Bis2A 5.6: Oxidative Phosphorylation and the Electron Transport Chain *

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1 OpenStax-CNX module: m 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 force which 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 as the nal/terminal acceptor for the electrons that were removed from the intermediate compounds in glucose catabolism. In eukaryotic cells the ETC is composed of four large, multiprotein complexes embedded in the inner mitochondrial membrane and two small diusible electron carriers shuttling electrons between them. The electrons are passed from enzyme to enzyme through a series of redox reactions. These reactions are couple 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 passing through the ETC gradually lose potential energy up until the point they are deposited on the terminal electron acceptor. The free energy dierence of this multistep redox process is -60 kcal/mol when NADH donates electrons or 45 kcal/mol when FADH 2 donates, for organisms using oxygen as the nal electron acceptor. Introduction to Red/Ox, oxidative phosphorylation and Electron Transport Chains In modules 5.1, we discussed the general concept of Red/Ox reactions in biology and introduced the Electron Tower, a tool to help you understand Red/Ox chemistry and to estimate the direction and magnitude of potential energy dierences for various Red/Ox couples. In modules 5.3 and 5.4 substrate level phosphorylation and fermentation were discussed and we saw how exergonic Red/Ox reactions could be directly coupled by enzymes to the endergonic synthesis of ATP. These processes are hypothesized to be one of the oldest forms of energy production used by cells. In this section we discuss the next evolutionary advancement in cellular energy metabolism, oxidative phosphorylation. First and foremost, oxidative phosphorylation does not imply the use of oxygen, it can, but it does not have to use oxygen. It is called oxidative phosphorylation because it relies on Red/Ox reactions to generate a electrochemical transmembrane potential that can then be used by the cell to do work. A quick summary of Electron Transport Chains The ETC begins with the addition of electrons, donated from NADH, FADH 2 or other reduced compounds. These electrons move through a series of electron transporters, enzymes that are embedded in a membrane, or * Version 1.1: Jan 25, :31 am

2 OpenStax-CNX module: m carriers that undergo Red/Ox reactions. The free energy transferred from these exergonic Red/Ox reactions is coupled to the endergonic movement of protons across a membrane. This unequal accumulation of protons on either side of the membrane "polarizes" or "charges" the membrane, with a net positive (protons) on one side of the membrane and a negative charge on the other side of the membrane. The separation of charge creates an electrical potential. In addition, the accumulation of protons also causes a ph gradient known as a chemical potential across the membrane. Together these two gradients (electrical and chemical) are called 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 reduction potential - sometimes called the "redox tower". As we discussed in Module 5.1, all kinds of compounds can participate in biological Red/Ox reactions. Making sense of all of this information and ranking potential Red/Ox pairs can be confusing. A tool has been developed to rate Red/Ox half reactions based on their reduction potentials or E 0' 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 tower ranks a variety of common compounds (their half reactions) from most negative E 0', compounds that readily get rid of electrons, to the most positive E 0', compounds most likely to accept electrons. The tower organizes these half reactions based on the ability of electrons to accept electrons. In addition, in many redox towers each half reaction is written by convention with the oxidized form on the left followed by the reduced form to its right. The two forms may be either separated by a slash, for example the half reaction for the reduction of NAD + to NADH is written: NAD + /NADH + 2e -, or by separate columns. An electron tower is shown in gure 1 below.

3 OpenStax-CNX module: m Figure 1: Common Red/ox tower note: Use the redox tower above as a reference guide to orient you as to the reduction potential of the various compounds in the ETC. Red/Ox reactions may be either exergonic or endergonic depending on the relative Red/Ox potentials of the donor and acceptor. Also remember there are

4 OpenStax-CNX module: m many dierent 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. For instance Cytochrome c ox/red. There only appears to be one form listed. Why? This is another example of language shortcuts (likely because someone was too lazy to write cytochrome twice) that can be confusing - particularly to students. The notation above could be rewritten as Cytochrome c ox /Cytochrome c red to indicate that the cytochrome c protein can exist in either and oxidized state Cytochrome c ox or reduced state Cytochrome c red. Review Red/Ox Tower video from Module 5.1 For a short video on how to use the redox tower in red/ox problems click here 1. This video was made by Dr. 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 left and the reduced form on the right. Notice that compounds such as glucose and hydrogen gas are excellent electron donors and have very low reduction potentials E 0'. Compounds, such as oxygen and nitrite, whose half reactions have relatively high positive reduction potentials (E 0' ) generally make good electron acceptors are found at the opposite end of the table. Menaquinone: an example Let's look at menaquinone ox/red. This compound sits in the middle of the redox tower with an half-reaction E 0' value of ev. Menaquinone ox can spontaneously ( G<0) accept electrons from reduced forms of compounds with lower half-reaction E 0'. Such transfers form menaquinone red and the oxidized form of the original electron donor. In the table above, examples of compounds that could act as electron donors to menaquinone include FADH 2, an E 0' value of -0.22, or NADH, with an E 0' value of 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 compound with a higher half-reaction E 0' value. Possible electron acceptors include cytochrome b ox with an E 0 ' value of ev; or ubiquinone ox with an E 0' 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 around a membrane that help couple to energetically couple a series of exergonic/spontaneous red/ox reactions to 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 whose energetically "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 FADH 2, which are generated during a variety of catabolic reactions like and including those associated glucose oxidation (review modules ). Depending on the complexity (number and types of electron carriers) of the ETC being used by an organism, electrons can enter at a variety of places in the electron transport chain - this depends upon the respective reduction potentials of the proposed electron donors and acceptors. Step 2. After the rst redox reaction, the initial electron donor will become oxidized and the electron acceptor will become reduced. The dierence in redox potential between the electron acceptor an donor is related to G by the relationship G = -nf E, where n = the number of electrons transferred and F = Faraday's constant. The larger a positive E the more exergonic a reaction. 1

5 OpenStax-CNX module: m Step 3. If sucient energy transferred during an exergonic redox step the electron carrier may couple this negative change in free energy to the endergonic process of transporting a proton from one side of the membrane to the other. Step 4. After multiple redox transfers, the electron is delivered to a molecule known as the terminal electron acceptor. 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 FADH 2. Many other compounds can serve as electron donors, the only requirements are that there exists an enzyme that can oxidize the electron donor and then reduce another compound and that the E 0' be positive (e.g. G<0). Even a small amounts of free energy transfers can add up. For example there are bacteria that use H 2 as an electron donor. This is not too dicult to believe because the half reaction 2H e - /H 2 has a reduction potential (E 0' ) of V. If these electrons are eventually delivered to oxygen then the E 0' of the reaction is 1.24 V which corresponds to a large negative G (- G). Alternatively, there are some bacteria that can oxidize iron, Fe 2+ at ph 7 to Fe 3+ with a reduction potential (E 0' ) of +0.2 V. These bacteria use oxygen as their terminal electron acceptor and in this case, the E 0' of the reaction is approximately 0.62 V. This still produces a - G. The bottom line is that depending on the electron donor and acceptor that the organism uses, a little or a lot of energy can be transferred and used by the cell per electrons donated to the electron transport chain. 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 integral) protein complexes (complex = more than one protein arranged in a quaternary structure) that move electrons from a donor source, such as NADH, to a nal terminal electron acceptor, such as oxygen - this donor/terminal acceptor pair is the primary one used in human mitochondria. Each electron transfer in the ETC 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, the complex 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 of linked red/ox reactions. These complexes are in fact multiprotein enzyme complexes referred to as oxidoreductases or simply reductases. The one exception to this naming convention is the terminal complex in aerobic respiration that uses molecular oxygen as the terminal electron acceptor. That enzyme complex is referred to as an oxidase. Red/Ox reactions in these complexes are typically carried out by a non-protein moiety 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, both the Quinone red and the Quinone ox 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 oxidoreductases. In general these prosthetic groups can be divided into two general types: those that carry both 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 the actual moiety that undergoes the oxidation/reduction reaction. FADH 2 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

6 OpenStax-CNX module: m Cytochromes are proteins that contain a heme prosthetic group. The Heme is capable of carrying a single electron. Iron-Sulfur proteins contain a non-heme iron-sulfur clusters that can carry an electron. The prosthetic group is often abbreviated as Fe-S Aerobic versus Anaerobic respiration In 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, specically aerobic respiration, we breath in oxygen, our cells take it up and transport it into the mitochondria where it is used as the nal acceptor of electrons from our electron transport chains. That is aerobic respiration: the process 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 process of respiration evolved at time when oxygen was not a major component of the atmosphere. Respiration or oxidative phosphorylation does not require oxygen at all; it simply requires a compound with a high reduction potential to act as a terminal electron acceptor; accept electrons from one of the complexes within the ETC. Many organisms can use a variety of compounds including nitrate (NO 3- ), nitrite (NO 2- ), even iron (Fe +++ ) as terminal electron acceptors. When oxygen is NOT the terminal electron acceptor, the process is referred to as anaerobic respiration. The ability of an organism to vary its terminal electron acceptor provides metabolic exibility and can ensure better survival if any given terminal acceptor is in limited supply. Think about this, in the absence of oxygen we die; but an organism that can use a dierent terminal electron acceptor can survive. A generic example of a simple, 2 complex ETC Figure 2 shows a generic electron transport chain, composed of two integral membrane complexes; Complex I ox and Complex II ox. A reduced electron donor, designated DH (such as NADH or FADH 2 ) reduces Complex 1 ox giving rise to the oxidized form D (such as NAD or FAD). Simultaneously, a prosthetic group within complex I is now reduced (accepts the electrons). In this example the redox reaction is exergonic and the free energy dierence is coupled by the enzymes in Complex I to the endergonic translocation of a proton from one side of the membrane to the other. The net result is that one surface of the membrane becomes more negatively charged, due to an excess of hydroxyl ions (OH - ) and the other side becomes positively charged due to an increase in protons on the other side. Complex I red can now reduce the prosthetic group in Complex II red while simultaneously oxidizing Complex I red. Electrons pass from Complex I to Complex II via thermodynamically spontaneous red/ox reactions, regenerating Complex I ox which can repeat the previous process. Complex II red reduces A, the terminal electron acceptor to regenerate Complex II ox and create the reduced form of the terminal electron acceptor. In this case, Complex II can also translocate a proton during the process. If A is molecular oxygen, water (AH) will be produced. This reaction would then be considered a model of an aerobic ETC. However, if A is nitrate, NO 3- then Nitrite, NO 2- is produced (AH) and this would be an example of an anaerobic ETC.

7 OpenStax-CNX module: m Generic electron transport chain Figure 2: Generic 2 complex electron transport chain. In the gure, DH is the electron donor (donor reduced) and D is the donor oxidized. A is the oxidized terminal electron acceptor and AH is the nal product, the reduced form of the acceptor. As DH is oxidized to D, protons are translocated across the membrane, leaving an excess of hydroxyl ions (negatively charged) on one side of the membrane and protons (positively charged) on the other side of the membrane. The same reaction occurs in Complex II 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 dierence in the electrical potential if (A) DH is NADH and A is O 2 and (B) DH is NADH and A is NO 3-. Which pairs (A or B) provide the most amount of usable energy? Detailed look at aerobic respiration The eukaryotic mitochondria has evolved a very ecient ETC. There are four complexes composed of proteins, labeled I through IV in Figure 3 (An Aerobic Electron Transport Chain), and the aggregation of these four complexes, together with associated mobile, accessory electron carriers, is called the electron transport chain. The electron transport chain is present in multiple copies in the inner mitochondrial membrane of eukaryotes and the plasma membrane of bacteria and arechaea.

8 OpenStax-CNX module: m An Aerobic Electron Transport Chain Figure 3: The electron transport chain is a series of electron transporters embedded in the inner mitochondrial membrane that shuttles electrons from NADH and FADH 2 to molecular oxygen. In the process, protons are pumped from the mitochondrial matrix to the intermembrane space, and oxygen is reduced to form water. 3.1 Complex I To start, two electrons are carried to the rst complex aboard NADH. This complex, labeled I, is composed of avin mononucleotide (FMN) and an iron-sulfur (Fe-S)-containing protein. FMN, which is derived from vitamin B 2, also called riboavin, is one of several prosthetic groups or co-factors in the electron transport chain. A prosthetic group is a non-protein molecule required for the activity of a protein. Prosthetic groups are organic or inorganic, non-peptide molecules bound to a protein that facilitate its function; prosthetic groups include co-enzymes, which are the prosthetic groups of enzymes. The enzyme in complex I is NADH

9 OpenStax-CNX module: m dehydrogenase and is a very large protein, containing 45 amino acid chains. Complex I can pump four hydrogen ions across the membrane from the matrix into the intermembrane space, and it is in this way that the hydrogen ion gradient is established and maintained between the two compartments separated by the inner mitochondrial membrane. 3.2 Q and Complex II Complex II directly receives FADH 2, 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 moves through the hydrophobic core of the membrane. Once it is reduced, (QH 2 ), ubiquinone delivers its electrons to the next complex in the electron transport chain. Q receives the electrons derived from NADH from complex I and the electrons derived from FADH 2 from complex II, including succinate dehydrogenase. This enzyme and FADH 2 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 FADH 2 electrons. As we will see in the following section, the number of ATP molecules ultimately obtained is directly proportional to the number of protons pumped 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), and cytochrome c proteins; this complex is also called cytochrome oxidoreductase. Cytochrome proteins have a prosthetic 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, uctuating between dierent oxidation states: Fe ++ (reduced) and Fe +++ (oxidized). The heme molecules in the cytochromes have slightly dierent characteristics due to the eects of the dierent proteins binding them, giving slightly dierent characteristics to each complex. Complex III pumps protons through the membrane and 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, cytochrome c can accept only one at a time). 3.4 Complex IV The fourth complex is composed of cytochrome proteins c, a, and a 3. This complex contains two heme groups (one in each of the two cytochromes, a, and a 3 ) and three copper ions (a pair of Cu A and one Cu B in cytochrome a 3 ). The cytochromes hold an oxygen molecule very tightly between the iron and copper ions until the oxygen is completely reduced. The reduced oxygen then picks up two hydrogen ions from the surrounding medium to make water (H 2 O). The removal of the hydrogen ions from the system contributes to the ion gradient used in the process of chemiosmosis. Helpful Links YouTubeElectron Transport Chain 2 YouTubeElectron Transport Chain # Chemiosmosis In chemiosmosis, the free energy from the series of redox reactions just described is used to pump hydrogen ions (protons) across the membrane. The uneven distribution of H + ions across the membrane establishes both concentration and electrical gradients (thus, an electrochemical gradient), owing to the hydrogen ions' positive charge and their aggregation on one side of the membrane

10 OpenStax-CNX module: m If the membrane were open to diusion by the hydrogen ions, the ions would tend to diuse back across into the matrix, driven by their electrochemical gradient. Many ions cannot diuse through the nonpolar regions of phospholipid membranes without the aid of ion channels. Similarly, hydrogen ions in the matrix space can only pass through the inner mitochondrial membrane through an integral membrane protein called ATP synthase (Figure 4). This complex protein acts as a tiny generator, turned by transfer of energy mediated 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 energy associated with the movement of protons down their electrochemical gradient to the endergonic addition of a phosphate to ADP, forming ATP. : Figure 4: ATP synthase is a complex, molecular machine that uses a proton (H + ) gradient to form ATP from ADP and inorganic phosphate (Pi). (Credit: modication of work by Klaus Homeier) note: Dinitrophenol (DNP) is a small chemical that serves to uncouple the ow of protons across the inner mitochondrial membrane to the ATP synthase and thus the synthesis of ATP. DNP makes the membrane leaky to protons. It was used until 1938 as a weight-loss drug. What eect would you expect DNP to have on the dierence in ph across both sides of the inner mitochondrial membrane? Why do you think this might be an eective 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 the process of photophosphorylation. Recall that the production of ATP using the process of chemiosmosis in

11 OpenStax-CNX module: m mitochondria is called oxidative phosphorylation. The overall result of these reactions is the production of ATP from the energy of the electrons removed from hydrogen atoms. These atoms were originally part of a glucose molecule. At the end of the pathway, the electrons are used to reduce an oxygen molecule to oxygen ions. The extra electrons on the oxygen attract hydrogen ions (protons) from the surrounding medium, and water is formed. Links How ATP is made from ATP synthase 4 : Figure 5: In oxidative phosphorylation, the ph gradient formed by the electron transport chain is used by ATP synthase to form ATP in a Gram - bacteria. note: Cyanide inhibits cytochrome c oxidase, a component of the electron transport chain. If cyanide poisoning occurs, would you expect the ph of the intermembrane space to increase or decrease? What eect 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 ETCs When we last discussed energy metabolism, it was in context of substrate level phosphorylation (SLP) and fermentation reactions. One of the questions in the Discussion points was what would be the consequences of SLP, both short-term and long-term to the environment? We discussed how cells would need to co-evolve mechanisms to remove protons from the cytosol (interior of the cell), which lead to the evolution of the F 0 F 1 ATPase, a multi-subunit enzyme that translocates protons from the inside of the cell to the outside of 4

12 OpenStax-CNX module: m the cell by hydrolyzing ATP as shown in gure 6 below. This arrangement works as long as small reduced organic molecules are freely available, making SLP and fermentation advantageous. As these biological process continue, the small reduced organic molecules begin to be used up and their concentration decreases, putting a demand on cells to be more ecient. One source of potential "ATP waste" is in the removal of protons 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 reactions as their energy source, as depicted in guire 7. In other words use the energy from a Red/Ox reaction to move protons. Such enzymes and enzyme complexes exist today in the form of the electron transport complexes, like Complex I, the NADH dehydrogenase. Figure 6: Proposed evolution of an ATP dependent proton translocator

13 OpenStax-CNX module: m Figure 7: As small reduced organic molecules become limiting organisms that can nd alternative mechanisms to remove protons from the cytosol may have had and advantage. The evolution of a proton translocator 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 translocate protons across the membrane, instead of an ATP driven proton pump. With protons being being translocated by Red/Ox reactions, this would now cause a build up of protons on the outside of the membrane, separating both charge (positive on the outside and negative on the inside; an electrical potential) and ph (low ph outside, higher ph inside). With excess protons on the outside of the cell membrane, and the F 0 F 1 ATPase no longer consuming ATP to translocate protons, the ph and charge gradients can be used to drive the F 0 F 1 ATPase "backwards"; that is to form or produce ATP by using the energy in the charge and ph gradients set up by the Red/Ox pumps as depicted in gure 8. This arrangement is called an electron transport chain (ETC).

14 OpenStax-CNX module: m Figure 8: The evolution of the ETC; the combination of the Red/Ox driven proton translocators coupled to the production of ATP by the F 0F 1ATPase.

15 OpenStax-CNX module: m Solutions to Exercises in this Module Solution to Exercise (p. 7)