 Hello, we're going to get started now. Yay Everybody say electron transport Okay, I am not with my child, so I will not talk to you like that. Okay So welcome back There was a phone left last time black case iPhone Nike on the back with the data turned off So find the phone didn't work. So if anybody found a phone And hopefully haven't sold it on eBay Please bring it up after class Okay, so today we we've set up a lot for this lecture and I kept saying aerobic Respiration you guys are like you're a liar man. There's no oxygen here today is the day man that we're gonna get some oxygen going And so we're gonna look at where this oxidative phosphorylation occurs some cool similarities between mitochondria and bacteria aerobic bacteria We're gonna be looking at the transfer of electrons and we'll see little chains of molecules They're used to transport these electrons You might imagine that there's some limitations the distances that an electron can just pass through space And so there's strategic placement of electron carriers With thin some of the protons or proteins that you'll see today And then we'll look at the way that we drive the formation of gradient of protons through the oxidation of NADH and then we'll have some loose ends and we'll do a Summing up at the end to get a sense for you know, how efficient aerobic respiration is and so we in the last two lectures we've covered an aerobic Respiration remember the end product of that is an acid lactic acid for us But in the presence of oxygen We are able to do the citric acid cycle and make lots of this NADH And now we're at the stage where we've made some of these reduced cofactors and we need to do something with them We need the currency of energy is not the oxidation of NADH the currency of energy is ATP oxidation of NADH Releases too much energy to be of general use across a variety of chemical transformations We need smaller quanta of energy So we're going to translate from this large amount of energy to a smaller amount a hydrolysis of ATP And so this all occurs in the mitochondria remember last time we're in the Krebs cycle in the mitochondria Remember I mentioned this outer membrane is somewhat porous molecules of 5kd or less are able to simply diffuse through these Pour-in channels, but then in the inner mitochondrial membrane that is impermeable To protons and other metabolites and so one of the important considerations today is that it not only Takes energy to make ATP molecules It also takes energy to move things into the appropriate place for us to do the reactions that we need to do So in general you can consider this inner membrane is impermeable to protons, but it's not absolutely perfect That's very small percentage of protons can leak through there. And so when you look at mitochondria It's like dude these look very similar There there's their size is similar to a bacteria They have both protein RNA encoding DNA. They have bacterial size ribosomes So you'll learn in lecture 20 that in eukaryotes. It's our ribosomes a little larger, but these bacteria have ribosomes that are Perfectly functional and they're 70 s in size And you also see These respiratory electron transport chains you can find those in a variety of aerobic bacteria as well and also this idea of Coupling a gradient to synthesis of ATP this chemo chemo osmotic Model that's also seen in bacteria. And so This leads people to propose the following model where an aerobic bacteria Infected a some sort of primordial anaerobic eukaryotic cell and these These bacteria then became what we see today as mitochondria, but then also in plant cells photosynthesis One one model of where that came from in plant cells is by Infection of plant cells with a cyanobacterial photosynthetic bacteria and so that conferred some of the useful abilities to perform photosynthesis so this is just an amazing similarity and strongly held belief that These these mitochondria that we see today are actually Primordial back aerobic bacteria, but they have changed over the eons and we're going to study what they are today And so this is the whole process. So what we're going to do here remember We left the last lecture making NADH in the matrix and this NADH needs to be Reoxidized right so all those enzymes those dehydrogenase enzymes that we've seen along the way will be of no use unless we have oxidized NAD so they can catch some more electrons So what we're going to do today is transfer these electrons directly from NADH and from F8 a DH2 Into this electron transport. So there's three different complex or four different complexes in this electron transport You can see each of these the Pink arrows you see the movement of protons from the matrix to the intermembrane space Now would you suppose that? Protons in the intermembrane space would stay necessarily in the inner membrane space So what did I just say about that outer membrane? It's got big fat holes in it and so what we're really doing here is we're Decreasing the concentration of protons in this matrix, right? So we're not probably not affecting very much the pH outside of the matrix, right? Because that can easily exchange with the environment around the mitochondria But we're evacuating or creating a very low concentration of protons here and that low concentration of protons and the movement of charged molecules in one direction this Electrogenic process will generate the energy necessary to make ATP molecules and so overall you have the transfer of electrons Starting from the NADH Ultimately being transferred through a series of these complexes in mobile electron carriers to an oxygen molecule Transforming that oxygen molecule for electrons at a time to water molecules. So that's the overall process here and so if you look at the energy here if you look at the redox potentials of NADH Or NAD in oxygen you'll see this transfer of electrons is highly exergonic If you do the calculations remember we set this up in an earlier lecture You get 220 kilojoules per mole and you guys remember ATP synthesis that only Takes maybe 30 kilojoules per mole You know if you consider Delta G maybe up to 50 kilojoules per mole because there's a higher concentration of ATP Than ADP it's about 10 to 1 and so really what we want to do is catch this energy So obviously we're going to need to make more than one ATP molecule per NADH And this might have evolved to just be a direct process you have some magical enzyme that oxidizes NADH to NAD and Somehow is able to catch that energy hold it for a while and then convert multiple ADP Molecules to ATP, but that's not how it evolved we it works through these gradients So there's a lot of energy here So potentially if this was absolutely a hundred percent efficient you can make potentially seven moles of ATP from that one mole of NADH oxidation and So if you look here you look at the pink arrows you see that a total of 10 protons are transferred Per NADH molecule oxidize. Okay, but to remember this molecule. What's the name of this enzyme? Does anybody remember this was in Krebs cycle this enzyme? member towards like the bottom 7 o'clock position in the circle Suction a it's transferring electrons. So it actually is a real Dehydrogenase right and so that comes in at a later stage. So the FADH to Results the oxidation of that results in only six Protons being transferred because it bypasses complex one Right, but the FADH to those two protons as you'll see Combine here and come out over here, right? And so you'll you'll see an example of this coming up Okay, and so if you do this stoichiometry, of course, we're making water molecules from oxygen molecules We need some hydrogens there. So We're taking for NAD and moving 10 moles per mole of NADH and so here we've labeled this Intermembering space in the matrix with an N side and a P side. What might you guess that means? N and P Just give it a guess. It's actually not written on a slide What might it be we're moving a charged proton Yeah, let it rip man. So there's a positive charge here is accumulation of charge And so we can use this gradient of both molecules and charge to drive The processes that we want to drive ATP Synthesis in the movement of metabolites across this membrane Okay, and so I'm not going to go This is the exact same slide that I showed you before and I'm just highlighting here again that there's two components The movement of these protons we're building up a gradient of molecules and also a gradient of charge and both of those Help the process to be more exergonic when you move molecules back into the mat matrix Especially if they're positively charged molecules, okay, or move negative charge net out of the mitochondria And so this is the overall process. So when we think about the movement of protons oftentimes you think about pH now pH is not Natural log right pH is a log base 10 so we can take that that original equation and Converted so if you look up in textbooks, you can see that you can express this as log base 10 if you just multiply by 2.3 So for every certain amount of change in pH that's going to affect how exergonic this is and so we're looking at the difference in pH between The negative side and the positive side, okay, and so we're moving charge molecules accumulating them on this positive side Okay, and so if you do all these calculations and you do some measurements a lot of times They use pH sensitive dyes that they load in the mitochondria so they can actually directly measure pH changes You see that this gradient that's formed by electron transport is about a change of pH about 0.75 pH units And so and if you do the calculation that's equivalent. So each mole of a proton or Cross gives you about 20 kilojoules of energy, right? So that's a little bit less than we need for ATP. So obviously when we're synthesizing ATP We're gonna have to move more than one proton. So any adh. Let's recap moves 10 protons f adh moves six right, but If we move one of those protons back, that's not enough to make ATP So we're actually gonna have to move as it turns out two and a half protons. Okay, and so Yep, so this is where we are right now. So what we're gonna do is go through this or not Let me just reconnect real quick Yeah Okay, so what we're gonna do is an actual electron transport chain and that the Magnetism they were going to apply to move electrons is this electron affinity Remember when I we talked earlier about this redox potentials I said you could also think of this table in terms of the affinity of each of these molecules for electrons So the the highest affinity For electrons is going to be the molecule that is the final resting place of the electrons makes sense But you can see electrons are going to be passed from one of these molecules to another now these are the redox potentials of of Various prosthetic groups that we'll see today in solution now the protein can modulate these things But the general processes each hop that an electron takes it's moving to a a position of higher Affinity and you can see this last hop generates a lot of there's a big delta here And so that's actually going to be used To to also push push the movement of some more protons in that last step. We'll see that at the end So here is the process. There's lots of cofactors. We have the proteins. We have this qh2 molecule And so that's lipophilic. It drifts around in the membrane carrying one Two electrons at a time between complexes Okay, and we've already seen FAD remember our succinate Dehydrogenase we can this is actually a one or a two electron carrier, but remember for succinate dehydrogenase. It is accepted to electrons and so These are our electron one of our electron carriers But the electron transport chains are not just these heme type prosthetic groups. They're also complexes of metal ions So we'll see metal and copper so copper is useful to store energy Iron here is useful to transfer these electrons and these We remember an econotaste. We saw another example of this of clusters of metal ions in that case It was more to provide that specific transformation Chemical transformation but here when you have a complex of metal ions in general the way nature is engineered this only one of these Metal ions is going to be changing oxidation state The other metal ions are precisely coordinating the position because the the distance between Where electrons are being transferred for is very very important But also these other metal ions are affecting the redox potential everything is exactly Precisely turns that every hop you're going to a higher electron affinity center Okay, and so here's an example where you have four iron atoms and only one goes to the oxidized state Okay, this is our mobile electron carrier You can see it's very lipophilic So we have ten of these isoprene units you guys now are very aware of the structure of isoprene and so This is attached to a Functional group this quinone this quinone can be Reduced by one electron to the semi-quinone. We'll actually see that today But also can have a two electron reduction to the ubiquinol form Okay, and so we abbreviate just from now on I'll just say Q Q H and Q H two these are the different Reduction states of this carrier and this thing just does Moves around in the bilayer. Here's the structure of some of the heme groups Remember some of the electrons are carried on heme groups others are carried on actual Just coordinated metal ions such as iron and copper and these also have tuned their electron affinity So this is increasing affinity here. So low higher higher And so each of these is our prosthetic groups So they're held tightly within the enzyme active site and actually remember when we saw heme before in my globin We had one histidine on one side and then the other side bound oxygen actually these hemes as they sit in the proteins You'll see today both sides are protected because we don't want to carry an oxygen molecule here We just want to have these change their Reduction state and so you know we want to control their environment very precisely by actually Ligating both sides of the iron Any questions so far All right, so each of these a complex this is a complex one two three and four has in assortment of different polypeptides So for example complex one has 43 different subunits it has a variety of different Prosthetic groups they're involved in the electron transport. We've already seen succinate dehydrogenase remember we saw FAD Electrons are actually waiting for us from the last lecture sitting on FAD as FADH2 on that protein And so some of these are just you know huge almost a mega Dalton in size if you look at the aggregate molecular weight and so And in you want one tidbit of information that might not be too surprising to you that each of you might think okay Well, this is very precise. We got transfer electrons But the efficiency of each of these is not identical So the Kcat over Km is not a good denicle for all these so for the ones They're a little bit slower a little less efficient We're going to need a higher abundance of those complexes So there's all of these complexes are not in the same amount, but they're in the perfect amount so that there's enough of them When you figure their catalytic efficiency, okay, so that's a little tidbit to throw in okay So let's we're going to discuss for these one at a time so complex one is responsible for moving four protons from the matrix to the intermembrane space and Then two of these protons are also strategically being Taking up from the matrix this ends I could have pulled them out from here But that would be kind of productive so that the two protons are taken up from here And then loaded on to our reduced qh to lipophilic mobile carrier. And so here's an NAD This is backwards isn't it? Yeah, it was backwards. Could you correct your slides, please? So we're taking electrons This one doesn't have much left to give I never caught that So so but look at this see the electrons It's like these strategically placed gauntlet of Different iron sulfur complexes in this FMN remember FMN is the head group of the FAD molecule, right? So it's not a it's a mononucleotide But that thing is just sitting there as a prosthetic group electrons are passed from one to the next according to electron affinity ultimately being deposited on Ubiquan you ubiquinone There and so Yep, and so we're converting this hydrophilic carrier in adh to a hydrophobic carrier So after this process the electrons end up on a hydrophobic carrier And so four protons go there and two go here And so let's look at this. So let's think about the logic here There's a linear relationship between the log of the rate of the transfer of the electron and the distance in each hop right, so if you had the total distance here is 74 angstroms and so if you look on this handy-dandy plot 74 angstroms It's just not going to happen. You need to transfer these, you know the way it works in nature. It's about I guess ten ten angstroms seven angstroms each pop, right? So it's not perfect, but it's you know This is what is necessary to move the electrons through here and so this movement of electrons and The the deposition of those electrons on our mobile electron carrier The Q molecule coenzyme Q is going to cause a Confirmational change and so some of that energy used to move these Electrons is going to cause a kick in this whole complex remember There's a large number of polypeptides here, and so this is actually a brand new hot off the presses This is the the the structure the crystal structure of the bacterial Complex one and so it sort of looks upside down because bacteria do their electron transport in a confusing way But the the assumption is that the eukaryotic Complex one structure is going to be very similar and so here you can see There's a variety. So this is the actual Membrane and so Throughout here you have a bunch of helices spanning this membrane But then you have this oddly placed shaft here So you have this one long helix that is touching all of these different polypeptides In this part of the protein and so the protons are going to make their way through here And so there's a total of one two three four different channels and so So you can see that along where these protons are there's these Hydrophilic pathways so you can imagine that a lot of the axial amino acids on these alpha helices are going to be Hydrophobic because this thing's sitting in a bilayer, but they have these Hydrophilic pathways the other cool thing that's going on here is there's this shaft of Hydrophilic amino acids that goes all the way down Alternating charge and so this is linking all of these complexes together So you have both the helix a structural link between all these different polypeptides, but you also have this this I guess you could say a salt bridge, so it's a long chain of Oddly, I mean you want to expect in the middle of a bilayer to have all these hydrophilic amino acids But this is useful because these are the things they're going to pull and push here And so when we transfer an electron that causes the the change of shape of This ubiquinone to ubiquinol reduce form shifts all of these things causing These transporters to change their confirmation. So here you have When you don't have molecules bound you have you're open to in our case it would be the matrix and eukaryotic cells and then when you reduce the molecule the thing pushes using this shaft of all these Polar amino acids in this helix and changes the confirmation here right moving it so that you release the molecules of Protons on the other side in our case it would be going to the intermembrane space. So it's a coordinate movement sort of like rowing teams So some members of the Brown crew team here and they can tell you all about how you have to be in perfect unison Right to make this the most efficient and so there's structural features that help us to do that It's really cool stuff Okay, so now we we've if we come in at complex to we bypass the movement of these four protons, right? And so let's look at that complex to real quick. So remember this is succinate dehydrogenase. It really is Dehydrogenating its substrates. You're forming a double bond not in that in this case a new bond to oxygen Any questions so far? I'm just sort of blazing blazing a trail questions All right, so we have our we left our electrons in this citric acid cycle on FADH2 And so what we're going to do here is a transfer those up to the same Lipophilic carrier this QH2 molecule is going to be the final resting place And so here's our FAD remember I left our electrons there and citric acid cycle And then those are as soon as they come off in the citric acid cycle They're immediately transferred up into this QH2 molecule and so on this also has lots of different same sort of idea you have a a series of electron centers that can transfer and minimize the distance that the electron has to hop Okay, and so we've We end up moving for less protons Okay, so now we've got all of our electrons on QH2 and now we need to Go into complex three now complex three is confusing because complex three is translating languages it's translating from the two electron language of QH2 Right to the one electron language of cytochrome C So as it turns out the next carrier for electrons could not accept two electrons at a time It has to accept just one But we need you know, so we're coming in with two and these are hot potatoes, right? You can't just sort of let the electrons float in space. They would immediately be dissipated So we need a safe place to store our electrons here So that we can translate and get one electron up to cytochrome C So obviously for each QH2 that comes in we're going to have two cytochrome C's In the reduced form coming out, right electrons are not disappearing in this process So Yeah, and then cytochrome C is the next thing but it can only accept one electron at a time And cytochrome C is actually a protein that's floating around in the inner membrane It's bigger than five kilodaltons, so it doesn't get out through those porans, but it's able to fuse there It's nice to have this mobile proton carrier that's floating around because it helps to buffer so if there's rapid changes and the Arrival of substrate in adh this thing you can have a little bit of buffering capacity, right? So if everything was a one-to-one linkage then that flexibility wouldn't be Available, so let's look at QH2 So this is something called the Q cycle and so we have QH2 remember QH2 could have come from complex one or Complex two remember both of those make a QH2 molecule now when this two electron carrier arrives at complex three One of the electrons does what you might guess it goes right on to cytochrome C Which is waiting there and that binding of the electron by cytochrome C induces a conformational change causing Cytochrome C to be released from complex three, but the other electron binds to another oxidized Q molecule Okay, and so the input here is QH2 reduced form and an oxidized form Okay, and so this single electron that's why it's cool that this thing can carry It doesn't have to carry two electrons at a time it can carry one electron, so it's a safe It's the semi-quinone form of the molecule and this is somewhat energized But it's a safe place to store electrons instead of just having it sitting around Floating around in space, so we have the semi-quinone Of the Q molecule coenzyme Q and it's storing our electron So now this is good, but you know we need to Figure this out like so we need to actually move another electron here We got to get this electron off of the Q molecule and put it back up here, and you might guess what would happen next It's somewhat logical. You have a second QH2 molecule come in And that QH2 molecule is going to do the exact same thing same move in electrons One electron goes to a second Cytochrome C molecule converting it from its oxidized to reduced form and the other Electron is going to go to the semi-quinone that's storing our first electron So now we've generated another QH2 molecule, but again, we're strategically taking The protons necessary here from the matrix So this is not a direct transport of a proton across the by their member complex one We have the seesawing of an actual transporter molecule here We're grabbing strategically grabbing protons from the matrix carry them on QH2 and then eventually Releasing those protons so here you can see there's two protons coming out But these came from these these QH2 is QH2, right? So we can't tell if it's something that was made by a complex three Complex one or complex two. It's a whole gamish, right? And so but this is the net process. So let's look at the overall process So you have a QH2 and a Q come in Split the electron one up one to make the semi-quinone and then you have a second QH2 come in You fully reduce the semi-quinone To a QH2 and the other electron goes to a second cytochrome C molecule So we've now translated through the Q cycle a two electron carrier into a one electron carrier Okay, you with me so far? Q cycle is a bit confusing. Yes There's no questions. You understand or it's just too painful All right, so we're going to move on. Okay So the last step well, here's the aerobic respiration part. Here's the oxygen molecule so oxygen is relatively able to pass through a Membrane so we have some amount of oxygen in the membrane the structural This is drawn in cartoon form because we really don't have an x-ray crystal structure yet That's very good for this But so the general thought is the oxygen molecule comes in here, but here we have another translation Problems so we have our one electron carrier Arriving cytochrome C docs like a space, you know lunar lander lands on a complex four But it's just it's insufficient to provide all the electrons for this Four electron reduction of oxygen so it delivers just one electron So we need a battery Right a copper top here is a copper atom That's waiting and this copper atom when it becomes reduced is still pretty stable And it's able to store some of these electrons There's actually a little tyrosine residue as well from one of these polypeptides and that tyrosine can become a tyrosyl radical It's sort of safe to put your radicals on something that's covalently attached To to a tyrosy or to the polypeptide because then you're not going to tend to release it So this is a very complex process You can read about all the details online or in your textbook But the net idea here is let's store up and wait till we get all four electrons So that means the docking the lunar landing of four total cytochrome C molecules and transfer those electrons storage on these copper atoms in these prosthetic groups and then all at once in a process that we don't fully understand yet exactly Like how does it know when it has four well you'd imagine that the changing the redox state of some of these metals might change the structure and so perhaps the binding this is just pure speculation perhaps the binding of oxygen Can only happen through the allosteric Changes that occur when these copper these copper ions change their oxidation state So then you bind oxygen and in one fell swoop transfer all four of those electrons to oxygen and also Transport for protons across here So remember that that last hop in a redox potentials table is like 0.5 Difference between like the last one which is here and oxygen. So there's a large amount of energy here It's enough to drive four protons across here Okay, but the final resting place of our electrons starting from NADH Was to land on oxygen. They're sitting out here in water molecules. Okay? and so we've transformed the chemical energy of the non aromatic reduced form of NADH and FADH2 to a gradient and so it took energy to make that gradient right so we have a Positive charge accumulating on the outside of this bilayer, and you have a higher concentration of protons Or you could consider it as a lower concentration of protons in the matrix And now we're just going to use transport in reverse to make our ATP molecules. Does this make sense so far? Cool Wow, so you might wonder like you just made this stuff up. Do you where did it come from? So these are actual experiments where people were able to Figure out, you know one of the general question is okay. I know there's a lot of different Prosthetic groups. What is the order of things like so here? We know the rule is that electrons only going to make that hop if it's going to an electron carrier that has higher affinity But you know a one necessary thing to understand the process is to say, okay? What what direction are these hops going in and so you can add inhibitors to different? complexes right so Here we have an inhibitor complex one and then you can look at the oxidation state of each of these prosthetic groups You can disrupt the protein using harsh conditions biochemically in rich for each of these prosthetic groups and then use spectroscopy to be able to measure their Oxidation states has when these prosthetic groups change oxidation state. You have a different set of absorbences show up So you can differentiate for each purified Prosthetic group the actual whether it's oxidized or reduced and so if you add the inhibitor that says okay Well, NADH comes before all of these other things say okay. That was not that it's shocking Antimysin a turns out to inhibit complex 3 and so that told them that these Factors can come before Complex 3 and then cyanide and carbon monoxide inhibit complex 4 and so they found oh man all of our cofactors are reduced can you Guess what the mechanism of this inhibition might be? So we use we learned some terms to describe inhibition when we thought about mackayless Minton What might you guess? Just looking at these inhibitors. What kind of inhibitors might they be? Competitive right so they structurally look very very similar right so carbon and oxide carbon Carbon monoxide and cyanide are literally competing for binding of oxygen on complex 4 Right, so these are just awful poisons And so all of all of this the whole electron transport will be shut off when we inhibit this complex 4 These are competitive inhibitors to the binding of oxygen to complex 4 So this is the type of way before you because these are not easy So I mean there one mega Dalton it takes a long time to get the structures We don't have the structures of any of the eukaryotic proteins Yes, just the prokaryotic proteins and so while we're waiting for those structures to be solved we can figure out the order of of Pristhetic groups and so this is a this is an illustration of how these electron carriers change absorbance is when they Oscillate between the oxidized and reduced form so that's how they the last experiment they were able to tell which molecules are oxidized But which molecules are reduced? Okay, and so we have a lot of energy built up here We've transferred this chemical energy and you can actually do some calculations Using our equation and so you can look I think the next slide or coming up eventually You can actually calculate how much energy has been Stored in this disequilibrium of both charge and protons Okay, and so we have both the chemical potential being stored up the aggregation of protons or the Disaggregation of protons in the matrix because the matrix now has a very low amount of protons We also have this charge so we can literally plug our USB cable directly into the membrane and tap out some of that energy Okay, so this is electron transport Any questions on that so far anything Alex in the forum the monitor Yeah, one about cytochrome C and complex BC one. Could you start again? I could there's one question about cytochrome C and complex BC one Yeah What causes the cytochrome C to bind to the third complex? It just has a set of cognate amino acids So there's a surface on the cytochrome C and it has a distribution of most likely positive And negative charges as well as Hydrophilic groups that causes a complementary binding on the complex So it's just a protein protein interaction But the change in the oxidation state causes cytochrome C to rearrange some of those amino acids and then be released And then second question why are four protons taken from the negative side if only one? Qh2 is formed for every qh2 coming into okay, so it is a confusing figure So we'll go through this one more time Okay So so the question is for for overall where are the other two coming from in the matrix What do you guys think? So here you like Okay So the overall summary Is here Right, so you got two protons actually literally being grabbed from the matrix where are the other two protons coming for four coming out to coming in man I'm not a genius, but that doesn't add up I'm not that smart So they're coming from somewhere else either from here or from here, right? So you could add two protons here because remember what is succinate? You're actually removing two protons for the succinate molecule to make a double doubly bond bonded fumarate So there's some protons or here in this complex. We're taking those protons So the same complex doesn't pick up the two of those protons As as the other so the other complex you have two protons here to do that or two protons here And those end up being two of the protons coming out It's a little confusing. I totally agree. I'm sorry the nature made it confusing That makes sense now Okay, so this is so now we've built up a huge amount of energy in this gradient and we're going to use this ATP synthase to be able to Transform that gradient energy, right? So this energy of the non Electrogenic transport of protons and we're going to use that to drive the synthesis of ATP molecules. So this is the F zero F one ATP synthase. So this is the F zero part Down here. It's the part in the membrane and this is the F one part up here and it literally is a a Rotating molecule. So this part in the membrane has the C subunits these things rotate So as protons come in they hitch a ride on aspartic acid, I believe Amino acid yep aspartic acid and as they ride that drives the rotation of this whole thing It's a cylinder rotating in the membrane. Isn't it amazing? And then you have this gamma subunit and that's attached to the rotating cylinder So the protons are pushing as there's a lot of gradient here There's a lot of energy of this protons coming back down across so here it's upside down, right? So here's the matrix and here's where our protons are if accumulated and so that high concentration of protons is pushing this whole Thing to rotate and this thing is attached the C subunits are attached to the gamma Subunit and this gamma subunit is a a cam camshaft thing And so this what this thing that the gamma subunit turns while this beta subunit is holding this whole top steady So if the whole thing just turned you wouldn't be able to harness energy So if this whole top and bottom everything turned, you know, there would be no energy being used here It would just be rotating be fun to look at Right and so but what we want to do is drive the synthesis of ATP from ATP and phosphate And so that synthesis occurs on these beta. So there's three beta subunits Which are the subunits that synthesize ATP the alpha subunits confusingly combined ATP? But they cannot or ADP but they cannot synthesize they're non-catalytic the alpha subunits just help to position the beta subunits Okay, and so this is steady this thing is sort of holding. It's a stator It's just like you'd see in an engine this stator and the camshaft is rotating and it's bonking each of these subunits So the the gamma subunit is sort of like a wedge and as it rotates it box each subunit So there's something that this has been engineered so that To to kick things out right because it's the rotation causes this Non-cylindrical thing up in here to hit each of the subunits And so one of the earliest experiments they did they wanted to figure out say how exactly is ATP being synthesized here one possible They probably I would have guessed the possibility a lot of people were going for was that well each of these beta subunits probably does its Confirmational thing right changing shape and and simultaneously and independently Synthesize ATP, but then they did a very simple experiment Where they they lopped off the top part of this so as it turns out this this is an enzyme that catalyzes ATP synthesis synthesis, but when you cut off the top part, it's able to hydrolyze ATP It's able to work in reverse and so when they did this experiment when you think about a hydrolysis reaction There should be one oxygen attached to your hydrolysis product this phosphate That is coming from a water molecule and so they're saying okay This is sort of a waste of time everybody everybody knows when we do this experiment hydrolyzing ATP We're going to get this inorganic phosphate and only one of the oxygen atoms are going to be labeled So why are you wasting our time? But the intrepid graduates didn't do this and just say dudes. They're all labeled And this was shocking when you think about ATP hydrolysis. What does this tell you about the thermodynamics of ATP? hydrolysis in this enzyme it's very surprising. What does it tell you about equilibrium? Think about so how many what is the standard change in free energy of hydrolysis of ATP? 30 kilojoules per mole. So should that be easily reversed So they're like to do this thing. They're all labeled. There's an equilibrium, you know up to the point of Synthesis of ATP or transformation between ATP and ADP this thing is basically binding being transformed coming off Binding coming off binding coming off and that's weird. They're like well wait a minute should be it's hydrolyzed It's lost so much free energy that there's not enough energy to go go for another round And so what this suggested is a shocking of free energy profile so here we have the the expected Free energy profile and they saw ADP is a lot less free energy than ATP and hydrolysis should be a one-way street There shouldn't be an equilibrium only one of those oxygens should be labeled But this result told them well There is an equilibrium between the enzyme-borne bound ADP and inorganic phosphate and the enzyme bound ATP They have Delta G it must necessarily be approaching zero because you have labeling throughout Which means this thing comes off and on and all of the oxygens get labeled eventually And so what this experiment told them is that the energy requiring step that this enzyme has designed is the kicking out of the phosphate So you can think of this as dust boot, right? So it just kicks out the phosphate and that and so if it's if this is greatly stabilized How would an enzyme stabilize a substrate? By making tons of bonds to the ATP molecule many more bonds to ATP than to ADP So it binds it very tightly. It's like dude. I like my ATP. You can't have it And so what this camshaft is doing is this providing the boot and it's kicking out the ATP molecules right and so that that's that's the that's what and so you can think about the The affinity here it turns out that the F1 subunit binds with this insanely high affinity To ATP compared to ADP right and so that's because there's just more bonds to that last phosphate There's all kinds of coordination going on there And so that provides you an ability to make the or to have the kicking out of the ATP be the energy requiring step They did one more experiment. So there's still this question. Okay, is it the are all these beta subunits? independently synthesizing ATP or is there some kind of coordination as the camshaft rotates around In other words as the camshaft have three blades or one blade that bumps the subunits And they didn't need a structure to figure this out So what they did is they threw in a nine non-hydrolyzable analog of ATP so it has this amino group Where in this phospho anhydride position that normally would have been an oxygen And so they stuck this in here and what they found is that this analog if each subunit was acting independently and the kicking out of the ATP is the hard step the expectation would be okay all three of these subunits should Have this this this inhibitor so this is going to block You know block this process and so all three should have this inhibitor if each one is acting independently But then they got the crystal structure and they say okay No, we have one of the three subunits has an ATP the other subunit has a ADP and phosphate and the other subunit is empty And they said ah ha The camshaft has one knob on it. So this is the structure. They got they had one subunit They're all sort of changes in confirmation Illustrated with these these different circles. So this is one conformational state. This is another Confirmational state. This is a third Confirmational state so they found this inhibitor was only in one position when they had one position the other positions were not able to just You know also have a Analog ATP analog there and so they found ATP here They found an ADP binding site and they found an empty site Okay, and so this led them to the current working model This is a little confusing you might want to enlarge these arrows because it's sort of important So this is the camshaft. So this is what a camshaft did is it has a round thing and every once in a while Has a knob remember this is like a pencil pointing up through the donut of that F1 subunit This pencil has a knob on one side. So every time it rotates every 120 degrees That knob is going to contact the beta subunit and literally kick the ATP molecule out so let's start from here and watch this so here we have the the the Gamma subunit the camshaft is pointing here that kicks an ATP out But then as this thing is rotating she's rotting rotating this case in counter clock wise of fashion So when it moves to the next position it kicks that out It actually has like some amino acids there that just crunch against that subunit and have it change its confirmation Kicking ATP out and then you rotate again again. We're rotating counterclockwise We're looking this is the top of the F1 subunit We're looking down the shaft of the gamma and this is the little Camshafty part of the gamma subunit. So you rotate to here. It kicks it out Okay, and so each And people have done a lot of work trying to study the stoichiometry here And they found for each movement three protons. So every ATP molecule that synthesized three protons need to be dissipated through the gradient now this totally depends on the type of organism you're working with you can imagine the exact lipid composition might affect the what percent of Protons are just able to dissipate so certain lipids perhaps the protons can so this is a general ballpark estimate takes about three protons Moving down their electrochemical gradient to move this camshaft by 120 degrees So this is like a nice model But they actually took a movie and this movie they could actually see the thing turn 120 stop stop Stop, I mean it's just so these dudes in Japan like hooked up some Acton to these subunits flipped them upside down and had this actin fluorescent whip going in circles Right and so each yes, so this sort of summarizes everything we've seen so far This thing this camshaft is rotating kicking it out every 120 degrees. It kicks out an ATP. Look at this insane Experiment so they separated this F1 complex They put what's called a his tag some of you have done some of these type of experiments where they put a tag on these Beta subunits and they put a nickel-coated plate and so the his tags this thing landed upside down Right so it can go in the opposite direction remember it can make ATP or can hydrolyze ATP in this case They were looking at ATP hydrolysis So they put this thing upside down on a cover slip and literally attached a whip to it On this side of the molecule they had another tag on the C subunits and the biotin Made specific interactions with the abitin labeled actin that had a fluorescent tag And so they're gonna actually watch this thing. They hold me. This is making new strong bonds here So now this thing as it rotates. It's just gonna whip around right So here's a movie 120 120 There it is, dude These things get biochemist quite excited But it stops. I mean it's you know sort of it's not exactly perfect in nature You don't have a whip going around another question that comes up often is like how fast is the rate? I don't know. It's probably pretty damn fast But it literally every time it stops. That's when it's like get out It's kicking out ATP molecules So there's direct experimental evidence of this mechanistic hypothesis So here in the next movie you can look at that we saw this in the first lecture You can look at the conformational changes that are occurring here And so see as that thing rotates see how everything is like moving around so look here comes the phosphate in That ADP look at all those bonds to the ATP It's like grabbing it and then that campshaft comes around and changes all of those amino acids and kicks it out So literally and see how this side thing is holding it in place preventing the top from rotating It's it's a little engine and this is how the majority of ATP is man. You're so I'm sorry Excuse my excitement Okay, all right, so let's do an accounting of costs here So so far we've transferred member 10 moles of protons per mole of NADH molecules or Six moles of protons for FADH for one mole of FADH 2 And then we saw I showed you that it takes three Protons to make one ATP molecule and but there's other things we have to do So we need another proton to transport molecules across the bilayer Right, so remember we're using where did we use the you've already seen us use a proton gradient to transport a molecule Do you remember where drink? Pirate so the movement of pyruvate was driven by protons. What else might you have to be moving across bilayers to? synthesize ATP inorganic phosphate and ADP have to come into the matrix and ATP has to come out that takes energy you're crossing a bilayer And so a total on average it takes about four protons four moles of protons To make an ATP molecule and so if you calculate that at four mole of protons that comes out to about 80 kilojoules That's way more than enough to make an ATP now ATP is usually about tenfold higher concentration ADP So in terms of the cellular state change in free energy, it's around 50 Right, but so this is you know up to the point of transport of protons across the bilayers about 90 percent efficient But then in the synthesis of ATP It's about 38 percent efficient. You say well, where did the rest of it go? Made heat right, so if it's not making molecules. It's making heat Okay, and so you are a warm-blooded person the heat that warms your body up is the excess from this process All right, so this keeps your core nice and toasty You have a nice layer of fat at least in case of the professor keep all that heat on the inside But if you're a bear you need a little bit more heat So you're just sitting in your cave a nice beer He's sitting there, and you have a nice dreams. Can you imagine sleeping for all winter? It'd be better than teaching I think or taking exams So the bear has to have test to tap into this heat So one of the reasons Evolutionarily that you know it wasn't a direct conversion of NADH to ATP is because we can also use heat And so bears they they uncouple their respiration. So they have this uncoupling protein thermogenin our little babies They have this brown fat that brown fat has expressed as some of this protein which allows Protons to come back down the gradient Right, so they have a futile cycle of protons Oxidation of pyruvate just like burning a tree generates heat, right? So if you're just allowing this to dissipate that's actually releasing heat keeping mr. Bear warm in the winter So that's another thing. There's also used to be a diet pill called dinitrofinol a poorly conceived diet pill this diet pill this molecule could take that proton and Wiggle its way through the into the matrix Right and deposit that proton on the other side So it's like, you know arsenic is a diet pill, right? So eventually people like why are you so pasty dude? It's like, you know, you just sit in there like a brown bear You're uncoupled you're not able to have enough ATP to make molecules, right? It's he makes you sick and so this is another molecule this FCCP which can bind a proton And is greasy enough to just slither its way across that inner membrane and deposit this proton on the other side So there's both a protein a protein that can in a gated fashion Control the transfer of protons when you're hibernating, but also you can have this small molecules can make this journey Disappating the gradient. So now I mentioned what about this one more proton? So it takes four protons moved across the bilayer to make one ATP Three to drive this camshaft around kicking out ATP molecules. The other one is right here, right? And so you're dissipating some of this. This is a Simport both of these are active transport so you're moving down the gradient with the proton and up the gradient with the inorganic phosphate and here you're Transporting or transferring ADPN or we need both ADP and inorganic phosphate and ATP out Why is this using the energy of the gradient? Or is it? Is it a good quicker question? I'll just write it right now Why? Is that using proton gradients? Look at the movement of charge Three negative charges going out Four negative charges coming in or I'm sorry four negative charges coming out three negative charges coming in So there's a net efflux of negative charge Is it helpful to have this plus and the minus the way they are? That's from the movement of protons. So the movement of protons is driving. This is a energy requiring step So this is co-transport and we're using just this Disequilibrium of charge on the bilayer So this is dissipating some more energy because we had to have this thing encapsulated in a mitochondria We're going to have to spend some energy to get get molecules moved across that bilayer Okay, so this is the whole process. We've deposited electrons on an oxygen molecule to make water and we've made ATP so let's Yeah, but we have one more problem We made an NADH molecule and where is that that was in glycolysis that was in the cytosol So we need a particularly scary pathway to get that NADH Into the mitochondria remember NADH on complex one is binding in the matrix It's not binding in the inner membrane space. And so we have this It gets better and better. So we have what's called the aspartate malate shuttle and this is electro neutral There's charged molecules moving But if you count everything up the number of charges moving in each direction, it's neutral Right, and so we have first we run this enzyme in reverse like dude. That's in the cytosol Yes, there's a cytosolic version of that enzyme and a matrix version of that enzyme So the cytosolic version same darn enzyme you saw it in the Krebs cycle Remember and so we're taking and running this in reverse We're depositing this electron from NADH on toxyl acetate taking the carbonyl to an alcohol That malate is then transferred in once in the we can have the malate dehydrogenase You know in the Krebs cycle, we know exactly what Krebs cycle is doing We're oxidizing and we transfer those electrons back on to NADH So the way this has evolved is that the electrons take a ride on a small metabolite Through into the matrix and then are released on to NADH and now we're in the right place now We can use NADH to an electron transport and there's all this other complexity So we're actually moving glutamic acid and alpha-ketoglutarate And there's this amino transferase. We're going to see the mechanism. This is a beautiful mechanism But it's really complicated. So we're going to skip that to a little bit later But here we're taking this carbonyl group and converting it to an amino group So this is aspartic amino aspartic acid amino acid that gets transported out and so that we have We don't want to have like net efflux of TCA Metabolites, so we have sort of both of these if you look at this process We don't have any net efflux anything comes in goes out So it's sort of making a circle here, but the net input is NADH and the output is NADH Okay, so this is called the aspartate malate shuttle There's a so this this is complicated not only to study But it takes time for this to happen in a cell So this typically this type of transport occurs mostly in heart tissue and in liver But in your muscle when you're making a ton of NADH, you know So when you start running on a lot you get those those pains that's lactic acid building up But the alternate place to put those electrons are on NADH in the mitochondria So instead of taking all this time and doing all these chemical transformations You can literally take a syringe and inject those electrons Directly into this inner membrane of the mitochondria. So there's this enzyme mitochondrial glycerol 3-phosphate Dehydrogenase which takes a glycerol a alcohol functionality and oxidizes it To a carbonyl group putting those electrons on FADH2 now remember succinate dehydrogenase that was hanging out in the matrix This guy is hanging out here So the advantages of having it on the outer surface here is that we don't need to transport molecules at all We could just inject those electrons directly in in the form of QH2, but it's less affinit efficient We took NADH and we just made QH2 that we lost the transport of four Protons, but we gain gained speed so when you're running around the lions behind you the bear is woken up from a slumber I mean you need to move fast and you're much more efficient when you can do aerobic respiration So in your skeletal muscle you have this process occurring so that you can rapidly it's not completely efficient You're losing four of those protons one ATP molecule per NADH, but it's still you know reasonably good Okay, so these are now. Let's do a whole counting of the cost where everything is so in glycolysis We're going to put everything together from taking our Glucose and converting it to carbon dioxide. What's the total number of ATP molecules? This is often asked on tests So pay attention that you can put a box around it if it's helpful so in glycolysis remember we made two NADHs because we split the molecule remember that part So per glucose two NADHs now if we're skeletal muscle and the bear is like chasing me around We're going to inject those electrons directly into the membrane or if we know the muscle your heart to start Doing the same thing very steady, then we have time to transport those electrons and get some more ATP molecules Right, so you have two NADHs for each NADH you make Either 2.5 if you fully take NADH into the matrix right so making ATP or The oxidation of NADH moves 10 protons takes four protons To make an ATP including the camshaft action and the transport of molecules So for 10 and 4 that's 2.5 per NADH molecule. We have two NADHs, so it comes out to five So you can convince yourself of that But if we shortcut and skip those first four protons by injecting the electrons directly in Bear escaping muscles, then we lose an ATP molecule and get 1.5 ATP molecules per NADH of course We've also made an ATP and that's in the cytosol remember pyruvate. We took each pyruvate molecule made one NADH We have two coming in from glucose, so it's two NADHs 2.5 ATPs per NADH remember four protons 10 total are moved and then we had in Krebs cycle. We had remember in the circle. It's like down here is one two three right times two right Is six that ends up you multiply by 2.5 you get 15 FADH We skip four protons again So you got three and of course you got two because you came in with part you came in with glucose Split into you get two pyruvates two pyruvates coming all the way in Remember there's that one step towards towards the bottom of the Krebs cycle where you make a GTP And I mentioned that GTP can be interconverted between ATP and GTP So if you sum all this together Depending on whether the lion is chasing you or not you have 30 or 32 ATP molecules So this is a good one to just make sure this is a Aggregation of knowledge type of slide so look through all the previous slide make sure that You believe this okay, and so this is pretty amazing This we're capturing that energy At about 32 to 34 percent efficiency the car then you might drive around go to Walmart or whatever That's only 30 percent efficient But some of that energy was wasted to keep us warm right to keep us warm-blooded animals Right and so it's even more efficient if you consider it's useful both to make ATP's and not be as cold as the room Okay All right, so here's some of you will be doctors hopefully and so in ischemic Injury you have like some kind of problem with your blood vessels being clogged and that ultimately results in Oxygen not being delivered to your cells. What might happen to the pH when oxygen is not delivered? What molecule would be synthesized? Black tit lactic acid Right, so if you're not getting oxygen it's all about glycolysis and you get acidic so there's this protein and it has a Transformation from a tetramer to a dimer depending on pH and So when the pH goes down when your blood's not flowing oxygen is cut off This makes a dimer and it's only able to bind so this is actually the F1 subunit of Two different F0 F1 ATP synthases and in the dimer form it literally is able to grab these two Because it's important if you don't stop them. They'll start going in reverse in hydrolyzing ATP Right and so that that that would be a bad thing So this thing says stop is the moment the pH changes your blood is Is restricted lactic acid builds up this thing changes into a dimer grabs these things they stop Grabs the camshaft actually you can see here. It's camshafty shaped So this is what happens to protect help protect you that way you can at least have a little bit of ATP to play around with Okay, so something that we'll say throughout the semester is this concept of energy charge And this is defined here with this equation You take all possible forms of these adenylates the triphosphate diphosphate monophosphate Right, and then you look at the fraction of the forms of that molecule that are generally used in biochemical reactions Generally, it's just ATP is used and so here it has ATP Okay, that's useful and it has one half ADP The reason it has one half ADP is because you can there's a Reaction that works close to equilibrium starting from the standard state where you can convert to ADP molecules into one ATP Useful molecule and one A and P not useful molecule. So you get half so this is the amount of Useful ATP equivalents in the cell and in general in healthy scent cells This is maintained through allosteric regulation of all the enzymes and glycolysis and Krebs cycle to be about point nine So it's about tenfold access in your cells of ATP over ADP that helps to push reactions for because the Cellular state is more exergonic ATP hydrolysis is more exergonic because it's ATP is tenfold higher in Concentration and so this Energy-charged level set point is affected by the allosteric regulation occurring throughout all of these pathways So remember allosteric regulators bind to a site other than the active site in that Regulatory site can have whatever binding affinity it wants and through evolution. It became useful To have a ratio of ATP to other forms of these adenylates There was about ten to one to help push processes forward and so all of these regulators are Precisely tuned so the flux through this pathway will maintain this charge if you start running around You need more flux through the pathway if you have you know a big meal You got to do something with all that energy if you're sitting on the couch. You need to regulate this pathway so we've now made it all the way through and We've synthesized 30 to 32 ATP molecules any questions so far This is sort of phase two of this class. There's a lot of information So nature we're just admiring nature's handiwork here. I didn't make it. Oh, it's full Thanks All right Test test test test test test test All right, you turn on everybody vote. Oh, no yet There's people coming through this collisions. It's bloody. It's it's just awful Everybody vote You turn it off All right, that's boot Which one is it You guys did very well on this Congratulations, even when it wasn't Google Bowl congratulations