Bis2A 5.6: Oxidative Phosphorylation and the Electron ... · OpenStax-CNX module: m59707 1 Bis2A 5.6: Oxidative Phosphorylation and the Electron Transport Chain * The BIS2A Team This

OpenStax-CNX module: m59707 1

Bis2A 5.6: Oxidative Phosphorylation

and the Electron Transport Chain*

The BIS2A Team

This work is produced by OpenStax-CNX and licensed under the

Creative Commons Attribution License 4.0�

Abstract

This module will discuss the fate of NADH which was produced in glycolysis and the TCA cycle.NADH donates high energy electrons to the electron transport chain to generate a proton motive forcewhich is then used by the cell to make ATP through oxidation phosphorylation.

1 Module Summary

The electron transport chain (ETC) is the portion of respiration that uses an external electron acceptor asthe �nal/terminal acceptor for the electrons that were removed from the intermediate compounds in glucosecatabolism. In eukaryotic cells the ETC is composed of four large, multiprotein complexes embedded in theinner mitochondrial membrane and two small di�usible electron carriers shuttling electrons between them.The electrons are passed from enzyme to enzyme through a series of redox reactions. These reactions arecouple the exergonic redox transfers to the endergonic transport of hydrogen ions across the membrane.This process contributes to the creation of a transmembrane electrochemical gradient. The electrons passingthrough the ETC gradually lose potential energy up until the point they are deposited on the terminalelectron acceptor. The free energy di�erence of this multistep redox process is ∼ -60 kcal/mol when NADHdonates electrons or 45 kcal/mol when FADH2 donates, for organisms using oxygen as the �nal electronacceptor.Introduction to Red/Ox, oxidative phosphorylation and Electron Transport ChainsIn modules 5.1, we discussed the general concept of Red/Ox reactions in biology and introduced the ElectronTower, a tool to help you understand Red/Ox chemistry and to estimate the direction and magnitude of po-tential energy di�erences for various Red/Ox couples. In modules 5.3 and 5.4 substrate level phosphorylationand fermentation were discussed and we saw how exergonic Red/Ox reactions could be directly coupled byenzymes to the endergonic synthesis of ATP. These processes are hypothesized to be one of the oldest formsof energy production used by cells. In this section we discuss the next evolutionary advancement in cellularenergy metabolism, oxidative phosphorylation. First and foremost, oxidative phosphorylation does notimply the use of oxygen, it can, but it does not have to use oxygen. It is called oxidative phosphorylationbecause it relies on Red/Ox reactions to generate a electrochemical transmembrane potential that canthen be used by the cell to do work.A quick summary of Electron Transport ChainsThe ETC begins with the addition of electrons, donated from NADH, FADH2 or other reduced compounds.These electrons move through a series of electron transporters, enzymes that are embedded in a membrane, or

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carriers that undergo Red/Ox reactions. The free energy transferred from these exergonic Red/Ox reactionsis coupled to the endergonic movement of protons across a membrane. This unequal accumulation of protonson either side of the membrane "polarizes" or "charges" the membrane, with a net positive (protons) on oneside of the membrane and a negative charge on the other side of the membrane. The separation of chargecreates an electrical potential. In addition, the accumulation of protons also causes a pH gradient knownas a chemical potential across the membrane. Together these two gradients (electrical and chemical) arecalled an electro-chemical gradient.

2 Review: The Electron Tower

Since Red/Ox chemistry is so central to the topic we begin with a quick review of the table of reductionpotential - sometimes called the "redox tower". As we discussed in Module 5.1, all kinds of compounds canparticipate in biological Red/Ox reactions. Making sense of all of this information and ranking potentialRed/Ox pairs can be confusing. A tool has been developed to rate Red/Ox half reactions based on theirreduction potentials or E0

' values. Whether a particular compound can act as an electron donor (reductant)or electron acceptor (oxidant) depends on what other compound it is interacting with. The redox towerranks a variety of common compounds (their half reactions) from most negative E0

', compounds that readilyget rid of electrons, to the most positive E0

', compounds most likely to accept electrons. The tower organizesthese half reactions based on the ability of electrons to accept electrons. In addition, in many redox towerseach half reaction is written by convention with the oxidized form on the left followed by the reduced form toits right. The two forms may be either separated by a slash, for example the half reaction for the reductionof NAD+ to NADH is written: NAD+/NADH + 2e-, or by separate columns. An electron tower is shownin �gure 1 below.



Figure 1: Common Red/ox tower

note: Use the redox tower above as a reference guide to orient you as to the reduction potentialof the various compounds in the ETC. Red/Ox reactions may be either exergonic or endergonicdepending on the relative Red/Ox potentials of the donor and acceptor. Also remember there are



many di�erent ways of looking at this conceptually; this type of redox tower is just one way.

note: In the Red/Ox table above some entries seem to be written in unconventional ways. Forinstance Cytochrome cox/red. There only appears to be one form listed. Why? This is anotherexample of language shortcuts (likely because someone was too lazy to write cytochrome twice) thatcan be confusing - particularly to students. The notation above could be rewritten as Cytochromecox/Cytochrome cred to indicate that the cytochrome c protein can exist in either and oxidized stateCytochrome cox or reduced state Cytochrome cred.

Review Red/Ox Tower video from Module 5.1For a short video on how to use the redox tower in red/ox problems click here1 . This video was made byDr. Easlon for Bis2A students.

2.1 Using the Red/Ox Tower: A tool to help understand electron transport chains

By convention the tower half reactions are written with the oxidized form of the compound on the leftand the reduced form on the right. Notice that compounds such as glucose and hydrogen gas are excellentelectron donors and have very low reduction potentials E0

'. Compounds, such as oxygen and nitrite, whosehalf reactions have relatively high positive reduction potentials (E0

') generally make good electron acceptorsare found at the opposite end of the table.Menaquinone: an exampleLet's look at menaquinoneox/red. This compound sits in the middle of the redox tower with an half-reaction

E0' value of -0.074 eV. Menaquinoneox can spontaneously (∆G<0) accept electrons from reduced forms of

compounds with lower half-reaction E0'. Such transfers form menaquinonered and the oxidized form of the

original electron donor. In the table above, examples of compounds that could act as electron donors tomenaquinone include FADH2, an E0

' value of -0.22, or NADH, with an E0' value of -0.32 eV. Remember the

reduced forms are on the right hand side of the red/ox pair.Once menaquinone has been reduced, it can now spontaneously (∆G<0) donate electrons to any com-

pound with a higher half-reaction E0' value. Possible electron acceptors include cytochrome box with an E0

'

value of 0.035 eV; or ubiquinoneox with an E0' of 0.11 eV. Remember that the oxidized forms lie on the left

side of the half reaction.

3 The Electron Transport Chain

The electron transport chain, or ETC, is composed of a group of protein complexes in and arounda membrane that help couple to energetically couple a series of exergonic/spontaneous red/ox reactionsto the endergonic pumping of protons across the membrane to generate a an electro-chemical gradient.This electrochemical gradient creates a free energy potential that is termed a proton motive force whoseenergetically "downhill" exergonic transfer can later be later coupled to a variety of cellular processes.

ETC Overview

Step 1. Electrons enter the ETC from a high energy electron donor, such as NADH or FADH2, which aregenerated during a variety of catabolic reactions like and including those associated glucose oxidation(review modules 5.3-5.5). Depending on the complexity (number and types of electron carriers) of theETC being used by an organism, electrons can enter at a variety of places in the electron transportchain - this depends upon the respective reduction potentials of the proposed electron donors andacceptors.

Step 2. After the �rst redox reaction, the initial electron donor will become oxidized and the electron acceptorwill become reduced. The di�erence in redox potential between the electron acceptor an donor isrelated to ∆G by the relationship ∆G = -nF∆E, where n = the number of electrons transferred andF = Faraday's constant. The larger a positive ∆E the more exergonic a reaction.

1http://youtu.be/HxbgKaTrxH8



Step 3. If su�cient energy transferred during an exergonic redox step the electron carrier may couple thisnegative change in free energy to the endergonic process of transporting a proton from one side of themembrane to the other.

Step 4. After multiple redox transfers, the electron is delivered to a molecule known as the terminal electronacceptor. In the case of humans and plants, this is oxygen. However, there are many, many, many,other possible electron acceptors, see below.

note: Electrons entering the ETC do not have to come from NADH or FADH2. Many othercompounds can serve as electron donors, the only requirements are that there exists an enzyme thatcan oxidize the electron donor and then reduce another compound and that the E0

' be positive (e.g.∆G<0). Even a small amounts of free energy transfers can add up. For example there are bacteriathat use H2 as an electron donor. This is not too di�cult to believe because the half reaction 2H+

+ 2 e-/H2 has a reduction potential (E0') of -0.42 V. If these electrons are eventually delivered to

oxygen then the ∆E0' of the reaction is 1.24 V which corresponds to a large negative ∆G (-∆G).

Alternatively, there are some bacteria that can oxidize iron, Fe2+ at pH 7 to Fe3+ with a reductionpotential (E0

') of +0.2 V. These bacteria use oxygen as their terminal electron acceptor and inthis case, the ∆E0

' of the reaction is approximately 0.62 V. This still produces a -∆G. The bottomline is that depending on the electron donor and acceptor that the organism uses, a little or a lotof energy can be transferred and used by the cell per electrons donated to the electron transportchain.

What are the complexes of the ETC?ETCs are made up of a series (at least one) of membrane associated red/ox proteins or (some are inte-gral) protein complexes (complex = more than one protein arranged in a quaternary structure) that moveelectrons from a donor source, such as NADH, to a �nal terminal electron acceptor, such as oxygen - thisdonor/terminal acceptor pair is the primary one used in human mitochondria. Each electron transfer in theETC requires a reduced substrate as an electron donor and an oxidized substrate as the electron acceptor.In most cases the electron acceptor is a member of the enzyme complex. Once the complex is reduced, thecomplex can serve as an electron donor for the next reaction.How do ETC complexes transfer electrons?As previously mentioned the ETC is composed of a series of protein complexes that undergo a series oflinked red/ox reactions. These complexes are in fact multiprotein enzyme complexes referred to as oxidore-ductases or simply reductases. The one exception to this naming convention is the terminal complex inaerobic respiration that uses molecular oxygen as the terminal electron acceptor. That enzyme complex isreferred to as an oxidase. Red/Ox reactions in these complexes are typically carried out by a non-proteinmoiety called a prosthetic group. This is true for all of the electron carriers with the exception of quinones,which are a class of lipids that can directly be reduced or oxidized by the oxidoreductases. In this case, boththe Quinonered and the Quinoneox is soluble within the membrane and can move from complex to complex.The prosthetic groups are directly involved in the red/ox reactions being catalyzed by their associated ox-idoreductases. In general these prosthetic groups can be divided into two general types: those that carryboth electrons and protons and those that only carry electrons.

The Electron and Proton carriers

• Flavoproteins (Fp), these proteins contain an organic prosthetic group called a �avin, which is theactual moiety that undergoes the oxidation/reduction reaction. FADH2 is an example of a Fp.

• Quinones, are a family of lipids which means they are soluble within the membrane.• It should also be noted that NADH and NADPH are considered electron (2e-) and proton (2 H+)

carriers.

Electon carriers



• Cytochromes are proteins that contain a heme prosthetic group. The Heme is capable of carrying asingle electron.

• Iron-Sulfur proteins contain a non-heme iron-sulfur clusters that can carry an electron. The pros-thetic group is often abbreviated as Fe-S

Aerobic versus Anaerobic respirationIn the world we live in, most of the organisms we interact with breath air, which is approximately 20%oxygen. Oxygen is our terminal electron acceptor. We call this process respiration, speci�cally aerobicrespiration, we breath in oxygen, our cells take it up and transport it into the mitochondria where it is usedas the �nal acceptor of electrons from our electron transport chains. That is aerobic respiration: theprocess of using oxygen as a terminal electron acceptor in an electron transport chain.

While most of the organisms we interact with use oxygen as the terminal electron acceptor, this processof respiration evolved at time when oxygen was not a major component of the atmosphere. Respiration oroxidative phosphorylation does not require oxygen at all; it simply requires a compound with a highreduction potential to act as a terminal electron acceptor; accept electrons from one of the complexes withinthe ETC. Many organisms can use a variety of compounds including nitrate (NO3

-), nitrite (NO2-), even

iron (Fe+++) as terminal electron acceptors. When oxygen is NOT the terminal electron acceptor, theprocess is referred to as anaerobic respiration. The ability of an organism to vary its terminal electronacceptor provides metabolic �exibility and can ensure better survival if any given terminal acceptor is inlimited supply. Think about this, in the absence of oxygen we die; but an organism that can use a di�erentterminal electron acceptor can survive.A generic example of a simple, 2 complex ETCFigure 2 shows a generic electron transport chain, composed of two integral membrane complexes; ComplexIox and Complex IIox. A reduced electron donor, designated DH (such as NADH or FADH2) reduces Complex1ox giving rise to the oxidized form D (such as NAD or FAD). Simultaneously, a prosthetic group withincomplex I is now reduced (accepts the electrons). In this example the redox reaction is exergonic and thefree energy di�erence is coupled by the enzymes in Complex I to the endergonic translocation of a protonfrom one side of the membrane to the other. The net result is that one surface of the membrane becomesmore negatively charged, due to an excess of hydroxyl ions (OH-) and the other side becomes positivelycharged due to an increase in protons on the other side. Complex Ired can now reduce the prosthetic groupin Complex IIred while simultaneously oxidizing Complex Ired. Electrons pass from Complex I to ComplexII via thermodynamically spontaneous red/ox reactions, regenerating Complex Iox which can repeat theprevious process. Complex IIred reduces A, the terminal electron acceptor to regenerate Complex IIox andcreate the reduced form of the terminal electron acceptor. In this case, Complex II can also translocate aproton during the process. If A is molecular oxygen, water (AH) will be produced. This reaction would thenbe considered a model of an aerobic ETC. However, if A is nitrate, NO3

- then Nitrite, NO2- is produced

(AH) and this would be an example of an anaerobic ETC.



Generic electron transport chain

Figure 2: Generic 2 complex electron transport chain. In the �gure, DH is the electron donor (donorreduced) and D is the donor oxidized. A is the oxidized terminal electron acceptor and AH is the �nalproduct, the reduced form of the acceptor. As DH is oxidized to D, protons are translocated across themembrane, leaving an excess of hydroxyl ions (negatively charged) on one side of the membrane andprotons (positively charged) on the other side of the membrane. The same reaction occurs in ComplexII as the terminal electron acceptor is reduced to AH.

Exercise 1: Thought question (Solution on p. 15.)

Based on Figure 2 above and using the electron tower in Figure 1, what is the di�erence in theelectrical potential if (A) DH is NADH and A is O2 and (B) DH is NADH and A is NO3

-. Whichpairs (A or B) provide the most amount of usable energy?

Detailed look at aerobic respirationThe eukaryotic mitochondria has evolved a very e�cient ETC. There are four complexes composed of pro-teins, labeled I through IV in Figure 3 (An Aerobic Electron Transport Chain), and the aggregation of thesefour complexes, together with associated mobile, accessory electron carriers, is called the electron transportchain. The electron transport chain is present in multiple copies in the inner mitochondrial membrane ofeukaryotes and the plasma membrane of bacteria and arechaea.



An Aerobic Electron Transport Chain

Figure 3: The electron transport chain is a series of electron transporters embedded in the innermitochondrial membrane that shuttles electrons from NADH and FADH2 to molecular oxygen. In theprocess, protons are pumped from the mitochondrial matrix to the intermembrane space, and oxygen isreduced to form water.

3.1 Complex I

To start, two electrons are carried to the �rst complex aboard NADH. This complex, labeled I, is composedof �avin mononucleotide (FMN) and an iron-sulfur (Fe-S)-containing protein. FMN, which is derived fromvitamin B2, also called ribo�avin, is one of several prosthetic groups or co-factors in the electron transportchain. A prosthetic group is a non-protein molecule required for the activity of a protein. Prosthetic groupsare organic or inorganic, non-peptide molecules bound to a protein that facilitate its function; prostheticgroups include co-enzymes, which are the prosthetic groups of enzymes. The enzyme in complex I is NADH



dehydrogenase and is a very large protein, containing 45 amino acid chains. Complex I can pump fourhydrogen ions across the membrane from the matrix into the intermembrane space, and it is in this way thatthe hydrogen ion gradient is established and maintained between the two compartments separated by theinner mitochondrial membrane.

3.2 Q and Complex II

Complex II directly receives FADH2, which does not pass through complex I. The compound connecting the�rst and second complexes to the third is ubiquinone (Q). The Q molecule is lipid soluble and freely movesthrough the hydrophobic core of the membrane. Once it is reduced, (QH2), ubiquinone delivers its electronsto the next complex in the electron transport chain. Q receives the electrons derived from NADH fromcomplex I and the electrons derived from FADH2 from complex II, including succinate dehydrogenase. Thisenzyme and FADH2 form a small complex that delivers electrons directly to the electron transport chain,bypassing the �rst complex. Since these electrons bypass and thus do not energize the proton pump in the�rst complex, fewer ATP molecules are made from the FADH2 electrons. As we will see in the followingsection, the number of ATP molecules ultimately obtained is directly proportional to the number of protonspumped across the inner mitochondrial membrane.

3.3 Complex III

The third complex is composed of cytochrome b, another Fe-S protein, Rieske center (2Fe-2S center), andcytochrome c proteins; this complex is also called cytochrome oxidoreductase. Cytochrome proteins have aprosthetic group of heme. The heme molecule is similar to the heme in hemoglobin, but it carries electrons,not oxygen. As a result, the iron ion at its core is reduced and oxidized as it passes the electrons, �uctuatingbetween di�erent oxidation states: Fe++ (reduced) and Fe+++ (oxidized). The heme molecules in thecytochromes have slightly di�erent characteristics due to the e�ects of the di�erent proteins binding them,giving slightly di�erent characteristics to each complex. Complex III pumps protons through the membraneand passes its electrons to cytochrome c for transport to the fourth complex of proteins and enzymes(cytochrome c is the acceptor of electrons from Q; however, whereas Q carries pairs of electrons, cytochromec can accept only one at a time).

3.4 Complex IV

The fourth complex is composed of cytochrome proteins c, a, and a3. This complex contains two hemegroups (one in each of the two cytochromes, a, and a3) and three copper ions (a pair of CuA and one CuBin cytochrome a3). The cytochromes hold an oxygen molecule very tightly between the iron and copperions until the oxygen is completely reduced. The reduced oxygen then picks up two hydrogen ions from thesurrounding medium to make water (H2O). The removal of the hydrogen ions from the system contributesto the ion gradient used in the process of chemiosmosis.

Helpful Links

• YouTubeElectron Transport Chain2

• YouTubeElectron Transport Chain #23

3.5 Chemiosmosis

In chemiosmosis, the free energy from the series of redox reactions just described is used to pump hydrogenions (protons) across the membrane. The uneven distribution of H+ ions across the membrane establishesboth concentration and electrical gradients (thus, an electrochemical gradient), owing to the hydrogen ions'positive charge and their aggregation on one side of the membrane.

2https://www.youtube.com/watch?v=j6_e39ueJBo3http://cnx.org/content/m59707/latest/hhttps://search.yahoo.com/yhs/search;_ylt=AwrT6V2ZoXBV_NQAav4nnIlQ;_ylc=X1MDMTM1MTE5NTY4NwRfcgMyBGZyA3locy1



If the membrane were open to di�usion by the hydrogen ions, the ions would tend to di�use back acrossinto the matrix, driven by their electrochemical gradient. Many ions cannot di�use through the nonpolarregions of phospholipid membranes without the aid of ion channels. Similarly, hydrogen ions in the matrixspace can only pass through the inner mitochondrial membrane through an integral membrane proteincalled ATP synthase (Figure 4). This complex protein acts as a tiny generator, turned by transfer of energymediated by protons moving down their electrochemical gradient. The movement of this molecular machine(enzyme) serves to lower the activation energy of reaction and couples the exergonic transfer of energyassociated with the movement of protons down their electrochemical gradient to the endergonic addition ofa phosphate to ADP, forming ATP.

:

Figure 4: ATP synthase is a complex, molecular machine that uses a proton (H+) gradient to formATP from ADP and inorganic phosphate (Pi). (Credit: modi�cation of work by Klaus Ho�meier)

note: Dinitrophenol (DNP) is a small chemical that serves to uncouple the �ow of protons acrossthe inner mitochondrial membrane to the ATP synthase and thus the synthesis of ATP. DNP makesthe membrane leaky to protons. It was used until 1938 as a weight-loss drug. What e�ect would youexpect DNP to have on the di�erence in pH across both sides of the inner mitochondrial membrane?Why do you think this might be an e�ective weight-loss drug? Why might it be dangerous?

Chemiosmosis (Figure 5) is used to generate 90 percent of the ATP made during aerobic glucose catabolism;it is also the method used in the light reactions of photosynthesis to harness the energy of sunlight in theprocess of photophosphorylation. Recall that the production of ATP using the process of chemiosmosis in



mitochondria is called oxidative phosphorylation. The overall result of these reactions is the production ofATP from the energy of the electrons removed from hydrogen atoms. These atoms were originally part of aglucose molecule. At the end of the pathway, the electrons are used to reduce an oxygen molecule to oxygenions. The extra electrons on the oxygen attract hydrogen ions (protons) from the surrounding medium, andwater is formed.LinksHow ATP is made from ATP synthase4

:

Figure 5: In oxidative phosphorylation, the pH gradient formed by the electron transport chain is usedby ATP synthase to form ATP in a Gram- bacteria.

note: Cyanide inhibits cytochrome c oxidase, a component of the electron transport chain. Ifcyanide poisoning occurs, would you expect the pH of the intermembrane space to increase ordecrease? What e�ect would cyanide have on ATP synthesis?

3.6 A Hypothesis as to how ETC may have evolved

A proposed link between SLP/Fermentation and the evolution of ETCsWhen we last discussed energy metabolism, it was in context of substrate level phosphorylation (SLP) andfermentation reactions. One of the questions in the Discussion points was what would be the consequencesof SLP, both short-term and long-term to the environment? We discussed how cells would need to co-evolvemechanisms to remove protons from the cytosol (interior of the cell), which lead to the evolution of theF0F1ATPase, a multi-subunit enzyme that translocates protons from the inside of the cell to the outside of

4https://www.youtube.com/watch?v=PjdPTY1wHdQ



the cell by hydrolyzing ATP as shown in �gure 6 below. This arrangement works as long as small reducedorganic molecules are freely available, making SLP and fermentation advantageous. As these biologicalprocess continue, the small reduced organic molecules begin to be used up and their concentration decreases,putting a demand on cells to be more e�cient. One source of potential "ATP waste" is in the removal ofprotons from the cell's cytosol, organisms that could �nd other mechanisms could have a selective advantage.Such selective pressure could have led to the �rst membrane-bound proteins that could use Red/Ox reactionsas their energy source, as depicted in �guire 7. In other words use the energy from a Red/Ox reactionto move protons. Such enzymes and enzyme complexes exist today in the form of the electron transportcomplexes, like Complex I, the NADH dehydrogenase.

Figure 6: Proposed evolution of an ATP dependent proton translocator



Figure 7: As small reduced organic molecules become limiting organisms that can �nd alternativemechanisms to remove protons from the cytosol may have had and advantage. The evolution of a protontranslocator that uses the energy in a Red/Ox reaction could substitute for the ATAase.

Continuing with this line of logic, there are organisms that can now use Red/Ox reactions to translocateprotons across the membrane, instead of an ATP driven proton pump. With protons being being translocatedby Red/Ox reactions, this would now cause a build up of protons on the outside of the membrane, separatingboth charge (positive on the outside and negative on the inside; an electrical potential) and pH (low pHoutside, higher pH inside). With excess protons on the outside of the cell membrane, and the F0F1ATPaseno longer consuming ATP to translocate protons, the pH and charge gradients can be used to drive theF0F1ATPase "backwards"; that is to form or produce ATP by using the energy in the charge and pHgradients set up by the Red/Ox pumps as depicted in �gure 8. This arrangement is called an electrontransport chain (ETC).



Figure 8: The evolution of the ETC; the combination of the Red/Ox driven proton translocators coupledto the production of ATP by the F0F1ATPase.



Solutions to Exercises in this Module

Solution to Exercise (p. 7)


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Bis2A 5.6: Oxidative Phosphorylation and the Electron ... · OpenStax-CNX module: m59707 1 Bis2A 5.6: Oxidative Phosphorylation and the Electron Transport Chain * The BIS2A Team This