Week 8 Lecture Biochemistry of Nutrition, 560B Dr. Charles Saladino The famous Krebs cycle is also known as the citric acid cycle and the tricarboxylic acid cycle. I will use the term Krebs cycle after Hans Krebs who most deservedly won the Nobel Prize for many important aspects of metabolism, including energy transformations and the urea cycle. Check out his extraordinary career on the internet. Many people incorrectly believe that it is the Krebs cycle itself that generates so much ATP. It, as a cycle alone generates no ATP. Rather, it is coupled to reactions that culminate in significant ATP formation. Overview When metabolism progressed from anaerobic to aerobic metabolism, a major increase in the ability to produce cellular energy in the form of ATP was realized. We see this wherein only two ATPs are formed in glycolysis for each glucose molecule oxidized (which occurs anaerobically in the cytosol), in contrast to the aerobic process of the Krebs cycle and those ATP-producing reactions to which it is coupled. Students often hear numbers like 36 ATPs formed in the Krebs cycle. However, by the end of the lecture, we shall have explored the Krebs cycle and have explained exactly how many ATPs are formed, where they are made, and the mechanisms by which ATP synthesis occurs. The remainder of this lecture will be devoted metabolic regulation germane to anaerobic vs. aerobic metabolism. Many consider the Krebs cycle the metabolic hub of cellular metabolism. Any molecule that can be converted into an acetyl group or a dicarboxylic acid could find its way into aerobic metabolism, which ultimately means amino acids (from proteolysis, the diet, or non-essential synthesis), many carbohydrates, and fatty acids from de novo synthesis, the diet, or triaglcerol breakdown This cycle is an important source of metabolic intermediates that can be utilized as storage forms of fuels, but also can serve as building blocks for amino acids, cholesterol, neurotransmitters, porphyrins, and nucleotides. We can say that fuels constitute those molecules that are oxidizable (lose electrons) carbon compounds. We can then look at the Krebs cycle as a series of oxidation-reduction reactions that oxidize two-carbon acetyl groups in the form of acetyl coenzyme A (acetyl CoA) by feeding them into that cycle where they are enzymatically oxidized to CO2. The energy that is released is preserved in the reduced electron carriers, NADH and FADH2. Finally, in the internal respiration stage (as opposed to extrernal respiration - breathing), these reduced coenzymes are themselves oxidized, releasing protons and electrons. In the end, the electrons are transferred to the final electron acceptor oxygen. During this electron transfer, we shall see a large amount of released energy preserved in the ATP molecule. This respiratory process is believed to have developed at a time much later than the less complex process of anaerobic glycolysis.

It is estimated that about 90% of all food-derived energy results from the oxidative process of the Krebs cycle in combination with oxidative phosphorylation. The Krebs cycle is diagramed with structures on page 554 of your text. Please refer to it as I outline some of the important features below that should be emphasized. You do not have to memorize all the steps, but you should understand the overall process, its significance, and some key steps that I might emphasize. Please examine the structures of the metabolites, especially those that I mention. First let us familiarize ourselves with one of the most important metabolites in the entire cell acetyl CoA. It is the fuel for the Krebs cycle and is formed from pyruvate, after that three-carbon glycolysis final product enters the mitochondria. Again, I need to clearly emphasize that, indirectly, through metabolic pathways, this critically-important molecule is eventually formed from the catabolism of glycogen (the storage form of glucose), amino acids, and lipids and alcohol (see page 552 of your text). Indeed, for example, when we study lipid metabolism, we shall see that fats contain polymers of two-carbon units that must first be oxidized to acetyl CoA and then completely oxidized to carbon dioxide by the Krebs cycle. The following is an important equation that illustrates the link between glycolysis and the Krebs cycle: The reduction of the coenzyme NAD+ to NADH takes place within the active site of the enzyme complex called pyruvate dehydrogenase, and the reaction is irreversible. Pyruvate + CoA + NAD+ ------------> acetyl CoA + CO2 + NADH If you look at Figure 14.15 of your text, we see that the preparation of the glucosederived pyruvate for the Krebs cycle involves an oxidative decarboxylation step, wherein high energy-level transfer electrons in the form of NADH are captured. Pyruvate dehydrogenase is a large oxidation-reduction enzyme complex utilizes the coenzyme NAD+. (Actually the enzyme is a huge multi-enzyme, multi-coenzyme complex, approximately 4600 kiloDaltons in size!) The reaction removes the carboxyl group from pyruvate to join that now-two-carbon fragment with CoA to produce the all-important acetyl CoA, as well as the CO2 by-product. The above reaction where acetyl CoA is formed is the critical one that can start the entire Krebs cycle. Further, that reaction is irreversible, and it occurs only after glycoysis has produced pyruvate in the cytosol, after which pyruvate is transported to the mitochondrial matrix where it is converted to acetyl CoA by the pyruvate dehydrogenase enzyme cpmplex and also where the Krebs cycle takes place. If you look at the Krebs cycle in the text, you get the definite and correct impression that this cycle turns in only one direction (clockwise, as per the text). One email assignment for this week: Why does it turn in one direction only? With the availability of acetyl CoA, this two carbon compound (two-carbon meaning

counting the two carbons attached to the CoA part) begins the cycle by condensing with a four carbon compound, oxaloacetate (OAA). Familiarize yourself with this name and structure.. (Remember the transamination of say, aspartate. The aspartate combines with -ketoglutarate to form glutamate and OAA?) You will hear about OAA again in gluconeogenesis. Anyway, this condensation forms a 6-carbon citrate molecule, which is isomerized to isocitrate. Now we will start to see the use of an important set of oxidation-reduction enzymes specifically dehydrogenases, three of which use the coenzyme NAD+, and one of which uses FAD+, by the time the Krebs cycle is complete. You see 6-carbon isocitrate converted to 5-carbon -ketoglutarate by decarboxylation (the removal of CO2). This is catalyzyed by isocitrate dehydrogenase. (You should remember -ketoglutarate from transamination.) The -ketoglutarate is then converted into 4-carbon succinyl CoA by another decarboxylation, catalyzed by an -ketoglutarate dehydrogenase enzyme complex. The CoA is then removed to form succinate. Please remember for the next lecture that in this step, as the CoA is removed, sufficient energy is given off so as to allow the formation of GTP from GDP. Then succinate dehydrogenase, utilizing the coenzyme FAD, converts succinate to fumarate, which rearranges (isomerizes) to malate. Catalyzed by malate dehydrogenase, malate is oxidized to regenerate OAA. That completes the cycle. We should note that in this last step where OAA is formed, the free energy for this reaction, unlike the other steps in the cycle, is significantly positive. However, the oxidation of malate is driven the use of the products (OAA by citrate synthase see step #1 in your text pg 554, as well as NADH by the electron transport system [ETS]). The ETS will be discussed in detail later in the lecture. Let us resynthesize a little of what we have said here: 1. Two carbon atoms enter the cycle and condense with OAA. Two carbons leave the cycle in the form of CO2 in successive decarboxylations, both catalyzed by dehydrogenations. 2. Four pairs of hydrogen atoms leave the cycle in four oxidation reactions, three utilizing the coenzyme NAD+ and one utilizing the coenzyme FAD. 3. One molecule with a high phosphoryl potential (high energy potential) GTP is formed by cleaving the thioester (S) linkage in succinyl CoA. 4. Two molecules of water are consumed one by the synthesis of citrate and one by the hydration of fumarate. We should note and of real significance that NADH is generated in the formation of acetyl CoA from pyruvate. This is important in accounting for every ATP formed from the total oxidation of glucose. So please keep on the back burner of your mind that 3 ATP molecules are produced as a result of this one step. If you think back, that is already more ATP than that we net from all of glycolysis. . We must also note the Krebs cycle operates only under aerobic conditions, and within the

mitochondria, even though molecular oxygen does not directly participate in this metabolic cycle. This will be explained next time as having to do with the fact that NAD+ and FAD can be regenerated within the mitochondria only by transferring electrons to molecular oxygen. We know glycolysis does proceed anaerobically, and the NAD+ can be regenerated when pyruvate is converted into lactate. In contrast, as we shall see shortly, in association with the Krebs cycle, NAD+ is generated within with mitochondria in association with the electron transport system. ATP formation (oxidative phosphorylation) Now, when we say that ATP is formed in conjunction with any step of the Krebs cycle or this reaction we just described, let us clearly understand that these oxidation-reduction steps are COUPLED to what we will call the electron transport system (ETS), which, in turn, is coupled to ATP formation, which we term oxidative phosphorylation (OP). It is now our job to explain exactly how this all occurs. Further, what we will describe will be compartmentalized to the mitochondria, with the Krebs cycle and OP actually subcompartmentalized to the matrix and the inner mitochondrial membrane, respectively. A description of any one of the three steps where a dehydrogenase utilizes the coenzyme, NAD+, will explain the other two reactions where NAD+ is utilized. At each of these three steps, 3 ATP will be formed. If you look at the figure, you will note that NAD+ is converted to NADH + a proton (H+). What is actually happening is that a hydride ion is being transferred from the substrate to the NAD+ coenzyme that is sitting in the active site of the dehydrogenase enzyme. Remember a hydride ion is a hydrogen atom with an extra electron (i.e. H-). That is why the second hydrogen removed from the substrate is in the form of a proton that is free to float away to serve some other purpose. The NAD+ is now NADH, the reduced form of the coenzyme. In the case where FAD is reduced to FADH2, both hydrogens obtained from the substrate, succinate, are transferred to the coenzyme as it becomes reduced. In fact, look at that step in the figure, and notice that the double bond of fumarate arose because of two hydrogens being removed (thus, a dehydrogenation reaction). This is why I said in the last lecture that in the Krebs cycle, four pairs of hydrogen atoms leave the cycle in four oxidation reactions (one pair from each dehydrogenation reaction that utilizes NAD+ and one that utilizes FAD). This all should be understood, not just memorized. I now refer you to Figure 14.40 of your text. This is a crude diagram of the electron transport system (ETS). Notice that each of its components is embedded to some degree within the inner mitochondrial membrane. Their order is critical to the function of the ETS. Of course, this arrangement is repeated many, many times along the membrane. Further, these constituents must be in close enough proximity to one another to accomplish electron transfer down the line, until those electrons reach oxygen, the final electron acceptor. Please notice that I said close enough to transfer electrons not touching, as that is not necessary. Why? Electrons can move through space, with that electron transfer decreasing

by a factor of 10 for each 0.8 Angstrom unit of separation. By comparison, for groups in contact, electron transfer reactions can be as fast as 1013 per second! The protein environment of the ETS helps the separation problem, and typically, electron-carrying groups are separated by about 15 Angstroms (1Ao = 10-8 cm) to allow for a transfer rate of about 104 electrons per second, all other factors being optimal. Not bad! However, without the proteins that are in the ETS, such a transfer of electrons would take all day literally. So why am I giving you this kind of detail. Well, in the beginning of the course, I told you that I was awe-struck by the design of how everything works, and that it works so well at all. This is what is on my mind, as I try to impart to you, not only the facts, but also the level of complexity we are witnessing here. At this point, before describing the ETS in some more detail, I would like to introduce a diagram whose picture is worth a thousand words. It is an electron energy diagram which shows the other determinant (besides distance) of electrons moving down ETS. If you look at the figure below, you will quickly notice the obvious. That is, the electrons are moving along the ETS, because they are moving from a higher to a lower energy state. You notice that oxygen is the final electron acceptor in the ETS, wherein it is used in the formation of water. This is one if not the most important reason why we breathe oxygen. It is the final electron acceptor in the ETS, because is has about the highest of what we call the standard reduction potential, meaning the ability to attract electrons or be reduced.

This utilization of oxygen is what is known as internal respiration or cellular respiration. It is primarily the aerobic part of metabolism, no matter what pathway (protein, carbohydrate or lipid oxidation) leads to the formation of acetyl CoA and the subsequent Krebs cycle. We can now examine the components of the electron transport system briefly. This ETS consists of four compartmentalized complexes: three proton pumps and a physical link to the Krebs cycle. Again electrons will make their way down the chain, because of the difference in reduction energy states demonstrated in the figure above. I t would not be a bad idea to read Section 14.7 in your text.

Lets now step back a few thoughts to where the dehydrogenase enzymes utilizing the coenzyme NAD+ were reduced to form NADH. The dehydrogenase is part of a very large molecular complex (880kD) consisting of at least 34 polypeptide chains. The electrons of NADH enter the ETS at complex I, which shares coenzyme Q (CoQ) with complexes II and III. Coenzyme Q is a quinone derivative (also called ubiquinone, because it is ubiquitous in biological systems). It can accept hydrogen atoms and electrons from both FADH2 and FMNH2 . Notice that the flavin mononucleotide (FMN) (a riboflavin derivative) is the first component to receive the electrons from the NADH, which then reoxidizes to NAD+ . Also notice the FeS-containing component in complex II. This is actually a non-heme Fe and S-containing cluster. Complex III is interesting, because it contains a series of cytochrome proteins, each containing a heme group containing Fe. The S-Fe cytochrome is not heme-containing. Please refer to pages 569 and 570 for more detail, if you wish, but you are not responsible for this detail. So anyway folks, with regard to the heme cytochromes, unlike hemoglobin, the cytochrome Fe is reversibly converted from the ferric (Fe+++) to the ferrous (Fe++) form during the reversible oxidation-reduction functions of the ETS. Maybe here I should emphasize this point. The entire ETS functions as a series of reversible oxidationreduction steps, as the electrons make their way from the first point where NADH contacts the ETS until those electrons reach oxygen, having just been passed from cytochrome a-a3. This a-a3 cytochrome protein is the only electron carrier wherein has a free ligand that can interact with oxygen. This cytochrome a-a3 ia also known as cytochrome oxidase (at complex IV in your text figures) and represents the site where oxygen, free protons, and electrons come together to form water, a by-product of internal respiration. The oxidase contains copper atoms that are part of the catalysis needed for the water-formation reaction to occur. Now you have a detailed explanation for one of the main reasons why we breathe oxygen into the lungs in the first place, that reason being, so that oxygen can act as an electron sink for electrons traveling down the ETS. Before we now go on to couple the ETS to oxidative phosphorylation (OP) where ATP is formed, lets briefly examine a few inhibitors of the ETS, which as you will understand shortly, also blocks OP. These inhibitors act by blocking electron transfer site-specifically. For example, rotenone prevents electron transfer between FMN and CoQ, whereas antimycin blocks electron passage between cytochrome b and cytochrome c. Interestingly, cyaninde ion, carbon monoxide, and sodium azide each prevent the final transfer of electrons to oxygen. What is really happening, therefore, is that all electron carriers before the block remain in the fully reduced state. Do you understand why? So far, we have turned a complete the Krebs cycle, wherein four dehydrogenase reactions have transferred hydrogens and electrons to the ETS. Compounds with a large Eo (located at the beginning of the ETS) are strong reducing agents. They have a substantial tendency to lose electrons, and, therefore, pass them along to the next compound that has a more positive Eo value, because that receiver of the electrons is in the oxidized state.

What now becomes important for us to recognize is that there is a free energy change that is directly related to the size in the change of Eo. Overall, the free energy change that comes from hydrolyzing the end phosphate from ATP is -7300 calories/mole. That is substantial! That means to created ATP from ADP, we need 7300 cal/mol worth of work available. Where does that work come from? The answer is the free energy change that comes from transferring a pair of electrons from NADH down the ETS to oxygen. This total free energy change is about 52, 500 calories, which is more than enough to form three ATP from three ADP (7300 x 3 = 21, 900 cal). The remaining calories are lost as heat, with about 40% of the energy drop from the ETS being used to make 3 ATP. So this translates into three ATP produced at each step in the Krebs cyclewhere NADH is the cofactor for the dehydrogenase enzyme. We have noted previously that where FAD is the dehydrogenase cofactor, only two (not three) ATP are produced. Why? If you look at the schematic of the ETS on page 571, you will see that FAD is located at complex two and thus enters the ETS at that point. Certainly, then, less free energy derived from the ETS by starting at that point. Why does this ultimately matter? The answer lies is the chemiosmotic hypothesis (Mitchell hypothesis), which is well accepted, and which is based upon coupling of the ETS to OP -ATP formation. Normally, the inner mitochondrial membrane where the ETS is located is impermeable to protons, which are made available in the colloidal matrix of the mitochondria from various reactions, including those involving the dehydrogenase enzymes. There are, however, three proton pumps which are located at complexes I, III, IV (see your text Figure). Because of the free energy changes at these complexes, protons are delivered across the inner mitochondrial membrane to the inter-membrane space (between the two mitochondrial membranes). This is an active transport process which allows protons to accumulate at a higher concentration within that space, vs. that of the matrix (located on in the inner side of the inner membrane). Thus, an electrical gradient is developed across the inner membrane (because of the charge differential). Actually, you also have a pH gradient, because pH is determined by the concentration of protons. Another way of saying it that you have developed a protomotive force by accumulating proteins into the space between the inner and outer mitochondrial membranes, such that the protons would like o get back to the matrix side of the inner mitochondrial membrane. Now next to multi-subunit complex IV is a enzyme complex known as ATP synthase, what we can certainly call the worlds smallest motor. It contains a catalytic unit and a proton-conducting unit, each composed of multiple subunits. The complex looks almost like a ball on a stick. The ball is called the F1 subunit, protrudes into the mitochondrial matrix, and contains the catalytic activity of the synthase enzyme. The Fo subunit is like the stick and spans the inner mitochondrial membrane. I have included a diagram below to help you visualize this. However, picture two functional units: a moving unit or rotor, and a stationary unit or stalk. What will move will be the membrane spanning stick (Fo unit).

The bottom line of what happens is that the only opportunity for the protons to return to the matrix (due to the proton concentration differential), is by their flowing through the ATP synthase. This will lead to the release of an ATP form the complex, according to the following reaction: ADP3- + HPO42- + H+ ATP4- - + H2O For simplicity sake, think of ADP plus a phosphate forming ATP. The actual binding to the ADP + phosphate to form ATP is facilitated by and requires a 120 degree counterclockwise rotation of the rotor. Yep! This has been proven. As far as I know, this is the worlds smallest molecular motor! What an incredible design. Finally we ask one more question. What powers the ATP synthesis? The answer is, apparently, the proton flow around the ring inside the stalk, as the protons are returning to the matrix. ADP/ATP Obviously, all the ATP formed in the mitochondrial world does no good if it is not made available to systems outside that organelle that require ATP to drive their reactions (as in glycogen formation, glucose-6-P formation etc.). Further, to engage in OP, we need a supply of ADP coming into the mitochondria. In simple terms, these needs are met by an adenine nucleotide translocase, which moves these highly charged molecules across the inner mitochondrial membrane, in order to traverse this permeability barrier. This translocase is what we call in biochemistry an antiporter, because it transports two molecules, each molecule in opposite directions. For historical reasons, such translocases are also called carriers. These translocases are very abundant, comprising about 14% of the protein in the inner mitochondrial membrane. There are many shuttles involved in moving molecules in and out of the mitochondria. You are responsible for reading pages 579 and 580 (for the next exam) on the glycerol phosphate shuttle. This contributes to the overall ATP tally in the complete oxidation of glucose.

The GTP-Generating Step Just a brief reminder: Remember the Krebs cycle step (succinyl CoA to succinate) wherein GTP was generated from GDP? The energy was supplied from the removal of the CoA. I also mentioned that the formation of one ATP is coupled to the GTP produced. Thus: GTP + ADP --------> ATP + GDP ATP Tally: I will tabulate the total possible ATP formation, assuming one glucose molecule is completely oxidized. Just remember that one glucose produces two pyruvate molecules, thus two acetyl CoA, thus two turns of the Krebs cycle (Any questions, let me know.): Net ATP Production: Glycolysis 2 Pyruvate to 2 Acetyl CoA Krebs cycle: 3 x 3 NAD+ 1 x 2 FAD 1 x 1 GTP 2 ATP (2 x 3 NAD+) 6 ATP 24 ATP =9 =2 =1 12 (x 2 turns of cycle via 2 pyruvate = 24) 4 ATP 36 ATP

Glycerol phosphate shuttle = 4 (approx) Total

Thus, we have a total of approximately 36 ATP molecules formed because of the complete oxidation of one glucose molecule. Of course, we remember that there are many feeder reactions into glycolysis and major side reactions, such as the pentose phosphate shunt. We can conclude quite safely, however, that then we look an anaerobic metabolism of carbohydrates, the addition of aerobic metabolism to the metabolic pathways greatly increases energy production in those cells that indeed have mitochondria. Certainly, a cell, such as the erythrocyte, that has no mitochondria depends solely on anaerobic metabolism. However, that makes sense when we think about it. The red blood cell exists primarily for oxygen transport. That mature cell does not exist to synthesize any major products. It is simply a transporter, requiring some only glucose sent to it from the liver. On the other hand, there are a large number of mitochondria (and hence aerobic metabolism) in cardiac and skeletal muscle, for what I am sure are obvious reasons. OK?

Uncoupling the ETS and OP I have already mentioned some specific ETS blocking agents which do not allow the electrons to proceed all the way through that ETS. This uncouples the ETS from OP, because there is not sufficient energy to drive the chemiosmotic proton pump. There are other uncoupling agents, such as the class 2,4-dinitrophenol (2,4-DNP). This lipophilic proton carrier, which readily diffuses through the inner mitochondrial membrane, increases the permeability of that membrane. Therefore, whereas it allows the ETS to function at a rapid rate, it does so without allowing the proton gradient to be established. The energy released from the ETS is dissipated solely as heat, instead of being used to synthesize ATP. Also, and interestingly, at high doses, aspirin can uncouple OP. This would explain the fever that accompanies toxic aspirin overdoses!