ATP SYNTHASE 1997 Nobel Prize was for the enzymatic mechanism underlying the synthesis of adenosine triphosphate 

Its function

There are several ATP synthase enzymes and they have been remarkably conserved during evolution. The bacterial enzymes are essentially the same in structure and function as those from mitochondria of animals, plants and fungi, and the chloroplasts of plants. The early ancestery of the enzyme is seen in the fact that the Archaea have an enzyme which is clearly closely related, but has significant differences from the Eubacterial branch. The H+ATPase found in vacuoles of the eucaryote cell cytoplasm is similar to the archaeal enzyme, and is thought to reflect the origin from an archaeal ancestor.

In most systems, the ATP synthase sits in the membrane (the "coupling" membrane), and catalyses the synthesis of ATP from ADP and phosphate driven by a flux of protons across the membrane down the proton gradient generated by electron transfer. The flux goes from the proton chemically positive side (high proton electrochemical potential) to the proton chemically negative side. The reaction catalyzed by ATP synthase is fully reversible, so ATP hydrolysis generates a proton gradient by a reversal of this flux. In some bacteria, the main function is to operate in the ATP hydrolysis direction, using ATP generated by fermentative metabolism to provide a proton gradient to drive substrate accumulation, and maintain ionic balance.

In mitochondria, the positive side is the intermembrane space, and the negative side is the mitochondrial matrix; in bacteria, the positive side is the outside the cell and the negative side the cytoplasm; in chloroplasts, the positive side is the thylakoid lumen and the negative side the stroma.

Subunit composition of the ATP synthase

There are minor differences between bacteria, mitochondria and chloroplasts in some of the smaller subunits, which leads to a confusing nomenclature. The simplest system is that from E. coli. The ATP synthase can be dissociated into two fractions by relatively mild salt treatments.

A soluble portion, the F1ATPase, contains 5 subunits, in a stoichiometry of 3a:3b:1g:1d:1e. Three substrate binding sites are in the b-subunits. Additional adenine nucleotide binding site in the a-subunits are regulatory. The F1 portion catalyzes ATP hydrolysis, but not ATP-synthesis.

Dissociation of the the F1 ATPase from the membranes of bacteria or organelles leaves behind a membrane embedded portion called FO. This consists (in E. coli) of three subunits a, b and c, with relative numbers of 1:2:9-12. The c-subunit is very hydrophobic, and forms a helix turn helix structure which spans the membrane twice, with a hydrophilic loop on the side of attachment of F1. There is a conserved acidic residue half-way across the membrane in the C-terminal helix.

After dissociation, the membranes are permeable to protons. The proton leak can be stopped by addition of inhibitors, which are also inhibitors of ATP synthesis in the functional complex. Two "classical" inhibtors are known. The antibiotic, Oligomycin, binds at the interface between F0 and F1; dicyclohexylcarbodiimide (DCCD) binds covalently to the conserved acidic residue in the c-subunit of F0. One DCCD per ATPase is sufficient to block turn-over, suggesting a cooperative mechanism. The action of these inhibitors indicates that the proton permeability of the F0 is a part of its functional mechanism.

Structure of the F1 ATPase

The structure of the soluble (F1) portion of the ATP synthase from beef heart mitochondria has been solved by X-ray crystallography. The pictures below are from Abrahams, J.P., Leslie, A.G., Lutter, R. and Walker, J.E. (1994) Structure at 2.8 resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621-628.

The protein was crystallized in the presence of ADP, and an ATP analogue, AMP-PNP, in which the the two terminal phosphates of ATP were replaced by the non-hydrolysible imidodiphosphate group. The three a-subunits each contained an AMP-PNP. The three b-subunits contained either ADP (bDP), AMP-PNP (bTP), or no nucleotide (bE).


Click on images for large version.


Left: The structure of the F1 ATP-ase, viewed from the side. The a-subunits are shown in yellow, the b-subunits in red, and the g-subunit in blue. The cartoon at the top left shows the orientation. Note that the a-b-subunits alternate in a ring around the g-subunit, which forms a rod up the middle. The a- and b-subunits are differentiated by subscript indicating the occupancy of the active site of the b-subunit of each a- b-pair: E - empty; DP - ADP; TP - ATP analogue, AMP-PNP. Scale bar is 20 .
Right: A vertical slice through the complex across the a-TP/b-DP diagonal highlighted in the cartoon.


Left: A vertical slice through the complex across the a-E/b-TP diagonal highlighted in the cartoon.
Right: A vertical slice through the complex across the a-DP/b-E diagonal highlighted in the cartoon.
Note how the "jaw" of the nutcracker swings open when the site is empty (arrow in picture on right).


View of the complex from the top (N-phase).


Left: A horizontal slice through the complex across the top, showing the b-sheet structure which provides a cap over the catalytic domain. Scale bar is 20 .
Right: A horizontal slice through the complex across the catalytic domain, which is predominantly helical.


Left: View of the calculated electrostatic surface potential of the a, b-sleeve formed by the structure below the b-sheet cap, showing regions of negative (red) and positive charge, and a predominantly neutral (hydrophobic) "hole" in the dough-nut, through which the top part of the g-subunit protrudes. The view is from inside the protein.
Right: Similar surface, but viewed from the side, of the g-subunit, showing a hydrophobic surface for most of the rod, but a marked neatively charged polar region half-way down. The upper part of the rod slides into the sleeve shown in the Fig. to the left, as indicated in cross-section by the ball-and-stick structure. 

Mechanism of the F1 ATP-ase

The ATP synthase operates through a mechanism in which the three active sites undergo a change in binding affinity for the reactants of the ATP-ase reaction, ATP, ADP and phosphate, as originally predicted by Paul Boyer. The change in affinity accompanies a change in the position of the g-subunit relative to the a, b-ring, which involves a rotation of the one relative to the other. In the direction of ATP synthesis, the rotation is driven by a flux of H+ down the proton gradient, through a coupling between the g-subunit, and the c-subunit of F0. This rotation has now been demonstrated experimentally.

Experimental support for rotational model

Biophysical approach

This rotational motion has been captured in dramatic videos from the laboratory of Masasuke Yoshida. In this work, the F1-ATPase was tethered to a glass surface by the b-subunit, using a His-tag engineered into the protein at the N-terminus, and NTA-ligand on the glass. (see illustration from Junge et al. TIBS article, below).

 
The motion was detected by attaching an actin filament to the g-subunit, which was tagged with fluorescent groups to make it visible, and recorded using a video camera attached to a microscope. The motion was seen only under conditions of ATP-hydrolysis, and the direction of motion was always counter-clockwise when viewed from the Fo portion, giving the sign of the catalytic mechanism.

Hiroyuki Noji, Ryohei Yasuda, Masasuke Yoshida & Kazuhiko Kinosita Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature, 386, 299 - 302.

An alternative approach using photometric methods has been explored in Wolfgang Junge's lab. Use of small chromophores attached directly to the g-subunit has the advantage of a time resolution unconstrained by the large torque associated with the movement of the actin filament above. Two methods have been used to explore the dynamics of the system.


Biochemical approach

(The pictures below are from CMB by Gerald Karp, 3rd edit, John Wiley & Sons, 2002.)

  In the above figure showing the binding-change mechanism of Paul Boyer, rotation of the gamma-subunit (dark blue) relative to the a, b-ring (the three a, b-pairs are represented by different shades of green, red, or blue) induces a change in the binding affinities of reactants, as represented here by a change in the conformation of the site on going from left to right in the diagram. ATP forms spontaneously from tightly bound ADP and Pi. The mechanism was proposed before the structure was known, so the structure provides a nice confirmation of the model. The Open site corresponds to the Empty site of the structure, the Tight site to the ATP site, and the Loose site to the ADP site.