ATP synthase, also called FoF1ATPase, is a rotary motor enzyme. This enzyme is found in the inner membrane of mitochondria, the analogous thylakoid membranes of chloroplasts, and in the cell membrane of bacteria. The enzyme consists of two parts, the membrane bound Fo which is a proton translocator, and the F1 part which has catalytic activity. The F0 part can be considered to be a rotary electrical motor powered by proton flow, while the F1 part acts as a rotary chemical motor powered (in reverse) by ATP hydrolysis. The enzyme is reversible. If protons flow down a concentration gradient through Fo, ATP is synthesized by F1. Alternatively, ATP hydrolysis by F1 leads to transport of protons through Fo and against a concentration gradient. Isolated F1 can only break down ATP, and not synthesize it.
Summer 2017: The following Jmol links contains multiple views of the complex. It is repeated below.
Jmol: FoF1 ATPase aka ATP Synthase Jmol14 (Java) | JSMol (HTML5)
The F1 subunit (with quaternary structure of a3b3 forming a hexagonal ringed structure with a central cavity,occupied by a gamma subunit)is about 80 angstroms from the Fo subunit and both are connected to the rod-shaped γ subunit which spans the center of the a3b3 ring. Energy transduction (necessary to capture the negative free energy change associated with the collapse of the proton gradient to drive the positive free energy change for ATP synthesis) occurs between the two subunits. Noji investigated the structural changes in the γ subunit, wishing to get direct experimental evidence for Boyer's three-state conformational model (L-O-T) for ATP synthesis.
Figure: Boyers three-state conformational model (L-O-T) for ATP synthesis
In this model, the F1 subunit exists in three states: an O - open - state with very low affinity for substrates and has no catalytic activity; a L - loose - state with low affinity for substrates and also no catalytic activity, and a T - tight - tight state with high affinity for substrates and with catalytic activity. The F1 subunit consist of three α and three β subunits, which can cycle between three conformations, bind substrate, and have catalytic activity. The collapse of the proton gradient (i.e. the proton-motive force) causes the γ subunit to rotate like a crankshaft relative to the F1 subunit, forcing the β subunit to change conformation from the T to the O (releasing ATP) and then the L (binding ADP and Pi). The γ subunit does not appear to undergo any significant conformational change on ATP hydrolysis as evidenced by tritium exchange studies of amide protons. To prove that the γ subunit rotates, you'd have to observe a single molecule. Since the γ subunit was too small to visually discern its rotation, Noji covalently attached a fluorescein-labeled protein filament called actin to the γ subunit (near where Fo would bind). He then fixed the whole F1 molecule to a glass slip through the a3b3 part, immobilizing that part of the molecule. The γ subunit was free to rotate, which could be detected by observing the fluorescence under a fluorescent microscope from the attached actin filament.
Figure: fluorescein-labeled protein filament called actin to the γ subunit
The actin filament rotated only in the presence of ATP. It rotated only counterclockwise, and continued for 10 minutes. This demonstrated that the motion was not random, but a specific motion of the γ subunit. At extremely low concentration of ATP, rotation occurred only in 120o increments, implying one step per molecule of ATP hydrolyzed. (Remember the β subunits are separated by 120o ). As the rotation occurs, there is viscous resistance to movement of the actin filament. He calculate that for a single 120o step caused by hydrolysis of a single ATP molecule, the amount of work was 80 piconewton which is about the free energy of hydrolysis of a single ATP molecule. Incidentally, Boyer was awarded the Noble prize in Chemistry in 1997 for his work. Noji and his colleagues (Nature, 410, 898 (2001), replaced the actin filament with a smaller colloidal gold bead (40 nm diameter) with less frictional resistance to movement and used laser light scattering to probe the rotation of the fixed F1 subunit through the γsubunit.
Figure: smaller colloidal gold bead (40 nm diameter) with less frictional resistance
At low [ATP], the motor rotates in 120o steps. At high [ATP], the rotation rate becomes continuous and saturates (with Michaelis/Menten kinetics) at 130 revolutions per second.
Figure: rotation rate becomes continuous and saturates
Recent experiments (Wantanbe, 2010) using immobilized ATPase and magnetic tweezers have addressed the timing of substrate binding and product release when the enzyme is run in reverse (ATP hydrolysis). On rotation of the gamma subunit, the three binding sites change properties. In hydrolysis, ATP binds to the open site, and helps promote the 120 degree rotation. In the next step, ATP is hydrolyzed. In final step, products dissociate. Pi dissociation occurs last from the third site. Hence each of the 3 beta binding sites have different roles. One binds substrate, one performs catalysis, and third releases products. Assuming the synthesis pathway is the reverse of the ATPase reaction, the final release of Pi in ATP cleavage predicts that Pi binds first in the synthetic direction. This would preclude the binding of ATP next which is critical since its concentration during synthesis can be 10x higher than that of ADP. As Pi is bound first, only ADP, not ATP can bind next.
The gamma subunit rotation plays a "catalytic" role as its rotation induces cyclic conformational changes in the beta subunit of the synthase. Can ATP synthesis occur without the gamma subunit by a mechanisms which involves a less proficient, but concerted set of cyclic change in beta subunit conformation? Apparently it can. Uchihashi et al have used high speed atomic force microscopy (AFM) to study the (alpha-beta)3 hexagon from the F1 subunit without the gamma subunit. They found that upon ATP hydrolysis, the beta subunits underwent conformational changes in the same counterclockwise rotary direction as when the gamma subunit was present.
Figure: AFM Study of Conformational Changes in F1 "gammaless" subunit
These experiments conclusively show that the F1 subunit is effectively a rotary motor with the gamma subunit acting as a rotor in the stationary hexagon ring composed of the 3 pairs of alpha/beta subunits which acts as the stator (stationary part of an electric rotary motor).
Jmol: FoF1 ATPase aka ATP Synthase Jmol14 (Java) | JSMol (HTML5)
The actual amino acids involved in the mechanism of ATP synthesis/hydrolysis are still not clearly defined but Glu 190 on the beta subunit clearly acts as a general base. The figure below shows bound ADP and the proximity of Glu 188. Ala 158 is thought to move towards the active site after a conformational change, with the nonpolar methyl side chain displacing an adjacent water molecule which could leave as a product of ATP synthesis.
Proton Gradient Collapse and ATP synthesis - Structure
The mechanism by which the proton gradient drives ATP synthesis involves a complex coupling of the F0 and F1 subunits. A more detailed image of the whole ATO synthase complex is shown below.
Closer views of the c subunits and a yellow rectangle representing the a subunit (missing in the combined crystal structure) comprise the Fo part of the complex are shown below. These subunits reside in the inner membrane of the mitochondria (or cell membrane of bacteria) and are involved in proton transport from matrix (or cytoplasm of a bacteria) to the inner membrane space (or periplasmic space of bacteria). The multiple c subunits consist of two very hydrophobic helices connected by a loop in a helix-loop-helix motif.
7/127/17: The following Jmol links contains multiple views of the FoF1ATPase. It is repeated several times below.
Two classic inhibitors (structures shown below) of ATP synthase interact with the Fo subunit. One, oligomycin A, binds between the a and c subunits and blocks proton transport activity of the Fo subunit. Oligomycin A sensitivity requires, paradoxically, OSCP (Oligomycin-Sensitivity Conferring Protein which is analogous to the bacterial delta subunit), a stalk protein subunit distal to Fo which couples Fo and F1. Another inhibitor, dicyclohexylcarbodiimide reacts with a protonated Asp 61 in c subunits of F0. It does so even at pH 8.0 which indicates that the pKa of the Asp 61 is much higher than usual. This might occur if the Asp is a very hydrophobic environment. The modification of one As 61 in only one c subunit is necessary to stop Fo activity. The protonated carboxyl group donates a proton to a N atom in DCCD, which then reacts with the deprotonated Asp to form an O-acyl isourea derivative.
The figures below shown the structure of the ac complex from E. Coli. Protons flow to the a chain Arg 210 which is between two Asp 61 on adjacent c chains. One of the Asp 61 is protonated allowing it to alter conformation and essentially rachet in the membrane domain in a motional faciliated by the development of a neutral protonated Asp.
Protons from the inner membrane space or in the periplasmic space (in the above figures) then flow from the periplasm by forming a "handshaking" proton transfer relay which delivers another proton to the deprotonated Arg 210 allowing the circular ratching of the c subunits in the membrane to continue. A set of polar residues entirely within subunit a, including Gln 252, Asn 214, Asn 148, Asp 119, His 245, Glu 219, Ser 144 and Asn 238 provide the path as illustrated below.
When a proton is passed to the unprotonated Asp 61, a conformational change in the protonated c subunit occurs. This leads to changes in c subunit interactions which seems to ratchet the c12 core. Since the c12 oligomer contacts the γsubunit connecting the Fo stalk and F1 ATPase units, the γ subunit rotates, leading to sequential conformational changes in each of the 3 contacted (αβ)2 dimers of the F1 enzyme. This leads to changes in ATP affinity through cycling each through the L, O, and T conformations.
Reprinted by permission of Nature. Rastogi & Girvin. Nature 402, 263-268 (1999) Copyright 1999 McMilllan Publishers LTD
XVIVO: Scientific Animations
ATPase animations from the MRC
In summary, FOF1ATPase (or synthase) is a rotary enzyme that ultimately couples collapse of a proton gradient (a chemical potential gradient which contributes to the transmembrane electrical potential) to a chemical (phosphorylation) step. The rotor, which is in contact with both the FO proton pore, and the F1 synthase, moves with respect to both subunits, which couples them. Motion of course is relative so the rotor can be thought of as static with the FO and F1 subunits as rotating. The FO pore can hence to be considered an electrical motor and the F1 synthase a chemical motor. Carrying the analogy of a motor even further, the FO electrical motor turns the F1 chemical motor into a generator, not of electricity but of ATP. The figure and link below, taken from the Protein Data Bank, go into more depth about this nanomotor.
Experimental evidence shows that it can. The FoF1ATPase complex can be removed from membranes and placed in a liposome into which ADP and Pi have been encapsulated. The pH of the outside of the vesicles is then lowered several pH units. Under these circumstances, ATP is generated inside the vesicle proving that a gradient alone can drive its synthesis.
Proton Gradient Collapse and ATP synthesis - Thermodynamics
Mathematical analyses show that it can as well. Consider a typical pH gradient (-1.4 pH units) across the inner membrane of respiring mitochondria (with the outside having a lower pH than inside making the inside more depleted in protons). Clearly there is a chemical potential difference in protons across the membrane. However, another factor determines the thermodynamic driving force for proton translocation across the membrane. A transmembrane potential exists across the inner membrane of the mitochondria, as it does across most membranes. The source of the membrane potential will be discussed in signal transduction chapter. The inside is more negative than the outside, giving the membrane a transmembrane electrical potential. of about -0.14 V. Clearly, protons would be attracted to the other side of the membrane (into the matrix) by this potential difference, which then augments the chemical potential difference as well. A simple mathematical derivation shows that indeed, a proton gradient can supply enough energy for ATP synthesis, especially when coupled to a transmembrane electrical potential.
Figure: A simple mathematical derivation
The sum of the electrical and chemical potentials are called the electrochemical potential, which when divided by nF gives the proton motive force.
Note: In the above discussion, we dealt with two different proton translocating methods:
- Complex I, III, and IV, which couple uphill proton movement (from the higher pH matrix to the lower pH intermembrane space) to oxidation (NADH + O2 to NAD+ + H2O).
- Downhill movement of protons through F0F1 ATPase which couples to ATP synthesis by the enzyme.