Chapter 12 Lecture PDF

All right, folks. I again I apologize because apparently my rst recording of a chapter 12 lecture was a le that ended up being corrupted. So it didn't couldn't actually play when I was trying to play it and and have. For you guys, so I have to rerecord everything. So I do apologi...

All right, folks. I again I apologize because apparently my rst recording of a chapter 12 lecture was a le that ended up being corrupted. So it didn't couldn't actually play when I was trying to play it and and have. For you guys, so I have to rerecord everything. So I do apologize for the late show up of our lecture. Now chapter 12 it's a chapter that most of you guys, at least on the fundamental aspects of energetics, you guys are familiar with. This was more to serve as a recap of those Topics and an expansion in a little bit of the molecular basis of energetics. So what enzymes are involved and perhaps some of the key elements of how These enzymes work. We're just gonna give one example towards the end of this lecture. And and just an overview of the bioenergetic processes occurring in in, in cells. Alright, now just go here. And just gonna brie y again, going up from the very basics when we looked at the overall energetic aspects of organisms in cells in general you're looking at three major stages that involves, of course, the the breakdown of these larger macromolecules into individual energetic units. And. These, of course, as we say, consider the breakfast you have this morning and the consequential breakdown of those food items into individual elements, right? So individual components that are serving are entering convergent pathways for. Breakdown and ultimately to fuel bioenergetic processes. Right? So initially, the metabolism in general breakdown and catabolic metabolism involves the rst of all, of course, the breakdown of those transcripts. Large micro molecules and and consequentially those through various digestive processes that you guys are likely familiar with they are broken down into much more simpler. Subunits or simple building blocks, right? So you're like, again, your glucose, your regular amino acids that are again imported from from protein ingested and of course your fatty acids, your glycerol and others that are also incorporated, right? Now. These building blocks are going to be a critical component because they could serve as precursors energetic precursors for various molecules that of course includes pyruvate and we'll talk a little bit about how can we get pyruvate, we guys know that pyruvate is used and is imported into the mitochondria to the body. To fuel bioenergetic processes. And and you can obtain pyruvate through a couple of various pathways. Similarly, glucose various entry points. So they can, these precursors, these subunits can in fact serve important bioenergetic or energy generation process. I like the word bioenergetic because it's really broad, but also it is a complex word. So again, think of bioenergetics as the ways that the cells is producing energy. So now these carbon molecules, so again, these are organic molecules that are going to consequentially be broken down. So these are Pyruvate gets what we call decarboxylated. We're going to talk about that in a moment. But again, the breakdown of pyruvate, the consequential breakdown of other molecules consequentially leads to the full oxidation. Of these molecules that essentially you're looking at a collection of a combustion reaction, right? Where the ultimate byproduct is energy released in the form of heat one way or another. And the consequential release of water and CO2 as byproducts of the combustion reaction, right? So now we can do that two ways, right? So combustion reactions can occur With the internal so the actual input of energy. So let's consider here example B. Let's consider. Let's say you have a cube of sugar. fl fi fi fi Like one of those sugar cubes you use for teas and you consequentially in done under oxy conditions, you apply heat and the system essentially by apply applying heat, you overcome this energetic barrier where pretty much you're converting and you're burning directly the sugar and all that to produce CO2 and water. And of course the energy that is stored in the molecules Of glucose or sucrose in this case will be released in the form of heat, right? Energy is not stored in this particular case, energy is released. Again, the entropy contributes to entropy by increasing the amount of heat of the system and the consequential releases CO2 and water. But when you think about this when you looked at this sort of step by step or stepwise or coordinated set of reactions where, for example, in biological systems, what you'll see is that the energy is actually, or. The energy contained within that sugar molecule undergoes a series of transformations. So in other words, the energy store in those molecules of sugar are going to be harnessed or actually transferred in the form of other molecules that are going to serve as intermediate. So all these, what is showing here is that. The amount of free energy between sugar and CO2 is pretty much the same. However, the harnessing of that energy storing those molecules will be able to be catalyzed on their biological conditions and physiological conditions. With the help of speci c enzymes that are going to be able to transfer that energy into intermediates. And these intermediate molecules, we'll know a few of them today, they're going to be important as those intermediate steps are going to fuel the energy production machinery. So by itself, what I'm trying to say here and the take home message is that sugars. Or let's say, for example, just glucose as you combust and glucose fuel bioenergetic processes they do not directly, they're not directly involved in the energy production part. In other words the breakdown of glucose, for example, directly, it's not involved chie y in the production of energy. However. Most of the intermediates that are going to come here, they're going to play an important role in fueling bioenergetic pathways, okay? I will come back to this I know in the glycolytic pathway there is a small amount of ATP generated but Majorly we feed our bodies or cells in our bodies from energy that is Generated by the modi cations of these intermediates. Now, of course the energy that it comes in and that is utilized in ourselves in the form of ATP, right? At the same time you need to have those intermediates. So you have the breakdown of, and the transformation of those subunits are going to be pretty much a key component. In other words, the breakdown of macromolecules into amino acids are perhaps fatty acids or of individual sugar molecules are going to be important, but they need to be modifying. That's what I mentioned earlier in our previous slide. So the breakdown of those are going to be critical. And and nally, of course, as those intermediates are modi ed by other enzymes, consequentially, they will be able to contribute to the nal step, which is the oxidation of those carbon molecules into 80 CO2 and water, and consequentially also the fueling of pathways that add. Or that increases the concentration of ATP that produces ATP in our cells. Now, the process can be, as you guys know, it could be oxygen dependent or oxygen independent, right? We're primarily today talking about aerobic metabolism. We'll talk brie y about the portion of it that is associated with oxygen consumption. fi fi fi fl fl fi fi However, there are various other ways by which you can chie y generate oxygen. A TP in our cells, in in independent of the concentration of oxygen in the cell. For example you can have here in your in the left of your of your panel here, you can clearly see that an example here for yeast cells glycolytic pathways and the presence of a specialized enzyme that is responsible for generating. A non toxic, relatively non toxic form of intermediate that is essentially ethanol. Again the fermentation pathway, so it's a pathway that, as you guys might expect, has important both economical and biological implications. Now the process here, and I'm not, I'm going to ask you guys to remember these pathways so anaerobic pathways, we're not talking too much about them, but I just want you guys to recognize that as these substrates are transformed, they are intermediates, and in the form of reducing agents here that are formed. And and those intermediates are going to play an important role in energy generation likewise occurring again from the glycolytic pathway, you end up with pyruvate, and in the presence of oxygen that pyruvate can go and transfer into the mitochondria, so it can be translocated. And consequentially fuel the citric acid cycle, which also produces intermediates in the form of reducing agents. And those reducing agents contribute to the electron transport system. Now glycolysis, of course, this is an energy, so this is an oxygen independent pathway. However I would like for you guys to at least be able to brie y explain what the glycolytic pathway is and what does it produce. We're just going to go over these steps that are shown here but for the most part, I'm not going to be asking you guys in the exam what chemical modi cations are occurring on each of these intermediate steps. Just just be aware of where. The stages, major stages of the glycolytic pathways are and for the most part, what are, what is the overall outcome of the glycolytic pathway? Now, of course, glycolytic glycolysis in general, it's a pathway that is ubiquitous. Glycolytic pathways engage in all cells and it's a, again, it's a, is a fundamental pathway for energy generation in organisms. Now the way that that this particular pathway is it's actually engaged is, of course glucose is the major initial substrates, nonetheless there are multiple entry points. In other words, you don't always have to start start with the glucose molecule, and that's, we'll see that later. And and that also has important implications into how much. How much ATP can be generated now, this is key. Of course, you can actually generate ATP, but it's also an ATP consuming process. So there is an energy investment phase that must be taken into account. Which is, of course, it reduces the e ciency of a system and the overall yield, but initially it is important to recognize that glucose molecule and in another intermediate steps here, they must be phosphorylated. The overall major stages include the activation stage the splitting, of course, as you can see here the activation requires a phosphorylation of these intermediates. Splitting involves creating isomers and overall moving or transforming that isomer into two glyceraldehyde 3 phosphate molecules and consequentially modifying those into into two pyruvate molecules. And through that process, you're also recovering. Some of those phosphates that were initially invested and additionally and consequentially produce ATP in the process. So if you looked at this, and this is again an overview, and I will show you some of the molecular basis, but we're not dwelling into that for the part of the exam, but again, major steps again for exam, major steps, major stages and what the outcomes are. ffi fl fl fi  Okay. Now in this particular rst stage or step, it's where you see that investment of energy in the form of ATP that has several purposes one of them is to actually trap The glucose molecule by phosphorylating it into the cell. So that way the glucose molecule can not exit the cell. All right. Now this energy, as we mentioned earlier, it's going to be a recovered later. Okay. So the third, the rst, again, these rst three steps. Involves that transformation into an intermediate, various intermediate molecules that are going to allow that glucose molecule to stay in inside the cell. Now, the splitting phase that breaking again, a six carbon molecule into two, three carbon molecules are also, it's also a critical and very characteristic stage of glycolysis. Again, you can see those phosphate groups. Derived from ATP in the investment, energy investment phase. Now, this glyceraldehyde 3 phosphate is going to further be modi ed. It's going to be phosphorylated. And as we will see later it's going to In an orderly fashion, it's going to participate in the formation of pyruvate molecules. That energy generation phase involves several modi cations that not only produces our ATP, but also will produce reducing agents such as NADH. NADH is very, it's critical. We'll see what the role of NADH is in a second. But just something to consider now going back to those three stages. So this is step one of stage one. So this is energy related. And again, I'm adding a little bit more information as we go through. This is for you guys to visualize and to see where those changes, those modi cations are occurring. I'm not going to ask you guys to draw this in our exam. I repeat, we're not, this is not part. Okay. Of what we what we were doing and we decided it's not going to be part of the nal in terms of, sketching this up and drawing these reactions. Okay, what I want you guys to realize is, rst of all, you have the actual phosphorylation of glucose into glucose 6 phosphate. So now we have a phosphorylated glucose which involves, again, modifying charges. So now the actual molecule is not able to exit. The cell through glucose transporter, right? So even if glucose concentration changes across the membrane. Now in this particular case, our step two modi cations involve the actual movement of the carbonyl group. As you can see, carbonyl group here, for those who have taken organic chemistry and biochem are essentially shifted into the second carbon, so it's not a terminal carbonyl, It's now a carbonyl group located in our carbon number two. Now, this excuse me, this isomerization forms forms an actual keto sugar instead of an aldose. So again, this is an aldehyde and ketose. Sugars are formed based on and thanks to the activity of these isomerase. Again, this is just simply altering the structure of the sugar as we will see creating that isomer it is going to trigger additional modi cations and that includes, again, the phosphorylation. that of that terminal carbon carbon in so the hydroxyl group located in our carbon number one. If you guys remember, again, we have hydroxyl group here, right? So that hydroxyl group now from again, as that keto sugars form then triggers and Leads to the phosphorylation. So that's second investment. That's second ATP molecule. And as you guys can see, the molecules now somewhat assuming a, an almost a symmetrical shape. Now, The molecule in this case by phosphate, right? So now we're talking about fructose one, six by phosphate. It's modi ed in such a way that now we have two phosphate groups. fi fi fi fi fi fi fi fi fi fi fi  And again, those are derived from the investment of phosphate group from ATP. All right now Now, in the, again the modi cation of this molecule now to fructose 1, 6 biphosphate it pretty much marks now the starting point where the actual glycolysis will take place. Again, glycolysis is a breakdown of the glycolysis. Glucose, right? So now we're splitting that sugar in that step number four, which is in charge or it is responsible this is a process where aldo and aldolase is responsible for its cleavage. Again, the the actual modi cation is an enzyme mediated process that produces one molecule of glyceraldehyde tri 3 phosphate and dehydroxyacetone. As you guys can see these two molecules are not identical so there's further modi cations that must occur to dehydroxyacetone in order for us to end up with two Glyceraldehyde triphosphate. So now the process and I'm just going to show you that in a moment. So this process involves the modi cation of the hydroxyacetone. So again, the hydroxyacetone right here. As I showed you earlier, so this is 3 DAP, which is shown here, Dehydroxyacetone phosphate, and it's modi ed by an isomerase, which consequentially produces the second glyceraldehyde 3 phosphate. Okay? In other words, 3 GDP, or glyceraldehyde 3 phosphate. It's this particular case, this one here, it will be the second GDP. Okay? Since you have a three GDP already form from the initial splitting o the sugar, right? So now you have two GDP molecules that are going to be able to further modify. Now what that modi cation is involved or what does the modi cation involves? It's the actual phosphorylation again. So there's A dehydrogenase that is in charge of adding that phosphate group into into our miseraldehyde phosphate. In this case, we're forming a what is called an hydride bond. And this is this is pretty much a high energy bond phosphate bond. Now, each of the Previous 3GD 3GDPs are going to be are going to have two phosphate groups attached, right? Now, you might expect, since these are phosphate groups, that an orderly or an enzyme mediated process that cleaves this phosphate group and this phosphate group later are going to consequentially participate in the ATP. Generation, the harvesting of ATP from these molecules later and you're correct. Also again, we produce on this step, also an ADH, which I mentioned, it's a reducing agent and is used Electrons as we will see in the, like the rst portion in this case Of the energy harvesting phase involves a kinase. If you guys remember kinases essentially can add or remove depending on the conditions, but these guys is actually able to add phosphate to your ATP, to your ADP through, or from the by phosphoglycerate now in this case, again, this is the rst one. Now, you end up with, back again, your 3 phosphoglycerate, and you only have the harvesting of a single ATP molecule from that process. Now, in this particular case, removing that 3 phosphoglycerate, which is in a terminal position, is a relatively, A di cult process. For that phosphate group to become highly reactive, that rst phosphate group must be moved to the second carbon. And as a result again it is readily able to hydrolyze. In other ways. In other words the movement or the translocation of the phosphate group to carbon number two facilitates the phosphate, the hydrolysis of that phosphate group into what consequentially will be the second a TP harvested from it. Now, there's also additional modi cations that are occurring, and this is an enzyme mediated process that removes water, and overall you essentially the process involves the removal of the fi fi fi fi fi fi fi fi fi ffi ff fi fi hydroxyl group to form a double bond between carbon 1 and 2, again, sorry, carbon 2 and 3, I should say, a And overall to form phosphoenolpyruvate from that process. Now in that, through that process you have the now the formation of these enolphosphate linkage in PEP. So this again becomes or makes that phosphate highly reactive or, Really able to be hydrolyzed and be able to be transferred with another kinase, it's called pyruvate kinase PK for short. It's involved in the formation of that second ATP, right now, since we have three two, three GDPs molecules. So since we have two, this process occurs twice. So there is a net formation of. The ATP and the energy harvesting of again, total four ATP, because we invested two in the energy investment phase. So there's a net gain of two ATP molecules after the whole glycolytic pathway has taken place. Now, this is pretty much a breakdown of that. You have, again, you end up with your pyruvate molecules and those will actually be imported. into the mitochondria under aerobic conditions to generate ATP. And overall, again, there is a net production, a net production of two ATPs since two are invested and four harvested, right? That's that's four. Very important. That's the net results. And additionally, those four electrons that are mentioned here are stored in the form of NADH. So those electrons are going to participate in other and fueling other processes. All right. Show you guys this video and see if it shows up. Hello and welcome to Biological organisms require energy to survive. Glycolysis is one of the pathways cells use to transform sugars like glucose. into biochemical energy in the form of ATP. In the cytosol of the cell, glycolysis converts glucose into pyruvate through a series of 10 enzymatic reactions. This process produces ATP, along with other products such as NADH that can be used later to produce even more ATP for the cell. Let's watch as these enzymes oxidize one glucose molecule into two pyruvate molecules. First, a kinase reaction adds a phosphate onto glucose to form glucose 6 phosphate. This is one of two energy consumption steps and is an irreversible reaction. Next, an isomerase reaction converts glucose 6 phosphate into fructose 6 phosphate by rearranging covalent bonds. Another kinase removes a phosphate group from ATP and gives it to fructose 6 phosphate to form fructose 1, 6 bisphosphate. This is the second energy consumption step and an irreversible reaction. In the fourth step of glycolysis, A lyase reaction splits the 6 carbon fructose 1, 6 bisphosphate into two 3 carbon sugars, dihydroxyacetone phosphate and glyceraldehyde 3 phosphate. The dihydroxyacetone phosphate is rearranged by another isomerase to form a second glyceraldehyde 3 phosphate. At this point in glycolysis, glucose has been metabolized into two glyceraldehyde 3 phosphates. And two ATPs have been consumed. The next ve steps of glycolysis are the energy producing phase in step six, both glyceraldehyde three phosphates are oxidized to one three bis phosphoglycerate by a dehydrogenase. This step produces one NADH for each oxidized glyceraldehyde, three phosphate for a total of two NADH. These NADHs are later used to produce more ATP for the cell. fi  In step 7, a kinase transfers a phosphate from 1, 3 phosphoglycerate to ADP to form ATP and 3 phosphoglycerate. This step is reversible. Even though a TP is formed, the next step involves a mutase reaction that moves the phosphate on the third carbon of three phosphoglycerate to the second carbon position to form two phosphoglycerate. In step nine, a liase reaction removes water from two phosphoglycerate to form phosphoenolpyruvate in the nal step of glycolysis. A kinase reaction removes the phosphate group from phosphoenolpyruvate and donates it to ADP to form ATP and pyruvate. Like reactions 1 and 3, this step is irreversible. At this point, 2 pyruvate molecules, 4 ATPs, and 2 NADHs are formed for each glucose that was broken down in glycolysis. 2 pyruvates and NADHs. could be used in aerobic respiration to produce more energy for the cell. Here, we depict glycolysis as a closed process. But in cells, substrates produced by other reactions can enter glycolysis at di erent points. For example, when an animal breaks down glycogen, glucose 6 phosphate is produced and can then enter the glycolysis pathway at the second step. Importantly, this means one less ATP is required for the pathway, because the rst ATP consuming step is skipped. Other sugars can also enter the glycolysis pathway at di erent points. each having a di erent e ect on the net number of ATPs that are produced by glycolysis. These ATPs are important energy molecules required for many biochemical pathways and ultimately life itself. Glycolysis is a major contributor to the pool of ATP used in these All right. So as you guys can clearly see, of course the convergent pathways are just for you guys as reference. I'm not going to ask you guys about all those enzymes and the substrates are required for these intermediate steps to overcoming that shown in the video, but just Sort of recapping, so we have pyruvate being formed and through glycolysis, and now there's obviously the Important aspect and of course that's close to my heart since that's actually what I studied as far as research. So actually how pyruvate is utilized in the mitochondria and how, in particular how mitochondrial ADP production drives organismal performance. That's pretty much what I do for research, but or asking questions related to those topics. But now let's take a look at how pyruvate is utilized in the mitochondria and look on over or get an overview of the decarboxylation pathways involved and and how ultimately pyruvate, the presence of pyruvate and the processing of pyruvate molecules can feed and can lead to the formation of ATP form in the mitochondria. So of course Going back to mitochondrial structure, you obviously remember you have an outer, more permeable membrane and inner membrane that is a bit tighter. It's actually more more impermeable than the outer membrane. And there is a reason for that, of course. Pourings usually makes those Smaller molecules pass more freely across and the outer membrane of the mitochondria, it's a bit more considerably more permeable than your inner membrane. Now, when we talked about the formation of ATP and the role of the electron transport system in energy generation at the mitochondria, we're looking at a system of protein complexes that are located in the inner. membrane of the mitochondria. Okay. So these inner mitochondrial membrane has an increased surface area given by foldings that are known as cristae. fi ff ff ff fi ff  So again, these these foldings are going to be increasing the surface area. Does it increases the number of proteins located in the inner membrane. As a result these results in the increase space that these electron transport system proteins can occupy, all right? So the more intercristae the more infoldings of that inner membrane, the more ETS proteins are present to catalyze their reactions. Now, of course, inside, the very inside of the mitochondria, it's known as the matrix, it's almost like a gel like environment. So it's very thick, just in viscosity, thanks to the high number of proteins in the matrix. And it, of course, it's the inner environment of the mitochondria. And you will see that it's packed with enzymes that are involved in the breakdown. of pyruvate and the consequential modi cations that are associated with the citric acid cycle. So now the citric acid cycle, it's it's a process by which carbon substrates are modi ed. However, you will see something very interesting that although the citric acid cycle, it's linked to energy production and the mitochondrion at the citric acid cycle, there is no ATP generation. So the citric acid cycle serves to modify carbon substrates. To produce reducing agents, but it doesn't re, it actually involve directly is not involved in the direct generation of a TP. Okay. So keep that in mind when you looked at the citric acid cycle. Now, last space, last environment we're talking about is the intermembrane space. So between the inner and outer membrane, you'll have a space. Space. It's also, relatively packed with proteins, but more importantly, can be packed with protons as they are pumped out from the matrix into the intermembrane space during the electron transport system function. All right, just graphically again, just see your. Inner membrane in magenta or red and you see those in foldings and again these in foldings here are showing you that the increased surface area then allows for proteins such as F F. O. F. 1 80 P. A. S. Are to be highly abundant in particular regions. So those orange Spots you see here are depicting ATP synthesis or F O F 1 ATPases. That's another name for these ADP synthesis. And again, the increased surface area are going to play an important role because the more surface area that inner membrane has, it's likely that there is going to be a more space for electron transfer system proteins to occupy, right? So the capacity of energy generation increases as those Proteins are present in high concentrations. Now, of course, in terms of function, you will see mitochondria assembling and and functioning in in regions of cells that are of particular energetic or particularly energy demanding regions. So we have talked at length about neuron cells, right? So neurons. I have synaptic terminals, and there's a lot of membrane or gradients across that membrane that needs to be restored. And those gradients are requiring energy in order for proteins to work, right? To, for those pumps to work, sodium, potassium, calcium pumps, all of those, they require energy. So as a result, For example, places like synaptic terminals are packed with mitochondria, reason being because there's a lot of energy needed in that area. It's a likewise, likewise, and if you look at a sperm tail, you have, colors of mitochondria surrounding the agellar core, and that's usually because of the energy that is required for locomotion to take place. Additionally, another example would be, of course, cardiac muscle cells, which are, of course, Always in need of energy. Now this is just an overview and a brief depiction of our citric acid cycle. Pyruvate gets modi ed. And as we will see, we're not going to dwell into the details of this process, like we fi fl fi fi  did for glycolysis. What I want you guys to take home from our citric acid cycle are the overall products of the process. Now, very important, pyruvate enters the citric acid cycle as again, it's modi ed to form acetyl CoA. If you look at this cycle, you're looking at a cycle that is involved with what is called a decarboxylation process. So where you are essentially combining acetyl CoA to form a side and pyruvate to form citrate. And this six carbon molecule, it's going to be consequentially decarboxylated. So you have a carbon being removed and the overall formation of. Co2, which as you guys remember, it's a common combustion reaction product, right? So these is, this is actually the Co2 that you guys exhale. In as an organism, and it is again, CO2 reaction here or a combustion reaction releasing CO2 removes carbon from those substrates intermediate. Now, additionally, CO2 is not utilizing the energetic pathway, but. NADH will serve as reducing agent. So you can see that both NADH and FADH2 are most of the products that are formed from the citric acid cycle. And that's critical. And we will see where these NADH And FADH2 are utilized in the electron transport system in a second. Now, additionally, you also have GTP form, which, that is an energy currency as well. Those tris phosphates can fuel other processes. And that's the take home message from this slide. And the overall what I wanted you guys to remember from the citric acid cycle, it's again, series of intermediates and it's, Overall in charge of producing, reducing agents. Okay. There's multiple steps that are decarboxylating for the most part. And the overall reaction triggers again, modi cations of that translocated pyruvate and consequential modi cations in moving, removing the carbon two carbon molecules from, or two carbon atoms from from the molecule intermediates. Now. Again, they're all tied in, and of course they also can work in other, serving other processes that involves the formation of intermediates which also includes again series of intermediates in the glycolytic and the citric acid cycle. You will see that some of these substrates are utilized. For other biosynthetic pathways, okay? Now, I'm not going to ask you guys to recognize and remember each of these intermediates. What I want you guys to recognize is that these intermediate molecules are not only involved in energetic pathways, but they also serve as precursors in the biosynthetic pathways of the cell. Okay, let's just take a message here and of course, there's a lot of important elements in the cell that are derived from these intermediate molecules. Now, when we think about the way that the energy from NADH and FADH2 is harnessed to produce useful cellular energy, we have to think about this as a way by which we chemically harness the energy in the form of ATP, right? So there is what is called a chemosmotic coupling of energy. And that chemosmotic Coupling of energy can be the de ned or divided in two stages. Primarily it's stage one where you have the usage of electrons donated by the reducing agents of NADH and FADH two are donated and shuttle and transported across the electron transport system, and that transport leads to the pumping of protons from. Enzyme complexes that will pump protons from the matrix into the intermembrane space. So pumping protons out of the mitochondrial matrix. And and that generation that that pumping of fi fi fi fi protons generate an electrochemical gradient, right? There's going to be a lot of protons are going to be in the intermembrane space versus the matrix. So that is a proton gradient. And that proton gradient is a harness or it is utilized. In that second stage by our ATP synthase, it's harnessed in by channeling those protons back into the matrix in a way that the ATP synthase is able to use the energy stored in that gradient to generate useful cellular energy in the form of ATP. Graphically looks in the very, very simplistic terms, it looks something like this where you have your proton pumps and that proton pumps are fueled by the electrons donated by an ADH or FADH2, that protons then act to pump sorry, that pump works to pump protons across the inner membrane into the extra intermembrane space. And as a result, this gradient of protons is harnessed by the ATP synthase to produce ATP in that second stage, okay? Those are the two stages I was talking about. Now, for the most part, you might think that The process of ADP production by the ATP synthase is relatively similar to glycolysis, but it's absolutely not because it's using an energy gradient stored by the proton gradient that I showed you guys earlier. So as a result it does produce a signi cant amount of ATP based on this again, the formation of those reducing agents and the consequential proton pumping action. Of the electron transport system. So as a result, the mitochondrial ATP production compared to glycolysis is signi cantly higher. So in single molecule glucose and the two pyruvate molecules that come as a product of the derived glycolytic pathway can fuel the formation or the pumping action of a proton such as in such a way that it can produce up to 36 give or take depending on the books depending on pathways but up to 36 ATP molecules compared to two molecules of ATP producing glycolysis right as a net product now of course this is a major energetic pathway so any departure from the proper function will result in problems associated with energetic energetic balance and the overall energy budget of the cell and metabolic metabolic diseases including diabetes or any neurodegenerative diseases are oftentimes tied into these malfunctioning mitochondria. Now, of course the molecular basis of energy production in the mitochondria a little bit complex, so I wanted to brief you, give you guys a brief overview of that and give you a written portion again, that NADH formed by the citric acid cycle and FADH2 are utilized. The form of electron donors or what is called reducing agents. Those again, this is what I just mentioned earlier. So that pro those electrons ow across the electron transfer system and that you have a generated gradient of protons across that membrane, that inner membrane, right? There's on stage. Two, you have a coordinated translocation of those protons across the membrane by ATP synthase, which of course lead to the formation of ATP from inorganic phosphate and ADP, right? Now, this is oxidative phosphorylation, so I'm going to show you guys in a brief animation how that looks, and hopefully talk a little bit more. I'm going to show you Complex one here is actually a proton pump that utilizes the electrons from NADH to pump protons. Complex two, it's another enzyme. It's a, again, it's a peripheral protein that utilizes electrons from FADH2. And this case, complex two, it's not a proton pump, but you have complex three acting as a proton pump. And cytochrome C, which translocates electrons to complex four cytochrome C oxidase, right? Now, there's a ubiquinone pool that takes place that is located in the inner membrane of the mitochondria. fi fl fi And those electrons are essentially owing across these and and essentially combined with oxygen by cytochrome C oxidase to form water and, of course, some of the protons that are present. Thanks. So oxygen is being consumed in that process. The gradient is generated. Some of that gradient can be dissipated through intrinsic leakage of the mitochondrial inner membrane. And, but most importantly, the FOF 1 ATPase or ATP synthase is in charge of coordinating the Movement of protons across that membrane to generate ATP, right? Now, let's look at the ATPase in a bit more detail, since it's the protein that is pretty much chie y responsible for ATP generation. And again, I'm just going to describe the basics of it, but the ATP synthase is a multi subunit complex. We're going to dissect this complex in a moment, but there is a front channel, a rear channel and a front channel. We'll talk about this in a moment. And there's a series of subunits that compose our FO subunit. This is not a zero. This is an O and it's O because it was known that the inhibitor oligomycin is responsible for inhibiting the functioning of this unit. And then you have your F1 unit here that is connected to a stock to the F0 unit. So this is a rotating stock or internal rod. I will see that in a moment. That's pretty interesting how it works, but the entire thing is pretty, it's like a molecular machine. So it rotates and it essentially translocate protons from the outer, from the intermembrane space into the matrix. And as it do it changes the shape of this F1 unit. Allowing the ADP and inorganic phosphate to get into special spaces, catalytic sites, and to essentially form ATP in that process. Okay. Now, of course this is a complex that because of its importance has been widely studied in yeast. And we know a lot of how they work again, just showing you here molecular kind of spatial model of the actual protein. But. Most importantly, you got, it's reversed right now. So it's showing you the internal portion. So you have your F1 and F0 at the bottom here, connected to a rotor that pretty much leads to the rotation of this F1 unit. And then a stator that controls and keeps the the connection and the integrity of the protein altogether. Of course, we're describing F1, F0, or sorry, F0, F1, this is typo. 80 P. A. S. But there are other 80 pieces that you guys know your sodium potassium pump and your hydrogen pumps that are located in the back fuels are speci c types of other 80 pieces that are present and can be described, but they've fairly made a mode of action are similar. Across now they are reversible. That's a very important aspect. So in the, depending on concentration of ADP and ADP, AD and inorganic phosphate, this particular molecular machine here can run in an opposite direction. Okay. Now from what I want for you guys to take into consideration is the fact that there are two main units. One that rotates and one that states pretty much static. So again, the stator, what it does is allow that stable a stable con rmation here. I think I mentioned this rotates and it's not, so I do apologize. So the rotation occurs at the F O. Unit as we will see that the actual F. O. Subunit F. O. Unit will rotate the rotor will actually which is in bed and it passes into these F. One unit will rotate as well and will create conformational changes in these subunits. Alfa and beta subunits. We'll show you a video of that in a moment. Okay? Now again, this is a little bit more information of what you need. fi fl fi fl  I'm gonna glance through it, but what I want you guys to see is your, again, your FO sub F FO unit connected to your F1 unit in the form of a rotational stalk. And. You also have a stator that pretty much keeps those two units connected. Now, most of the interactions are going to occur into this C ring form here by 10 subunits. So there are 10 subunits conforming this C ring and there is a gamma subunit here that is going to be rotating, right? That's our rotor. Now as this rotor moves, this gamma subunit works it's rotating and it's an eccentric rotation. So it leads to changes in the shape of this. Of this F1 unit. So these subunits are going to change its shape. I'm going to show you an animation of that in a moment. But the idea is that as the subunits changes their shape, it's going to allow the exit or the entry. Of either ADP, inorganic phosphate, or ATP from these subunits. Okay now, how do we know how the rotation takes place, which is pretty cool. At this, I'm going to show you guys something real quick here, but it's it's again, a deviation. But how do we know that the actual C ring rotates and they, what they did at sexual, a couple of researchers were able to anchor a C ring sorry, the stock that normally connects the C ring with the F1 subunit. This is the F1 unit here composed of alpha and beta. Subunits. Oops. And so as you can see they, what they did is they were able to anchor modi ed stock here. The gamma subunit, they were able to anchor an actin lament. And that actin lament had uorescence. Capabilities. So there were uorescent tags in it, and they were able to document how the actual gamma subunit rotates, and it do so in essentially in three stages. So every so it's a full 360 degree turn, but it is done in 120 degree increments. So the rotations are taking place in 120 degree increments. Now this is pretty cool. I just, the rotation shown here is actually 130 revolutions per second. So you don't get the picture of how the orientation takes place, but I just wanted you guys to see that rotation right there. So this is again, that's what you're seeing there is the actin lament spinning. Of course, there is no active lament on the real protein. The acting lament was placed there to visualize the rotation. The gamma subunit is connected to your f your fo unit, which composes composed of your C rings and other proteins. It's pretty cool now the 120 degree rotation, if you were to look at the F1 unit. Again, this is the F1 unit that is composed of alpha alpha and beta subunits. And in the inside embed within those subunits, you have your gamma star. That is rotating eccentrically. So out of center, that's what I mean. And as it rotates, it changes the shape of these subunits. These alpha and beta subunits are bulging out, which allows ADP to come in. As you can see here. And as the actual. As the actual stock, as the actual gamma subunit rotates eccentrically, it changes the other shape of other subunits. Again, in other words, so every 120 degrees, everything is doing something di erent. Which it is forced by the action, the rotating action of the gamma subunit. That's what I wanted for you guys to take here. So this, the arrows are pointing to the rotation of the gamma stock and how in the right position there is either ADP and inorganic phosphate coming in. There might be the consequential closing of that subunit and the catalytic activity in one of the sites, the active sites, or consequently the actual trapping and catalytic function of the enzyme by the Squeezing ADP and inorganic phosphate together to form ATP. That nal step is where ATP is actually released, right? So just want to show you that video real quick. Hold on. And with that, we'll nish the lecture, right? This is a pretty cool video. Life fl fi fi fi fl fi ff fi fi fi fi  requires energy. The universal biological fuel is a molecule called ATP or adenosine triphosphate. ATP stores chemical energy in the form of a high energy phosphate bond. As the name suggests, it consists of a dexenazine ring with a chain of three phosphate groups attached. The last of these phosphates can be split o to release the energy within the bond. resulting in adenosine diphosphate and pre inorganic phosphate. This energy is needed to keep cells alive and is used from processes from muscle contraction to reducing the thoughts your brain is having right now. The hydrolysis of ATP can be reversed by the addition of inorganic phosphate to ADP. However, this of course requires energy and is performed by a molecular power generator called ATP synthase. Here we see an atomic model of F1 FO ATP synthase sitting in a membrane. This molecular motor is found in the mitochondria of eukaryotic cells and is responsible for making most of the ATP. Mitochondria, the powerhouses of the cells, turn over around 60 kilograms of ATP every day in an adult person. A long chain of biochemical processes convert the energy stored in food into a proton gradient across the inner mitochondrial membrane. These protons drive a rotation of the turbine within ATP synthase, resulting in the synthesis of ATP. Like all generators, ATP synthase consists of two separate motors coupled together. Within the membrane, we have the F O motor, named after the binding of antibiotic oligomycin. And at the top, we have the F I motor from Factor I. The F I motor is a proton powered motor. It is thought that protons ow through a channel open to just the intermembrane space. Where they bind into a ring of protein sub rotate 360 degrees and exit through another channel exposed only to the matrix. The net ow protons driven by the proton motor force provides the energy for the generation of rotation. The torque generated in the FO motor is transferred the F1 motor by a central stalk or shaft. The F1 motor is responsible for generating the a TP by additional phosphate to a DP. The top of the central stalk acts similar to a camshaft, so that as it rotates within the F1 motor, it causes conformational changes of the catalytic subunits. The catalytic unit is made of a dimer of subunits, and there are three of these arranged in a ring. Catalysis occurs at the interface between the dimers. If we concentrate on one dimer, we can observe three distinct states. First, ADP and phosphate bind to the catalytic site. The central star then rotates 120 degrees to rearrange the molecules. Next, the enzyme undergoes a further 120 degree rotation, and the a DP and phosphate are fused together to create a TP. The enzyme then rotates again to return to the starting position where a DP is released and a DP and phosphate can be bound for the next cycle of catalysis. One key aspect of this relationship is that catalytic subnets must remain stationary with respect to a rotating central shaft. This task is performed by a sca old on the outside of the complex, referred to as the peripheral stalk. Recent work has shown that what was once thought of as a rigid sca old is actually dynamic and is able to accommodate changes necessary for most e cient function. Looking at this machine in its natural environment by electron chromatography of intact mitochondria has shown that ATP synthase dimerizes to shape the mitochondrial inner membrane into their signature cristae shape. This turbocharges ATP synthesis by focusing the proton gradient near ATP synthase. fl fl ff ff ffi ff  All right, guys. So that's that was chapter 12. And again, only giving you guys a detail example of a single protein within all this realm which is our ATP synthase. So if you have questions, I will have my o ce hours on Tuesday as usual. And it will be there's also a remote option if needed, but. I'll be in my o ce if you need to chat with me for any particular purpose. All right. Until then catch up with you guys later. ffi ffi