Cellular Respiration

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:

Objectives:

By the time you complete this unit, you should be able to explain the core concepts on this page:

  • ATP Synthesis is powered by an electrochemical proton gradient
  • Chemical energy is harvested from food molecules through redox reactions
  • The electron transport chain produces the proton gradient

For more detailed objectives, see the review section at the bottom of this page.

On this page, I’m going to give you an overview of what I consider to be the most fundamental concepts of cellular respiration. Rather than following the sequence in the chapter, I’ll start with the big idea that I think is most important. I think this is a good way to learn complex topics: put the most important ideas first.

Introduction

On this page, I’m going to give you an overview of what I consider to be the most fundamental concepts of cellular respiration. Rather than following the sequence in the chapter, I’ll start with the big idea that I think is most important. I think this is a good way to learn complex topics: put the most important ideas first.

Cellular respiration is the most important energy transformation that happens in eukaryotic cells; it's the way you produce almost all the ATP used in your body. In Bio 6B, we're going to go into this process in considerable detail, not just because generating ATP is important, but because the specific mechanisms of cellular respiration shape many other aspects of biology.

ATP Synthesis is powered by a proton gradient

I made a 10-minute video lecture for this topic. (Hint: you can increase the playback speed! I'm not a fast talker.)

Here's the most amazing thing about cellular respiration: at its core, ATP synthesis is powered by electrical energy. As a biology student, you've probably taken some chemistry, and you may be tempted to view cellular respiration as a series of chemical reactions, but that isn’t quite right. There are a lot of chemical reactions involved, but at the heart of cellular respiration there is a completely different kind of energy: an electrochemical proton gradient across the inner mitochondrial membrane.

The process starts with chemical energy in food, uses some of that energy to create an electrochemical proton gradient, then uses the energy of the proton gradient to create a new form of chemical energy: ATP. This whole process is called oxidative phosphorylation (OXPHOS), because it uses exergonic redox processes to phosphorylate ADP, producing ATP.

Proton transport creates the electrochemical gradient

Cells contain a lot of H2O molecules. As you know from your chemistry classes, some of those molecules will spontaneously dissociate:

H2O ↔ H+ + OH-

The H+ is a hydrogen ion, or simply a proton; OH- is a hydroxide ion. It's not likely to remain free; protons will temporarily stick to some other molecule, such as another H2O (forming H3O+). In pure water, dissociation can be represented like this:

H2O + H2O ↔ H3O+ + OH-

However, the protons can also interact with other molecules, including proteins in cells. For the purposes of this page, I'll just regard them as free protons. Wherever there is water, there are free protons. There's no change in free energy as some water molecules dissociate and others re-associate.

"Proton

In cellular respiration, protons are actively transported out across the inner membrane of the mitochondrion, creating an electrochemical gradient. Transporting the protons creates a concentration gradienta difference in concentration from one side of the membrane to the other. Also, when protons are transported across the membrane, hydroxide ions are left behind. This creates a separation of charge across the membrane, which is a voltage gradienta form of stored electrical energy. The positive and negative charges are strongly attracted to each other across the membrane, but, as ions, they can't cross the phospholipid bilayer without help from a protein. Overall, it's called an electrochemical gradient because it's a gradient (difference) of both concentration and charge.

In OXPHOS, the electrochemical gradient is generated across the inner mitochondrial membrane. Protons are pumped out of the mitochondrial matrix to the intermembrane space, with hydroxides left behind in the matrix. ("Intermembrane space" simply means the space between the inner and outer membranes.)

mitochondrion overview; high proton concentratiion in intermembrane space.

The electrochemical gradient is a form of stored energy. The protons will have a strong tendency to move back across the membrane if they can, and this tendency can be used to perform work in the cell. Cells use electrochemical gradients in many different ways; in OXPHOS, the gradient powers ATP synthesis.

ATP Synthase couples proton flow with ATP synthesis

Most of the ATP in cellular respiration is produced by the enzyme ATP Synthase. Once the proton gradient has been generated (by the electron transport chain, which is described below), the flow of protons back into the matrix will be highly exergonic, as the positive charges move toward the negative charges inside. ATP synthase provides a mechanism that allows this exergonic proton flow to occur, but only if ATP is simultaneously synthesized in this reaction:

ADP + inorganic phosphate (Pi) → ATP

This reaction is highly endergonic, which is why ATP is a good energy source. ATP Synthase couples exergonic proton flow with endergonic ATP synthesis; neither of these events can happen without the other.

ATP synthase mechanisms

The process couples mechanical movements of the protein with catalysis:

  1. A proton is pushed into an open binding site on ATP synthase.
  2. There are multiple binding sites for protons. Each time a new proton enters the open binding site, the central part of the enzyme is forced to rotate.
  3. Each proton is eventually released into the matrix. A new binding site is opened up. The overall proton flow is strongly exergonic, due to the electrochemical gradient.
  4. The proton flow forces the enzyme's central rotor to turn.
  5. As the rotor turns, active sites bind ADP and inorganic phosphate, then force them together until a new high-energy phosphate bond is formed. Formation of this phosphate bond is highly endergonic. ATP is released, and the cycle starts over.

Overall, the complete process is exergonic; enough energy is released by the exergonic flow of protons to power the endergonic formation of the phosphate bond. ATP synthase isn't simply a catalyst; it's a mechanical motor connected to a catalyst. (The short video ATP synthase: Structure and Function gives an excellent explanation of the mechanics of this amazing molecular structure.)

So far, I've said that a proton gradient is created, and it powers ATP synthesis by ATP synthase. Next, I need to explain how the proton gradient is generated. The process begins with chemical energy in food molecules.

Chemical energy is harvested through redox reactions

Work is required to generate the proton gradient that powers the phosphorylation of ADP. The work is done by the electron transport chain (ETC), and the energy is supplied by high-energy electrons harvested from food molecules. Glycolysis, pyruvate oxidation, and the citric acid cycle capture those electrons.

It takes energy to make a proton gradient; in cellular respiration, that energy comes from food. The electron transport chain (ETC, which I'll describe below) does the work of actively transporting the protons, and the ETC is powered by high-energy electrons that are harvested from food molecules. The harvesting of electrons begins with glycolysis and the citric acid cycle.

Glycolysis

Glycolysis literally means "sugar splitting" (glyco = sugar and lysis = splitting), but for our purposes, it makes more sense to focus on the electrons, rather than the molecules that get split.

At this point, I don’t think it will help you to memorize every step of glycolysis and the citric acid cycle. Instead, I recommend focusing on the kinds of reactions that need to occur. Glycolysis consists of 10 steps, but we can put those reactions into a few categories:

  • Phosphorylation (phosphate transfer). The enzymes that do this are called kinases. In glycolysis, kinases phosphorylate the glucose molecule, increasing its free energy. Later, other kinases transfer phosphates to ADP to make ATP.
  • Isomerization. Reorganizing the molecule without adding or subtracting anything. By reorganizing the molecule, the energy of individual bonds can be changed, even if the molecule’s overall energy is unchanged.
  • Splitting. The sugar is broken in two by an aldolase, which cleaves an aldol bond.
  • Oxidation. This step is done by a dehydrogenase, which transfers electrons from an intermediate (G3P) to NAD+, forming NADH. (In Campbell, this enzyme is referred to as triose phosphate dehydrogenase; it is also called GAPDH, or glyceraldehyde -3-phosphate dehydrogenase. You don't need to memorize that.)

Overall, glycolysis can be summarized like this:

Glycolysis summary, with inputs & outputs.

Glucose gets split into two pyruvates and partially oxidized, generating some NADH. A small amount of ATP is produced. However, keep in mind that this simple diagram only shows the net inputs and outputs. It ignores the energy investment phase, in which ATP is used as an input to get the process started.

I won't go into more detail on glycolysis here, because I want to focus on the concept I think is most important: oxidative phosphorylation. I made a separate page on glycolysis  to provide a fuller explanation of how this pathway works.

Citric Acid Cycle

Glycolysis begins the process of harvesting high-energy electrons from food, and that process continues with the citric acid cycle (CAC). As with glycolysis, it's best to focus first on why this process is important, rather than memorizing the steps. The citric acid cycle harvests high-energy electrons derived from pyruvate (an end product of glycolysis), and feeds these electrons to the electron transport chain. I'll show this process in the next section, in the context of the electron transport chain.

You don't need to memorize the names of the intermediates or the enzymes of the CAC for Bio 6B; we're not going to look closely at the reaction mechanisms, and simply knowing the names of the molecules won't enhance your understanding.

What is this cycle called?

This cycle has several names. I'm calling it the citric acid cycle (CAC) because that's what you'll see in Campbell. Older editions of Campbell called it the Krebs cycle; I don't know why they changed it. Many people prefer to call it the tricarboxylic acid cycle (TCA). In my experience, many people insist that the first name they learned is the only correct name.

The electron transport chain produces the proton gradient

The electron transport chain is a form of active transport, using energy to move protons across the inner mitochondrial membrane. The energy comes from a series of exergonic redox reactions, using high-energy electrons from food.

Complexes of the ETC couple electron flow with proton pumping. Electrons are passed through a series of acceptors and donors, losing some energy each time they are transferred. Thus, the flow of electrons down the ETC is exergonic.

As the electrons are passed from one donor to another, the proteins of the ETC are forced to change shape slightly. This causes protons to be picked up in the matrix and transported across the membrane to the intermembrane space. Thus, the exergonic flow of electrons is coupled to the endergonic pumping of protons. The exergonic process is allowed to happen only if the endergonic process occurs at the same time. The net process, including endergonic and exergonic events, is exergonic, so the whole process is energetically favorable and moves forward. The coupling of exergonic and endergonic processes is one the core concepts of biological energetics, and occurs at multiple points in cellular respiration.

The ETC couples redox reactions with proton transport

ETC and ATP Synthase.

Here is an extremely simplified view of oxidative phosphorylation:

  1. Some H2O molecules spontaneously dissociate to form H+ (protons) and OH-. The protons are then available to be pumped across the membrane.
  2. High-energy electrons harvested from food molecules such as glucose provide energy for the electron transport chain (ETC). These electrons are delivered by NADH or by the citric acid cycle.
  3. High-energy electrons are passed down the ETC through a series of electron carriers, losing energy at each step. At the end of the ETC, the electrons are passed to O2 and H+, forming H2O as a byproduct.
  4. As the electrons are passed down the ETC, some carrier proteins are forced to change shape and actively transport protons (H+) from the matrix to the intermembrane space. This creates an electrochemical proton gradient across the inner mitochondrial membrane. The ETC consists of a series of complexes, each composed of multiple proteins.
  5. The energy of the proton gradient forces protons to pass through the ATP synthase enzyme complex. This forces the rotor to turn and brings ADP and inorganic phosphate (Pi) together until they form a covalent bond, making ATP.

Overall, this process is exergonic. Some energy is captured in the endergonic synthesis of ATP, but even more energy is given off as electrons lose free energy in a series of redox reactions. To understand the mechanisms behind these processes, we need a more detailed look at some of the complexes.

ETC complex I pumps protons

ETC complex I couples electron flow with proton pumping.

Here's a detailed look at complex I of the electron transport chain, one of the three proton-pumping complexes in the ETC. This complex comprises multiple protein subunits, which work together to couple electron flow with proton pumping.

  1. NADH delivers high-energy electrons derived from food molecules (from glycolysis, pyruvate oxidation, or the citric acid cycle).
  2. The electrons make their way though a series of electron carriers within Complex I. Each carrier is more electronegative than the one before it, so this series of redox steps is exergonic.
  3. As the electrons are being passed through the redox centers, the proteins change shape slightly. This shape change forces the transmembrane part of the complex to grab protons from the mitochondrial matrix and then release the protons into the intermembrane space. These two processes are coupled: electrons can't flow down the chain without protons being pumped.
  4. After the last redox center, the electrons are passed to Q, an electron carrier. The electron carrier Q is called ubiquinone in its oxidized form; when it receives a pair of electrons from Complex I, it gets reduced to become ubiquinol. Ubiquinone/ubiquinol is also called coenzyme Q10, but in the diagrams I will just call it Q, in both the oxidized and the reduced forms. Q then delivers the electrons to Complex III of the ETC. Q is a mobile carrier, meaning that it is free to diffuse from one molecule to another. It's hydrophobic, so it stays in the interior of the membrane.

Complex II is part of the Citric Acid Cycle

Complex II connected to the citric acid cycle.

The citric acid cycle completes the oxidation of the carbons derived from pyruvate, the 3-carbon end product of glycolysis, completing the process of capturing high-energy electrons for the ETC. One step of the citric acid cycle is catalyzed by an enzyme that is part of Complex II of the ETC.

  1. Pyruvate, which is an ion, crosses the inner mitochondrial membrane by way of the mitochondrial pyruvate carrier (MPC).
  2. In the matrix, pyruvate is partially oxidized by pyruvate dehydrogenase, which captures some electrons and forms NADH. One fully oxidized carbon is lost as CO2, and the remaining two carbons, in the form of acetyl, are attached to a CoA molecule.
  3. CoA, or coenzyme A, simply binds to the acetyl group from pyruvate dehydrogenase and releases it to an enzyme in the CAC.
  4. The citric acid cycle completes the oxidation of the carbons from the acetyl group. Some of the high-energy electrons are passed to NADH. The NADH can then pass electrons to Complex I of the ETC. Some ATP is produced by the CAC, but much less than the amount produced by ATP synthase.
  5. One step of the citric acid cycle is catalyzed by an enzyme that is part of Complex II of the ETC. This step captures more electrons for the ETC. The electrons are passed to Q, which can then deliver them to Complex III.

Unlike Complexes I, III, and IV, complex II does not transport protons across the membrane.

FADH2 is not a product of the CAC

Most diagrams of the CAC in biology textbooks (including Campbell) are misleading in a couple of ways. First, the diagrams show the CAC occurring as a free-floating pathway inside the mitochondrial matrix. That's not quite right, because one step (conversion of succinate to fumarate) is catalyzed by Complex II of the electron transport chain. In addition, that step is usually shown producing FADH2 as a product. There is FADH2 inside Complex II, and it plays a catalytic role, but it's not a product. It's part of the enzyme complex, and it immediately passes on the electrons it gains. It's more realistic to simply say that this step harvests electrons and feeds them on to Q for the next step in the ETC. There is no net gain of FADH2. Thus, the diagram I'm showing you is a bit different from the one in Campbell. Where I'm showing electrons (e-) being harvested by the CAC and passed directly to Complex II, Campbell is showing electrons passed to FADH2, which later passes the electrons to Complex II.

 OXPHOS in detail

Detailed view of whole ETC.

This diagram integrates the previous diagrams with the rest of OXPHOS. The steps in brief:

  1. Pyruvate from glycolysis enters the mitochondrial matrix via the mitochondrial pyruvate carrier.
  2. Pyruvate dehydrogenase generates NADH & Acetyl-CoA.
  3. The citric acid cycle oxidizes the acetyl, capturing more high-energy electrons for the ETC and producing NADH. The CAC also produces ATP. The fully-oxidized carbons that originally came from glucose are released as CO2.
  4. Complex II catalyzes one step of the CAC, and passes some electrons directly into the ETC by way of Q.
  5. NADH passes electrons to Complex I, which uses a series of redox events to power proton transport across the inner mitochondrial membrane. After passing through Complex I, these electrons are passed to Q.
  6. Complex III accepts electrons from Q, derived from both Complex II and Complex I. Complex III uses these electrons to power the transport of protons across the inner membrane. Eventually, the electrons are passed to the protein Cytochrome C. (Because it passes electrons to Cytochrome C, Complex III is also called Cytochrome Reductase).
  7. Cytochrome C carries electrons from Complex III to Complex IV.
  8. Complex IV uses the electrons from Cytochrome C to power more proton transport. Eventually, the electrons are pulled away by O2, in a reaction that combines oxygen, protons, and electrons to form H2O. The strong oxidizing power of O2, set against the strong reducing power of NADH and other electron carriers, drives the electron transport chain. (Because it takes electrons from Cytochrome C, Complex IV is also called Cytochrome Oxidase.)
  9. Proton transport by complexes I, III, and IV creates a strong electrochemical gradient. ATP synthase harnesses this gradient to synthesize ATP.

This diagram might seem complex, but remember that you can summarize the overall process of cellular respiration in simple terms:

In cellular respiration, high-energy electrons are harvested from food molecules. The electrons are used in a series of exergonic redox reactions to provide energy for proton pumping. The proton transport creates an electrochemical gradient, which is used by ATP synthase to power the endergonic synthesis of ATP.

Each event on this diagram is part of that overall process.

Compare this diagram to the one in Campbell, and you'll see some differences. For one thing, I didn't put the complexes in numerical order. That's because the numbers of the complexes don't exactly reflect the steps of electron flow. Electrons don't move from Complex I to Complex II; they flow from I or II to III. I switched the order to reflect that. In reality, the complexes are all connected together in supercomplexes; it's not realistic to say that they occur in a specific order. Also, I'm showing the citric acid cycle connected to Complex II.

Although this diagram shows numerous steps, it still leaves out some important points, which I'll address elsewhere:

  • How do cells balance their rate of ATP production with their rate of ATP consumption?
  • How does the ATP get from the matrix, where it is produced, to the cytosol, where most of it is used?
  • What happens to the NADH from glycolysis?

Cristae & respirasomes

There are multiple levels of organization in cellular respiration. First, individual proteins carry out specific functions. Second, many of those proteins are grouped into large functional complexes in the ETC and elsewhere. Those complexes are arrayed in membranes, allowing them to work together by way of an electrochemical gradient. Finally, the various large complexes of OXPHOS are structured into respiratory supercomplexes, sometimes called respirasomes, which are located on the folds of the inner mitochondrial membrane.

These folds, called cristae, are the main sites of both the ETC and ATP synthase. As researchers have developed techniques for ultra-high resolution imaging of cells, a new model has emerged that shows that the proton gradient is generated primarily across the membranes of the cristae, and ATP synthases are grouped into complexes at the creases of the folds. This organization is likely to make the proton gradient function much more efficiently, by maximizing the strength of the gradient for a given number of protons pumped.

Respirasomes in mitochondrial cristae from Wu et al.

Image credit: Wu et al. 2020, Research journey of respirasome, Protein & Cell. CC-BY 4.0

In fact, ATP synthase units seem to bind together and help create the folds of the cristae:

ATP synthase dimer in fold of crista.

Image credit: Image credit: David Goodsell, PDB-101, ATP Synthase. CC-BY-4.0.

So far, the functional importance of these higher-level structures isn't fully understood, but it seems clear that this approach will lead to a deeper understanding of how cellular respiration works.

Image credits

I made the ETC diagrams and pathways using molecular graphics of the ETC complexes and ATP synthesis from PDB-101. These images of protein complexes were created by David Goodsell and are used under a CC-BY-4.0 license. My modified images are, in turn, shareable under the same CC-BY-4.0 license. See PDB-101 in the references section below for specific links relevant to cellular respiration.

Review

Terms & concepts: Respiration

  • What is a redox process, and what makes it exergonic?
  • Mitochondrion: inner and outer membranes and intermembrane space.
  • Glycolysis:
    • Where does it happen (in eukaryotes and prokaryotes)?
    • Inputs and outputs
    • What is the energy investment phase? Why is it important?
    • When glucose (or its product) gets oxidized, what is the oxidizer? What gets reduced?
    • ATP made by substrate-level phosphorylation. Total ATP production vs. net ATP production.
    • Pyruvate
    • Kinases. What gets phosphorylated, when and why?
    • Isomerases
    • Dehydrogenase & oxidation
    • Phosphofructokinase (PFK) is allosterically regulated as part of a feedback inhibition system. ATP is both a substrate and an allosteric inhibitor of PFK. How is this possible?
    • Phosphates: Inorganic phosphate is PO4 with a variable number of negative charges and protons attached when it's free in solution. When phosphate is attached to an organic molecule, it's called a phosphate group.
  • Pyruvate dehydrogenase
  • Citric acid cycle:
    • Where does it happen (in eukaryotes and prokaryotes)?
    • Acetyl in, CO2 out
    • Why it’s a cycle
    • NADH, ATP produced
    • Why does the citric acid cycle stop if no O2 is present?
    • Complex II is attached to the membrane; compare this to the book’s citric acid cycle diagram.
  • Electron transport chain & ATP synthase:
    • Electrochemical proton gradient. It's also a pH gradient.
    • Where electrons come from & where they go.
    • What is the energy source for making the proton gradient? Where do the protons come from?
    • Why is O2 needed? Could there ever be an electron transport chain without O2?
    • Structure of mitochondrion: inner & outer membranes, intermembrane space, matrix
    • How ATP synthase works
    • Mitochondrial Pyruvate Carrier (MPC): what does it do?
    • Role of NADH/NAD+. Where does it get oxidized, and where does it get reduced?

Review questions: respiration

  1. What is an electrochemical gradient? (What does gradient mean, and how is it electrochemical?)
  2. What is oxidative phosphorylation, and why is it called that? (You should be able to describe multiple oxidative events, and one phosphorylation event.)
  3. What are the inputs and outputs of glycolysis? (If you consider only the net, or overall inputs and outputs, you're ignoring the energy investment phase. What essential input is needed for the energy investment phase? Why isn't this one counted as a net input?)
  4. Diagram and describe how eukaryotic cellular respiration works. Include a labeled diagram of the appropriate cell structures. Show where each of the main stages of respiration occurs. (Note that there isn’t a single diagram in Campbell that shows it all.)
  5. Compare and contrast the OXPHOS diagrams on this page and in Campbell. Why do you think I didn't just copy the diagram from Campbell?
  6. What parts of cellular respiration are endergonic? What parts are exergonic? Overall, is the whole process endergonic or exergonic?
  7. For each step in cellular respiration, what gets oxidized and what gets reduced?
  8. For the proton gradient in OXPHOS, where do the protons come from? What is the source of energy for making the gradient?
  9. How is the proton gradient used as a source of energy?
  10. Consider the whole process carried out by ATP synthesis? What events are coupled together by this enzyme? Are these events endergonic or exergonic? Is the overall process endergonic or exergonic?
  11. What happens to O2 molecules in the ETC? What's special about O2 that makes this process work?

References & further reading

Videos: EdX

A series of excellent videos from an EdX course (Cell Biology: Mitochondria) covers cellular respiration in detail:

Other videos:

ATP synthase: Structure and Function Stewart lab. This video covers the fundamentals of OXPHOS and adds one interesting fact: ATP synthase forms dimers, which help control the structure of the inner mitochondrial membrane. 

PDB-101

PDB-101: This excellent website from the Protein Database includes structure and function information for many of the proteins that feature prominently in Bio 6B. This site has the best molecular graphics on the web. Cellular respiration examples include:

Deep reading: Beyond Bio 6B

Structure of the dimeric ATP synthase from bovine mitochondria. Spikes et al., 2020. This research article has fantastic atomic-level animations and images of ATP synthase. Clear and well-written.

Structure and function of mitochondrial membrane protein complexes. Kuhlbrandt, 2015, BMC Biology (alternate link here). Oxidative phosphorylation is the subject of ongoing study, and there is still much to learn. This article discusses some recent research, including the methods that have been used to determine the structures, and points out some important unknowns.

Nick Lane: This researcher and writer deserves a whole section to himself for his work on energy and deep evolution. I highly recommend reading any of his books or articles.

 

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