 41.
Kimball, J. W. (2014, 17 April). Cellular respiration. In Kimball’s biology pages. Retrieved from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellularRespiration.html.
Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., and Darnell, J. (2000). Electron transport and oxidative phosphorylation. In Molecular cell biology (4th ed., section 16.2). New York, NY: W. H. Freeman. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK21528/.
Purves, W. K., Sadava, D. E., Orians, G. H., and Heller, H.C. (2003). Cellular pathways that harvest chemical energy. In Life: the science of biology (7th ed., pp. 125-144). Sunderland, MA: Sinauer Associates.
Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). Cellular respiration and fermentation. In Campbell biology (10th ed., pp. 162-184). San Francisco, CA: Pearson.
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