in the previous lecture we focused on step one of the citric acid cycle and we saw that in step one we basically take an acetyl group and attach it onto an oxaloacetate molecule to form a six carbon intermediate known as the citrate molecule and so in this lecture I'd like to focus on what happens next so we're going to focus on steps two three and four of the citric acid cycle and so let's begin with step number two now the entire point of step number two is basically to take the citrate molecule and to prepare it for oxidative decarboxylation that will take place in step three and step four of the citric acid cycle so in these two steps were basically going to produce carbon dioxide molecules and we're going to abstract those high-energy electrons that we're going to use on the electron transport chain but before steps three and four take place we have to prepare this situate molecule and the way that we prepare that citrate molecule is by actually changing the position of this hydroxyl group so citrate and isocitrate are actually isomers they have the same exact molecular formula but they differ in the position of this hydroxyl group on the citrate the hydroxyl is attached on sin this carbon let's call it carbon three and on this molecule the hydroxyl isn't that attached onto this carbon here and we see that to go from this reactant to this product we have to go through an intermediate and so this step two is actually a two-step process so in process one of step two we have a dehydration reaction why well because we want to basically remove this hydroxyl group and in addition we remove this H to form the water molecule and form the double bond between this carbon and this carbon here and once we form this double bond this water molecule that comes in in step two will basically undergo a hydro Station reaction the water will act as a nucleophile and instead of attacking this carbon it will attack this carbon because if the water molecule attack this carbon we would have simply reformed the citrate molecule but if the water attacks this carbon which is basically less hindered because it contains a smaller group on this side compared to this large group here the water molecule is able to actually attack from this side because of less hindrance and so once it attacks that side we form the isocitrate molecule so the entire point of this step is to basically prepare the citrate molecule for oxidative decarboxylation that takes place in step 3 as well as step 4 now this double bonded intermediate molecule is known as sis Akana Tait and because of this sis Akana Tait the enzyme that catalyzes step 2 is known as a connotates so once again once citrate is formed in step one of the citric acid cycle it must be transformed into its isomeric form isocitrate and this reaction we basically transfer a hydroxyl group on the third carbon onto the Jason carbon shown here and what this process does once again is it prepares the molecule for a decarboxylation reaction that we'll talk about in the next step now the enzyme that catalyzes this step is known as a connotates and this econo taste actually contains an iron sulfur component and that's why this molecule that connotates enzyme is known as an iron sulfur enzyme and iron sulfur protein now actually contains a ratio of four iron to four sulfur inorganic sulfide atoms and this complex is found an active site and it binds until the hydroxyl granata hydroxyl onto the carboxylate ion group of the citrate and that holds the citrate molecules within the active site and allows the catalysis to actually take place so once again step two is a two-step process so we have this dehydration and hydration that is catalyzed by the connotates which is and I am self a protein because it uses the iron sulfur complex to carry out these two reactions now once we form the isocitrate molecule now this six carbon molecule is ready to undergo the first oxidative decarboxylation step of the citric acid cycle and this is what happens in step 3 so once the isocitrate is formed it is ready to undergo the first oxidative decarboxylation step and this reaction is catalyzed by isocitrate dehydrogenase Y dehydrogenase will remember a dehydrogenase is an enzyme that basically abstracts those electrons attached onto the H ion to basically form that reduced NADH molecule so in this particular case in the same exact way that we have a two-step process here we also have a two-step process here and in the first step of step 3 we take the isocitrate and we reacted with the nicotine and nicotine amide adenine dinucleotide in the oxidized form and so in this process the energy plus is actually reduced into the NADH and the isocitrate molecule is oxidized to form the oxy lo succinate and we also release this H+ ion so the first reaction involves the abstraction of a pair of high-energy electrons to form the NADH and this high-energy intermediate known as oxalá succinate and ox lo succinate is unstable because it is a beta keto acid so remember from organic chemistry that beta keto acids are generally unstable molecules now the NADH that we produced will be used by the electron transport chain as we'll discuss in a future lecture so now let's move on to step 2 of this process that takes place in step 3 so in the next step we take that axl Oh succinate and by the activity of the same enzyme isocitrate dehydrogenase we mix it with an H+ ion and we basically form a molecule known as alpha keto glue to rate so the highly unstable oxy low succinate can now undergo a decarboxylation reaction so this was the oxidation reduction reaction and this is the decarboxylation step and actually as we'll discuss in much more detail in a future lecture this essentially is the step the formation of the alpha ketoglutarate is the step that actually determines the rate at which the citric acid cycle actually takes place so this is a very important step and if we sum up these two steps these two reactions of step three this is the net reaction that we're going to get noticed that the oxalá succinate molecules don't appear on either sides because they cancel out so if we sum up this reaction with this reaction this cancels out and so does this as well as the h+ here and h+ here so this is the net reaction that we get on the reactant side the isocitrate that we produced in step two and the nad plus that acts as the carrier and picks of those two electrons that we abstract from the isocitrate we form the NADH the carbon dioxide molecule is removed from the isocitrate and we form this alpha ketoglutarate molecule now let's move on to step four in step four once again we have another oxidative decarboxylation step we're going to remove yet another carbon dioxide in the process abstracting the pair of high-energy electrons to basically form the reduced NADH molecule which eventually will be used by the electron transport chain to generate those high-energy adenosine triphosphate molecules and in this step we actually use the coenzyme a the same coenzyme we used in pyruvate decarboxylation so the next step is a second oxidative decarboxylation reaction of the citric acid cycle this step involves the conversion of the alpha ketoglutarate into the succinylcholine zhan a and this is what the reaction looks like so this is the net reaction on the reactant side we have the product of step 3 the alpha ketoglutarate in the presence of the nad plus we need this because this acts as the carrier to actually extract those electrons and we have the co enzyme the the coenzyme a co a and on the product side we essentially attach we remove this component that produces the carbon dioxide and we attach the coal ends on a onto this bond to form the high-energy thio ester bond and this is the bond that will be broken in the steps to come as we'll discuss in the next several lectures now this succinylcholine sign a basically is the product of step 4 and the enzyme that catalyzes step 4 is known as alpha ketoglutarate because this is a substrate molecule that binds into the enzyme dehydrogenase complex and in fact this complex is very similar to the complex that catalyze step 1 of the citric acid cycle how is it similar well this complex just like that complex also consists of 3 different types of enzymes and it also uses many different types of cofactors so we have the e1 enzyme that is known as alpha ketoglutarate dehydrogenase that uses the TPP so thiamine pyrophosphate cofactor we have the e2 known as dihydrolipoyl sucks and they'll transfer rates that uses the lipoic acid derivative and we have III dihydrolipoyl dehydrogenase that uses the fa G so this large complex the Alpha k-y-t's of glue dehydrogenase complex consists of these three enzymes and it uses different types of call enzymes to carry out the steps of step 4 and if you want to learn more about the mechanism of this particular step go back and watch my lecture on step 1 because these two steps are actually very similar to one another