Round and Round We Go, Making ATP

The mechanism of the proton energy pump that lies deep within, and gives us ATP.

One of the more elegant and dynamic structures in biology is that of the ATP synthase, which lies at the heart of the mitochondrion's conversion of its proton / electromotive gradient into ATP. This large enzyme is not static, but functions like a carousel, whirling around as it lets in groups of H+ ions. Naturally, it has been heavily studied to learn the secrets of why such motion is necessary, and how it works, in detail.

Basic view of ATP synthetase components. The lower complex (a,b,c, epsilon,) is  often called Fo, and is embedded in the membrane bilayer, which otherwise keeps H+ protons out. Top is the internal side (of the bacterium or mitochondrion) and bottom is the outside, where H+ has been pumped by the processes of oxidative phosphorylation. The c subunits comprise the spinning rotor, causing the gamma subunit, which reaches up into the F1 (alpha, beta), to crank the non-spinning F1 proteins through a series of shape changes that prompt them to synthesize ATP.

The primary product of mitochondiral respiration, which burns our food in a controlled way using oxygen, is a transmembrane proton gradient, which is a mechanism that mitochondria inherited from their free-living bacterial ancestors. While it may seem odd that pumping protons out of the cell into the vast outside is a way to efficiently store energy, it was the original battery technology, a charged state that can later be used by many other processes, like transporters that couple the energy-releasing import of H+ with the energy-using import of K+, (a symporter), or with the energy-using export of Na+ (and antiporter). Yes, life is all about chemistry!

ATP quickly became an important chemical currency for life, but only small amounts can be made directly from breaking up food molecules like glucose by glycolysis. Much more can be made by carefully tuning the respiratory chain to export protons (or import electrons) during the stepwise transformations of glucose to smaller molecules, and then later using that electrical / proton gradient for other needs such as making ATP.

Thus the ATP synthetase was born, but it was not born in a vacuum. Rather it seems to have been derived from prior structures that used protons to drive flagellar rotation. The tails of bacteria do not wave side to side, but rather rotate, which, given their particular semi-rigid structure, can drive bacteria forward. At the flagellar base is a rotating motor which lets in H+ ions as its energy source. This structure was evidently married with what seems to have originally been a DNA helicase- a donut-shaped ATP-using enzyme that travels along DNA, prying open the double-helix. Such enzymes are necessary during DNA replication and meiosis, of which at least replication was an ancient process. The ATP-using character of this helicase was reversed to be ATP-generating in its new setting, which is biochemically easier than it seems. Indeed, the whole ATP synthetase can still today run in reverse to use up ATP when needed.

More detailed representation of the ATP synthetase. The plasma membrane is in yellow, inside the cell (or mitochondrion) is above, and outside is below. The outside has a higher concentration of tiny water triads, which represent H3O+, or H2O plus a proton. They dock to the blue transmembrane portion (Fo) of the enzyme, as shown in green and red at the key interface. The protons are handed off to coordinate with portions of the protein in highly regulated fasion. Above, ADP comes into the synthetase part of the enzyme (F1), gets a phosphate group added (yellow and red) to become ATP. 

Two videos of this process are linked above. #2 is accompanied by a great submersible soundtrack, and shows greater detail for the ATP synthesis mechanism. And a video from the paper discussed has even higher detail, showing particularly the extensive structural reshaping that goes on within the F1 subunits that are making ATP. That mechanism could be the subject of another post.

The mechanical details are that H+ is let in only at the interface of the rotating (blue) and stationary (red) parts of the Fo membrane portion of the enzyme. It is allowed in to bind only at one site per segment (green dots), which then rotate around and eventually come back to another part of the stationary part where the H+ is finally let out via a different channel, into the cell. This specific directionality of binding and release is a sort of ratchet which lets the chemical energy in the H+ gradient drive rotational motion.

This energy is then coupled to the second element, the top (F1) complex, where the six red/pink ATP synthase subunits surround the blue shaft which comes up from the rotating Fo component. While those subunits are held stably by the orange stator element at the outside, the blue shaft is like a washing machine rotor that wrenches around, distorting each of the ATP synthase subunits in turn in ways that induce them to carry out the reaction of adding a phosphate group to ADP to form ATP.

A recent paper describes new structural and mutational studies on this enzyme complex, to look for some further mechanistic details. It used primarily cryo-electron micoscopy, which is sufficient to resolve shapes of helices in proteins, though not detailed atomic locations. Yet one can combine this with mutations, prior X-ray structural studies, selective inhibitors, and other modeling to make some interesting inferences. The key one which these authors make is that there is a critical arginine (R) amino acid in the (a) subunit, or the static red part of Fo above (orange in the diagram below), which seems to be the key to H+ conveyance. This amino acid tends to be positively charged, so it would readily bind with an aspartic acids coming along as part of the (c) subunits, popping off their bound hydrogens. It is also genetically essential, as mutations are lethal.

The proposal is that this arginine is specially exposed to the internal side of the membrane, via a channel, (curved arrow leading upward to the cytoplasm in the diagram below), and also positioned such that it can latch onto aspartic acids the blue (c) subunits after rotation. These aspartic acids (D) are carrying the protons that came in via the complementary channel from below (outside) before they started their trip around the carousel. All that time, the membrane has protected those protons from displacement by other chemicals, such as water, other proteins and amino acids, etc.

Image of the proton-conducting interface within the Fo subunit of the ATP synthase. The static (a) subunit is portrayed in orange at rear, while the passing set of c subunits (there are ten of them) are going by in front as a pink band, though dimmed for clarity. The c subunits are progressing from right to left, with hydrogens coordinated on the key aspartate (D) residue #61 on each (c) subunit of the rotating set. As they pass, they are captured by the arginine (R) residue on the static A subunit and released in the only direction possible, up the chute into the cell. Immediately thereafter, the c subunit band passes to another channel where protons can load up again from the external solution.

Those protons are portrayed above by the band containing "c D61", indicating an aspartic acid (D) on a (c) subunit's 61st coded position, whose proton could be displaced by the arginine on the other (a) subunit as it is going by. The gray band of (c) subunits is travelling towards the left, so the idea is that aspartic acid-coordinated protons coming in from the right hit the blue arginine first, where the aspartate and arginine, with their different and complementary charges, bind directly and immediately, releasing the coordinated H+ which then shoots right up the channel to the cytoplasm. At the very next position, protons come in from the outside (periplasm) to bind to the just-vacated D61 spot on the (c) subunit. It is an elegant and very spare, atomic and electrochemical ratchet.

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