Dr. Michelle Pozzi: So in the second part of this lecture, I wanted to go back and give more details on pyruvate dehydrogenase complex. So as a reminder, the pyruvate dehydrogenase complex is composed of three different enzymes that I will tell you the names of in just a second, but we'll call them E one, E two, E three. And these three enzymes function together, utilizing one, two, three, four, five, different cofactors. And what pyruvate dehydrogenase complex does is to allow for the oxidation of pyruvate by NAD plus, in order to allow for the formation of acetyl-CoA, which allows for this thioester group to be generated. When we look at this overall reaction, it has a standard free energy change of about negative 33.4 kilojoules per mole. So this is going to be a spontaneous reaction. And automatically, what do you start thinking of when you see values like this? That this is going to be regulated. When we look at the purpose of this, remember, as a reminder, pyruvate dehydrogenase complex, this is going to exist in the mitochondrial matrix. And producing acetyl-CoA is going to be what fuels the citric acid cycle. So we want this to be operating when we need ATP, and this is what functions when we have high enough levels of O2 to do what's called oxidative phosphorylation. So a little bit more in terms of introducing some of these new cofactors that you probably have never heard of before. So the first one we're going to look at is coenzyme A. And I like to point out this structure, because when you we write it out a lot of times and we're going to see CoA as part of other molecules as well, we see that we just call it this S-CoA. And the S is important because this is the reactive thiol group that we're going to see that can form this high energy bond that's so important for driving different processes and producing the energy that's necessary. But we say CoA and the CoA is the entirety of this molecule, which is actually quite large. So the reactive thiol group is part of what is called beta mercaptoethanolamine. That is linked by pantothenic acid to what is called three-prime-phosphoadenosine diphosphate. And all three totals of these parts together are what make up the entire CoA structure. When we look at CoA, it's going to function as a carrier of acetyl as well as other acyl groups. Acetyl-CoA is a high energy compound. So we know that when we allow for CoA to be attached to any type of molecule that there is going to be energy associated with that molecule, which becomes very important in metabolism. We know that the standard free energy change for the hydrolysis is again 33-- negative 33-- negative 31.5 kilojoules per mole. Why do I point that out? What other molecule do you know of that exists, that's important that releases energy in that range? ATP. So we know that there is about as much energy in this high energy thio bond-- thioester bond as what we would see in ATP. So the pyruvate dehydrogenase, and I want to highlight to this complex, okay, these enzymes that are going to come together to work to allow for pyruvate to be converted to acetyl-CoA. And we know that this requires multiple enzymes, and also that it's irreversible. As we said, anytime you see the word reversible, you think about the fact that it's going to be regulated. And why would it be so important that we allow for regulation of the pyruvate dehydrogenase complex? Well think of it as the gateway to the citric acid cycle. So this is a way that we can move carbon skeletons from the cytosol into the mitochondrial matrix and allow for ATP to be generated. So again, the regulation of this complex is going to be important, but we're going to put a pin in it. So I'm going to discuss regulation later. The purpose of this lecture is to get through the chemistry that's happening with pyruvate dehydrogenase complex. So when we look at pyruvate dehydrogenase complex, remember I kept saying go ahead and call it complex-- get used to thinking of it as a complex, and a lot of times figures will shorthand it and they'll just call it pyruvate dehydrogenase. I personally don't like that, because when you actually look at the name of E one, it's pyruvate dehydrogenase. So when we're talking about all of the different enzymes complexed together, that would include pyruvate dehydrogenase as E one, dihydrolipoyl transacetylase is E two, and dihydrolipoyl dehydrogenase as E three. So when we say complex, this is going to show that it's composed of all three of these enzymes. Now these three enzymes are going to make use of five different cofactors. So TPP, you've looked at before, we've seen this one before. This is what helps allow for movement of carbon skeletons, particularly when you need resonant-- resonance stabilization because of added electrons. We have NAD plus. And we've looked at that before going through redox chemistry and the importance of being able to add or remove a hydride, does the to electron transfer. We introduced last time FAD. When we look at FAD, and we look at the ring structures, remember that we have the ability not only to do two electron transfers, but also single electron transfers. And we just talked about the structure of CoA which I have shown down here and again, showing shorthand for how we draw out. And then the last one we're gonna introduce is lipoic acid. And that's what's shown here, and you look at the disulfide bonds, and this is also going to be a cofactor, that's going to be capable of doing redox chemistry. And when we look at what's happening, we're seeing that we call it the complex because yes, there's individual enzymes that are catalyzing their reactions, but they do it in a concerted manner. So as E one goes, and E two goes, and E three goes in order to finish the final product. So we can think about it in terms of pyruvate to acetyl-CoA, that's going to be the role of the pyruvate dehydrogenase complex functioning, in which you have concerted effort between E one, E two, and E three together. So this is a nice figure to help map out in your head to understand what exactly is happening. And I'm going to I'm going to break it down over the next few slides as well. Because this gives us an idea of really thinking through, in terms of mechanisms, all the way to watching electrons. And you can ask yourself, why do electrons matter so much? Well, what is the driving force for the proton motive force to allow for ATP to be made in our body? Movement of electrons through the electron transport chain, so electrons matter a lot. So let's go through what we're having. We start here with pyruvate. This is what is our substrate. When we look at the reaction with pyruvate, the first thing that we see is a facilitation of a decarboxylation. Now, this decarboxylation is important because this is a source of energy. Remember, decarboxylation events give off energy. Now when we have a decarboxylation event, we're going to have these electrons that have to be dealt with. This is where the cofactor hydroxyethyl TPP, sorry, the cofactor TPP becomes important is because it can form hydroxyethyl TPP, and have a place for these electrons to go. So pyruvate dehydrogenase is what offers TPP. So this is the first enzyme, and we're going to see that E one is going to facilitate the decarboxylation event by allowing for a bond to be formed between what's called the hydroxyethyl moiety and TPP. And this is what we call then the intermediate, hydroxyethyl TPP. Notice we've lost the CO2, we still have the electrons retained, and we still have the carbon skeleton that we need to make acetyl-CoA. Next what happens is that we have a reaction in which the hydroxyethyl portion is going to be transferred over to lipoamide, the cofactor that is used by dihydrolipoyl transacetylase. So we have here E two. So this carbon skeleton and the electrons are going to get transferred over to lipoamide. This cofactor lipoamide and E two is its oxidized state. So it can pick up electrons as well as the carbon moiety to be in its reduced state. And this is important here because what we're going to see is, we're going to have a separation event, we're going to have a way for the acetyl portion to be attacked by CoA. So we form acetyl-CoA and the other part we see are the electrons that are going to be moved. So notice we have acetyldihydrolipoamide nucleophilic attack by CoA, that's going to generate for us acetyl-CoA and this is one of our products from the pyruvate dehydrogenase complex. Then we have our electrons. Notice we still have lipoamide in the reduced form. This is where we now have enzyme three come in this is dihydrolipoyl dehydrogenase, E three. And it has as part of its structures sulfhydryl groups and an FAD molecule that are going to allow for the transfer of these electrons. So these electrons are going to move. And we're going to see that we have the oxidized form of lipoamide regenerated. Electrons are going to move, we're going to allow for the reduction of-- dihydrolipoyl dehydrogenase transfer the electrons. And where do those electrons end up? Well notice here NAD plus gets fully reduced to make NADH. This is the second product of the reaction. We could use Cleland notation and call it Q and we're going to see that we have our substrate converted to product, acetyl-CoA, and NADH. So what happens is we have this moiety becomes part of our acetyl-CoA. And we have the electrons contained within this bond that are going to end up with NADH. And those electrons are important because we can utilize those to drive the electron transport chain. So here's a maybe a little bit more of a simple view. So I'm going to break this down, what we just covered. I'm gonna go through it one more time slowing it down to look at the details, because again, when it comes to metabolism, every electron matters. So what we have here is a simple real-- view of the role pyruvate dehydrogenase plays. So again, the first thing that we have to facilitate is a decarboxylation event, this decarboxylation event is going to produce electrons, these extra electrons left on the acetyl moiety. Then we have to facilitate the nucleophilic attack with CoA so that we can generate acetyl-CoA. So we need to have a nucleophilic attack CoA with acetyl moiety. And we also have to have a place for the electrons to go, which is where we end up seeing them with NADH, ultimately where these electrons go. So by the end of the reaction, we generate acetyl-CoA, and NADH. So if we go back and look at the mechanism, and I just want you all to know, this is what we're looking at. And we're just going to put a gray box over the centerpiece. This is what we're looking at. This is the strategy for pyruvate dehydrogenase, when it comes to the complex reaction. So here is pyruvate, we're going to see that CO2 is going to be released first. So if you look here, we have a decarboxylation event that's going to facilitate CO2 is release. So step one that we've been calling the decarboxylation, is going to allow for CO2 to be released. The second thing we have to do is to set up the reaction between the acetyl group and CoA in order to form acetyl-CoA. That's what's happening. So here's our acetyl moiety, we have CoA, our cofactor. And these come together through nucleophilic attack to generate acetyl-CoA and again, remember this is what's going to fuel the citric acid cycle. And the last thing that we have to deal with are those electrons. So we have the electrons here in this bond. There's two electrons that can be dealt with. And what happens to those electrons is they end up being transferred to NAD plus forming NADH. And this is going to serve as our electron carrier. And this is going to fuel the electron transport chain. So this is the strategy of what is being done with pyruvate dehydrogenase complex. So let's walk through as we think about these reactions that are happening. So again, we have our decarboxylation event, we have our transfer with CoA, and we look at oxidation, allowing those electrons that are going to eventually end up as NADH. So these are the three major things that have to happen. So let's look at this and think about it in terms of the cofactors and the chemistry that's being facilitated. So we know E one is what utilizes TPP. TPP is important because it facilitated decarboxylation, we have the cofactor that can help stabilize through resonance having these extra electrons associated with the carbon moiety that's got to be transferred. The next we have is lipoic acid. This is going to be important poly-- lipoamide is part of the structure because of redox chemistry. We have to move the carbon skeleton, but we also have to deal with the electrons as we have the reduced versus oxidized form. Now when we look at this, I want to show for every cofactor that's used as its changing configuration, or its has something added to its changing its redox state, what ends up happening is that it has to be regenerated. So as TPP facilitates decarboxylation, we have here our carbon skeleton transferred to TPP. As E two comes in and picks up that same carbon skeleton, notice TPP gets regenerated. So this is what we're going to see with E two, we have our lipoamide, we're going to allow for nucleophilic attack by CoA. That's going to release our acetyl-CoA moiety but we still have it in the reduced form, holding the electrons. So to get to the oxidized form. Now we have to have a place for those electrons to go. When we look at lipoic acid as you see it has to allow for a chemistry center with CoA as well as the chemistry center where it transfers electrons to FAD. You may think how can all that happen in the same active site? Well remember the structure of lipoamide, shown here, we look it has this long arm like structure. And again, when we think about that it can literally serve to help swing the arm that transfers from the chemistry that happens at E two to form acetyl-CoA, to the active site that involves E three, where we can transfer electrons from the reduced lipoamide, to FAD, forming oxidized lipoamide and FADH. Now, then we have CoA, the importance of CoA setting up is because of the thioester linkage. We're going to need energy to allow for the driving of the citric acid cycle that makes it readily readily favorable to happen because our ability to make ATP is dependent upon flux through the citric acid cycle. That thioester linkage is important. And we can think of it as a high energy bond. So then we have our FAD, this is part of E three. And we know that it has a reduction potential, which we're going to discuss in greater detail, that allows for the spontaneous flow of electrons from lipoamide to FAD to NAD plus to ultimately form NADH. When we look at NADH this is going to be the final electron carrier that's going to deliver those electrons to the ETC, so electron transport chain. So this is the role each of these cofactors are going to play. Now, I oftentimes like to put in some historical context as well. And one of the things that y'all may have heard of before is arsenic poisoning. Why does arsenic-- why is it so poisonous? And also how did we determine what was going on? So it turns out that arsenic compounds are toxic. And the reason they are toxic is because they bind to sulfhydryl compounds. So when we think of sulfhydryl compounds, we think about dihydrolipoamide, and we can see here in a reaction with arsenate, that we formed this complex with arsenic that's not going to allow for the chemistry to continue. It can't be regenerated. So it turns out that this is a way of thinking about it in terms of inactivation of lipoamide. So any compounds, enzyme complexes that require the action of lipoamide will essentially be inactivated in the presence of arsenic. So we already know that pyruvate dehydrogenase of the pyruvate dehydrogenase complex relies on it, we also are going to see alpha ketoglutarate dehydrogenase is also going to require a dihydrolipoamide. So in the presence of arsenic, were stopping the functions of these enzymes, which is going to halt oxidative phosphorylation, halting your respiration rates so that you're not able to utilize oxygen as part of generating ATP, and can obviously be deadly for the individual. Now, organic arsenic compounds are more toxic to micro-- microorganisms than they are to humans. So did you know a couple 100 years ago, arsenic was utilized often as a way to treat different types of infections caused by microorganisms. And one of the common ones that you was utilized for is what is called syphilis. This is a type of STD and as well as other bacterial and parasitic diseases, and what was given was an amount of arsenic that would hopefully be more toxic to the microorganisms, getting rid of it without killing the person. Of course, you can imagine getting the dosaging right for those issues, because arsenic does pose a issue to the human as well. And turns out that this treatment with arsenic was successful, I guess, in many regards at that time point for syphilis. And there is actually what's called Dr. Ehrlich's magic bullet, and this is the first modern antimicrobial coined the term chemotherapy. And they looked at the compound arsphenamine which has arsenic as part of it, and they started marketing it as Salvarsan. It also had the number 606 associated with it and as early as 1910. So start to-- as we understand metabolism and we start to understand what's going on, and how we have these treatments, why we can have intended effects, why dosaging can matter. But anyway, this became the huge wave of research in terms of what kinds of things that we can give to people in order to treat whatever disease state that they're in. And in this particular case, it ties to lipoamide, the cofactor. So we can inhibit infections such as syphilis and other bacterial diseases by stopping reactions that require lipoamide. Now when we look at pyruvate dehydrogenase, it's one of the key reactions and we now know that it needs TPP. If you'll recall, somewhere in the first week or two of class we talked about TPP. And how this is thiamine pyrophosphate. It is also called vitamin B 12. When you have to get from your diet, and when you have a deficiency in TPP, there's an associated disease state. One of them is called Beriberi. And when we look at deficiencies of TPP, it causes pain, paralysis wasting away, and also heart failure. Well, now that you see the role pyruvate dehydrogenase is part of the pyruvate dehydrogenase complex has with TPP, it starts to make sense how we can see these conditions. So pain as the body doesn't have a way to generate enough ATP. Paralysis, when things quit working, there's not enough energy to allow. Wasting away as your muscles no longer work and of course, heart failure because your heart is a muscle. So TPP is very important in humans, when we think about it in terms of getting energy to allow for cells to be able to do the things that they need to do. People again, who are susceptible to Beriberi are those who only eat white rice, not enough variety in their diet in terms of getting vitamins, and alcoholics, because alcohol is-- really messes with a lot of things metabolically. We'll we'll look at this throughout the semester, but it gets the best way to say it, it metabolically gets things out of whack, and gets things out of the mode that they should be in for how we typically will regulate. And we'll we'll touch upon that later too, it deals with digestion issues, how well your body can break down the things that you're eating. A lot of times when somebody is an alcoholic, they don't even have an appetite. So there's a lot of negative things that come from alcoholism, that impact diet, and therefore your nutrition level. So when we look at TPP, it's used exactly the same way in pyruvate dehydrogenase as what we saw, that we've seen previously. So TPP, remember, as we have a decarboxylation event, we're going to have these extra electrons that are going to be associated with this bond. So decarboxylation is going to leave those electrons. And as we add the carbon skeleton to make hydroxyethyl TPP, we can allow for resonance stabilization that can exist within this cofactor. And so it makes this otherwise highly unfavorable reaction much more favorable by lowering the energy. We saw TPP and pyruvate decarboxylase. Remember, this is something that's done in yeast as part of fermentation. This isn't a reaction humans have. But hydroxyethyl TPP is going to be cleaved in a very similar way we saw with pyruvate decarboxylase dealing with pyruvate to produce acetaldehyde. So if you remember this reaction mechanism, we set up here a nucleophilic attack that allows for the attachment of TPP and pyruvate. And in this case, what we're going to facilitate is the elimination of CO2. So the extra bonds that come-- or sorry, the extra electrons that come from the bond as CO2 breaks, its resonance stabilized allowing for stable stabilization of the carbanion. Now, where do we differ in terms of looking at pyruvate dehydrogenase versus pyruvate decarboxylase? Well, pyruvate dehydrogenase is going to transfer the carbon moiety over to E two with lipoamide. We're not going to allow for it to dissociate so we won't see this portion of the reaction when it comes to pyruvate dehydrogenase as part of pyruvate dehydrogenase complex. Instead, we're going to see E two come in and play a role. So TPP is important because it facilitates decarboxylation of pyruvate. Next, we're going to transfer a hydroxyethyl group from TPP to lipoic acid. This is where oxidation has to occur. And we're going to have the loss of electrons as well as facilitate nucleophilic attack by CoA. So again, TPP is the starting point. As the decarboxylation event unfolds, we allow for the intermediate hydroxyethyl TPP. Remember, we're not going to let the carbon skeleton leave, instead we're going to transfer it to E two. So this regenerates TPP and now the carbon skeleton is transferred over here to lipoamide forming acetyldihydrolipoamide. This then is going to set up nucleophilic attack by CoA, while allowing for-- so that will release the carbon skeleton, so I'm gonna put that here. This is the carbon skeleton of pyruvate, what's left of it. The carbon skeleton gets transferred over to make acetyl-CoA, we also can see the electrons retained in the reduced lipoamide. When we look at the structure of lipoamide, we're again having to think about not just the fact that it can do redox chemistry because of the sulfur groups that are in close proximity to each other and able to form disulfide bonds in the oxidized form or sulfhydryls in the reduced form. So it not only does redox chemistry, but also that it has a very long structure. And the reason that that's important is we're going to have to facilitate chemistry and with CoA as part of E two but we're also going to have to facilitate chemistry in the form of electron transfers with E three. So this arm structure is what allows for it to transfer these substrates from one active site to the next. And it turns out lipoic acid is also a vitamin, this is not something that your body can make. So the lipoic acid portion, you get this from your diet. Leafy greens are going to have be a good source of lipoic acid, and we call it lipoamide, because we utilize lysine and we have that epsilon amine group, that's going to allow for the attachment. So lipoic acid attached to the lysine is what we call lipoamide, this is how it's anchored to E two. When we look then at what's going to occur, the transfer, we're looking here at E two. So here's my hydroxyethyl TPP. And we're going to see the carbon skeleton get transferred. We're going to need that carbanion to do nucleophilic attack to lipoamide E two, and this is going to allow then for a deprotonation event that will release TPP, E one. And I always like to think of this, this is ready for the next round. Sorry, ready for next round of catalysis. So we have regenerated, E one. Now we have to think about what's happening with E two. Well, in this case, we still have our carbon moiety acetyl connected to our dihydrolipoamide E two. So acetyl tells us about the carbon moiety that's still attached. Dihydro means that we have it in its reduced form. And we do not have the disulfide bonds, so we still contain the electrons. So this starts reduced form for hydroxyethyl TPP, we had the oxidized form of lipoamide. By the end of this reaction, we're going to have the reduced form of our dihydrolipoamide. And we have the oxidized acetyl portion that's now primed for nucleophilic attack. So now CoA comes in, it's going to do nucleophilic attack, we have the oxidized portion of our molecule here. So you have that partial positive charge of your carbon opposite of electronegative oxygen. It's going to do nucleophilic attack. And this is going to allow for the release of acetyl-CoA, which is again, the first product. Yes, we've already lost CO2, but it's a waste product. It's not something we we track. So we also still have our reduced lipoamide. So at the end of E two's action, it has produced acetyl-CoA. And now that arm of lipoamide in it's reduced state is going to have to move to the active site of-- with E three so that these electrons can then be transferred. So acetyl-CoA is what's generated by E one and E two. If you're following this, and you're thinking through what is the purpose of E three, if we are taking pyruvate and converting it to acetyl-CoA. Why do we need E three? E two is in a reduced form. What does it need to be in order to be active? We need it to start in the oxidized form. So we need E three because E three is what's going to accept these electrons in the reduced form of lipoamide to regenerate it for the next round of catalysis. So what do we have? The reduced form, it's got to go back to the oxidized form of lipoamide for E two to continue its path, we have to allow for electrons to get picked up in E three. And it's going to ultimately transfer those electrons to NADH, well sorry two NAD plus producing NADH. And this then becomes the second product. Now both acetyl-CoA and NADH are going to tie to energy production. Remember acetyl-CoA is the fuel for the citric acid cycle, it's going to generate electrons to drive the electron transport chain, NADH. This is a mobile electron carrier that takes electrons to the electron transport chain. It's all about getting electrons to the electron transport chain. So we must have an enzyme that's capable of catalyzing the next round of catalysis. So why is E three necessary? Because we have to take the reduced form of E two and get it back to the oxidized form. What serves is the electron acceptor? FAD of E three is going to go from its oxidized form to the reduced form of E three. And remember when we look at the structure, we have the ability of the FAD to transfer these are what we call single electron transfers. Or likely in the case of this, it just does two electron transfers at a time so we're just going to move it in the form of a hydride. And we're going to allow for it to be attached with both electrons and do a single electron transfer, or sorry to do a two electron transfer, at the end of that we regenerated our oxidized form. And we have our electrons still in the form of FADH two within E two. So where do these electrons go? FADH two is not actually a mobile electron carrier, it has advantages, because it can do single electron transfers or two electron transfers. But it's not mobile. And what that means is that it has to be associated with an enzyme. Now, where do these electrons go? To NAD plus to form NADH. NADH is a mobile electron carrier. So it can flow through solution within the mitochondrial matrix and drop off electrons at the electron transport chain, it can only do two electron transfers. That's what it's capable of. Not single electron transfers, but it's mobile. So you start to see how we have different types of electron carriers for different reasons in terms of their function. Once we form NADH, transfer electrons are complete, everything has been oxidized, and it's now ready to go through the next round of catalysis. So this is a slide that you can utilize to study. You know, there's there's multiple ways to think about this. If you're a person who likes following the electrons in chemistry like I do, you can go in and draw out the mechanism utilizing this. Do you know the structure of pyruvate? Can you show the actions of enzyme E one pyruvate dehydrogenase with TPP? How do you set up the reaction to allow for attack by CoA? To form acetyl-CoA? Can you then go in and show the involvement of lipoamide? How does it get reduced? How do we take these electrons transfer them to FAD? Do you know the reactive structure of FAD? How to generate FADH two then to transfer the electrons and you can think about it piecewise. The other thing that you can do is go in and utilize this in terms of a big picture strategy, draw out the different carbon skeletons show the cofactors. And think about in terms of what's happening here we are facilitating a decarboxylation event. Here we are generating acetyl-CoA. Here, we are generating NADH as the final electron acceptor. And you can look at those details as well. So go back and review. This is a lot of the idea of how the pyruvate dehydrogenase complex works. And the reason that I go through it so slow, is as we get to the citric acid cycle, we're going to see the alpha ketoglutarate dehydrogenase complex, which is an important important enzyme in the citric acid cycle is going to do the exact same mechanism just with a different carbon skeleton. Instead of pyruvate it's going to use alpha ketoglutarate. So we're going to see this idea of strategy again. And both of these enzyme complexes are highly important when it comes to regulation. So go through, can you remember the names of these enzymes? Do you remember the different cofactors they use? What order are the cofactors used in what type of chemistry are those cofactors being used for? So use this as a study slide as a review for yourself to go through this material. The other thing that I wanted to do is to give you this table, this table is actually helpful as well, some of you not as much visual drawing stuff out, you like looking at things that are organized. This gives you a way to look at it from the cofactor. And you're going to see that it's going to tell you what enzyme it's bound to. And it does this in order of pyruvate's conversion to acetyl-CoA. And it's also going to give you an idea of the function. So this table can also be helpful in terms of going through the material and reviewing making sure that you can read these words and understand based on what we've had drawn out already. And this is a mechanism that helps allow for you to practice, again allowing for activation of TPP, you have to have it in the ylide form or need it to function for nucleophilic attack. We have to allow for pyruvate to go through the decarboxylation reaction. And we need to have resonance stabilization, because of the fact that we have a carbanion that is highly unfavorable it's what TPP allows. Then we're going to allow for the transfer of the carbon skeleton from hydroxyethyl TPP to lipoamide that's going to regenerate TPP and now we're going to have our lipoamide that's going to set up nucleophilic attack by CoA. And the purpose of that is to generate a high energy thioester bond. So this is a high energy bond. And we're going to make use of that energy as we take acetyl-CoA and we put it through the citric acid cycle to liberate electrons. And then we're going to see that we have our lipoamide in the reduced form. And ultimately you have to allow for lipoamide to be oxidized by FAD. FADH two is formed and it gets reduced. Then we have NAD plus that's oxidized to pick up those electrons, forming NADH, regenerating FAD. So by the end of this, all of our cofactors are ready for the next round of catalysis. So hopefully this kind of helps look at it. Another thing I would like to point out are dietary needs. We talked today about cofactors. And I'm sure a lot of you know vitamins, take your vitamins, and eat balanced diets. Think about what things you're filling your body with. When it comes to dietary needs. one of the things that we do have to consider are the different types of vitamins sources, just like we know, thiamine deficiencies can come from people who rely too much on the rice diet and not getting enough. So I just wanted to put this list together to help you think about in terms of pyruvate dehydrogenase, what types of foods that you eat give you the necessary vitamins so that pyruvate dehydrogenase complex can function. So thiamine is also called vitamin B one, it's one of the eight essential B vitamins and good sources of thiamine are meat, nuts, and whole grains. Lipoic acid is made from a fatty acid precursor that's found in animals. Many foods contain this alpha lipoic acid. I think I said earlier about eating a lot of greens. So things like spinach, broccoli, yams, potatoes, brussel sprouts, all of those are going to be good sources of lipoic acid. If you're a person who likes meat, red meat, particularly organ meat, things like liver. Coenzyme A, requires cysteine as well as the pantothenate. And when we look at that structure, that's kind of that arm structure in the middle when you look at it, is what's called vitamin B five. Vitamin B five is a lesser known vitamin because its deficiencies are very rare. It turns out that we get pantothenate from almost anything we eat. So as long as you're eating food, you're probably getting a source of vitamin B five. FAD and FMN. Remember we talked about the riboflavin component. It's called vitamin B two. So you have to have riboflavin from your diet, eggs, organ meats, lean meats low fat milk, green vegetables, fortified cereals, bread and grain products. All of these are going to be sources of FAD or FMN. When we talk about fortified that's just a way we can add things to our foods that enhance their nutritional value. Sometimes we talk about it in a negative way, like putting in high amounts of high fructose corn syrup. But there's also other things we can do like fortifying cereals so that people can get their daily amount of riboflavin. And NAD plus is actually biosynthesized from tryptophan, but it relies on salvage pathways that use what's called niacin, which is vitamin B three. We get our vitamin B three from things like liver, tuna, salmon, some vegetables like mushroom and green peas. Other fortified foods are another way we'll add in vitamin B three source as well. So these give an idea of your essential B vitamins and half of them are listed here. And how important it is that you have these vitamins. That you have good health and diet. Because this is again what your body's going to rely on to be able to make energy how you use the food that you eat, in order to allow for the energy to be obtained. So that completes this lecture and we'll get finished with pyruvate dehydrogenase complex. So now we'll be leading into the citric acid cycle.