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How a Rotating Enzyme Makes ATP |
April 1, 1999. |
Every day, your body synthesizes approximately its own weight in ATP, a molecule which serves as the energy currency of its cells, fueling biosynthesis, muscle contraction, and all the other work that cells do. The enzyme which carries out this ATP synthesis has long been a mystery to biochemists, who have only recently began to understand its very unusual mode of action.
The Bacterial Flagellar Motor. To understand the very unusual enzyme that manufactures ATP in your cells, it helps to first consider what may seem a very different kind of protein, the ATP-powered flagellar motor of bacteria. Bacteria swim about by rotating one or more long ropes of protein called flagella, whipping them round and round in the water like propellers. Each flagellum is attached at its base to the "drive shaft" of its motor. The drive shaft extends through the cell wall, ending in a ring of proteins. This ring is set against a second similar ring anchored in the cell wall, like a ring of ballbearings, each protein of one ring touching an opposite partner in the other. The flagellar shaft rotates when the ring at it's base turns with respect to the fixed ring embedded in the cell wall. What powers this rotation? It turns out that the shaft ring proteins are proton (H+) channels. The passage of a proton inward through one of the protein channels of the shaft ring causes the ring to move counterclockwise, each of its proteins sliding past its opposite fixed-ring partner to the next adjacent one. This movement of one protein ring past the other produces true rotary motion.
ATP synthase. When scientists in recent years succeeded in isolating the enzyme that carries out ATP synthesis in your cells, they found that this enzyme, called ATP synthase, is actually a complex of proteins, a molecular machine remarkably similar to the bacterial flagellar motor. Like the flagellar motor, the complex consists of two rings of proteins. An exposed F1 ring is composed of six alternating protein subunits, three alpha and three beta. A second F0 ring is embedded in the mitochondrial membrane. The two rings are connected by a protein shaft called the gamma subunit. Also like the flagellar motor, one of the rings (in this case, the embedded F0 one) acts as a proton channel. ATP Synthesis: Protons passing down a concentration or electrochemical gradient through the embedded F0 ring empower the exposed F1 ring to synthesize ATP from ADP + Pi. Proton Pumping: In the reverse reaction, protons are pumped through the embedded F0 ring up a concentration or electrochemical gradient. This transport is powered by ATP hydrolysis into ADP + Pi by the exposed F1 ring.
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Two rotary motors. (a) The bacterial flagellar motor consists of two rings of protein that slide over one another; a shaft is attached to one of the rings. (b) Mitochondrial ATP synthase is also composed of two opposing rings of protein, one connected to a shaft. Both motors act as proton channels associated with the hydrolysis of ATP.
How does movement of protons down a gradient through the F0 ring cause the F1 ring to synthesize ATP? How does hydrolysis of ATP by the F1 ring cause the F0 ring to transport protons up a concentration or electrochemical gradient? How is the energy communicated between the two rings? The obvious place to look for an answer to these questions is in the gamma subunit that connects the two rings. In 1993, on the basis of extensive data collected by many researchers, Boyer proposed that energy was transmitted from one ring to the other by rotation of the gamma subunit within the center of the F1 ring. Boyer's "binding change mechanism" proposed that proteins of the gamma subunit shaft slid past members of the F1 ring, binding to opposite partners only to have energy cause them to pass along counterclockwise to the next partner in the ring. In effect, Boyer was proposing that the ATP synthase enzyme was a first cousin to the bacterial flagellar motor.
Testing Boyer's proposal is no simple matter. One cannot detect rotation by looking at the average behavior of many molecules. Also, even if you were successful in detecting motion of the gamma subunits, this does not prove that they actually rotated. What is required is a way of watching a single APT synthase complex so that rotation can be directly observed, or ruled out.
The demanding requirements of this test were met in 1996 by a Japanese graduate student, Hiroyuki Noji, as part of his doctoral thesis, reported in 1998. In a very clever series of experiments, Noji first glued F1 rings with their associated gamma subunit shafts to a glass coverslip, using a trick of protein chemistry in which the amino acid histidine is attached to the bottoms (amino-terminal ends) of three of the six proteins of the F1 ring, creating tags that can be made to adhere to the glass. Because the F1 rings are fixed in position, any rotation observed cannot be the result of Brownian (random) movement.
Since rotation of the gamma subunit would be far too small to be observed directly, Noji attached a long filament of fluorescently-labeled actin to the portion of the gamma subunit protruding on the F0 side of the F1 complex. Any movement of these very long actin filaments can be observed directly using a fluorescent microscope.
As you can by now appreciate, Noji had in a very real sense converted his mitochondrial F1 complexes into the analog of flagellar motors. Just as ATP fuels rotation of the bacterial flagellar motor, and so spins its attached flagella, so ATP would be expected to fuel the rotation of the gamma subunit within the F1 ring, spinning the gamma-attached actin filament. The test was to perfuse ATP into the experimental chamber, and see if the actin filaments rotated.
They did. The actin filaments attached to the gamma subunits rotate in the presence of ATP, and only in the presence of ATP. The direction of rotation was without exception counterclockwise. At very low ATP concentrations, the rotation occurred in increments of 120 degrees, one step per molecule of ATP hydrolyzed, just as would be predicted if the F1 beta subunits each had an ATPase catalytic site, as X-ray diffraction structural studies had suggested. These results provide direct proof of Boyer's rotational model.
The amount of rotational torque requires to rotate the actin filament through the viscous resistance of water can be estimated from the length of the filaments and the rate of their rotation. Noji found that the work required to move the filament through a single 120 degree rotation step, presumably powered by a single ATP hydrolysis, was 80 pN nm, in close agreement with the amount of free energy released by the hydrolysis of a single ATP molecule.
A motor protein is one that converts chemical energy to mechanical energy, and vice versa. The cells of your body contain many kinds of motor proteins, including the myosin in your muscle, the dynein that powers the waving of cilia, and the kinesin that powers organelle transport along microtubules. In all of these cases, movement is produced by changes in protein shape induced by ATP hydrolysis. Note, however, that in all of these cases the motors produce linear motion. What is remarkable about ATP synthase is that it produces rotary motion. This sort of rotary motor is very rare among living systems. No other rotary motor is known among vertebrates, for example. Indeed, the only other rotary motor described in any animal drives the spinning mouthparts of an obscure group of arthropods!
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Proof the gamma subunit rotates. Fluotescent actin filaments were attached via streptavidin to gamma subunits of F1 complexes immobilized by His tags. When ATP was infused into the chamber, the actin filaments always rotated counterclockwise. At very low ATP concentrations, they rotated in discrete 120 degree steps.
The ATP synthase enzyme is found in the membranes of mitochondria and chloroplasts, and in the plasma membranes of aerobic bacteria, where this enzyme almost certainly first evolved (recall that mitochondria and chloroplasts are thought to be the endosymbionic descendants of aerobic bacteria taken up by early eukaryotic cells). Because of its similarity to the bacterial flagellar motor, and the great rarity of rotary motors among living organisms, it is tempting to speculate that ATP synthase evolved from the flagellar motor. When the DNA sequences of the genes become available, it will become possible to test this hypothesis.