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Oxidative phosphorylation

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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 FADHstart subscript, 2, end subscript. Reduced electron carriers (NADH and FADHstart subscript, 2, end subscript) 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 NADstart superscript, plus, end superscript 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 Hstart superscript, plus, end superscript 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 Hstart superscript, plus, end superscript to form water.
  • Gradient-driven synthesis of ATP. As Hstart superscript, plus, end superscript 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 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 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 FADHstart subscript, 2, end subscript 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 NADstart superscript, plus, end superscript. 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.
  • FADHstart subscript, 2, end subscript 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 FADHstart subscript, 2, end subscript molecule causes fewer protons to be pumped (and contributes less to the proton gradient) than an NADH.
Beyond the first two complexes, electrons from NADH and FADHstart subscript, 2, end subscript 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 QHstart subscript, 2, end subscript and travels through the membrane, delivering the electrons to complex III. As electrons move through complex III, more Hstart superscript, plus, end superscript 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 Hstart superscript, plus, end superscript ions is pumped across the membrane. Complex IV passes the electrons to Ostart subscript, 2, end subscript, which splits into two oxygen atoms and accepts protons from the matrix to form water. Four electrons are required to reduce each molecule of Ostart subscript, 2, end subscript, and two water molecules are formed in the process.
Overall, what does the electron transport chain do for the cell? It has two important functions:
  • Regenerates electron carriers. NADH and FADHstart subscript, 2, end subscript pass their electrons to the electron transport chain, turning back into NADstart superscript, plus, end superscript 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 Hstart superscript, plus, end superscript 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 Hstart superscript, plus, end superscript 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, Hstart superscript, plus, end superscript 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, Hstart superscript, plus, end superscript 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 Hstart superscript, plus, end superscript 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 ATPstart superscript, 2, comma, 3, comma, 4, end superscript. This range is lower than previous estimates because it accounts for the necessary transport of ADP into, and ATP out of, the mitochondrion.
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 Hstart superscript, plus, end superscript 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 Hstart superscript, plus, end superscript ions are pumped from the matrix to the intermembrane space, so each NADH yields about 2.5 ATP. Electrons from FADHstart subscript, 2, end subscript, which enter the chain at a later stage, drive pumping of only 6 Hstart superscript, plus, end superscript, 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 FADHstart subscript, 2, end subscript3 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 FADHstart subscript, 2, end subscript. 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:
  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:


Want to join the conversation?

  • mr pink red style avatar for user Maulana Akmal
    how does the nadh from glycolisys gets into the matrix so its electron could be used?
    (16 votes)
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  • blobby green style avatar for user na26262
    if the volume of the intermembrane space was increased, what effect would this have on the function of a mitochondrion?
    (8 votes)
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    • starky ultimate style avatar for user Richard Wu
      Hm.... A cell stays small to allow easier transport of molecules and charged particles from organelles. If the intermembrane space of the mitochondria was increased, I would think that respiration would be less efficient, because now the electrons have to cross a larger space and lose much more energy. So.... That's my guess and it would probably be wrong,
      (11 votes)
  • blobby green style avatar for user Herukm18
    What does substrate level phosphorylation means?
    (7 votes)
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  • starky sapling style avatar for user Chaarvee Gulia
    I don't quite understand why oxygen is essential in this process. I get that oxygen serves as an electron acceptor at the end of the electron transport chain, but why is having this electron acceptor so important? Why would ATP not be able to be produced without this acceptor (oxygen)?
    (6 votes)
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  • aqualine ultimate style avatar for user Ashley Jane
    Where do the hydrogens go? I mean in glycolysis, one glucose is oxidised into two pyruvic acid and two NADHs. But technically there should be net two protons left in cytosol and that's where I am puzzled. Are the protons tansported into mitochondria matix and later pumped out by ETC or intermembrane space to form electrochemical gradient, or are they left in cytosol? According to the amont of water molecules generated in chemiosmosis, all the hydrogen from the glucose should be used to form water, so do protons go into the mitochondria or mitochondria has extra protons itself?
    (6 votes)
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    • female robot grace style avatar for user tyersome
      Remember that all aqueous solutions contain a small amount of hydronium (H₃O⁺) and hydroxide (OH¯) due to autoionization.

      This means that processes in cells can use water to get rid of or grab "protons" (H⁺) as needed.

      This could have a small local effect on the pH, but cells are well buffered. Also, in the end the various processes that create and use H⁺ end up balancing each other out.

      The only situations where we really need to keep an account of H⁺ are processes involving membranes (e.g. oxidative phosphorylation) where H⁺ gradients are created and used.
      (11 votes)
  • leaf green style avatar for user richie56rich
    How much H2O is produced is the electron transport chain?
    (6 votes)
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    • winston baby style avatar for user Ivana - Science trainee
      C6H12O6 + 6O2 → 6CO2 + 6H2O + energy


      6 molecules of water :D


      This is the overall formula for cell respiration, however, you ask how many water molecules are produced specifically during electron transport chain?

      Knowing that in the end, you get 6 molecules of water from one glucose molecule.
      In the electron transport chain, hydrogen atoms are being added to available oxygen in order to produce water.


      So you are confused about 6Co2, too? well, you know that after glycolysis, you get 3C molecules and bot undergoes Krebs cycle. Now, what you get from one Krebs cycle and proceeding ETC is doubled.

      While it is true that via Krebs cycle we get 2 CO2 (giving 4 in total), do not forget decarboxylation prior to entering Krebs cycle (in between Glycolysis and Krebs cycle) there are is 1CO2. 1x2 = 2, 2+4 = 6

      only 2NADH are reduced during ETC, giving a total number of 2, and also one FADH2 giving 1. It is 3x2 =6

      as for Oxygen, it comes from the red blood cells which carry oxygen to absolutely every cell. Once in the cell, it enters mitochondria and acts as final acceptor of electrons. if not, water would not be produced.


      http://www.dbriers.com/tutorials/2012/04/the-electron-transport-chain-simplified/
      (5 votes)
  • leaf green style avatar for user yejikwon00
    Where did all the hydrogen ions come from? If NADH becomes NAD+, it releases H+ and if FADH2 becomes FAD and would release 2H+. So are the hydrogen ions released by those electron carriers are going to be used for the gradient and also for the water formation? Or are the Hydrogen ions that just came back through the ATP synthase going to be used for forming H2O??
    (5 votes)
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    • female robot grace style avatar for user tyersome
      The individual reactions can't know where a particular "proton" came from.

      Remember that all aqueous solutions contain a small amount of hydronium (H₃O⁺) and hydroxide (OH¯) due to autoionization.

      This means that processes in cells can use water to get rid of or grab "protons" (H⁺) as needed.

      This does have a small local effect on the pH, but forming that NADH or FADH₂ used protons so it all balances out in the end.
      (6 votes)
  • piceratops ultimate style avatar for user Dallas Huggins
    The new Campbell Biology textbook updated the ATP yield totals to be 26-28 (instead of 30-32). What does this mean for your table on the 'breakdown of one molecule of glucose'? Where did the net yield go down?
    (5 votes)
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  • duskpin ultimate style avatar for user SanteeAlexander
    I thought it was 38 ATPs from the previous videos
    (2 votes)
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  • leaf blue style avatar for user Abdul Mannan
    How much electron NADH & FADH2 donates?
    (2 votes)
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