ATP Biosynthesis

I’m baaaack! And I bring to you what I consider to be the most interesting thing we’ve covered in this test period—ATP synthesis!

If you’re like me, you’re thinking, “It’s about freaking time… isn’t that the whole purpose of all of this stuff we’ve been talking about?” To which I reply, right you are, dearest reader! This whole thing is really important!

My thought process seems really disjointed right now. It’s probably Firefox’s fault, for taking ten minutes to open up WordPress properly…

(That, and there’s a distinct lack of sunlight today. I’m a little ticked off at a certain yellow orb. Not naming names…)


If you think back to the ETC, you’ll remember that, for every two electrons we moved from NADH to oxygen, we moved ten protons from the mitochondrial matrix to the inner membrane space. I did say, then, that that would be relevant later.

Well, later has finally come.

As you know, the whole function of the ETC is essentially to move those protons from the matrix to the inner membrane space using the energy we generated from oxidizing glucose into carbon dioxide. Now, the fact that we’re taking all of the energy from our food and using it to pump those protons would imply that they have a pretty vital role in the generation of ATP.

How, you ask, is that possible? Well, moving those protons from one side of the membrane to the other generates energy in two forms—a concentration gradient, and an electrical gradient. The energy in the concentration gradient is dependent on the ratio of protons on one side of the membrane to the other side, while the energy in the electrical gradient is dependent on the charge of the ions and the potential difference between the different sides of the membrane. Together, these two values describe something called the proton-motive force (PMF), which is about 200 mV for a mitochondrion (170 mV electrical and 30 mV chemical).

When we plug in real numbers (which I’m not showing here because formatting on WordPress isn’t fun), what we find is that, for every mole of protons that move from the outside of the matrix to the inside, we release 20 kJ of energy. Where do you suppose that energy is going? Yup! Straight into ATP synthesis! This is called the chemiosmotic hypothesis, and it’s quite different from what you might expect—instead of manipulating chemical properties of intermediates to accomplish large-scale substrate-level phosphorylation, our mitochondria use an electrochemical gradient to force ADP and Pi to condense into ATP straight-up.

Okay, wait, slow down. How is this accomplished? Well, wonderful reader, there’s this amazing enzyme called ATP synthase, and it does a really cool thing.

On a broad scale, what happens is that protons flow through ATP synthase from the inner membrane space to the mitochondrial matrix. This releases 20 kJ/mol of energy, which is harnessed by the enzyme to make ATP through condensation of ADP and Pi in a way that’s completely spontaneous.

If you’re feeling kind of skeptical about this, allow me to tell you about these things called uncouplers. These are organic acids that are soluble in the inner mitochondrial membrane. When these are present, they can pick up protons from the inner membrane space and move them to the matrix without the protons having to travel through ATP synthase. When this occurs, oxygen is still consumed (since the ETC is still pumping protons into the inner membrane space), but ATP isn’t made.

(By the way, this is actually something that occurs in brown fat, which is present in hibernating animals. Decouplers make it so that the energy from the proton gradient isn’t put into making ATP, which means it gets released in a different way—as thermal energy, which keeps the animal warm.)

Now, you’re probably sitting there trying to reconcile the energy released by a proton moving from the outside to the inside with the energy that’s required to make a molecule of ATP. If you’re using that number I just gave you, you’re coming up with a minimum energy requirement of three protons moving from the outside of the matrix to the inside to make one ATP (assuming that ATP is worth 50 kJ/mol). That number’s right, but we more often go with four protons instead. Why?

Well, keep that number in the back of your mind, ’cause I’ll come back to it. It’ll make a lot more sense once we talk about the mechanism of this reaction. However, before we do that, we should really talk about what makes up ATP synthase.

ATP synthase is made up of two domains, the F0 domain and the F1 domain. The F0 domain, also called the shaft, contains fifteen subunits of three types: an a subunit, two b subunits, and twelve c subunits. The c subunits are helical, and are arranged in a barrel-like apparatus within the inner mitochondrial membrane. The a subunit contains separate proton entry and exit ports, and interacts with the c subunits. The b subunits will come in later, so hang tight for a minute.

The F1 domain contains nine subunits of five types: three α, three β, and one δ, ε, and γ. The three α and β subunits are joined together into three α/β subunits that carry out ATP synthesis. These are held stationary (whaaaaat?) by a stator, which is made of b and δ subunits. Finally, a γ/ε stalk, which is positioned in the center of the c subunits, is embedded in the α/β dimers.

Now, I mentioned that the α/β dimers are the site of ATP synthesis. To understand how this occurs, we first have to understand the binding change mechanism.

You see, here’s what’s up: there are three α/β dimers in each ATP synthase, but they are all in different conformations at any given time. This is important because every conformation has a different affinity for ATP and ADP + Pi. The Open (O) conformation binds ADP and Pi loosely, and has very little affinity for ATP. The Loose (L) conformation binds ADP and Pi reasonably well. The Tight (T) conformation binds ATP very well. When protons flow through ATP synthase, each dimer changes its conformation; open becomes loose, loose becomes tight, and tight becomes open.

So, how do protons manage to cause such a large-scale conformation change? Well, that’s where the complicated setup of the enzyme comes in. Here’s how it works:

A highly conserved Asp61 (negative charge) on a subunit near the subunit interacts with a highly conserved Arg210 (positive charge) on the a subunit, forming a salt bridge. A proton enters the entry port of the a subunit and binds to the aspartate, neutralizing its negative charge (COO becomes COOH). This weakens (for our purposes, eliminates) the interaction between the c subunit and the a subunit. Meanwhile, a proton bound to the c subunit nearest the a subunit’s exit port (this subunit neighbors the one that just received the proton) exits into the mitochondrial matrix, regenerating a negative charge on Arg. This c subunit is attracted to the Asp on the a subunit, and the whole ring of c subunits turns 30º so that it can form a salt bridge with the Asp.

Now, as the ring of c subunits rotates, the lower part of the γ/ε stalk also rotates. However, since the top of the stalk is embedded in the α/β dimers, it doesn’t turn as freely as the c ring. It’s only  after four protons flow through the enzyme that the stalk rotates a full 120º (at all once). This causes the crook in the stalk to point to a different α/β dimer, and the conformations of all of the dimers change.

When the conformational change occurs, each dimer performs its function. The O-site first ejects its ATP (which was formed when the dimer was in the T conformation), then binds ADP and Pi. The T site (previously the L site) forms ATP through the elimination of water. This occurs due to the conformation of the enzyme; the enzyme stabilizes water and correctly positions both ADP and Pi in such a way that the condensation reaction becomes fast and reversible as long as the dimer is in the T conformation. Once four more protons flow through the enzyme, the conformations change again, and this ATP is released.

Has your mind been smashed into a million little pieces? If not, here’s a video that animates the whole thing. It’s amazing enough that it’s got its own slide in my professor’s notes.

Okay, I trust that now you are sufficiently awestruck. You see now why I like this enzyme so much—it makes electrochemical energy (a proton gradient) into mechanical energy, and it uses the mechanical energy to make an environment in which the chemical reaction that we want to take place requires no chemical energy.

This is the whole picture. This is how it all fits together. Glucose to pyruvate, pyruvate to Acetyl-CoA, Acetyl-CoA to carbon dioxide and NADH (and GTP), NADH to proton pumping. Freaking amazing, huh?

Now, let’s put all of it together. Assuming that each mitochondrial NADH pumps ten protons from the matrix to the inner membrane space, and assuming that it takes four protons to make one ATP, each NADH is worth 2.5 ATP. Knowing that each CoQH2 from FADH2 results in six protons being pumped into the matrix, we find that each FADH2 is worth 1.5 ATP.

Okay, let’s add it all together. In converting glucose to two pyruvate, we make two NADH and two ATP. Converting each pyruvate to Acetyl-CoA produces another NADH (for a total of four NADH per glucose molecule). Running each Acetyl-CoA through the citric acid cycle produces three more NADH (2*3 + 4 = 10 NADH), an FADH2 (two per glucose), and a GTP (two per glucose).

Assuming 2.5 ATP/NADH, we get 25 ATP when we cash in all of our NADH. When we add in our ATP from glycolysis, our GTP (which is basically an ATP) from the citric acid cycle and the 1.5 ATP we get for the FADH2 (2*1.5 = 3), we end up with 25 + 3 + 2 + 2 = 32 ATP per glucose.

Turn out this isn’t entirely accurate. More recent measurements suggest that, since some of the electrons from the NADH produced in glycolysis are brought to the mitochondrion by CoQ, the real number we should go with is something closer to 30 ATP per glucose molecule.

Still, can I get a heck yeah! for aerobic respiration? Or, alternatively, a lol, glycolysis, you just got rekt!? After all, that’s 15 x more energy than we get from glycolysis alone. And, get this: we know from combustion that this reaction is 50% energy efficient. That’s a lot better than whatever the heck glycolysis is doing over there in the cytosol.

[ahem] Right, so, as awesome as this whole thing is, we don’t want it running all the time. After all, when our cell has enough energy, there’s really not a reason to be making more of it. (I like to think this is much like me trying to drink caffeine recreationally—it’s just a bad idea.) Turns out, the citric acid cycle, the ETC, and ATP synthesis are all stimulated by low energy charge (high ADP) and low reducing potential (high NAD+). When ATP and NADH levels rise, respiration slows down, and so does energy production.

And, of course, if you’re worried about all of that ATP getting out of the mitochondrial matrix, don’t be; our cells have got it covered. ADP-ATP Translocase facilitates movement of ATP out of the matrix (and ADP into the matrix) by taking advantage of the charge difference between the two species. (I mean, it’s not that surprising that ATP, the more negative species, wants to move into the inner membrane space, anyway.) We also have a Phosphate Translocase, which moves Pi into the matrix by moving a proton with it (again, taking advantage of that gradient). Pyruvate/-OH and Citrate/Malate have transporters, too; in both cases, the charge gradient is what drives each species to the side of the membrane where it’s needed.

[breathes deeply, inhales Mountain Dew in the process] It’s all so amazing. So freaking amazing.

There it is, everyone. There you have the whole picture of aerobic respiration. I hope you enjoyed it, because I certainly did. Unfortunately, I’m afraid that I’m going to enjoy writing about the pentose phosphate pathway a lot less.

Oh well. I guess it’s kind of important in its own right.


Ironically enough, I’ve written more than enough words to have finished my writing for NaNoWriMo for today. Maybe I can incorporate it somehow.

Questions? Comments? Fangirl-squeeing? Put it below!

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