BIOCHEMISTRY DISCUSSION 7

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So this is where it all comes down to - the proton-motive force. Now that we’ve generated this proton gradient in the mitochondria, how do we turn that into ATP?

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Biochemistry: A Short Course

Fourth Edition

CHAPTER 21 The Proton-Motive

Force

Tymoczko • Berg • Gatto • Stryer

Itapúa Binacional, on the border between Brazil and Paraguay, is one of biggest hydroelectric dams in the world. The dam transforms the energy of falling water into electrical energy. Analogously, the mitochondrial enzyme ATP synthase transforms the energy of protons falling down an energy gradient into ATP. [Christian Heeb/AgeFotostock.]

A useful analogy might be the flow of water through a dam. And we know that water up at the upper level has more potential energy than the water at the lower level because it’s higher. And if you let it go, it’ll flow from the upper level to the lower level. And that’s basically what’s happening with the protons flowing from outside of the matrix to the inside of the matrix. That potential energy is the voltage between them. And just like when you have water flowing through a dam, that water can be used to turn a wheel. And that wheel can be used to power other things, to generate electricity, to ... whatever. You can make energy with it, basically - harness that energy. And that is exactly what’s happening with the proton-motive force. Protons are coming through the membrane (the membrane is your land) and they’re turning a wheel. And that wheel is being used to harness that energy and put it into ATP so that it can be used by the rest of the cell.

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CHAPTER 21 The Proton-Motive Force

So that’s the first thing that we are going to talk about (21.1), and then we’ll talk about how certain shuttles allow things to get across mitochondrial membranes (21.2), and then how the respiration of the cell is actually regulated by the need for ATP (21.3).

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Chapter 21: Outline

21.1 A Proton Gradient Powers the Synthesis of ATP

21.2 Shuttles Allow Movement Across Mitochondrial Membranes

21.3 Cellular Respiration Is Regulated by the Need for ATP

So the proton gradient generated by the oxidation of NADH and FADH2 is called the proton-motive force. And that is what allows the synthesis of ATP, that’s basically the battery that drives these motors. And this was a very controversial idea in the beginning. People didn’t believe that it was really possible. And Peter Mitchell, the one who discovered it and developed this work, actually was the subject of a great deal of derision by his colleagues. But he ultimately won the Nobel Prize for it in 1978, and it’s one of the most important contributions in biology. It’s just an amazing system. And it’s been demonstrated to be true with a number of different experimental systems.

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Section 21.1 A Proton Gradient Powers the Synthesis of ATP

Learning objective 3: Describe how the proton-motive force is converted into ATP.

• The proton gradient generated by the oxidation of NADH and FADH2 is called the proton-motive force.

• The proton-motive force powers the synthesis of ATP.

• Heterologous experimental systems confirmed that proton gradients can power ATP synthesis.

DID YOU KNOW? Some have argued that, along with the elucidation of the structure of DNA, the discovery that ATP synthesis is powered by a proton gradient is one of the two major advancements in biology in the 20th century. However, Mitchell’s initial postulation of the chemiosmotic theory was not warmly received by all. Efraim Racker, one of the early investigators of ATP synthase, recalls that some thought of Mitchell as a court jester, whose work was of no consequence. Peter Mitchell was awarded the Nobel Prize in chemistry in 1978 for his contributions to understanding oxidative phosphorylation.

Figure 21.1 Chemiosmotic hypothesis. Electron transfer through the respiratory chain leads to the pumping of protons from the matrix to the cytoplasmic side of the inner mitochondrial membrane. The pH gradient and membrane potential constitute a proton-motive force that is used to drive ATP synthesis.

So this much you already know - it’s a review basically. Protons get pumped out of the matrix and into the intermembrane space - and then, it establishes a gradient with positive charges on the outside and negative charges on the inside.

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Diagram of the Chemiosmotic Hypothesis

Figure 21.2 Testing the chemiosmotic hypothesis. ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a light-driven proton pump) and ATP synthase are illuminated. The orientation of ATP synthase in this reconstituted membrane is the reverse of that in the mitochondrion.

And here’s a really simple system that gives you a good example of how the protons are converted into ATP. Up at the top we see a bacteriorhodopsin, which is a proton pump which is powered by light. So they put some light on it and it starts pumping protons into the inside of the vesicle (this is a synthetic vesicle that they’ve made). And then you see at the bottom, ATP synthase, that’s the magical molecule - well it is not just a molecule, it’s a huge complex of molecules. But hydrogen ions pass into ATP synthase causing it to rotate, and in its rotation it turns ADP and inorganic phosphate into ATP.

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Diagram of Testing the Chemiosmotic Hypothesis

Figure 21.3 The structure of ATP synthase. A schematic structure of ATP synthase is shown. Notice that part of the enzyme complex (the F0 subunit) is embedded in the inner mitochondrial membrane, whereas the remainder (the F1 subunit) resides in the matrix. [Drawn from 1E79.pdb and 1COV.pdb.]

So let’s look at this ATP synthase. It’s a pretty big and fairly complicated complex, but we’ll take it apart and look at it individually, and I think it will all make sense. There’s basically two different parts:

1. There is the c ring and the gamma and epsilon units, that you see (the red and the green units attached to the blue c ring there). Those all rotate – and the rotation of those is driven by the protons, the proton-motive force.

2. The other part, which consists of the purple part on the left - the a, b2, delta, and then also the orange and yellow parts on the bottom, the alpha and beta - all of that is a separate unit which more or less stays in place. And you see how that c ring would rotate? It rotates that gamma subunit down there in the middle (that’s the red one). And it’s got a long arm that stretches into the alpha-beta complex - and then when it rotates, it kind of nudges the alpha and beta subunits into various configurations which make the ATP get synthesized out of ADP and phosphate. The purple alpha a, b2, and delta parts on the left are very important because they basically hold the alpha and beta subunits in place so that when the c ring spins it

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Diagram of the Structure of ATP Synthase

won’t just spin the whole thing, it actually spins relative to the rest of it. If you take a close look at the “a” purple unit up at the top, looks like a half moon, you can see two channels in it that go from the outside of the matrix to the inside of the matrix. And that’s where the protons go - they go in through that channel, then they move from one channel to the other; and when they move, they drive the c ring to rotate. And then they come out of that channel into the matrix, and that’s how the protons get back into the matrix. Another way of describing this complex is calling it the F0 and F1 subunits, you’ll see that terminology as well. The F0 subunit would be the portion of the complex that’s embedded in the membrane, and the F1 component would be the part that is sticking out into the matrix.

Ok, so at this point I am going to recommend that you pause the presentation and take a look at these two videos. The first one is by a colleague of mine named Graham Johnson, and it’s a great explanation of how this ATPase works. ATPase, by the way, is the same thing as ATP synthase. ATPase means that it breaks apart ATP, and ATP synthase means that it puts together ATP. It actually can do both, so sometimes you might hear it called one or the other. But it is a great explanation of how it works, and is very clear and actually has laboratory footage, amazing footage of the ATPase in action moving around. The second one will really show you the molecular level mechanisms that’s going on inside. And sometimes it’s running in one direction making ATP, and sometimes it is running backwards breaking apart ATP. You just have to understand that’s what is going on while you are watching it. But yeah, it’s incredible, incredible stuff.

So the beta subunits of the F1 component, that’s the part that’s sticking out into the matrix, those are the catalytic parts. Those are the parts that actually interact with ADP and Pi, or inorganic, phosphate and convert them into ATP. And they have three different forms. We can call them the open, loose, and tight (you know, just whatever - names). In the open form, the elements can come in and bind, the loose form sort of closes around them so that they are trapped, and in the tight form, it squeezes them together into the transition state.

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Proton Flow Through ATP Synthase Leads to the Release of Tightly Bound ATP (1/2)

• The three catalytic β subunits of the F1 component can exist in three conformations:

– In the O (open) form, nucleotides can bind to or be released from the β subunit.

– In the L (loose) form, nucleotides are trapped in the β subunit.

– In the T (tight) form, ATP is synthesized from ADP and Pi.

No two subunits are ever in the same conformation because their conformational changes are driven by the rotation of the gamma subunit, and the gamma subunit only points to one of the beta subunits at a time.

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Proton Flow Through ATP Synthase Leads to the Release of Tightly Bound ATP (2/2)

• No two subunits are ever in the same conformation.

• Each subunit cycles through the three conformations.

• The rotation of the γ subunit interconverts the β subunits.

Figure 21.5 ATP synthase nucleotide-binding sites are not equivalent. The g subunit passes through the center of the a3b3 hexamer and makes the nucleotide-binding sites in the b subunits distinct from one another.

So here is a diagram showing a top view of the catalytic beta subunits and alpha subunits and the gamma subunit there in the middle; and the open, loose and tight conformations. The open is in green on the top left, and the loose is in blue on the top right, and the bottom is the tight conformation in yellow. And you can see the gamma subunit has three different faces. Now, I don’t know how accurate that is, as far as, you know, actual mechanism goes, but the point that they’re trying to make, I think, is that it’s the shape of the gamma subunit that changes the interaction with these beta subunits. So there’s one part that sticks out that’s forcing the beta subunit into the tight conformation. There’s one part that is kind of a pit and that kind of lets the beta subunit open up. And there’s a flat part, which closes the door, so to speak, and traps the substrates in that beta subunit. The gamma subunit then rotates, in this case it would be counterclockwise, and changes the conformations of the individual subunits.

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Diagram Depicting How ATP Synthase Nucleotide-Binding Sites are Not Equivalent

Figure 21.6 A binding-change mechanism for ATP synthase. The rotation of the g subunit interconverts the three b subunits. The subunit in the T (tight) form converts ADP and Pi into ATP but does not allow ATP to be released. When the g subunit is rotated counterclockwise (CCW) 120 degrees, the T-form subunit is converted into the O form, allowing ATP release. New molecules of ADP and Pi can then bind to the O-form subunit. An additional 120-degree rotation (not shown) traps these substrates in an L-form subunit.

Here’s a diagram showing what that rotation would look like. And it’s important to keep in mind that these subunits are labeled with color, and that has nothing to do with their conformation. The green subunit is always the green subunit, and it starts out in open conformation in which the ADP and inorganic phosphate are able to diffuse in and out of the open conformation. ATP could also drift in there. Other molecules could also drift in there – so, you know, it’s open. But then, the gamma subunit rotates 120 degrees and it traps ADP and phosphate in there. And if there were another molecule in there that didn’t quite fit, it probably would not be able to rotate because the subunit would not be able to reach the correct conformation. It’s only when ADP and Pi are bound in there that it’s able to reach that correct conformation. Now let’s take a look at the blue. In the upper left hand corner, it’s got ADP and Pi in it already, trapped. After a rotation of the gamma subunit, it forces them into the transition state. This is where the energy of the proton-motive force goes - this is the whole crux of the issue. This is an endergonic process, which means

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Diagram of a Binding-Change Mechanism for ATP Synthase

it takes energy to drive the ADP and Pi together to make ATP, and that energy is the stored energy in ATP that is able to be used later. So that’s what this whole proton- motive force and electron transport chain is all about - getting enough energy to push these two guys together. Now let’s take a look at the yellow subunit. On the upper left we have the ATP / ADP and inorganic phosphate in the transition state. The gamma subunit rotates, and now it’s in an open state. And I am not entirely sure, but I think that it’s set up in such a way that it can only end up with ATP there. I don’t think that the gamma subunit would be able to do a counterclockwise rotation if it were still ADP and Pi. But you end up there in the middle with the ATP in an open conformation. And it’s an open conformation, so the ATP is free to drift out, and that’s what you see over on the top right. And then more ADP and inorganic phosphate will drift in and then the whole process will start over.

Figure 21.7 Direct observation of ATP-driven rotation in ATP synthase. The a3b3 hexamer of ATP synthase is fixed to a surface, with the g subunit projecting upward and linked to a fluorescently labeled actin filament. The addition and subsequent hydrolysis of ATP result in the counterclockwise rotation of the g subunit, which can be directly seen under a fluorescence microscope.

So what we have here is a molecular motor that’s driven by hydrogen ions and it moves the subunits to create ATP. And as we’ve seen, it is possible to see that rotation of the gamma subunit directly, and I hope you watched the video because you got to see that. It is a very famous experiment. In that experiment, I am not sure if this was clear in the video, but they attached the F1 part of the complex to the glass and then had the gamma subunit spin around driven by ATP hydrolysis, so this was actually the reverse reaction that they used to show this.

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Rotational Catalysis Is the World’s Smallest Molecular Motor

• It is possible to observe the rotation of the γ subunit directly.

• Cloned α3β3γ subunits were attached to a glass slide that allowed the movement of the γ subunit to be visualized as a result of ATP hydrolysis.

• The hydrolysis of a single ATP powered the rotation of the γ subunit 120°.

Now let’s talk about proton flow - and this is the one part of this which is kind of mysterious. The protons flow through the F0 component of the ATP synthase, and in the process they turn the c ring. I pointed out in the previous diagram - there is a channel, or rather two half-channels, in that “a” subunit. Protons enter one half- channel from the outside of the matrix, and then they actually travel all the way around the c unit until they get to the other half channel. It’s not that short path, but rather the long way around. And while they’re traveling around, they are interacting with an aspartate residue, which you might remember is negatively charged. So the protons come in, they attach to the aspartate and make it neutral, and they travel around and then they come out the other side. As the c ring rotates, it rotates the gamma subunit and that changes the conformation of the beta subunits allowing ATP to be synthesized.

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Proton Flow Around the c Ring Powers ATP Synthesis (1/2)

• Proton flow occurs through the F0 component of the ATP synthase.

• Subunit a, which abuts the c ring, has two channels that reach halfway into the a subunit. One half-channel opens to the intermembrane space and the other to the matrix.

• Protons enter the half-channel facing the proton-rich intermembrane space, bind to a glutamate residue on one of the subunits of the c ring, and then leave the c subunit once they rotate around to face the matrix half channel.

• The force of the proton gradient powers rotation of the c ring.

• The rotation of the c ring powers the movement of the γ subunit, which in turn alters the conformation of the β subunits.

Figure 21.8 Components of the proton-conducting unit of ATP synthase. The c subunit consists of two helices that span the membrane. A glutamic acid residue in one of the helices lies on the center of the membrane. The structure of the a subunit appears to include two half-channels that allow protons to enter and pass part way but not completely through the membrane.

Here’s a diagram of subunit c on the left, and that’s one of the ring subunits, the c ring. And you can see, in the middle, the glutamic acid sticking out with its two little oxygens. That’s negatively charged. And then, another look at subunit a, with its two half-channels.

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Diagram of Components of the Proton- conducting Unit of ATP Synthase

Figure 21.9 Proton motion across the membrane drives the rotation of the c ring. A proton enters from the intermembrane space into the cytoplasmic half-channel to neutralize the charge on an aspartate residue in a c subunit. With this charge neutralized, the c ring can rotate clockwise by one c subunit, moving an aspartic acid residue out of the membrane into the matrix half-channel. This proton can move into the matrix, resetting the system to its initial state.

Now here’s the trick - and it is a little tricky. But if you look on the upper left, what you’ve got is the a subunit, two empty half channels, and the c units that are associated with those half channels are both negatively charged. The other c subunits of the c ring are all neutral because they all have a hydrogen attached to them. Now, if you look at the c subunits, you’ll see that they’re all embedded in the membrane. And you’ll recall that the membrane is a hydrophobic area.

So in this configuration, the c ring is being buffeted by thermal motions and it’s trying to turn one way or the other, but there’s an energetic barrier. It cannot turn because of the negative charges. Those negative charges don’t want to be out there in the oily interior of the membrane. And so the ring can spin a little bit left, a little bit right, but can’t rotate very far in either direction {see the link for a great illustration of this}.

Now if you look at the second picture, you’ll notice there’s a whole bunch of hydrogen ions on the outside in the inter membrane space, and very few in the

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Diagram Depicting how Proton Motion Across the Membrane Drives the Rotation of the c Ring

inside. Now, hydrogen ions are able to diffuse into either one of these channels. But if you look at the concentration of hydrogen on the outside, you’ll see it’s much more likely that you‘re going to get a hydrogen ion diffusing into the top channel. Also, remember there is a membrane potential - so it’s highly positively charged on the outside and negatively charged on the inside. So there’s actually a force that is driving the hydrogens on the outside toward the matrix. So that hydrogen in the left hand top channel there is most likely to enter.

Now we’ll take a look at the top right diagram, and you can see the hydrogen is now inside the channel at the bottom, and it has neutralized the aspartate. And now the forces, the thermodynamic forces, are buffeting that either way, but it can actually rotate because the negative charge has been neutralized. So it does. It rotates one spot and then exposes the hydrogen attached to the next c subunit to the other channel. In the bottom right, you can see what that looks like.

And there’s still a proton motive force. There’s still a membrane potential. And so, that hydrogen ion is driven out of that channel and into the interior of the matrix, as you can see in the fifth diagram in the bottom center. And then, it diffuses out and we’re back to the place that we started in the sixth diagram, and the process starts over again.

So each time a hydrogen ion enters a channel, the c ring will rotate. It looks like 8 subunits there, or 10 actually, yeah, there’s 10 subunits pictured, so it would rotate 1/10th of the way around. After 10 hydrogens pass through, it would make a complete circuit.

Figure 21.10 Proton path through the membrane. Each proton enters the cytoplasmic half-channel, follows a complete rotation of the c ring, and exits through the other half-channel into the matrix.

So yeah - the hydrogen ions, they travel all the way around the c ring, instead of just going from the one channel to the next.

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Diagram of the Proton Path Through the Membrane

Now remember, we said in that diagram that there were 10 subunits of the c ring, so it took 10 hydrogens to drive it around one complete turn. And if you can remember from before, 1 complete turn would generate 3 ATP. But you can have a different number of subunits in the c ring. If you had 20 subunits in the c ring, then it would take 20 hydrogen ions to drive it around 1 turn and generate 3 ATP.

We vertebrates, all of us, have the c rings consisting of 8 subunits, and therefore it only takes 8 hydrogen ions to generate 3 ATP. And that’s the most efficient one that we’ve come across so far.

Now, a fascinating figure is included in the blurb here, that a resting human being requires about 116 watts of energy to run. So that’s to keep the blood pumping, to generate heat throughout the body, for all of your thinking and your breathing, is about the same amount of energy as a 100 watt light bulb. And I just find that really remarkable. And also a little bit comforting, because, if you recall in the movie The Matrix, they used human beings as their energy source. Well, we don’t really provide very much energy - so that’s probably unlikely to happen.

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Proton Flow Around the c Ring Powers ATP Synthesis (2/2)

• The number of c ring subunits determines the number of protons required to synthesize a molecule of ATP.

• The c rings of vertebrates consist of 8 subunits, making vertebrate ATP synthase the most efficient known.

DID YOU KNOW? A resting human being requires surprisingly little power. Approximately 116 watts, the energy output of a typical incandescent lightbulb, provides enough energy to sustain a resting person.

Figure 21.11 An overview of oxidative phosphorylation. The electron-transport chain generates a proton gradient, which is used to synthesize ATP.

So here’s just a diagrammatic overview of this entire process. And we’re looking at the internal portion of one of these cristae. So even though it looks kind of like the center part is the inside – rather the center part is the outside of the mitochondrion, and the grey bar represents the membrane, and the outside of this is the matrix. Electrons go through the electron transport chain, Complexes I, II, III, and IV. In the process, it’s driving protons into the intermembrane space. And these protons then re-enter the matrix through ATP synthase, in what I think is a poorly drawn diagram, since it looks like the hydrogen ions are going through the center of ATP synthase (which they’re not).

But at the top, you can see ATP synthase rotating (or it would be rotating) and driving the formation of ATP. And that’s about it. This is oxidative phosphorylation. This is where/this is how the electron transport chain generates a proton gradient which is then used synthesize ATP.

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Diagram of an Overview of Oxidative Phosphorylation

So this next bit, I think probably could be a separate chapter. But, you know, they don’t want the chapters to be too small. But it’s a pretty different topic. There are a lot of things that need to get through the mitochondrial membrane. And how do they do that? Well talk about NADH for one thing. If you’ll recall, NADH is generated during glycolysis by the action of glyceraldehyde 3-phosphate dehydrogenase. And, if you recall, we discussed how the NADH needs to be oxidized so that it can regenerate NAD+ and keep glycolysis going. So what happens with the NADH? It’s got two high energy electrons on it, and it’s got to get those over to the mitochondrion so that they can be used to make ATP. This is accomplished by the glycerol 3-phosphate shuttle, and the high energy electrons are transferred to E-FAD, to make E-FADH2.

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Electrons from Cytoplasmic NADH Enter Mitochondria by Shuttles (1/2)

• In muscle, electrons from cytoplasmic NADH can enter the electron-transport chain by using the glycerol phosphate shuttle.

• The electrons are transferred from NADH to FADH2 and subsequently to Q to form QH2.

Figure 21.12 The glycerol 3-phosphate shuttle. Electrons from NADH can enter the mitochondrial electron-transport chain by reducing dihydroxyacetone phosphate to glycerol 3-phosphate. Electron transfer to an FAD prosthetic group in a membrane- bound glycerol 3-phosphate dehydrogenase reoxidizes glycerol 3-phosphate. Subsequent electron transfer to Q to form QH2 allows these electrons to enter the electron-transport chain.

The first step in this process is the processing of the NADH to NAD+ by cytoplasmic glycerol 3-phosphate dehydrogenase. Now, this is not glyceraldehyde 3-phosphate dehydrogenase. That’s the one that generated the NADH to begin with. But it’s very similar. Glycerol 3-phosphate dehydrogenase can transform DHAP (dihydroxyacetone phosphate) into glycerol 3-phosphate. This is a reduction of DHAP, which stores a little energy into the glycerol 3-phosphate.

And then, those two hydrogens and the associated electrons are shifted to E-FAD in the mitochondrial glycerol 3-phosphate dehydrogenase complex, which is associated with the cytoplasmic side of the mitochondrial membrane. Now if you recall, that mitochondrial membrane is the inner mitochondrial membrane, is where the electron transport chain takes place. And the electrons and hydrogens from the reduced E-FAD then get passed along to coenzyme Q, which then goes along and enters the electron transport chain as we’ve studied before.

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Diagram of the Glycerol 3-Phosphate Shuttle

The product of this is dihydroxyacetone phosphate, again. So it’s able to repeat this cycle as many times as necessary. But if you’ll notice, the NADH has been - the electrons have been passed to E-FADH2 and to QH2 and therefore they would continue into the electron transport chain starting with Complex III. That is not as much energy as you would get out of these electrons if they were able to start the process at Complex I. Because Complex I pumps a lot of protons. So this is sort of analogous to Complex II, which takes 2 electrons from FADH2 and turns them into QH2. But the key ingredient here is that this is on the outside of the matrix, and therefore it’s accessible to the cytoplasm. And that’s why this particular complex is necessary.

But is it actually necessary? Well, in the heart and liver there’s a different way for cytoplasmic NADH to get into the mitochondrial matrix. And this is called the malate aspartate shuttle. The glycerol 3-phosphate shuttle is fast, but it comes at the cost of one ATP per NADH. The malate-aspartate shuttle is more efficient, because no energy is lost, so it is better for slow and steady energy consumption.

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Electrons from Cytoplasmic NADH Enter Mitochondria by Shuttles (2/2)

• In the heart and liver, electrons from cytoplasmic NADH are used to generate mitochondrial NADH through the malate– aspartate shuttle.

• The malate–aspartate shuttle consists of two membrane transporters and four enzymes.

So here’s a diagram of the essential elements of the malate-aspartate shuttle. I’m not a big fan of the diagram in the book - I think it’s a little confusing. So this will show you basically what happens.

In the cytoplasm, you have an aspartate molecule. And, if you’ll recall, that’s simply an amino acid with a negative charge. And it can be turned into oxaloacetate. You might recall oxaloacetate from the citric acid cycle, and when we studied the citric acid cycle, we talked about how oxaloacetate can be turned into aspartate, oxaloacetate being one of the raw ingredients that is necessary for aspartate synthesis. Well, this is just the reverse reaction, and it doesn’t require the input of any energy.

So oxaloacetate can then be reduced to malate by the oxidation of an NADH to NAD+. Malate is able to be transported into the mitochondrial matrix because of a malate transport protein. This is actually an antiporter, as they’ll show you in the textbook. It’s simultaneously pumping alpha ketoglutarate out of the matrix, but the important thing is that you’ve got your two high energy electrons attached to your malate. And once they get inside of the mitochondrial matrix, they can give up those two high energy electrons and turn back into oxaloacetate and NADH. And so, the NADH can be processed by the electron transport chain just like any other NADH and you’ve successfully gotten those two electrons across the membrane. Oxaloacetate is then turned back into aspartate, which is then pumped out of the mitochondrial matrix by

another antiporter, which is simultaneously pumping glutamate into the mitochondrial matrix.

So we’ve talked about how you can get electrons into the matrix - how about getting ATP out of the matrix? Obviously a very important thing to be able to do. And also, getting ADP into the matrix. Well there is a single protein that handles both of these, and it is called the ATP-ADP translocase. The first thing it does is that it pumps out an ATP, and then it pumps in an ADP, alternately. So they need to actually alternate. And this reaction, this transporter, is actually facilitated because of the positive membrane potential. You’re going from a -3 charge in the cytoplasm and a -4 charge in the matrix, to a -3 charge in the matrix and a -4 charge in the cytoplasm. And you’ll recall, due to the proton gradient that is created by the pumping of protons out of the matrix, there is a positive charge on the outside. So ATP, with 4 negative charges, is more attracted to the outside than ADP. So this actually drives this reaction.

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The Entry of ADP into Mitochondria Is Coupled to the Exit of ATP

• The ATP–ADP translocase enables the exchange of cytoplasmic ADP for mitochondrial ATP.

• ADP must enter the mitochondria for ATP to leave.

• The translocase is powered by the proton-motive force,

Figure 21.14 The mechanism of mitochondrial ATP–ADP translocase. The translocase catalyzes the coupled entry of ADP into the matrix and the exit of ATP from it. The binding of ADP (1) from the cytoplasm favors eversion of the transporter (2) to release ADP into the matrix (3). Subsequent binding of ATP from the matrix to the everted form (4) favors eversion back to the original conformation (5), releasing ATP into the cytoplasm (6).

Here’s a pretty simple diagram showing how it works. Let’s start in the middle. The cytoplasm is on the top, and this protein is opened up to the cytoplasm. And ADP is then able to go into the active site of the protein, and it causes a conformational change, which opens up the matrix side of the protein and closes the cytoplasmic side. Thus ADP will then diffuse out, and there’s an open spot. ATP can then diffuse in, and when ATP diffuses in, it causes a conformational change to go back to the other conformation such that the protein is open to the cytoplasm again.

So basically, when ADP is bound to this protein, it takes one conformation and when ATP is bound, it takes another confirmation. And in one conformation, the ADP is then exposed to the interior, and in the other conformation, the ATP is exposed to the exterior. So doesn’t really take a whole lot of intelligence or whatever to make this thing work. It’s kind of just very straightforward.

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Diagram of the Mechanism of Mitochondrial ATP-ADP Translocase

Figure 21.15 Mitochondrial transporters. Transporters (also called carriers) are transmembrane proteins that carry specific ions and charged metabolites across the inner mitochondrial membrane.

There are many other transport proteins that work in a similar way. This diagram shows a few of them. The molecules that are listed in the intermembrane space are the ones that are being pumped out of the matrix, and the ones that are listed on the matrix side are the ones that are being pumped in. So obviously, to make ATP, you need to have ADP and phosphate. And you can get those into the inner mitochondrial membrane in exchange for ATP and malate. You can get malate into the matrix by exchanging it with citrate. Phosphate is carried into the matrix in exchange for a hydroxide ion. So those are two other exceptionally important things. And remember, the hydroxide ions will be attracted to the outside because of the positive charge, but you’ll lose a little bit of your proton motive force, because you’re negating the positive charge on the outside there.

So it actually turns out in order to make ATP, you’ve got to burn a little bit of this proton motive force. And so there are some disagreements about what the actual ATP yield of a glucose molecule is when you take into account all of these other incidental costs.

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Mitochondrial Transporters Allow Metabolite Exchange Between the

Cytoplasm and Mitochondria • The inner mitochondrial membrane has many transporters

or carriers to enable the exchange of ions or charged molecules between the mitochondria and cytoplasm.

But just counting the reactions that we’ve covered in glycolysis, the citric acid cycle, and the electron transport chain, we say that there’s about 30 molecules of ATP formed for every glucose that’s completely combusted (turned into carbon dioxide and water). 26 of these are from oxidative phosphorylation and the electron transport chain and ATP synthase. So that’s where you get the majority of your ATP.

By comparison, when glucose undergoes fermentation, in anaerobic situations, only 2 molecules of ATP are generated per glucose molecule, and those are generated directly through the process of glycolysis. That’s enough to keep those organisms alive. So it actually is not an insignificant amount of energy. But our mammalian cells need a lot more energy to run all the things that they do. As a matter of fact, our bodies are using up ATP so fast that we consume about 3 pounds of ATP per hour. That’s 1.36 kilograms. So each ATP that’s created only sticks around for about a minute or two before it’s used, and ATP needs to be continually made and sent out into the cytoplasm for use.

Now all of that transport of ATP takes time. And as a matter of fact, the anaerobic glycolysis is much faster than aerobic glycolysis. Anaerobic breakdown of glucose produces ATP about 100 times faster than aerobic breakdown. Also, you need a continuous supply of oxygen to make it work. So this is one of the reasons that your fast muscles, those that you use when you’re running, for example, rely a great deal on anaerobic glycolysis. The slow muscles, which are the ones that we use when

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Section 21.3 Cellular Respiration Is Regulated by the Need for ATP

Learning objective 4: Identify the ultimate determinant of the rate of cellular respiration. The Complete Oxidation of Glucose Yields About 30 Molecules of ATP

• Of the 30 molecules of ATP formed by the complete combustion of glucose, 26 are formed in oxidative phosphorylation.

• The metabolism of glucose to two molecules of pyruvate in glycolysis yields the remaining four ATP.

• When glucose undergoes fermentation, only two molecules of ATP are generated per glucose molecule.

we’re just standing around - to just keep us upright, basically - those muscles use aerobic metabolism, which means they have to have a lot of mitochondria. One other point I’d like to make is that if you were to light a mole of glucose on fire, it would produce 3000 kilojoules of energy. The process of breaking down that mole of glucose in aerobic metabolism produces around 30 moles of ATP, and that’s less than 1000 kilojoules of energy. So you’ve lost 2000 kilojoules of energy by breaking it down in this way. But you’re controlling the breakdown very carefully, and capturing that energy in chemical ways you can use.

So that’s one of the reasons that you need to use aerobic metabolism. If we were to burn sugar at maximum efficiency, then we’d all burst into flames.

Figure 21.15 Mitochondrial transporters. Transporters (also called carriers) are transmembrane proteins that carry specific ions and charged metabolites across the inner mitochondrial membrane.

Here’s just a little chart - no need to go through it all right now, probably, but it just shows the ATP yield of all the different stages of glucose being broken down. One slightly interesting point, is that this is calculated assuming the transport of NADH by the glycerol 3-phosphate shuttle, which is not always the case. So it’s not easy to say that there is exactly X number of ATP generated for each glucose molecule. It does vary.

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Table 21.1 ATP Yield From the Complete Oxidation of Glucose

Now, there are a lot of places that oxidative phosphorylation can be inhibited. The first one that we’ll talk about is the inhibition of the electron transport chain. So if you break down one of those elements, you won’t be able to pump protons as well out into the intermembrane space, and that will slow down the whole process.

If you were to somehow break down the ATP synthase, that would prevent proton flow from occurring, and then you would have a very strong membrane potential. Remember, things may work - the electron transport chain may be trying to pump protons out, but if you create too much of a gradient, then that’s not going to be working anymore. And so the whole system gets backed up. Uncouplers are a specifically interesting way of screwing up your oxidative phosphorylation. Basically what they do is they just let the protons back in. They do not require them to go through ATP synthase and the proton gradient is immediately destroyed. As soon as the protons go out, they come back in.

So the electron transport chain continues pumping them out, and then they come back in, and this actually generates heat. Usually the force of them coming back in is channeled into the formation of ATP. But when that’s not happening, that force is then turned into heat and this is a strategy that animals can use to keep themselves warm.

You may have heard of brown fat. That’s a kind of fat that we also have in our bodies,

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Clinical Insight: Oxidative Phosphorylation Can Be Inhibited at Many Stages (1/2)

CLINICAL INSIGHT Oxidative Phosphorylation Can Be Inhibited at Many Stages

• Inhibition of the electron-transport chain prevents oxidative phosphorylation by inhibiting the formation of the proton-motive force.

• Inhibition of ATP synthase by inhibiting proton flow prevents electron transport.

• Uncouplers carry protons across the inner mitochondrial membrane. The electron-transport chain functions, but ATP synthesis does not occur because the proton gradient can never form.

• Inhibition of the ATP–ADP translocase prevents oxidative phosphorylation.

in which these uncouplers are functioning. And they, in fact, generate a great deal of heat. But the most important use of this is by animals in hibernation in the winter. They’re not moving around, they’re not generating a lot of heat through their exercise, so they need to use this mechanism to just keep themselves warm so they won’t freeze in the winter.

And then finally, in the end, we see the inhibition of ATP-ADP translocase. That makes a lot of sense. Obviously, if the ATP can’t get out of the mitochondrion, and the ADP can’t get back in, then your ATP-synthase won’t be able to turn anymore. And if it’s not turning, it’s not making any more ATP, and the whole process comes to a halt.

Figure 21.21 Sites of action of some inhibitors of electron transport.

The electron transport chain can be inhibited in several places as well. If you inhibit, somehow, the production of QH2, using molecules like rotenone or amytal, that’s one place you can stop the electron transport chain. Antimycin will stop it after Complex III, and if you look at the end - that’s cyanide, azide, and carbon monoxide - those will block cytochrome c oxidase, which is Complex IV. All three of these interact with the iron in the middle of the heme in Complex IV and interfere with its conduction of electrons. Incidentally, they also interact with the heme in hemoglobin and cause problems there.

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Diagram of the Sites of Action of Some Inhibitors of Electron Transport

Remember - Complex I is one of the most important elements of the electron transport chain, and also one of the largest, and most complicated. So it’s kind of natural that disruptions of Complex I would be very common, more common than disruptions of the other elements of the electron transport chain. And remember, if the electron transport chain’s not working, you’re not only going to stop making ATP, but you’re going to make a bunch of reactive oxygen species which can do a whole bunch of damage to your mitochondria. The results of which we have discussed before.

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Clinical Insight: Mitochondrial Diseases Are Being Discovered in Increasing Numbers

CLINICAL INSIGHT Mitochondrial Diseases Are Being Discovered in Increasing Numbers

• Disruption of Complex I is the most common cause of mitochondrial disease.

• Defects in the components of the electron-transport chain not only reduce ATP synthesis but also increase the amount of reactive oxygen species formed, leading to increased mitochondrial damage.

Figure 21.22 The proton gradient is an interconvertible form of free energy.

Well now that we’ve studied proton gradients, you’ll be happy to know that they don’t apply only to ATP production, but they’re in fact a very common biological process. They occur in heat production, NADPH synthesis, flagellar rotation, active transport, and several other mechanisms. So this is one of the fundamental mechanisms of biochemistry.

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Diagram Depicting the Proton Gradient as an Interconvertible Form of Free Energy