Electron Transport and Oxidative Phosphorylation Notes
Outline HERE

Oxidation of glucose may be broken into two phases:

1. Oxidation of the glucose carbon atoms (for example glycolysis and citric acid cycle)

2. Reduction of molecular oxygen

Overview: The electron transport system (ETS) is a mechanism for capturing in the form of ATP, energy from the transfer of electrons from NADH and FADH2 to O2. During this process, the mitochondrion expels protons, creating a pH gradient across the mitochondrial membrane. When the proton gradient energy is harnessed (oxidative phosphorylation), ATP is produced from ADP and Pi.

Electrons are carried to the electron transport chain by the molecules NADH and FADH2, which are produced in many cellular processes, included glycolysis and citric acid cycle. Oxidation of one molecule of glucose yields 30-38 ATPs from glycolysis and the citric acid cycle (depends on how you count - see below).

The Mitochondrion

Electron transport and oxidative phosphorylation occur in the mitochondrion, the cell's "power plant". The citric acid enzymes are housed there as is pyruvate dehydrogenase, enzymes for fatty acid oxidation, and all of the enzymes and proteins of the electron transport/oxidative phosphorylation system.

Mitochondria have a smooth outer membrane and a complicated inner membrane, with many folds inside the organelle. This infolding material is called cristae, the number of which vary with the respiratory needs of the cell. Cells with low respiration rates have fewer cristae; those with higher rates have more. Proteins of electron transport and oxidative phosphorylation are bound to the inner mitochondrial membrane. The "matrix" of the inner mitochondrial compartment (a gel-like aqueous material) contains high concentrations of oxidative metabolic enzymes (citric acid cycle, etc.) as well as the mitochondrial DNA, RNA, and ribosomes for making mitochondrial proteins. Mitochondrial DNA is evidence of a previous endosymbiotic event.

The outer membrane of the mitochondrion contains porin (also called VDAC), a pore-forming protein that allows diffusion of up to 10 kD (10,000 Dalton molecular weight) molecules. The inner membrane is much richer in proteins (up to 75% protein by mass) than the outer membrane and allows only O2, CO2 and H20 to freely pass.The inner membrane is the anchor for electron transport and oxidative phosphorylation proteins, and also contains transport proteins for passing common metabolites, such as ATP, ADP, pyruvate, Ca++, and phosphate. The integrity of the inner membrane is important for operation of the mitochondrion, which harnesses energy from the chemical gradients it creates across the membrane.

Electron Transport Oxidations/Energy

Electron transport involves transfer of the reducing equivalents of electrons through several biological compounds. As a compound accepts electrons, it is reduced, and as it passes the electron on to the next molecule in the chain the molecule is oxidized.

This may be written as a reaction:

Reduced molecule #1 + Oxidized Molecule #2 Oxidized Molecule #1 + Reduced Molecule #2

One example of this is:

Fe(+2) + Cu (+2) Fe(+3) + Cu(+1)

Standard Reduction Potential

The tendency of a molecule to act as an electron donor is referred to as the Reduction Potential, and is given the symbol 'E'. The Standard Reduction Potential at pH 7.0 is given the symbol . The scale for is set so that the potential of a hydrogen electrode is 0.00 volts. The Standard Reduction Potential voltages shown in the table in the book can be viewed as the tendency of electrons to flow towards the molecule of interest. Molecules with a more positive Standard Reduction Potential will pull electrons away from molecules with a more negative Standard Reduction Potential.

As an example,

Fe(+3) + Cytochrome a3 (+2) Fe(+2) + Cytochrome a3 (+3)

The Standard Reduction Potentials shown in the table predict that this reaction will go to the right if the concentration of all the molecules is the same. Students should note a few things here. First of all, a reaction such as this is called a Reduction/Oxidation reaction (or Redox reaction for short). Each side of the equation has a reductant and an oxidant. The reaction is an equilibrium, and is governed by the Gibbs free energy rules. Thus, under some conditions, the reaction will move to the left, and under other conditions it will move to the right. can be related to by the following equation:

= -nF,

where 'n' is the number of electrons transferred, F is Faraday's constant (96.5 kJ/mol/Volt), and
= (acceptor molecule) - (donor molecule)

Rearranging into the G equation you've seen previously,

G = -nF + RTln (Products/Reactants)

The flow of electrons through the cell's electron transport chain is in the direction of molecules with increasing Standard Reduction Potentials, as one would expect from above. Transfer of electrons in this manner releases energy that can be used to do the work of the cell.

Energy Capture

The free energy released during the entire electron transfer process is sufficient, in theory, to yield 7 ATPs for each NADH. In reality, cells appear to synthesize 2.5- 3ATPs for each NADH. (Note: FADH2, another electron carrier, enters the electron transport system at a point different from NADH, and yields only 1.5-2 ATPs for each FADH2.) The transfer of electrons from NADH to O2 does not occur directly, but rather by a series of small steps three of which yield ATP. We call the process of making ATP by the capture of energy produced during electron transport Oxidative Phosphorylation.

Sequence of Electron Transport

In electron transport, electrons from NADH and FADH2 are passed through four complexes (I, II, III, IV). Note that I prefer to use simple names for the complexes. Your book calls Complex I NADH-Qoxidoreductase, it calls Complex II Succinate-Qreductase, it calls Complex III Q-cytochrome c oxidoreductase, and it calls Complex IV Cytochrome c reductase. Another complex, sometimes called Complex V refers to an enzyme in the process of oxidative phosphorylation, not electron transport. The sequence of events in ETS is as follows. Note that electrons enter EITHER through Step A below OR through Step B, but that they do NOT pass through both steps.

Components of the ETS

The complexes of the ETS are embedded in the inner mitochondrial membrane, where they have move independently of each other.

ETS Complex I

Complex I is an enormous cluster of proteins (at least 34) that passes electrons from NADH to Coenzyme Q (CoQ). The complex contains one molecule of flavin mononucleotide (FMN) - similar to FAD - and several iron-sulfur proteins (nonheme iron proteins). The sulfurs in these proteins come from Cys residues and from free sulfide. The iron atoms are surrounded each by 4 sulfur atoms. Remember that iron can exist as Fe++ and Fe+++. The difference is a single electron. Despite the presence of as many as four Fe atoms, the total charge difference between the oxidized and reduced forms of the cluster is one electron. For example, each four FeS cluster in ferredoxin has one Fe++ and three Fe+++ (total charge = +11) when oxidized, and two Fe++ and two Fe+++ (total charge = +10) when reduced.

FMN and CoQ can have three oxidation states. NADH passes pairs of electrons. Both FMN and CoQ can accept and donate either one or two electrons. FMN and CoQ buffer the flow of electrons downstream because Complex III can only handle electrons one at a time. The tail of CoQ is hydrophobic, and is soluble in the inner mitochondrial membrane.

ETS Complex II

Complex II contains succinate dehydrogenase (citric acid enzyme), which passes electrons from succinate (through FADH2) to CoQ. Other components of the system include a four Fe-S cluster, two 2 Fe-S clusters, and a cytochrome b560. No protons are pumped in passing electrons from complex II to CoQ.


CoQ acts as a traffic cop for electrons in the transport system. It is capable of accepting electrons singly or in pairs, but, importantly, passes them off to cytochrome C via Complex III singly in the Q cycle. CoQ is capable also of accepting electrons from either Complex I or Complex II and is thus the only branch point in the ETS. The reduced form of CoQ is called ubiquinol and the oxidized form is called ubiquinone. Ubiquinol/ubiquinone is a small molecule anchored by a hydrophobic tail that can readily move in the inner mitochondrial membrane. Note that when ubiquinone accepts two electrons, it also takes two protons from the matrix. The protons accompany the electrons when they are passed to Complex III.

ETS Complex III

Complex III contains two cytochrome b's, a cytochrome c, and a single 2Fe-S cluster. The complex is scattered across the inner mitochondrial membrane. Cytochrome c1 and the Fe-S protein are on the membrane's outer surface, while cytochrome b is transmembranous. The cytochromes hold Fe with N instead of S, and the different cytochromes here have different configurations of the hemes. The rings containing the N are called porphyrins, and the ring of cytochrome b (contains two) is called protoporphyrin IX, which is also used in hemoglobin. Note that Complex III binds to both cytochrome C AND CoQ and serve to facilitate the transfer of electrons from CoQ to cytochrome C.

Cytochromes are ubiquitous among aerobic organisms. The cytochrome proteins contain heme groups that alternate between Fe++ and Fe+++ as above. Complex III transfers electrons to cytochrome C and, in doing so, transfer four protons outside the mitochondrion. Remember that two protons came with the electrons from CoQ. Complex III transports this pair of protons, as well as two other protons from the matrix to outside the mitochondrion as the electrons pass through it to cytychrome C.

Cytochrome C

Cytochrome C (Cyt C) is a peripheral membrane protein (different from cyt c1 above). It is only loosely bound to the outer surface of the inner membrane. It alternately binds cyt c1 of complex III and cyt c oxidase in Complex IV (passing electrons between them). The heme group of cyt c is buried in the protein. Lysine residues appear to mediate the binding to both cyt c1 and to cyt c oxidase. Cytochrome C is a ubiquitous protein found in most living cells on earth and the its structure and amino acid sequence can give a measure of evolutionary relationship/distance.

Complex IV (Cyt c Oxidase)

Complex IV catalyzes one electron oxidations of four consecutive reduced cytochrome c molecules and the four electron reduction of O2. Mammalian complex IV is a large transmembrane protein with 6-13 subunits. The complex exists as a dimer. Subunits I and II contains all four redox centers (two hemes (a and a3) changing between Fe++ and Fe+++ and two Cu atoms (Cua and Cub) that vary between Cu+ and Cu++. Reductions of O2 to 2H2O takes place on the cytochrome a3-Cub complex. Note in the figure HERE the steps in the reduction of molecular oxygen to water - binding of electrons, binding of O2, formation of a peroxide bridge, cleavage of the O2 bond, reduction of the ferryl group, and release of water. Note that four protons are pumped by the complex outside of the mitochondrion during the electron transfers and that all of the protons come directly from the mitochondrial matrix. In addition, four protons are taken from the matrix to complete the reduction of oxygen to water, so a total of eight protons are removed from the matrix for the four electrons passing through complex IV en route to reduction of oxygen.

Reactive Oxygen

Oxygen is readily reduced and is thus a good terminal electron acceptor. So long as each oxygen receives four electrons, the products of the reduction (two molecules of water) are safe to the cell. If, however, less than four electrons are transferred, free radical oxygen molecules, such as superoxide or peroxide, are formed. These molecules are very chemically reactive and can wreak havoc in cells. To guard against this damage, cells have enzymes to 'deactivate' reactive oxygen species, or ROS. One of these enzymes is superoxide dismutase that catalyzes the conversion of superoxide to oxygen and hydrogen peroxide. Another enzyme, catalase, converts hydrogen peroxide to water. Other cellular protectors against free radical damage are the antioxidants, such as vitamins E and C. We will talk about other antioxidants, such as uric acid, later this term.

Oxidative Phosphorylation

The actual synthesis of ATP resulting from energy transfer of the ETS is catalyzed by proton translocating ATP synthase (also called PTAS, F1F0ATPase, or Complex V). One clue to the mechanism of the synthesis is that Complex V is physically separate from the ETS. Other than being in the inner mitochondrial membrane, there is no direct linkage between Complex V and the ETS. Complex V does, however, utilize the proton gradient built up by the ETS as a means of making ATP.

Definition: The P/O ratio is the number of ATPs produced per pair of electrons traveling through the electron transport system (ETS). Depending upon the point at which electrons enter the chain, the P/O ration will change (See Figure 15.12). As discussed earlier, electrons entering from NADH produce 3ATPs per pair of electrons, from FADH2 produce 2 ATPs per pair of electrons, and from ascorbate produce 1 ATP per pair of electrons. Since the production of ATP is not directly linked to each step in the transport of electrons, but rather by the gradient arising from pumping of protons during the electron transport, it is not necessary that an exact integral number of ATPs be produced per entry point. In fact, it appears that it is not exactly 3, 2, or 1 ATP produced per pair of electrons, but it is useful conceptually to think in this way.

The Chemiosmotic Coupling Hypothesis

Peter Mitchell proposed that the ETS generates potential energy by pumping protons out of the mitochondrial matrix into the intermembrane space. The energy of the proton gradient is used to synthesize ATP.

Observations Consistent with Chemiosmotic Hypothesis Proton Gradient Generation

The ETS pumps protons across the inner membrane as part of its operation. This action serves to store energy from the ETS process as potential energy in the form of a proton gradient (high H+ in the intermembrane space, low H+ inside the mitochondria). As noted, pumping protons from a region of low concentration to a region of high concentration requires energy, and this energy is supplied by the ETS. Remember that protons are charged, so the action of pumping positive charges outside of the mitochondria causes the inner membrane's inner surface to become negatively charged relative to its outer surface.

Complex V Structure.

The enzyme that uses the energy of the proton gradient to synthesize ATP from ADP + Pi is called proton-translocating ATP synthase (PTAS or Complex V), as noted above. This complex system is composed of two major components and numerous other subunits. Close examination of the mitochondrial cristae reveals a knobby set of structures sitting on top of what appear to be stalks. The knobs are known as F1 spheres. The stalk and its base are referred to as F0. F0 itself is composed of many subunit proteins. It is transmembranous (insoluble in water) and creates a channel for protein translocation.

F1 is water soluble and a peripheral membrane protein. It has several subunits and interacts with F0 weakly. Solubilized F1 can hydrolyze ATP, but cannot synthesize it. PTAS from which F1 has been removed no longer exist as knobby "lollipops" and further cannot synthesize ATP. Addition back of F1 to PTAS containing F0 restores both the lollipop appearance and the ability to synthesize ATP. The antibiotic oligomycin inhibits ATP synthase by binding one of F0's subunits and preventing H+ transport through F0. Dicyclohexylcarbodiimide (DCCD), a lipid soluble compound also inhibits H+ transport through F0.

ATP Synthesis Mechanism

Complex V catalyzes the synthesis of ATP from ADP and phosphate. As seen, the chemical mechanism involves formation of a pentacovalent intermediate followed by removal of a hydroxyl by a proton to form water and ATP. Further analysis of the mechanism of formation of ATP revealed that ATP can readily form in the enzyme, but cannot leave it in the absence of a proton. Thus, the proton supplied by the gradient are essential for release of the ATP.

The mechanism of ATP synthesis consists of three phases on F1; 1) binding of ADP + Pi; 2) Catalysis of formation of ATP; 3) Release of the ATP. Three beta subunits of the F1 change conformations (Open, Tight, and Loose) facilitating the release of ATP, formation of ATP, and binding of ADP+Pi, respectively. Note that the changing forms of the beta are caused by a rotating gamma subunit that causes the beta forms to be altered as the gamma spins under them. Spinning of the gamma has been demonstrated.

Spinning of the gamma subunit is caused by movement of protons through the c ring of the F0. Tight links between the c ring and the gamma and epsilon subunits cause them to rotate as well. Each 360 degree rotation of the c ring generates 3 ATPs.

Ultimate ATP Yield from ETS

Remember first that ETS does not generate ATP, but instead generates the proton gradient that PTAS uses to make ATP. The proton gradient is an amazingly flexible means of storing energy. From the electrons transported through the ETS, we discover that entry of electrons through Complex I (from NADH) generates enough energy (from protons pumped) to synthesize approximately 2.5-3 ATP per pair of electrons. Entry of electrons through Complex II (from FADH2) generates sufficient energy to synthesize approximately 1.5-2 ATP per pair of electrons. Note that ATP yields are approximate, because not every proton transported outside the mitochondrion necessarily makes it back into the matrix via the PTAS for a variety of reasons. Consequently, ATP yield estimates vary from 30 to 38 per molecules of glucose being completely oxidized.

Mitochondrial Transport Systems

Inner mitochondrial transport systems move the following molecules across the inner membrane:

NADH Shuttle Systems

Oxidation of pyruvate produced by glucose in glycolysis is accomplished by citric acid enzymes in the mitochondrion. This yields NADH, whose electrons can enter the electron transport system directly. NADH from glycolysis, however is in the cytosol. Since the mitochondrion does not have a direct transfer mechanism for NADH, "shuttles" are used instead, which ferry the electrons in. The glycerol-3-phosphate shuttle uses an enzyme in the outer layer of the inner mitochondrial membrane - glycerol-3-phosphate dehydrogenase (Gly3PDH). In this series of reactions, dihydroxyacetone phosphate (DHAP) in the cytosol is reduced to glycerol-3-phosphate as NADH is oxidixed to NAD+ (The electrons from NADH are transferred to DHAP to form glyerol-3-phosphate). In the second step, the electrons of glycerol-3-phosphate are transferred to FAD (yielding FADH2) and DHAP is produced. The glycerol-3-phosphate dehydrogenase enzyme for this reaction is embedded in the outer mitochondrial membrane, and the electrons in FADH2 are transferred directly to the electron transport chain. This process, because it goes through FADH2, yields 1.5-2 ATP for each original NADH.

Malate Aspartate Shuttle

Another shuttle (mammals) is the malate-aspartate shuttle. This system relies on transport systems for malate, aspartate, glutamate, and alpha-keto-glutarate. Electrons from NADH outside the matrix are transferred in as follows:

ATP/ADP Movement

ATP and ADP do not freely diffuse across the mitochondrial membrane. They too must be shuttled and the protein involved is called the ATP-ADP translocase.

Other translocases involved in moving metabolites across the innter mitochondrial membrane have common structural features.

Coupling of ETS and Oxidative Phosphorylation

The most efficient generation of ATP by cells occurs when ETS and oxidative phosphorylation are 'tightly coupled.' Essentially this occurs when the mitochondrial inner membrane is intact. Under these conditions, stoppage of either ETS or oxidative phosphorylation can lead to the stoppage of the other. If ETS stops, the proton gradient will quickly be dissipated by complex V and ATP synthesis will grind to a halt. Conversely, if one stops ATP synthesis via complex V, the ETS stops, but it may not be apparent at first why. The reason is that the ETS is constantly pumping protons OUT of the mitochondrion. Normally the complex V allows them to return. If there is no return mechanism, the ETS will run low on protons AND the gradient of protons will become so large as to make pumping of additional ones virtually impossible. Because the ETS and Complex V are co-dependent in this way, we have what is known as respiratory control.

For example, when we rest, ATP concentrations build high. This leads to a lack of ADP in the mitochondrion, which leads to stoppage of complex V. Stoppage of complex V stops ETS which causes NADH and FADH2 concentrations to rise. These, in turn, cause the citric acid cycle to slow or stop. As the citric acid cycle slows, acetyl-CoA and citrate accumulate. High levels of acetyl-CoA and citrate combined with high NADH levels can lead to synthesis of fat.

Another scenario - normally oxygen is the terminal electron acceptor. When oxygen is abundant, ETS and complex V can function (if there is sufficient ADP), but if we exercise faster than oxygen can be delivered, we go anaerobic. When this happens, ETS stops (no terminal electron acceptor), the proton gradient rapidly dissipates and oxidative phosphorylation stops. Thus NADH and FADH2 concentrations rise. Remember that NAD is needed for glycolysis, so it is for this reason that our cells make lactate - to regenerate NAD+ because the ETS is stopped.

You should be able to predict what will happen if ETS is stopped by an ETS or Complex V inhibitor described below is used and also what would happen if ETS and complex V are UNCOUPLED, as described below.

Control points of glycolysis (PFK), TCA cycle (pyruvate DH, citrate synthase, isocitrate DN, and alpha keto glutarate DH) are responsive to adenine nucleotides (ADP, ATP), NADH, or both as well as other metabolites. All the major paths for production of ATP (glycolysis, TCA cycle, oxidative phosphorylation) must be coordinated for production of ATP as needed. Oxidative phosphorylation is dependent on the ETS, which is dependent on electrons carried by NADH/FADH2. Glycolysis and the TCA cycle are readily producers of NADH and FADH2. It is easy to see how what I call feed forward activation of ATP synthesis (glycolysis turns on TCA, and both turn on oxidative phosphorylation by turning on the electron transport system) is coordinated. Feed backwards deactivation of ATP synthesis is also important, because cells need to turn off ATP synthesis, as well as turn it on. One important metabolite that helps to turn off the coordinated feed forward system is citrate, which acts allosterically to deactivate PFK, the major control point of glycolysis. When the body moves from exercising to rest, the concentration of ATP increases (because the ATP being produced rapidly by oxidative phosphorylation is not being burned to ADP + Pi). Increasing ATP inhibits alpha keto glutarate DH and slows isocitrate DH (activated by ADP). The combined effects of the inactivation or slowing of these enzymes slows the TCA cycle, and increases citrate concentration. Increasing citrate in the inner mitochondrion is transported to the cytosol where it interacts with PFK, shutting down carbohydrate breakdown (glycolysis).

ETS and Complex V Inhibitors

Several compounds are known that inhibit the transfer of electrons through the ETS. Rotenone and amytal inhibit ETS in complex I. The ETS can be restarted in the presence of rotenone if an FADH2 generating system is added (this bypasses the block in complex I). Antimycin blocks complex III (passage of electrons past cytochrome b). Addition of cyanide, azide, or carbon monoxide stops electron transport through complex IV. Inhibitors of complex V include DCCD and oligomycin.

Uncoupling of Oxidative Phosphorylation

Remember that oxidative phosphorylation is usually tightly coupled to electron transport. This means that blocking of the ETS system will prevent oxidative phosphorylation and, importantly, that the reverse is true also - blocking oxidative phosphorylation stops electron transport. It is possible to "uncouple" these two processes as noted above, using agents that permeabilize the inner mitochondrial membrane to protons. Two such foreign agents are 2,4 dinitrophenol (DNP) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Remember that permeabilizing the inner mitochondrial membrane to proton dissipates the electrochemical gradient in which energy from electron transport is stored. Uncontrolled uncoupling the ETS system from oxidative phosphorylation is potentially lethal. In an uncoupled system oxidative phosphorylation does not occur because the proton gradient is destroyed, but ETS continues wildly, causing burning of much energy in the citric acid cycle and glycolysis, with very little ATP synthesis. turn it on).

Biological Uncoupling

Uncoupling the ETS from oxidative phosphorylation speeds metabolism, and generates heat. Some mammals lacking fur use this function in brown adipose tissue as a way of generating heat. One such process is called nonshivering thermogenesis that occurs in cells in the neck and upper back. The mitochondria of brown adipose tissue cells contain a protein called thermogenin (or uncoupling protein - UCP). Thermogenin acts as a channel to permeabilize these cells' inner mitochondrial membrane to protons. Normally, ADP, ATP, GDP, and GTP are present in high enough concentrations to block the flow of protons through it. However, thermogenin in the mitochondria of these cells is activated to uncoupling by the presence of free fatty acids. Free fatty acids can be generated in these cells by the hormone norepinephrine, which through second messengers (including cAMP) activates hormone-sensitive triacylglycerol lipase to cleave fats to release fatty acids. Thus, brown adipose tissue cells respond to norepinephrine by uncoupling the ETS from oxidative phosphorylation, speeding metabolism and generating heat, at the expense of metabolic energy.