13.17: S2023_Bis2A_Singer_Electron_Transport_Chains

Electron Transport Chains

An electron transport chain, or ETC, is composed of a group of protein complexes in and around a membrane that help energetically couple a series of exergonic/spontaneous red/ox reactions to the endergonic pumping of protons across the membrane to generate an electrochemical gradient. This electrochemical gradient creates a free energy potential that we call a proton motive force whose energetically "downhill" exergonic flow can later be coupled to a variety of cellular processes.

ETC overview

Step 1: Electrons enter the ETC from an electron donor, such as NADH or FADH₂, which are generated during a variety of catabolic reactions, including those associated glucose oxidation. Depending on the 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. Entry of electrons at a specific "spot" in the ETC depends upon the respective reduction potentials of the electron donors and acceptors.

Step 2: After the first red/ox reaction, the initial electron donor will become oxidized and the electron acceptor will become reduced. The difference in red/ox potential between the electron acceptor and donor is related to ΔG by the relationship ΔG = -nFΔE, where n = the number of electrons transferred and F = Faraday's constant. The larger a positive ΔE, the more exergonic the red/ox reaction is.

Step 3: If sufficient energy is transferred during an exergonic red/ox 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 usually multiple red/ox transfers, the electron is delivered to a molecule known as the terminal electron acceptor. With humans, the terminal electron acceptor is oxygen. However, there are many, many, many other possible electron acceptors in nature; see below.

Note: NADH AND FADH₂ ARE NOT THE ONLY ELECTRON DONORS

Electrons entering the ETC do not have to come from NADH or FADH₂. Many other compounds can serve as electron donors; the only requirements are (1) that there is an enzyme that can oxidize the electron donor and then reduce another compound, and (2) that the ∆E₀' is positive (e.g., ΔG<0). Even a small amount of free energy transfers can add up. For example, there are bacteria that use H₂ as an electron donor. This is not too difficult 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 ΔE₀' of the reaction is 1.24 V, which corresponds to a large negative ΔG (-ΔG). Alternatively, there are some bacteria that can oxidize iron, Fe²⁺ at pH 7 to Fe³⁺ with a reduction potential (E₀') of + 0.2 V. These bacteria use oxygen as their terminal electron acceptor, and, in this case, the ΔE₀' of the reaction is approximately 0.62 V. This still produces a -ΔG. 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 comprise 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 final terminal electron acceptor, such as oxygen. This specific 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 itself. 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 multi-protein 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. 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.

 

Note

This use of prosthetic groups by members of ETC 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. Both the Quinone_(red) and the Quinone_(ox) forms of these lipids are soluble within the membrane and can move from complex to complex to shuttle electrons.

 

The electron and proton carriers

• Flavoproteins (Fp), these proteins contain an organic prosthetic group called a flavin, which is the actual moiety that undergoes the oxidation/reduction reaction. FADH₂ is an example of an Fp.
• Quinones are a family of lipids, which means they are soluble within the membrane.
• We also note that we consider NADH and NADPH electron (2e^-) and proton (2 H⁺) carriers.

Electron carriers

• Cytochromes are proteins that contain a heme prosthetic group. The heme can carry a single electron.
• Iron-Sulfur proteins contain a nonheme iron-sulfur cluster that can carry an electron. We often abbreviate the prosthetic group as Fe-S

Introduction to Mobile Energy Carriers

Section Summary

Energy moves and transfers within the cell in a variety of ways. One critical mechanism that nature has developed is the use of recyclable molecular energy carriers. While there are several major recyclable energy carriers, they all share some common functional features:

Properties of Key Cellular Molecular Energy Carriers

• We think of the energy carriers as existing in "pools" of available carriers. One could, by analogy, consider these mobile energy carriers analogous to the delivery vehicles of parcel carriers—the company has a certain "pool" of available vehicles at any one time to pickup and make deliveries.
• Each individual carrier in the pool can exist in one of multiple distinct states: it is either carrying a "load" of energy, a fractional load, or is "empty". The molecule can interconvert between "loaded" and empty and thus can be recycled. Again by analogy, the delivery vehicles can be either carrying packages or be empty and switch between these states.
• The balance or ratio in the pool between "loaded" and "unloaded" carriers is important for cellular function, is regulated by the cell, and can often tell us something about the state of a cell. Likewise, a parcel carrier service keeps close tabs on how full or empty their delivery vehicles are—if they are too full, there may be insufficient "empty" trucks to pick up new packages; if they are too empty, business must not be going well or they shut it down. There is an appropriate balance for different situations.

In this course, we will examine two major types of molecular recyclable energy carriers: (1) the adenine nucleotides: nicotinamide adenine dinucleotide (NAD⁺), a close relative, nicotinamide adenine dinucleotide phosphate (NADP⁺), and flavin adenine dinucleotide (FAD₂⁺) and (2) nucleotide mono-, di-, and triphosphates, with particular attention paid to adenosine triphosphate (ATP). These two types of molecules participate in a variety of energy transfer reactions. We primarily associate adenine nucleotides with red/ox chemistry, and nucleotide triphosphates with energy transfers linked to the hydrolysis or condensation of inorganic phosphates.

 

Red/ox chemistry and electron carriers

The oxidation of, or removal of an electron from, a molecule (whether accompanied with the removal of an accompanying proton or not) results in a change of free energy for that molecule—matter, internal energy, and entropy have all changed. Likewise, the reduction of a molecule also changes its free energy. The magnitude of change in free energy and its direction (positive or negative) for a red/ox reaction dictates the spontaneity of the reaction and how much energy it transfers. In biological systems, where a great deal of energy transfer happens via red/ox reactions, it is important to understand how these reactions are mediated and to consider ideas or hypotheses for why these reactions are mediated most times by a small family of electron carriers.

Note: BURNING BEAR

Relate the burning of (the full oxidation of the sugar in) a gummy bear with the last paragraph above. What does that demonstration have to do with our upcoming discussion on red/ox carriers? There is some mention above already—can you find it?

Note: DESIGN CHALLENGE

The problem alluded to in the previous discussion question is a great place to bring in the design challenge rubric. If you recall, the first step of the rubric asks that you define a problem or question. Here, let's imagine that there is a problem to define for which the mobile electron carriers below helped Nature solve.

***Remember, evolution DOES NOT forward-engineer solutions to problems, but in retrospect, we can use our imagination and logic to infer that what we see preserved by natural selection provided a selective advantage, because the natural innovation "solved" a problem that limited success.***

Design challenge for red/ox carriers

• What was a problem(s) that the evolution of mobile electron carriers helped solve?
• The next step of the design challenge asks you to identify criteria for successful solutions. What are criteria for success in the problem you've identified?
• Step 3 in the design challenge asks you to identify solutions. Well, here Nature has identified some for us—we consider three in the reading below. It looks like Nature is happy to have multiple solutions to the problem.
• The penultimate step of the design challenge rubric asks you to test the proposed solutions against the criteria for success. This should make you think/discuss why there are multiple different electron carriers. Are there different criteria for success? Are they each solving slightly different problems? What do you think? Be on the lookout as we go through metabolism for clues.

NAD⁺/H and FADH/H₂

In living systems, a small class of compounds function as electron shuttles: they bind and carry electrons between compounds in different metabolic pathways. The principal electron carriers we will consider derive from the B vitamin group and nucleotides. These compounds can both become reduced (that is, they accept electrons) or oxidized (they lose electrons) depending on the reduction potential of a potential electron donor or acceptor that they might transfer electrons to and from. Nicotinamide adenine dinucleotide (NAD⁺) (we show the structure below) derives from vitamin B₃, niacin. NAD⁺ is the oxidized form of the molecule; NADH is the reduced form of the molecule after it has accepted two electrons and a proton (which together are the equivalent of a hydrogen atom with an extra electron).

We are expecting you to memorize the two forms of NAD⁺/NADH, know which form is oxidized and which is reduced, and be able to recognize either form on the spot in a chemical reaction.

NAD⁺ can accept electrons from an organic molecule according to the general equation:

Here is some vocabulary review: when electrons are added to a compound, we say the compound has been reduced. A compound that reduces (donates electrons to) another is called a reducing agent. In the above equation, RH is a reducing agent, and NAD⁺ becomes reduced to NADH. When electrons leave a compound, it becomes oxidized. A compound that oxidizes another is called an oxidizing agent. In the above equation, NAD⁺ is an oxidizing agent, and RH is oxidized to R. Put another way, the reducing agent gets oxidized and the oxidizing agent gets reduced.

You need to get this down! We will (a) test specifically on your ability to do so (as "easy" questions), and (b) we will use the terms with the expectation that you know what they mean and can relate them to biochemical reactions correctly (in class and on tests).

You will also encounter a second variation of NAD⁺, NADP⁺. It is structurally very similar to NAD⁺, but it contains an extra phosphate group and plays an important role in anabolic reactions, such as photosynthesis. Another nucleotide-based electron carrier that you will also encounter in this course and beyond, flavin adenine dinucleotide (FAD⁺), derives from vitamin B₂, also called riboflavin. Its reduced form is FADH₂. Learn to recognize these molecules as electron carriers.

Figure 1. The oxidized form of the electron carrier (NAD⁺) is shown on the left, and the reduced form (NADH) is shown on the right. The nitrogenous base in NADH has one more hydrogen ion and two more electrons than in NAD⁺.

The cell uses NAD+ to "pull" electrons off of compounds and to "carry" them to other locations within the cell; thus we call it an electron carrier.  Many metabolic processes we will discuss in this class involve NAD(P)⁺/H compounds. For example, in its oxidized form, NAD⁺ is used as a reactant in glycolysis and the TCA cycle, whereas in its reduced form (NADH), it is a reactant in fermentation reactions and electron transport chains (ETC). We will discuss each of these processes in later modules.

Energy story for a red/ox reaction

***As a rule of thumb, when we see NAD+/H as a reactant or product, we know we are looking at a red/ox reaction.***

When NADH is a product and NAD⁺ is a reactant, we know that NAD⁺ has become reduced (forming NADH); therefore, the other reactant must have been the electron donor and become oxidized. The reverse is also true. If NADH has become NAD⁺, then the other reactant must have gained the electron from NADH and become reduced.

Figure 2. This reaction shows the conversion of pyruvate to lactic acid coupled with the conversion of NADH to NAD⁺. Source: https://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/sequential_reactions

In the figure above, we see pyruvate becoming lactic acid, coupled with the conversion of NADH into NAD⁺. LDH catalyses this reaction. Using our "rule of thumb" above, we categorize this reaction as a red/ox reaction. NADH is the reduced form of the electron carrier, and NADH is converted into NAD⁺. This half of the reaction results in the oxidation of the electron carrier. Pyruvate is converted into lactic acid in this reaction. Both sugars are negatively charged, so it would be difficult to see which compound is more reduced using the charges of the compounds. However, we know that pyruvate has become reduced to form lactic acid, because this conversion is coupled to the oxidation of NADH into NAD⁺. But how can we tell that lactic acid is more reduced than pyruvate? The answer is to look at the carbon-hydrogen bonds in both compounds. As electrons transfer, they are often accompanied by a hydrogen atom. Pyruvate has a total of three C-H bonds, while lactic acid has four C-H bonds. When we compare these two compounds in the before and after states, we see that lactic acid has one more C-H bond; therefore, lactic acid is more reduced than pyruvate. This holds true for multiple compounds. For example, in the figure below, you should be able to rank the compounds from most to least reduced using the C-H bonds as your guide.

Figure 3. Above are a series of compounds than can be ranked or reorganized from most to least reduced. Compare the number of C-H bonds in each compound. Carbon dioxide has no C-H bonds and is the most oxidized form of carbon we will discuss in this class. Answer: the most reduced is methane (compound 3), then methanol (4), formaldehyde (1), carboxylic acid (2), and finally carbon dioxide (5).

 

Figure 4. This reaction shows the conversion of G3P, NAD⁺, and P_i into NADH and 1,3-BPG. This reaction is catalyzed by glyceraldehyde-3-phosphate dehydrogenase.

Energy story for the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase:

Lets make an energy story for the reaction above.

First, let us characterize the reactants and products. The reactants are glyceraldehyde-3-phosphate (a carbon compound), P_i (inorganic phosphate), and NAD⁺. These three reactants enter a chemical reaction to produce two products, NADH and 1,3-bisphosphoglycerate. If you look closely, you can see that the 1,3-BPG contains two phosphates. This is important since a chemical reaction should lose no mass between its beginning and its end. There are two phosphates in the reactants, so there must be two phosphates in the products (conservation of mass!). You can double check the book keeping of mass for all other atoms. It should also tabulate correctly. An enzyme called glyceraldehyde-3-phosphate dehydrogenasethat catalyzes this reaction. The standard free energy change of this reaction is ~6.3 kJ/mol, so under standard conditions, we can say that the free energy of the products is higher than that of the reactants and that this reaction is not spontaneous under standard conditions.

What can we say about this reaction when it is catalyzed by glyceraldehyde-3-phosphate dehydrogenase?

This is a red/ox reaction. We know that because we have produced a reduced electron carrier (NADH) as a product and NAD⁺ is a reactant. Where did the electron come from to make NADH? The electron must have come from the other reactant (the carbon compound).

 

Aerobic versus anaerobic respiration

We humans use oxygen as the terminal electron acceptor for the ETCs in our cells. This is also the case for many of the organisms we intentionally and frequently interact with (e.g. our classmates, pets, food animals, etc). We breathe in oxygen; Our cells take it up and transport it into the mitochondria, where it becomes the final acceptor of electrons from our electron transport chains. We call the process where oxygen is the terminal electron acceptor aerobic respiration.

While we may use oxygen as the terminal electron acceptor for our respiratory chains, this is not the only mode of respiration on the planet. The more general processes of respiration evolved when oxygen was not a major component of the atmosphere. As a result, many organisms can use a variety of compounds, including nitrate (NO₃^-), nitrite (NO₂^-), even iron (Fe₃⁺) as terminal electron acceptors. When oxygen is NOT the terminal electron acceptor, we refer the process to as anaerobic respiration. Therefore, respiration or oxidative phosphorylation does not require oxygen at all; It requires a compound with a high enough reduction potential to act as a terminal electron acceptor, accepting electrons from one complex within the ETC.

The ability of some organisms to vary their terminal electron acceptor provides metabolic flexibility 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 other organisms can use a different terminal electron acceptor when conditions change to survive.

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Possible NB Discussion Point

Nature has figured out how to use different molecules as terminal electron acceptors of ETCs.  Yet humans seem limited to using only oxygen. Can you offer any hypotheses why humans have not evolved to use multiple different terminal electron acceptors?  Why do you think it might be advantageous for an organism to use oxygen as a sole terminal electron acceptor?

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A generic example: A simple, two-complex ETC

The figure below depicts 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 FADH₂) reduces Complex I_(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 red/ox reaction is exergonic and the free energy difference 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, because of an excess of hydroxyl ions (OH^-), and the other side becomes positively charged because of an increase in protons on the other side. Complex I_(red) can now reduce a mobile electron carrier Q, which will then move through the membrane and transfer the electron(s) to the prosthetic group of Complex II_(red). Electrons pass from Complex I to Q then from Q to Complex II via thermodynamically spontaneous red/ox reactions, regenerating Complex I_(ox), which can repeat the previous process. Complex II_(red) then reduces A, the terminal electron acceptor to regenerate Complex II_(ox) and create the reduced form of the terminal electron acceptor, AH. In this specific example, Complex II can also translocate a proton during the process. If A is molecular oxygen, AH represents water and the process would be considered being a model of an aerobic ETC. If A is nitrate, NO₃^-, then AH represents NO₂^- (nitrite) and this would be an example of an anaerobic ETC.

 

 

Figure 1. Generic 2 complex electron transport chain. In the figure, DH is the electron donor (donor reduced), and D is the donor oxidized. A is the oxidized terminal electron acceptor, and AH is the final 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.

Attribution: Marc T. Facciotti (original work)

Detailed look at aerobic respiration

The eukaryotic mitochondria have evolved a very efficient ETC. There are four complexes composed of proteins, labeled I through IV depicted in the figure below. The aggregation of these four complexes, together with associated mobile, accessory electron carriers, is called an electron transport chain. This electron transport chain is present in multiple copies in the inner mitochondrial membrane of eukaryotes.

Figure 2. The electron transport chain is a series of electron transporters embedded in the inner mitochondrial membrane that shuttles electrons from NADH and FADH₂ to molecular oxygen. In the process, protons are pumped from the mitochondrial matrix to the intermembrane space, and oxygen is reduced to form water.

Complex I

To start, NADH delivers two electrons to the first protein complex. This complex, labeled I in Figure 2, includes flavin mononucleotide (FMN) and iron-sulfur (Fe-S)-containing proteins. FMN, which is derived from vitamin B₂, also called riboflavin, is one of several prosthetic groups or cofactors in the electron transport chain. Prosthetic groups are organic or inorganic, nonpeptide molecules bound to a protein that facilitate its function; prosthetic groups include coenzymes, which are the prosthetic groups of enzymes. We also call the enzyme in Complex I NADH dehydrogenase. This protein complex contains 45 individual polypeptide chains. Complex I can pump four hydrogen ions across the membrane from the matrix into the intermembrane space helping to generate and maintain a hydrogen ion gradient between the two compartments separated by the inner mitochondrial membrane.

Q and Complex II

Complex II directly receives FADH₂, which does not pass through Complex I. The compound connecting the first 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 reduced, (QH₂), 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 FADH₂ from Complex II, succinate dehydrogenase. Since these electrons bypass and thus do not energize the proton pump in the first complex, fewer ATP molecules are made from the FADH₂ 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.

Complex III

The third complex is composed of cytochrome b, another Fe-S protein, Rieske center (2Fe-2S center), and cytochrome c proteins; we also call this complex cytochrome oxidoreductase. Cytochrome proteins have a prosthetic group of heme. The heme molecule is like 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, fluctuating between different oxidation states: Fe²⁺ (reduced) and Fe³⁺ (oxidized). The heme molecules in the cytochromes have slightly different characteristics because of the effects of the different proteins binding them, giving slightly different 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).

Complex IV

The fourth complex is composed of cytochrome proteins c, a, and a3. This complex contains two heme groups (one in each of the two Cytochromes, a, and a3) and three copper ions (a pair of CuA and one CuB in Cytochrome a3). The cytochromes hold an oxygen molecule tightly between the iron and copper ions until it completely reduces the oxygen. The reduced oxygen then picks up two hydrogen ions from the surrounding medium to make water (H₂O). The removal of the hydrogen ions from the system contributes to the ion gradient used in the process of chemiosmosis.

Chemiosmosis

In chemiosmosis, the free energy from the series of red/ox reactions just described is used to pump 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 proton's positive charge and their aggregation on one side of the membrane.

If the membrane were open to diffusion by protons, the ions would tend to diffuse back across into the matrix, driven by their electrochemical gradient. Ions, however, cannot diffuse through the nonpolar regions of phospholipid membranes without the aid of ion channels. Similarly, protons in the intermembrane space can only traverse the inner mitochondrial membrane through an integral membrane protein called ATP synthase (depicted below). 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 3. ATP synthase is a complex, molecular machine that uses a proton (H⁺) gradient to form ATP from ADP and inorganic phosphate (Pi).

Credit: modification of work by Klaus Hoffmeier

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Possible NB Discussion Point

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 effect would cyanide have on ATP synthesis? How would this affect the rates of reactions in glycolysis and the TCA cycle?

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In healthy cells, chemiosmosis (depicted below) 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 mitochondria is called oxidative phosphorylation and that a similar process can occur in the membranes of bacterial and archaeal cells. The overall result of these reactions is the production of ATP from the energy of the electrons removed originally from a reduced organic molecule like glucose. In the aerobic example, these electrons ultimately reduce oxygen and create water.

Figure 4. In oxidative phosphorylation, the pH gradient formed by the electron transport chain is used by ATP synthase to form ATP in a Gram-bacteria.

 

Helpful link: How ATP is made from ATP synthase