Reactive Oxygen Species & Uncoupling

Eukaryotic aerobic cellular respiration is the process of producing ATP by capturing energy that is released when food molecules are oxidized, with mitochondria playing a central role and oxygen acting as the final oxidizer. This is one of the core topics of Bio 6B. We'll go into this in detail, with explanations from the textbook, lecture videos, and other sources. I've created several pages related to cellular respiration:


By the time you complete this unit, you should be ablle to explain:

  • How the electron transport chain produces reactive oxygen species (ROS).
  • How uncoupling proteins can reduce ROS formation.

OX-PHOS produces ROS

Oxidative phosphorylation relies on huge differences in redox potential as electrons are passed from one molecule to another. Unfortunately, this flow of electrons can be hazardous to your health. In particular, if a large amount of electrons are fed into OX-PHOS while ATP usage remains low, the excess electrons may escape from the electron transport chain and form oxygen radicals.

The ETC produces reactive oxygen species

ETC Complex I

Compare this diagram to the previous version showing Complex I on the cellular respiration page. Two things are different in this diagram: the intermembrane space is more crowded with protons, and some of the electrons fail to make it to Q.

  1. NADH delivers electrons to Complex I, as usual. The supply of NADH depends on the rate of oxidative processes, including glycolysis, pyruvate oxidation, and the citric acid cycle.
  2. Electrons are passed through a series of redox centers within Complex I. Each redox center is slightly more electronegative than the previous one, so the electrons are pulled through a series of exergonic redox steps.
  3. The movement of electrons forces the protein complex to change shape. This causes protons to be bound on the mitochondrial matrix side and released into the intermembrane space. This active transport of protons requires energy, which is supplied by the redox steps. However, if the proton gradient becomes extremely strong (as shown in the diagram), even more energy is required to move the protons. At some point, the flow of electrons through Complex I doesn't supply enough energy to keep pumping protons.
  4. The processes of electron flow and proton transport are tightly coupled, so if protons can't be transported, the electrons can't be passed all the way to the electron acceptor Q, their normal destination.
  5. If electrons continue to be fed into the complex, they have to escape somewhere. Rather than being passed through the redox centers in controlled jumps, the electrons can be taken up by any available molecule with high electronegativity. This often turns out to be O2, the most electronegative thing around. When O2 accepts an extra electron, it becomes O2-, which is called superoxide. Since this extra electron is unpaired, it's a radical, written O2-.. (The little dot signifies the unpaired electron.)

Superoxide is highly reactive. Electrons almost always occur in pairs, so the unpaired electron configuration is unstable. Superoxide readily attacks other molecules, oxidizing them to gain a new electron and complete the pair (oxygen doesn't readily give up its extra electron, because it's so electronegative). I'm showing ROS formation in Complex I, but it happens at other points in the ETC as well.

Several factors can contribute to increased ROS formation in mitochondria, including:

  • The proton gradient is too strong (as shown above).
  • Electron acceptors of ETC are unavailable. For example, if O2 is not present at the end of the chain, Complex IV can't pass on its electrons and the whole chain gets backed up.

Each mitochondrion contains thousands of copies of the ETC complexes, and it's critical that the flow of electrons remains balanced. For example, if a mitochondrion makes too many copies of Complex I and not enough copies of Complex IV, there could be a bottleneck in the flow of electrons, resulting in increased ROS production. This may be one reason why mitochondria have their own genomes, which allows the expression of ETC proteins to be controlled at the level of individual mitochondria, rather than at the level of the whole cell.

Every oxygen-using cell faces the problem of ROS. The earliest cells evolved in a low-oxygen environment, but as the O2 level on earth increased (as a result of photosynthesis), some cells adapted to make use of this powerful oxidizer. Oxygen presents an opportunity, because its strong oxidizing power allows for the highly exergonic oxidation of reduced carbon molecules, but it also presents a hazard in terms of uncontrolled oxidation. Thus, cells that live in oxygen-rich environments need protection against ROS. There are two aspects to these defenses: systems for safely removing ROS (such as the enzyme superoxide dismutase), and systems for reducing the formation of ROS (such as uncoupling proteins, which I'll describe below).

ATP synthase couples proton flow with ATP synthesis

Strong proton gradient causes ROS formation

Cells don't stockpile ATP. Each cell has a limited supply of ATP/ADP, which gets constantly recycled as ATP is used for energy. If energy use is low, all the ADP is converted to ATP, and the supply of ADP runs low. ATP synthase allows protons to flow down their gradient only if the enzyme can bind to ADP and phosphate and produce ATP. An excess of ATP might sound like a good thing, because the cell has plenty of energy available. However, if ATP synthase is stopped and the ETC keeps going, the proton gradient can become dangerously strong, resulting in increased ROS production (as described previously).

Uncoupling proteins allow proton flow without ATP synthesis

There is a solution to the problem of an excessively strong proton gradient: uncoupling proteins. While ATP synthase couples proton movement with ATP synthesis, uncoupling proteins allow proton flow without ATP synthesis, uncoupling the two processes.

ROS causes UCP to open

Uncoupling proteins (UCP) provide an alternate route for protons to go down their electrochemical gradient, back into the matrix. There are several different UCPs, operating in different contexts. The diagram above represents UCP2, which responds to the formation of ROS (such as superoxide) by allowing proton flow. In the absence of ROS, UCP2  is closed, allowing for more efficient ATP production.

This example shows that ROS are not only dangerous byproducts of respiration; they also send important signals within cells. In addition to controlling UCP activity, ROS are also responsible for sending other important signals that help cells adapt to changes in energy usage and redox states.

Uncoupling proteins reduce ROS formation

When it opens for proton flow, the uncoupling protein weakens the proton gradient, making it easier for electrons to be passed down the ETC. Thus, UCP reduces ROS formation.

Uncoupling protein weakens gradient, reducing ROS formation.

Uncoupling proteins are important for managing the strength of the proton gradient and the rate of ROS formation, but that's not their only role.

Uncoupling proteins function in thermogenesis

Wasting energy might not sound like a good thing, but it has its advantages. When an uncoupling protein allows proton leakage, food molecules are oxidized and energy is consumed, but little ATP is produced. The cell responds by increasing the rate of glycolysis, the citric acid cycle, and the ETC. All those exergonic processes release energy. If the energy isn't captured in the form of ATP, it is released as heat. Heat could be seen as wasted energy, an inevitable consequence of thermodynamics. On the other hand, heat keeps us warm, and sometimes that's more important than generating ATP.

Mammals sometimes warm their bodies by way of a specialized tissue called brown adipose tissue, or brown fat. Brown adipose is very different from standard white adipose tissue. It's brown because it's tightly packed with mitochondria, more like muscle than fat. Those mitochondria allow the tissue to sustain a high rate of oxidative metabolism. However, all that oxidation doesn't result in a large amount of ATP production. Instead, the mitochondria are abundantly supplied with the uncoupling protein UCP1, which dissipates the proton gradient, allowing the energy of catabolism to be released as heat. In other words, brown adipose tissue performs thermogenesis: using energy to generate heat.

Structurally, UCP1 is similar to UCP2, but with one key difference. UCP1, the thermogenesis protein, is activated in response to low temperatures, helping the body generate heat when necessary. UCP1 is specifically expressed in brown fat, while UCP2 is ready to manage ROS production in all kinds of cells.


Terms & Concepts

  • ROS (Reactive Oxygen Species)
  • Superoxide (one kind of ROS)
  • Uncoupling proteins (I won't ask you to memorize which one is called UCP1 vs. UCP2)

Review questions

  1. Why does a stronger proton gradient contribute to an increased rate of ROS formation?
  2. What does "uncoupling" mean with respect to uncoupling proteins? What gets uncoupled?
  3. How do uncoupling proteins reduce ROS formation?
  4. How does uncoupling protein UCP1 contribute to thermogenesis?
  5. UCP1 contributes mostly to thermogenesis, while UCP2 is involved in controlling ROS formation. How are the proteins functionally different?
  6. How does the [ATP]/[ADP] ratio affect ROS formation?

References & further reading

Brown adipose tissue (Wikipedia).

Uncoupling Protein 2 in Cardiovascular Health and Disease. Tian et al, 2018. Frontiers in Physiology. This scientific review article explains the important roles of ROS and uncoupling in normal physiology and in cardiovascular disease. The authors point out that there is a complex web of interactions in which ROS formation helps to regulate many aspects of metabolism.

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